Dendritic cells restore CD8+ T cell reactivity to autologous HIV-1.

Dendritic Cells Restore CD8+ T Cell Reactivity to Autologous HIV-1

Updated information and services can be found at: http://jvi.asm.org/content/88/17/9976 These include: SUPPLEMENTAL MATERIAL REFERENCES

CONTENT ALERTS

Supplemental material This article cites 79 articles, 41 of which can be accessed free at: http://jvi.asm.org/content/88/17/9976#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://jvi.asm.org/ on August 23, 2014 by NEW YORK MEDICAL COLLEGE

Kellie N. Smith, Robbie B. Mailliard, Brendan B. Larsen, Kim Wong, Phalguni Gupta, James I. Mullins and Charles R. Rinaldo J. Virol. 2014, 88(17):9976. DOI: 10.1128/JVI.01275-14. Published Ahead of Print 18 June 2014.

Dendritic Cells Restore CD8ⴙ T Cell Reactivity to Autologous HIV-1 Kellie N. Smith,a,b* Robbie B. Mailliard,b Brendan B. Larsen,d Kim Wong,d Phalguni Gupta,b James I. Mullins,d Charles R. Rinaldob,c Departments of Microbiology and Molecular Genetics,a Infectious Diseases and Microbiology,b and Pathology,c University of Pittsburgh, Pittsburgh, Pennsylvania, USA; Department of Microbiology, University of Washington, Seattle, Washington, USAd

Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk peripheral blood mononuclear cells (PBMC) yet ignore professional antigen-presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of major histocompatibility complex (MHC) class I-restricted epitopes from HIV-1 Gag and Env were used as antigens in gamma interferon (IFN-␥) enzyme-linked immunosorbent spot (ELISpot) and polyfunctional cytokine assays. Here we show that dendritic cells (DC) enhanced T cell reactivity at all stages of disease progression but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type 1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of DC immunotherapy that aims to awaken a dormant, autologous, HIV-1-specific CD8ⴙ T cell response. IMPORTANCE

Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition and that CD8ⴙ cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.

H

uman immunodeficiency virus type 1 (HIV-1)-specific CD8⫹ T cell responses are effective at imposing immunological pressure in acute infection, as evidenced by the high turnover and mutation rates in virus populations (1–3). However, the failure of cytotoxic T lymphocytes (CTL) to control virus in chronic infection results in progression to AIDS, which can be attributed to several factors. Viral evolution, specifically in CTL epitopes, can interfere with recognition by naive CD8⫹ T cells, resulting in a limited repertoire of T cell-mediated immune responses against the mutated regions (4). In the absence of an effective CTL response that is specific for these mutated epitopes, the virus persists, and disease progression continues (5, 6). To more fully understand the mechanisms of viral pathogenesis and develop effective treatments for HIV-1-infected subjects, we must evaluate how mutations within regions of T cell recognition affect HIV-1specific T cell responses. Alterations in T cell homeostasis during chronic infection largely impact naive T cell subsets and partially result from decreases in thymic output (7–9). Progressive infection is also accompanied by decreases and dysfunction in the naive CD8⫹ T cell subset despite increases in numbers of total CD8⫹ T cells (10, 11). These perturbations in the naive CD8⫹ T cell repertoire could reduce the number and likelihood of mutated epitopes being recognized. This may explain the observed decreases in HIV-1-specific CTL activity in chronic infection, presumably as a result of viral mutations (5, 12). It remains to be elucidated, however, if these responses are not generated or if they are generated but are

9976

jvi.asm.org

Journal of Virology

not detected due to insufficient antigen presentation and/or stimulation in readout assays. While many aspects of the immune system become dysfunctional in chronic HIV-1 infection and remain dysfunctional even when subjects receive combination antiretroviral therapy (cART), myeloid dendritic cells (DC), the most potent antigen-presenting cells (APC), retain the ability to process and present antigen (13, 14) and to stimulate HIV-1-specific gamma interferon (IFN-␥) production in CD8⫹ (15–17) and CD4⫹ (18) T cells. These DC require activation with proinflammatory cytokines and additional stimulation such as that provided by CD40 ligand (CD40L) on activated CD4⫹ T cells. The resulting mature DC express high levels of the T cell costimulatory molecules CD80 and CD86 and the maturation marker CD83 and secrete the proinflammatory

Received 7 May 2014 Accepted 12 June 2014 Published ahead of print 18 June 2014 Editor: G. Silvestri Address correspondence to Kellie N. Smith, [email protected]. * Present address: Kellie N. Smith, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.01275-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01275-14

p. 9976 –9990

September 2014 Volume 88 Number 17


ABSTRACT

DC Enhancement of HIV-1-Speciﬁc T Cell Responses

MATERIALS AND METHODS Study participants. Three HIV-1-infected subjects (designated subjects S2, S3, and S8) were chosen from the Multicenter AIDS Cohort study (MACS), a natural-history study of men who have sex with men, for which the methodologies have been described previously (25, 26). Human subject approval was obtained from the University of Pittsburgh Institutional Review Board. These subjects were chosen based on their prolonged enrollment in the study (⬎10 years), their typical course of disease progression, their favorable response to combination antiretroviral therapy (cART), and the presence of at least one common HLA allele. Positivity for HLA A*2402 was confirmed by high-resolution PCR genotyping (Tissue Typing Laboratory, University of Pittsburgh Medical Center). All three subjects were enrolled in the MACS prior to seroconversion to HIV-1. Seropositivity was confirmed by an enzyme-linked immunosorbent assay (ELISA) positive for the presence of HIV-1 p24 and by Western blotting with bands corresponding to at least two of the Gag, Pol, and Env proteins (25). Blood specimens and epidemiological and clinical data were collected at each visit, as described previously (27). Clinical and virologic characteristics. At each biannual visit, plasma samples and peripheral blood mononuclear cells (PBMC) were collected from the study subjects and were stored at ⫺80°C and ⫺140°C, respectively. T cell phenotypes were determined by flow cytometry, as previously described (28, 29). HIV-1 plasma viremia was determined by extracting RNA using a Cobas Ampliprep instrument (Roche Diagnostics, Indianapolis, IN) and performing reverse transcription-PCR (RT-PCR) on a Cobas TaqMan 48 analyzer (Roche Diagnostics) using the Cobas Ampliprep/ Cobas TaqMan HIV-1 test. This assay is capable of detecting 20 to 1 ⫻ 106 HIV-1 RNA copies/ml of plasma. Negative, low-positive, and high-positive controls were used for each extraction and amplification according to the manufacturer’s instructions. Sequencing of autologous plasma- and cell-derived HIV-1 gag and env. Five postseroconversion (post-SC) time points for subjects S2 and S8, six postseroconversion time points for subject S3, and one post-cART time point for subjects S2 and S3 were chosen for HIV-1 gag p17-p6 and env gp120 sequencing. HIV-1 RNA was obtained from freeze-thawed


plasma for all pre-cART time points. Under cART, plasma viremia was reduced to ⬍20 copies/ml in all subjects. We therefore sequenced HIV-1 cultured from latently infected CD4⫹ T cells obtained during cART using a previously described method (30–32). The presence of HIV-1 in culture supernatants was evaluated every 3 days by a p24 ELISA (Zeptometrix, Buffalo, NY). Cultures were terminated, and supernatants were collected when the concentration of p24 reached or exceeded 20,000 pg/ml. While this technique was attempted using cells derived after cART in subject S8, we were unable to induce sufficient virus production for sequencing. Viral RNA was extracted from plasma or cell culture supernatants by using a viral RNA minikit (Qiagen, Valencia, CA). cDNA synthesis was performed by using primers Nef3 (33) and RT2 (34) with SuperScript III reverse transcriptase (200 U/ml; Invitrogen, Carlsbad, CA). Endpoint dilution methodology was used to dilute reaction mixtures to ⬃1 copy per 3 to 5 reactions, followed by direct sequencing to avoid detection of PCRinduced mutations (35). Multiplex first-round PCR was performed with primers Gag1 (34) and RT2 to amplify gag and primers Ed3 (36) and Nef3 (33) to amplify env-gp120. Singleplex second-round PCR was performed with primers Gag2 (34) and RSP15R (37) for gag and gp120 forward (38) and reverse (38) primers for env. PCR products were run on a QIAxcel automated electrophoresis system (Qiagen, Valencia, CA), and Sanger sequencing was performed on samples with positive bands (High Throughput Genomics Center, Seattle, WA). Identification of epitopes and peptide synthesis. Six known HLA A*24-restricted HIV-1 Gag and Env epitopes were identified for each subject by using the Los Alamos Database (http://www.hiv.lanl.gov/). The combined epitope prediction model within the Immune Epitope Database (39, 40) was used to identify seven additional predicted HLA A*2402restricted epitopes within autologous sequences from the three study subjects. This prediction model ranks potential epitopes within an input sequence based on predicted proteasomal cleavage, transporter associated with antigen processing (TAP) binding, and MHC class I affinity by using the netMHCpan prediction method (39–41). A PEPscreen custom library representing the dominant epitope variants (frequency, ⬎0.25) that evolved in each study subject was synthesized (Sigma-Aldrich). Each peptide was resuspended in 100 ␮l dimethyl sulfoxide (DMSO) and further resuspended in AIM V medium at a final concentration of 1 mg/ml or 100 ␮g/ml. Peptides were stored at ⫺80°C. Generation of monocyte-derived dendritic cells. PBMC from each study subject under cART were obtained by leukapheresis. Monocytes were isolated from PBMC by Percoll (GE Healthcare Life Sciences, Uppsala, Sweden) density separation. Immature DC were generated by culturing monocytes in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum (FBS) with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 (both 1,000 U/ml; R&D Systems, Minneapolis, MN). On day 5, immature DC were treated with 0.5 ␮g/ml soluble CD40L (Enzo, Farmingdale, NY) for 48 h. The maturation status of the DC was confirmed by expression of CD83, CD86, and CCR7, as determined by flow cytometry. IFN-␥ ELISpot assay. IFN-␥ production was measured by a standard overnight enzyme-linked immunosorbent spot (ELISpot) assay. Briefly, 96-well nitrocellulose plates (EMD Millipore, Billerica, MA) were coated with anti-IFN-␥ monoclonal antibody (10 ␮g/ml; Mabtech, Stockholm, Sweden) and incubated overnight at 4°C. Plates were washed and blocked with IMDM supplemented with 10% heat-inactivated FBS for 2 h at 37°C. PBMC (1 ⫻ 105 cells/well) obtained early post-SC (⬍6 months post-SC), late post-SC (⬎7 years post-SC and ⬍6 months before cART), early postcART (⬍6 months post-cART), and late post-cART (⬎13 years postcART) were tested in singlet or duplicate for reactivity to peptides representing the autologous HIV-1 epitope variants that evolved by the time point of PBMC sampling. Responders were stimulated overnight at 37°C with peptide alone (5 ␮g/ml) or with 1 ⫻ 104 autologous DC prepulsed with peptide (5 ␮g/ml) in IMDM supplemented with 10% heat-inactivated FBS. Responders with medium alone or with DC alone served as negative controls, and a peptide pool consisting of cytomegalovirus

jvi.asm.org 9977


molecule interleukin-12p70 (IL-12p70) (15, 16, 19, 20). These cells may be a valuable tool for analysis of the antigen recognition repertoire of CD8⫹ T cells or in immunotherapies that strive to enhance a dysfunctional HIV-1 recall CD8⫹ T cell response. Indeed, we have previously shown that CD40L-matured DC stimulation reveals CD8⫹ T cell responses to consensus major histocompatibility complex (MHC) class I-restricted HIV-1 epitopes that are otherwise masked in subjects on cART (15, 21). It is currently unclear if DC can also reveal responses to the subject’s own, unique (autologous) virus and if this DC enhancement changes with treated HIV-1 infection. As a successful immunotherapy will likely enhance the breadth and magnitude of the autologous HIV1-specific T cell response (22–24), it is pertinent to ascertain the best method of detecting and enhancing these responses. The aim of this study was to longitudinally evaluate CD8⫹ T cell reactivity to a broad array of autologous HIV-1 Gag and Env MHC class I founder and variant epitopes pre- and post-cART and to determine the effects of DC addition on the breadth and magnitude of these responses. We also determined the effects of epitope mutations on the generation of HIV-1-specifc immune responses and analyzed how DC can reveal otherwise masked responses for these mutated epitopes. The findings presented here demonstrate a unique ability of DC to reveal HIV-1-specific responses to a multitude of autologous epitope variants and to enhance CD8⫹ T cell cytokine profiles after long-term treatment, thus supporting the use of these cells in immunotherapy for HIV1-infected subjects on cART.

Smith et al.

(CMV), Epstein-Barr virus (EBV), and influenza virus (CEF) peptides was used as a positive control for both responder conditions. ELISpot plates were washed and processed as described previously (15, 42). Spots were counted by using an automated ELISpot plate reader (AID, Strasberg, Germany) and are shown as the number of background-subtracted antigen-specific spot-forming cells (SFC) per 106 cells. Background was calculated as the mean number of SFC/106 cells in duplicate control wells without peptide plus 2 standard deviations. Intracellular cytokine staining. PBMC were also evaluated for polyfunctional cytokine secretion in response to peptide alone or peptideloaded DC. PBMC obtained from each subject during late infection (⬍6 months pre-cART), early post-cART (⬍6 months post-cART), and late post-cART (⬎13 years post-cART) were thawed and resuspended in AIM V medium with CD28/CD49d FastImmune costimulatory reagent (BD Biosciences), Golgistop (BD Biosciences), Golgiplug (BD Biosciences), and CD107a-fluorescein isothiocyanate (FITC) H4A3 (BD Pharmingen). Peptides representing variants of known HLA A*24 HIV-1 Gag and Env epitopes that were in circulation at the time of PBMC sampling (“contemporaneous variants”) were added to PBMC or preloaded into DC before being added to PBMC at a final concentration of 5 ␮g/ml. Cells were incubated for 6 h at 37°C, washed with phosphate-buffered saline (PBS), and stained with Live/Dead Aqua Viability dye (Invitrogen) for 30 min at room temperature in the dark. Cells were washed and stained for surface expression of CD3-peridinin chlorophyll protein (PerCP) clone SK7 (BD Biosciences), CD4-allophycocyanin-Cy7 clone SK3 (BD Biosciences), and CD8-PerCP-Cy5.5 clone SK1 (BD Biosciences). Cells were again washed and fixed with 100 ␮l Cytofix/Cytoperm (BD Biosciences) and then incubated with 1⫻ Perm/Wash buffer (BD Biosciences) for 30 min at room temperature in the dark. Cells were washed with 1⫻ Perm/Wash buffer and resuspended in 50 ␮l 1⫻ Perm/Wash buffer containing IFN␥–Alexa Fluor 700 clone B27 (BD Pharmingen), tumor necrosis factor alpha (TNF-␣)-eFluor 450 clone MAb11 (eBioscience), and macrophage inflammatory protein 1␤ (MIP-1␤)-allophycocyanin clone D21-1351 (BD Pharmingen) and incubated for 20 min at room temperature in the dark. Cells were washed, resuspended in PBS, and analyzed on a BD LSR Fortessa flow cytometer using BD FACSDiva software. Data were analyzed by using FlowJo version 9.6.4. The gating strategy is show in Fig. S3 in the supplemental material, and multifunctional CD8⫹ T cells were identified by using Boolean gating. Polyfunctional data were analyzed by using SPICE version 5.3 (43). Statistical analyses. Evaluation of the differences in IFN-␥ production with and without DC in response to individual peptides and the differences in mean IFN-␥ production of all peptides combined was performed by using two-way analysis of variance (ANOVA) with Šídák’s multiplecomparison posttest. All graphs and statistical analyses were generated by using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA).

RESULTS

Clinical characteristics of the study participants. In the present study, 3 HIV-1-infected subjects from the Multicenter AIDS Co-

9978

jvi.asm.org

hort study (MACS) were chosen for longitudinal analysis. These subjects were chosen based on their prolonged enrollment in the study (⬎15 years), their typical course of disease progression, and the presence of at least 1 common HLA allele, which we determined to be HLA A*2402. For each subject (subjects S2, S3, and S8), HIV-1 plasma viral loads and CD4⫹ and CD8⫹ T cell counts were determined biannually for ⬎15 years postseroconversion (Fig. 1). All three subjects progressed to AIDS (⬍200 CD4⫹ T cells/␮l) between 7.0 and 8.25 years after infection and subsequently received and responded favorably to cART. Subject S2 had several rebounds in viral load shortly after implementation of cART, which possibly indicated treatment adherence issues. At most other post-cART time points, however, the viral load was below the limit of detection (⬍20 copies/ml) in this subject as well as in subjects S3 and S8. Changes in HIV-1 Gag and Env epitopes in chronic infection. A proposed mechanism by which infected subjects fail to control HIV-1 replication is the lack of cytotoxic T lymphocytes (CTL) that recognize the patient’s own virus (“autologous virus”) due to mutations within CTL epitopes that ablate MHC class I affinity and/or T cell recognition (4, 44). We therefore longitudinally assessed the changes in predicted MHC class I affinities of Gag and Env epitope variants derived from the autologous HIV-1 sequences of subjects S2, S3, and S8. HIV-1 was sequenced from plasma obtained from 5 to 6 postseroconversion, pre-cART time points in all three subjects. Additionally, HIV-1 was isolated from CD4⫹ T cells at one post-cART time point in subjects S2 and S3. We were unable to isolate HIV-1 from CD4⫹ T cells in subject S8, potentially due to the low HIV-1 load in this subject at all stages of disease progression. The source of HIV-1 likely did not affect the resulting sequences, as virus recovered from plasma has been shown to be identical to virus obtained from CD4⫹ T cells after short-term culture (45). We performed single-genome HIV-1 gag p17-p24 and env gp120 sequencing on the virus obtained from each subject at each time point. Nucleotide sequences were then translated to their corresponding amino acid sequences, and we identified 6 known Gag and Env HLA A*24-restricted epitopes that were documented in the Los Alamos HIV Immunology Database (http://www.hiv.lanl.gov /content/immunology) and 7 HLA A*2402-restricted epitopes predicted by using the Immune Epitope Database combined predictor with the netMHCpan MHC class I prediction method (39–41). Using the autologous Gag and Env sequences from each subject, we were able to generate a library of epitope variants that evolved throughout infection. To narrow the number of epitope variants

Journal of Virology


FIG 1 Clinical characteristics of the study subjects. HIV-1 plasma RNA and CD4⫹ and CD8⫹ T cell counts were determined biannually following seroconversion to HIV-1. CD4⫹ and CD8⫹ T cell counts and viral loads are shown longitudinally for subjects S2 (left), S3 (middle), and S8 (right). All three subjects progressed to AIDS (CD4⫹ T cell count below 200 cells/mm3) (solid lines) and received cART (dotted lines).


TABLE 1 Autologous variants of known and predicted HIV-1 Gag epitopes that evolved throughout infection Epitope locationa

Amino acid sequenceb

Subjectc

Time of evolution (YPS)e

Predicted MHC class I affinity (nM)f

Gag p1728–36

KYKLKHIVW

S2

KYKLKHIVW KYRLKHIVW KYKLKHIVW QYKLKHIVW RYRLKHLVW

0.2 1.2 0.2 0.2 0.5

954.7 1,164.5 954.7 1,616.7 233.0

RFAVNPGLL RFAVNPSLL RFAVNPGLL RFALNPGLL RFAVNPGLI RFAVNPGLL

0.2 3.4 0.2 6.4 0.5 2.4

626.9 245.9 626.9 650.0 293.8 626.9

S3 S8

NYPIVQNI NYPIVQNL NYPIVQNL NYPIVQNL

0.2 9.6 0.2 0.5

74.0 214.7 214.7 214.7

S3 S8 Gag p1743–51

RFAVNPGLL

S2 S3 S8

Gag p17131–132/p241–6

NYPIVQNI

S2

Gag p2431–40

AFSPEVIPMF

S2 S3 S8

AFSPEVIPMF AFSPEVIPMF AFSPEVIPMF

0.2 0.2 0.5

274.3 274.3 274.3

Gag p24129–138

IYKRWIILGL

S2 S3 S8

IYKGWIILGL IYKRWIILGL IYKRWIILGL IYKRWIILGL

0.2 0.2 0.2 0.5

107.3 78.9 78.9 78.9

S2 S3 S8

DYVDRFYKT DYVDRFYKT DYVDRFYKT

0.2 0.2 0.5

19,856.1 19,856.1 19,856.1

Gag p24163–171

DYVDRFYKT

a

HXB2 location of each HIV-1 epitope within Gag p17 and p24. HXB2 consensus amino acid sequences for known and predicted HLA A*24-restricted HIV-1 Gag and Env epitopes. Known epitopes are in boldface type, and the HXB2 sequences of predicted CTL epitopes are in lightface type. c Subjects S2, S3, and S8 were chosen from the Multicenter AIDS Cohort study for analysis. d Autologous epitope variants were identified by single-genome gag p17-p6 and env gp120 sequencing of autologous plasma-derived HIV-1. Underlined variants were the dominant form of the epitope (⬎55% of the variant pool) that was observed at the last time point sequenced. e The time at which the epitope variant was detected by single-genome sequencing. YPS, years postseroconversion. f Predicted affinity for HLA A*2402, as determined by using netMHCpan. A lower value indicates a higher predicted affinity. b

and to eliminate focus on the dominant variants that evolved throughout infection, only variants that had a frequency of ⬎0.25 within the variant pool at any given time point were included in our study. The Gag epitopes studied and the variants that evolved in vivo are shown in Table 1. In all Gag epitopes that evolved throughout infection, there was a switch in the dominant form of the epitope, whereby the founder epitope became less frequent than the variant that evolved later in infection (Fig. 2A). In subjects S2 and S3, for whom we obtained post-cART sequences, the dominant variant at the last pre-cART time point was the only variant in circulation post-cART; accordingly, this last time point (⬎20 years post-SC) is not shown in Fig. 2. Interestingly, not all known and predicted epitopes evolved throughout infection, despite each subject being infected for ⬎7 years without receiving cART. Of note, the predicted AFSPEVIPMF (AF10) and known DYVDRFYKT (DT9) Gag p24 epitopes did not evolve in any of the subjects, and the predicted Gag p17 RFAVNPGLL (RL9) epitope presented major variants in all 3 subjects. Not surprisingly, most but not all variants had a lower predicted affinity for the HLA A*2402 molecule than the founder variant that was present at the first postseroconversion time point.


We then evaluated amino acid changes in the 3 known and 4 predicted Env epitopes (Table 2). In Env epitopes that mutated to produce variants in subjects S2 and S8, the founder epitope was less frequent than the variant by the last sequencing time point (Fig. 2B). This was true for the mutated epitopes in subject S3 as well, except for the variant of the SI8 epitope, which made up only 31% of the SI8 epitope pool at its maximum. Two variants comprising ⬎25% of the epitope pool at a given time point, but not more than 50%, were also noted in subjects S3 and S8. We next used computational prediction algorithms to evaluate the potential effects of these amino acid substitutions on the affinity of these epitope variants for the common HLA allele among the 3 subjects, HLA A*2402. Of the 4 epitopes that evolved in subject S2, 2 evolved to variants with lower predicted affinity (KF9 and RI10) and 2 evolved to variants with higher predicted affinity (SI8 and MI10). In subject S3, 6 epitopes produced major variants throughout infection. Of these epitopes, 3 mutated to produce variants with lower predicted MHC class I affinity (SI8, KF9, and MI10), 2 epitopes produced variants with higher MHC class I affinity (RI10 and LI9), and 1 epitope produced variants with lower and higher predicted MHC class I affinities (FF9). In subject

jvi.asm.org 9979


Autologous variant sequenced

Smith et al.

S8, 4 epitopes contained amino acid changes, with 2 producing variants with lower predicted MHC class I affinity (LY10 and KF9) and 2 producing variants with higher predicted MHC class I affinity (FF9 and MI10). Therefore, these findings suggest that Gag and Env epitope variants with lower MHC class I affinity were not specifically selected for during disease progression or were at a high cost to viral fitness and were therefore removed from the viral pool. Escape mutations in Gag, but not Env, may have incurred significant fitness costs, as described previously (46). Nonetheless, within the quasispecies that remained in circulation, variants with a higher MHC class I affinity than the founder epitope were as predominant as variants with a lower affinity than the founder epitope.

9980

jvi.asm.org

DC-mediated enhancement of autologous HIV-1-specific T cell responses. As described above, we showed that HIV-1 Gag and Env epitope variants may evolve to higher and lower MHC class I affinities. This has obvious implications for the generation of T cell responses to these variants. However, we have shown previously that CD8⫹ T cell responses generated in vivo to HIV-1 peptide antigens may not be detected in standard in vitro assays without the addition of mature, antigen-loaded DC (47). We therefore confirmed the recognition of the known and predicted epitopes and assessed the impact of autologous HIV-1 epitope evolution on longitudinal T cell responses and on the ability to detect them with the addition of DC at early postseroconversion (early post-SC) (⬍6 months post-SC), late post-SC (⬎7 years

Journal of Virology


FIG 2 Evolution and changes in frequency of autologous HIV-1 Gag and Env epitope variants. HIV-1 gag and env single-genome sequencing was performed at multiple postseroconversion time points for subjects S2, S3, and S8 and at one post-cART time point for subjects S2 and S3. Known and predicted epitopes were identified by using the Los Alamos Database and netMHCpan, respectively (Tables 1 and 2), and the frequency of each variant within each epitope was determined for subjects S2, S3, and S8. (A) Changes in the frequency of variants of Gag p17 and p24 epitopes. (B) Changes in the frequency of variants of Env epitopes. The founder variant, or the variant that was present at the first postseroconversion time point, is shown in black. Major variants that evolved after the first postseroconversion time point and that have a frequency of ⬎0.5 at at least one time point are shown in red. Minor variants that evolved after the first postseroconversion time point and have a frequency of ⬎0.25 at at least one time point but ⬍0.5 at all time points are shown in blue. The Gag p24 AFSPEVIPMF and Gag p24 DYVDRFYKT epitopes did not evolve in any of the subjects and are therefore excluded from this graph.


TABLE 2 Autologous variants of known and predicted HIV-1 Env epitopes that evolved throughout infection Epitope locationa

Amino acid sequenceb

Env gp12052–61

LFCASDAKAY

Env gp120158–165

SFNISTSI

Time of evolution (YPS)e

Predicted MHC class I affinity (nM)f

S2 S3 S8

LFCASDAKAY LFCASDAKAY LFCASDARAY LFCASDAKAY

0.2 0.2 0.5 5.0

29,256.5 29,256.5 27,869.6 29,256.5

S2

TFNITTSI TFNITTNI SFNITTNI SFNITTDI SFNVTTSI

0.2 9.6 0.2 5.3 0.5

1,515.1 1,382.9 1,321.7 3,088.4 1,862.3

KMQKEYALF KVQKEYALF KVQKEYALF KVRQEYALF KMKREYAFF KIKREYATF KRRREYATF

0.2 3.4 0.2 5.3 0.5 5.0 6.6

17.9 139.7 139.7 768.5 132.5 1,256.3 9,284.2

MGPGGAFYAT IGPGRAFYAT IGPGRAFYAT IGPGRAFYAA IGPGRAFYTT

0.2 3.4 0.2 6.4 0.5

26,383.6 27,231.5 27,231.5 19,176.8 21,484.4

FYCNTTQLF FYCNSTQLF FYCNTTQLF FYCNTTRLF FYCNTAQLF FYCNTSQLF

0.2 0.2 1.5 8.3 0.5 2.4

8.9 11.5 8.9 28.5 11.9 9.4

MYAPPIRGQI MYAPPIKGLI MYAPPISGLI MYAPPIKGLI MYAPPNKGLI MYAPPIRGEI MYAPPISGVI

0.2 3.4 0.2 8.3 8.3 0.5 5.0

89.0 52.9 21.4 52.9 82.5 99.5 46.8

LYKYKVVKI LYKYKVVKI LYKYKVVEI LYKYKVVKI

0.2 0.2 8.3 0.5

301.8 301.8 237.6 301.8

S3 S8 Env gp120168–176

KVQKEYAFF

S2 S3 S8

Env gp120311–320

RGPGRAFVTI

S2 S3 S8

Env gp120383–391

FYCNSTQLF

S2 S3

S8

Env gp120434–443

MYAPPISGQI

S2 S3

S8

Env gp120483–491

LYKYKVVKI

S2 S3 S8

a

HXB2 location of each HIV-1 epitope within the Env protein. b HXB2 consensus amino acid sequences for known and predicted HLA A*24-restricted HIV-1 Gag and Env epitopes. Known epitopes are in boldface type, and the HXB2 sequences of predicted CTL epitopes are in lightface type. c Subjects S2, S3, and S8 were chosen from the Multicenter AIDS Cohort study for analysis. d Autologous epitope variants were identified by single-genome gag p17-p6 and env gp120 sequencing of autologous plasma-derived HIV-1. Underlined variants were the dominant form of the epitope (⬎55% of the variant pool) that was observed at the last time point sequenced. e The time at which the epitope variant was detected by single-genome sequencing. YPS, years postseroconversion. f Predicted affinity for HLA A*2402, as determined by using netMHCpan. A lower value indicates a higher predicted affinity.

post-SC and ⬍6 months before cART), early post-cART (⬍6 months post-cART), and late post-cART (⬎13 years post-cART) time points. To do this, the founder peptides and variants shown in Tables 1 and 2 were synthesized and used to represent autologous MHC class I-restricted HIV-1 peptide antigens, with “founder” epitopes, designated autologous epitope variants, that were in circulation early post-SC and “variants,” which evolved after the early post-SC time point. PBMC obtained from each time


point were stimulated with peptide alone or peptide-loaded mature, autologous DC in an overnight IFN-␥ ELISpot assay. Only peptides representing the autologous HIV-1 epitope founders and variants that evolved by the time of PBMC sampling were used. At both postcART time points, PBMC from subjects S2 and S3 were tested against autologous epitope variants, as determined by sequencing of reactivated cell-associated virus derived from the early post-cART time point. We were unable to reactivate virus in subject S8, so we there-

jvi.asm.org 9981


Autologous variant sequenced

Subjectc

Smith et al.

early post-SC (⬍6 months after seroconversion), late post-SC (⬎7 years after seroconversion and ⬍6 months prior to cART), early post-cART (⬍6 months after cART administration), and late post-cART (⬎13 years after cART administration) were stimulated with peptide antigen or antigen-loaded DC in an overnight IFN-␥ ELISpot assay. PBMC alone (no DC) and DC-stimulated PBMC (DC) were evaluated for reactivity to autologous founder (black lines) and variant (red lines) peptide sequences representing known and predicted Gag and Env epitopes. Response data are shown as spot-forming cells (SFC) per 106 PBMC and are the means ⫾ standard deviations from duplicate ELISpot wells with the average background plus 2 standard deviations subtracted. P values comparing ELISpot responses with and without DC are shown in Tables S1 and S2 in the supplemental material.

fore tested PBMC against the epitope variants that were in circulation immediately prior to cART, as HIV-1 derived from the cART reservoir has been shown to be phylogenetically similar to the virus that was in circulation prior to treatment (48). PBMC responses to all founder epitopes and all epitope variants of various magnitudes were detected at at least one time point in subject S2 (Fig. 3). Included in these were responses to peptides predicted to be epitopes, thereby confirming the recognition of these peptides by PBMC. The use of antigen-loaded DC significantly increased the PBMC reactivity observed early post-SC to 13 out of 14 founder epitopes (P values are shown in Tables S1 and S2 in the supplemental material). By late post-SC, the effect of DC on

9982

jvi.asm.org

IFN-␥ production had waned, with increased reactivity to only 6/14 founder epitopes and only 3/7 variants being detected. This was not unexpected, as the subject had progressed to AIDS. The effect of DC on PBMC reactivity decreased even further by the early post-cART time point, with DC enhancing responses to only 3 founder epitopes and 2 variants. In total, 12 of 14 founder epitopes exhibited the lowest PBMC reactivity at the early postcART time point. Subsequently, in 11 of 14 founder epitopes, DC-enhanced IFN-␥ production rebounded after many years of treatment at the late post-cART time point, coincident with recovery during long-term cART. We observed a similar pattern of reactivity in subject S3. PBMC

Journal of Virology


FIG 3 Representative longitudinal HIV-1-specific IFN-␥ production in response to autologous antigen with and without DC. PBMC obtained from subject S2


responses to all founder epitopes and all variants were detected at at least one time point (see Fig. S1 in the supplemental material), indicating that priming to these variants occurred in vivo. At the early post-SC time point, the presence of antigen-loaded DC in the ELISpot assay significantly increased IFN-␥ production in response to 12 out of 13 founders (P values are shown in Tables S1 and S2 in the supplemental material). DC enhancement of the response to 11 founders decreased by the late post-SC time point, and significant enhancement of the response to only 5 founders and only 4 of 10 variants was observed. Unlike subject S2, the effect of DC on PBMC reactivity rebounded early after treatment, at the early post-cART time point, showing a significant enhancement in the response to 12 founders and 9 variants. This effect and IFN-␥ production were largely maintained after many years of treatment, at the late post-cART time point, with 11 founders and all 10 variants inducing significantly greater reactivity with antigen-loaded DC than with PBMC stimulated with peptide alone. Analysis of subject S8 also produced similar findings. At the early post-SC time point, antigen-loaded DC significantly increased IFN-␥ production in response to 11 out of 13 founders (see Fig. S2 in the supplemental material; P values are shown in Tables S1 and S2 in the supplemental material). We observed decreases in DC enhancement of various magnitudes for 9 of 13 founders at the late post-SC time point, with DC significantly increasing IFN-␥ production in response to only 6 of 13 founders. This was the first time point at which late-evolving epitope variants were evaluated, and DC were unable to increase PBMC reactivity to any of these 6 variants. However, DC enhancement was restored to reactivity that was equal to or greater than that observed early post-SC in response to all founders for which we observed a decrease after the early post-SC time point. Additionally, DC-enhanced responses to 5 of 6 variants were increased late post-cART. These increases were mostly maintained late postcART.


There were no correlations identified between IFN-␥ production and predicted MHC class I affinity for any subject or time point, with or without DC (data not shown). In summary, the data presented here suggest that HIV-1-specific responses are generated in vivo to founder epitopes and late-evolving epitope variants. Recall PBMC reactivity was enhanced by the use of antigen-loaded DC in the ELISpot assay and was detected during chronic infection and after many years of suppressive cART. These responses and the enhancing effect of DC were detected at all time points but were of a lesser efficacy late post-SC, when the subjects had progressed to AIDS and were in poor immunological health, and to an extent at the early post-cART time point in subject S2, when this patient experienced a rebound in viral load while under cART. Nevertheless, our findings show that autologous HIV-1 epitope variants induce primary immune responses in vivo, regardless of minute changes in affinity for MHC class I, but may not be detected in immunological assays without the use of potent APC. PBMC obtained after cART are restored in their ability to respond to HIV-1-specific DC stimulation. We then longitudinally evaluated average PBMC reactivity with and without DC to founder and variant epitopes (Fig. 4), as determined by our IFN-␥ ELISpot assay described above. Evaluating the data in this fashion enabled us to assess the overall change in the effect of DC on antigen-specific immune responses at each time point in infection. The mean PBMC reactivities to founder epitopes without DC were similar in subjects S2 and S3 (Fig. 4, top left and top middle), with moderate mean IFN-␥ production early post-SC followed by a decrease late post-SC. The low response was maintained early post-cART and rebounded to early post-SC levels at the late post-cART time point. In subject S8, however, the early post-SC PBMC response to founder epitopes was nearly undetectable. This was followed by an increase in mean reactivity late post-SC that was maintained early post-cART and slightly increased late post-cART (Fig. 4, top right).

jvi.asm.org 9983


FIG 4 cART restores the ability of DC to enhance HIV-1-specific reactivity. The average IFN-␥ ELISpot responses of all founder (top) and variant (bottom) epitopes are shown at each time point for PBMC alone and DC-stimulated PBMC in subjects S2 (left), S3 (middle), and S8 (right). Data shown are the average background-subtracted IFN-␥ production levels of all variants evaluated at the early post-SC, late post-SC, early post-cART, and late post-cART time points ⫾ standard deviations (ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001; ⴱⴱⴱⴱ, P ⬍ 0.0001).

Smith et al.

9984

jvi.asm.org

early post-SC time point). For this analysis, we used only variants of previously known Gag and Env epitopes and excluded the predicted epitopes (Tables 1 and 2). Following a 6-h coculture, PBMC were evaluated for their expression of the type 1-associated molecules IFN-␥, CD107a, MIP-1␤, TNF-␣, and IL-2 by flow cytometry. The gating strategy for identifying cytokine-secreting cells is shown in Fig. S3 in the supplemental material. All three subjects produced similar findings, with results from subject S2 shown here. Late post-SC, DC often increased the percentage of HIV-1specific monofunctional CD8⫹ T cells (Fig. 5) and specifically enhanced the breadth and magnitude of IFN-␥ production and CD107a translocation. We also observed increases in the percentage of CD8⫹ T cells producing MIP-1␤ and TNF-␣ following DC stimulation as well as in the number of peptide antigens that induced this bifunctional response. Trifunctional CD8⫹ T cells that stained positive for CD107a, MIP-1␤, and TNF-␣ were also more prevalent following DC stimulation and were observed in response to a higher number of autologous HIV-1 antigens. The emergence of CD8⫹ T cells producing 4 immune mediators in response to the DYVDRFYKT variant was observed following DC stimulation, and this was the only variant to induce CD8⫹ T cell production of 4 immune mediators. We observed an overall “shift to the left,” whereby more polyfunctional responses were seen with DC than without DC and a greater breadth of peptides were inducing these polyfunctional responses. We observed similar findings shortly after the initiation of cART (Fig. 6). The breadth and magnitude of CD107a-positive CD8⫹ T cells were again increased following DC stimulation, as were those of bifunctional CD8⫹ T cells that produced MIP-1␤ and TNF-␣. There were also DC-mediated increases in the breadth and magnitude of IFN-␥⫹/MIP-1␤⫹/TNF-␣⫹/CD8⫹ T cells and CD107a⫹/MIP-1␤⫹/TNF-␣⫹/CD8⫹ T cells. We again observed the emergence of CD8⫹ T cells staining positive for CD107a, IFN-␥, MIP-1␤, and TNF-␣ when DC were used, again in response to DYVDRFYKT and now also in response to KYRLK HIVW. At this time point, polyfunctional responses were more pronounced than at the late post-SC time point. After many years of suppressive cART, at the late post-cART time point, we observed a pronounced shift to the left in the cytokine profile of HIV-1-specific CD8⫹ T cell reactivity (Fig. 7). We noted increases in the percentage of monofunctional CD8⫹ T cells that stained positive for CD107a and IFN-␥ and in the number of peptides that induced the production of these mediators following DC stimulation. As seen during the late post-SC and early postcART time points, the breadth and magnitude of MIP-1␤⫹/TNF␣⫹/CD8⫹ T cells were increased with the use of DC, as were a myriad of other bi- and trifunctional cytokine profiles. The CD107a⫹/IFN-␥⫹/MIP-1␤⫹/TNF-␣⫹ cytokine profile was again revealed and enhanced by DC. In summary, the addition of DC to the HIV-1 variant-specific intracellular cytokine assay revealed CD8⫹ T cells that were producing multiple immune mediators at all three time points but with a more pronounced polyfunctional profile after initiation of cART. These observations therefore show the capacity of DC to reveal otherwise masked recall T cell responses specific for autologous virus even after many years of treatment, at a time point when DC immunotherapy would likely be implemented.

Journal of Virology


In all 3 subjects, the mean reactivity with antigen-loaded DC was greater than the reactivity of PBMC with peptide alone at the early post-SC time point (P ⬍ 0.001 for subjects S2 and S3, and P ⫽ 0.0012 for subject S8) (Fig. 4, top). This enhancement was eliminated late post-SC in all three subjects, with the average DCenhanced reactivity showing no significant differences from that of PBMC stimulated with peptide alone. This low DC-stimulated response to founder epitopes was maintained in subject S2 early post-cART but rebounded in subjects S3 and S8 to show a significant difference from PBMC alone (P ⫽ 0.0008 for subject S3 and P ⫽ 0.0006 for subject S8). By the late post-cART time point, all 3 subjects exhibited robust DC-enhanced IFN-␥ production in response to the founder epitopes that was of a greater magnitude than that exhibited by peptide-stimulated PBMC (P ⬍ 0.0001 for subject S2, P ⫽ 0.0003 for subject S3, and P ⫽ 0.008 for subject S8). A similar pattern was observed for mean IFN production in response to variant epitopes that were not evaluated for PBMC reactivity until the late post-SC time point (Fig. 4, bottom). In subject S2, DC had no significant effect on the mean PBMC reactivity late post-SC or early post-cART but induced significant increases in IFN-␥ production after many years of treatment (P ⬍ 0.0001) (Fig. 4, bottom left). In subject S3, DC induced no significant increases in the mean PBMC response late post-SC but induced gradual, significant increases in this response at the early post-cART (P ⫽ 0.0077) and late post-cART (P ⫽ 0.0053) time points (Fig. 4, bottom middle). This same pattern was seen in subject S8 (Fig. 4, bottom right), with DC inducing significant increases in mean PBMC reactivity to variant epitopes at the early post-cART (P ⫽ 0.0002) and late post-cART (P ⫽ 0.0023) time points. Taken together, these data show a stark trend in the ability of DC to reveal HIV-1-specific T cell responses to Gag and Env founder and variant peptide epitopes. In all three subjects, DC enhancement of antigen-specific responses to founder epitopes was high early post-SC and then dropped in chronic HIV-1 infection. There was then a rebound in the DC-mediated increase in reactivity to founder and variant epitopes after cART, when partial immune reconstitution and a reduction in viremia occurred. DC-mediated increases in CD8ⴙ T cell production of type 1 immune mediators. As described above, we showed that DC enhance IFN-␥ secretion specific for autologous epitope variants at multiple stages of HIV-1 infection. Control of HIV-1 has been associated with a broad polyfunctional CD8⫹ T cell response to HIV-1 antigens (49), particularly Gag (6, 50–52). DC immunotherapy would aim to induce or reactivate such a response. These immunotherapies would be implemented in subjects who are likely to have been on cART for many years, so it is imperative to determine if an endogenous polyfunctional CD8⫹ T cell response was generated, was retained during long-term cART, and is capable of being enhanced or reactivated by DC. We therefore evaluated intracellular polyfunctional CD8⫹ T cell reactivity in response to the autologous epitope variants that were in circulation at the late post-SC and post-cART time points and determined if this effect increased as subjects regained their health under suppressive treatment. We cocultured PBMC obtained from each subject at the late post-SC, early post-cART, and late post-cART time points with autologous HIV-1 epitope variants alone or loaded onto DC (insufficient cell numbers were available for the


loaded with autologous HIV-1 epitope variants. PBMC derived late post-SC were incubated with peptide alone (no DC) or peptide-loaded DC (DC). For practicality, only the variants of known Gag and Env epitopes were used. PBMC with and without DC were also incubated with staphylococcal enterotoxin B (SEB) or medium alone for positive and negative controls, respectively. PBMC were then stained for CD107a, IFN-␥, IL-2, MIP-1␤, and TNF-␣. Background was determined as the percentage of CD8⫹ T cells staining positive for the relevant cytokine under negative-control conditions. Polyfunctional data were analyzed by using SPICE version 5.3 (43). Data shown are the percentages of CD8⫹ T cells that are antigen specific in response to each peptide evaluated for each cytokine/chemokine profile as well as the overall numbers of immune mediators detected in response to each variant.

DISCUSSION

CTL play a vital role in controlling HIV-1 infection (53, 54). The failure to control viremia in chronic infection has been attributed to a decline in CTL responses that are specific for the autologous virus (5, 12, 55–60). Indeed, mutations within CTL epitopes that ablate T cell recognition or MHC class I affinity would therefore prevent the generation of a primary CD8⫹ T cell response against that epitope. In this study, we employed a method of detecting and enhancing T cell responses specific for autologous HIV-1 epitope variants at multiple stages of infection, before and after cART. Here we show that DC reveal broad and robust IFN-␥ ELISpot responses regardless of disease progression but most specifically after long-term suppressive cART. These results were expanded by using a DC-enhanced, polychromatic flow cytometry assay to detect multiple immune mediators in response to autologous HIV-1 epitope variants that were in circulation immediately prior to or after cART (contemporaneous variants). By using this approach, we observed DC enhancement of the production of multiple cytokines by HIV-1-specific CD8⫹ T cells that was associated with suppression of viremia subsequent to cART. The known and predicted HLA A*24-restricted epitopes used in our study exhibited various patterns of evolution. While many of the epitopes incurred amino acid changes throughout infection, there was no universal effect of these changes on the predicted MHC class I affinity, indicating that HIV-1 evolution did


not specifically evade MHC class I loading by antigen-presenting cells (APC) or expression on the surface of infected cells or that the mutations affected the proteolytic processing required to result in peptide loading. This observation suggests that APC priming of naive CD8⫹ T cells could have occurred in vivo if the T cell receptor (TCR) repertoire was sufficient to recognize these antigens. Indeed, we detected PBMC responses to ⬎95% of variants irrespective of the predicted MHC class I affinity, showing that amino acid mutations within epitopes did not ablate priming to these variants in vivo. Moreover, the magnitude of IFN-␥ production in response to variants of novel predicted epitopes was similar to that in response to variants of known epitopes, therefore highlighting the potential of these predicted epitope regions to be classified as HLA A*2402-restricted epitopes. We observed no correlation between the predicted MHC class I affinity of known or predicted epitope variants and IFN-␥ production. These findings are not surprising, as we have previously reported the detection of recall T cell responses to autologous HIV-1 antigens with high, medium, and low experimental MHC class I affinities and multiple time points in disease progression (61). While decreased binding of mutated epitopes to MHC class I is thought to be a primary mechanism by which HIV-1 and simian immunodeficiency virus (SIV) evade CD8⫹ T lymphocyte responses (4, 12, 62–70), our detection of IFN-␥ production in response to the epitope variants in this report would challenge this

jvi.asm.org 9985


FIG 5 DC-mediated increase in polyfunctional CD8⫹ T cell cytokine responses late post-SC. Autologous monocyte-derived DC were matured with CD40L and

Smith et al.

and loaded with autologous HIV-1 epitope variants. PBMC derived early post-cART were incubated with peptide alone (no DC) or peptide-loaded DC (DC). For practicality, only the variants of known Gag and Env epitopes were used. PBMC with and without DC were also incubated with staphylococcal enterotoxin B (SEB) or medium alone for positive and negative controls, respectively. PBMC were then stained for CD107a, IFN-␥, IL-2, MIP-1␤, and TNF-␣. Background was determined as the percentage of CD8⫹ T cells staining positive for the relevant cytokine under negative-control conditions. Polyfunctional data were analyzed by using SPICE version 5.3 (43). Data shown are the percentages of CD8⫹ T cells that are antigen specific in response to each peptide evaluated for each cytokine/chemokine profile as well as the overall numbers of immune mediators detected in response to each variant.

conclusion. We do recognize, however, that detection of IFN-␥ production is not necessarily indicative of a cytolytic CD8⫹ T cell response that can control infection (71–74). The 3 subjects used in our study exhibited conventional characteristics of HIV-1 disease progression, and therefore, resident CTL were unable to control viral replication in chronic infection. A CTL response with great breadth and magnitude, specifically to Gag proteins, has been associated with SIV and HIV-1 control (53, 54, 75–77). However, high-avidity CD8⫹ T cells have been detected in progressive infection and therefore point to poor immune selection pressure exerted by these cells (72). Indeed, the PBMC responses to autologous epitope variants described in this study were broad and within a wide range of magnitudes despite the continuation of disease progression, thus indicating ineffective selective pressure on the virus. We postulate that a primary response to these epitope variants was generated in vivo, although this response was likely of variable efficacy, as disease progression continued and many of the epitope variants persisted throughout infection. Future studies should focus on changes in cytolytic effector function against a select number of epitope variants throughout HIV-1 infection. Nonetheless, these findings provide an in-depth understanding of the breadth of T cell responses that are generated against autologous HIV-1 antigens regardless of MHC class I affinity. We previously reported on the ability of mature, autologous DC to reveal and enhance HIV-1-specific T cell responses in subjects on cART (21) and on their usefulness in detecting and gen-

9986

jvi.asm.org

erating responses specific for HIV-1 variants with variable MHC class I affinities (61). Not much is known, however, about the ability of DC to enhance responses during progressive infection. After detecting moderate responses to autologous epitope variants in PBMC, we determined the effects of DC addition on longitudinal CD8⫹ T cell IFN-␥ production throughout untreated and treated HIV-1 infections. The addition of DC to PBMC did not enhance the number of epitope variants that were recognized, as the breadth was already ⬎95% when PBMC were evaluated, but the magnitude was significantly enhanced at most time points in each subject. Regardless of disease progression, DC were functional at enhancing IFN-␥ production against autologous epitope variants, as judged by our overnight ELISpot assays. More importantly, we observed similar responses to variants that evolved early and late postseroconversion, thus supporting the ability of DC to present mutated epitopes to their cognate CD8⫹ T cells. When we evaluated the “effect” of DC enhancement on the PBMC response over time, we saw striking similarities between the three subjects. There was an enhancing effect of DC observed early post-SC, followed by a drastic reduction late post-SC, and a restoration at the early post-cART or late post-cART time point. There are multiple mechanisms by which DC enhance recall T cell responses to HIV-1 (15, 16, 19), including high-level IL-12p70 production, expression of the T cell costimulatory molecules CD80 and CD86, and expression of the DC maturation marker CD83. Our current

Journal of Virology


FIG 6 DC-mediated increase in polyfunctional CD8⫹ T cell cytokine responses early post-cART. Autologous monocyte-derived DC were matured with CD40L


CD40L and loaded with autologous HIV-1 epitope variants. PBMC derived late post-cART were incubated with peptide alone (no DC) or peptide-loaded DC (DC). For practicality, only the variants of known Gag and Env epitopes were used. PBMC with and without DC were also incubated with staphylococcal enterotoxin B (SEB) or medium alone for positive and negative controls, respectively. PBMC were then stained for CD107a, IFN-␥, IL-2, MIP-1␤, and TNF-␣. Background was determined as the percentage of CD8⫹ T cells staining positive for the relevant cytokine under negative-control conditions. Polyfunctional data were analyzed by using SPICE version 5.3 (43). Data shown are the percentages of CD8⫹ T cells that are antigen specific in response to each peptide evaluated for each cytokine/chemokine profile as well as the overall numbers of immune mediators detected in response to each variant.

findings show that DC-mediated enhancement of T cell responses is restored after many years of cART. These results suggest that DC immunotherapy that enhances endogenous HIV-1 reactivity could be effective in persons on cART. Such a treatment would involve the ex vivo generation of monocyte-derived DC followed by antigen loading and injection back into the patient to reactivate quiescent recall T cell populations or to prime new CTL from naive precursors (78). We hypothesize that these findings are indicative of the immunological health of the subjects during infection. Within the first 6 months after seroconversion, which is the time point at which our early post-SC PBMC were derived, T cell counts in our three subjects were within normal ranges. It can be assumed, then, that immunological function was relatively intact, and therefore, T cells at this time point could respond to DC stimulation. Late post-SC, however, an environment of immune dysfunction persisted, and T cells were less able to respond to antigen-specific DC stimulation. In two of the three subjects, this dysfunction was reversed early after cART, and T cell responses to DC stimulation were restored. Interestingly, we did not see this in subject S2 until late post-cART. This may be due to the delay in the reduction of the viral load and consequently in the reconstitution of immunological function following the administration of cART (Fig. 1). In all three subjects, however, cells regained or retained the ability to respond to DC stimulation late post-cART, thus suggesting that DC-mediated therapy for subjects on long-term treatment may be successful in reinvigorating quiescent memory responses.


Production of multiple cytokines by CD8⫹ T cells has been associated with CTL effector function and control of HIV-1 infection (6, 49, 50, 52, 79). We therefore expanded our analysis of DC-mediated enhancement of HIV-1-specific CD8⫹ T cell responses by evaluating intracellular cytokine secretion late postseroconversion and during early and late cART time points with and without DC. Even after many years of cART, DC revealed monofunctional and polyfunctional HIV-1-specific CD8⫹ T cell responses. These findings again support the notion that DC immunotherapy aimed at reinvigorating the dysfunctional CTL response can be effective. Our IFN-␥ ELISpot analysis showed variable time-associated effects of DC addition as subjects received cART, but staining for multiple immune mediators gave a broader picture of how suppressive cART aids in the ability of T cells to respond to HIV-1-specific stimuli with a phenotype most associated with CTL effector function. For example, at all three time points, DC stimulation enhanced the breadth and magnitude of CD8⫹ T cells that stained positive for the degranulation marker CD107a, which is associated with CTL function. The data presented in this study highlight the ability of T cells to respond to autologous HIV-1 epitope variants at all stages of disease progression and support the use of DC immunotherapy in subjects on cART. These data should be viewed with caution, however, as we have recently shown that recall HIV-1-specific CTL may produce multiple immune mediators without effector function if stimulated with an antigen that is related to, but slightly different than, the one to which it was originally primed (71). Not

jvi.asm.org 9987


FIG 7 DC-mediated increase in polyfunctional CD8⫹ T cell cytokine responses after long-term cART. Autologous monocyte-derived DC were matured with

Smith et al.

10.

11.

12.

ACKNOWLEDGMENTS We thank John Mellors for the TaqMan assay; Kelly Gordon, Ronald Fecek, and Weimin Jiang for technical assistance; and the volunteers at the Pittsburgh site of the Multicenter AIDS Cohort study. This work was supported in part by NIH grants R01-AI-40388, R37AI-41870, and U01-AI-35041 and by a T32-AI065380 fellowship to K.N.S.

13.

REFERENCES 1. Streeck H, Jolin JS, Qi Y, Yassine-Diab B, Johnson RC, Kwon DS, Addo MM, Brumme C, Routy JP, Little S, Jessen HK, Kelleher AD, Hecht FM, Sekaly RP, Rosenberg ES, Walker BD, Carrington M, Altfeld M. 2009. Human immunodeficiency virus type 1-specific CD8⫹ T-cell responses during primary infection are major determinants of the viral set point and loss of CD4⫹ T cells. J. Virol. 83:7641–7648. http://dx.doi.org/10.1128 /JVI.00182-09. 2. Vanderford TH, Bleckwehl C, Engram JC, Dunham RM, Klatt NR, Feinberg MB, Garber DA, Betts MR, Silvestri G. 2011. Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques. PLoS Pathog. 7:e1002048. http://dx.doi.org/10.1371/journal.ppat.1002048. 3. Dong T, Zhang Y, Xu KY, Yan H, James I, Peng Y, Blais ME, Gaudieri S, Chen X, Lun W, Wu H, Qu WY, Rostron T, Li N, Mao Y, Mallal S, Xu X, McMichael A, John M, Rowland-Jones SL. 2011. Extensive HLA-driven viral diversity following a narrow-source HIV-1 outbreak in rural China. Blood 118:98 –106. http://dx.doi.org/10.1182/blood-2010-06-291963. 4. Chen ZW, Craiu A, Shen L, Kuroda MJ, Iroku UC, Watkins DI, Voss G, Letvin NL. 2000. Simian immunodeficiency virus evades a dominant epitope-specific cytotoxic T lymphocyte response through a mutation resulting in the accelerated dissociation of viral peptide and MHC class I. J. Immunol. 164:6474 – 6479. http://dx.doi.org/10.4049/jimmunol.164.12.6474. 5. Allen TM, Altfeld M, Geer SC, Kalife ET, Moore C, O’Sullivan KM, Desouza I, Feeney ME, Eldridge RL, Maier EL, Kaufmann DE, Lahaie MP, Reyor L, Tanzi G, Johnston MN, Brander C, Draenert R, Rockstroh JK, Jessen H, Rosenberg ES, Mallal SA, Walker BD. 2005. Selective escape from CD8⫹ T-cell responses represents a major driving force of human immunodeficiency virus type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution. J. Virol. 79:13239 –13249. http: //dx.doi.org/10.1128/JVI.79.21.13239-13249.2005. 6. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, Asher TE, Samri A, Schnuriger A, Theodorou I, Costagliola D, Rouzioux C, Agut H, Marcelin AG, Douek D, Autran B, Appay V. 2007. Superior control of HIV-1 replication by CD8⫹ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J. Exp. Med. 204:2473– 2485. http://dx.doi.org/10.1084/jem.20070784. 7. Douek DC, McFarland RD, Keiser PH, Gage EA, Massey JM, Haynes BF, Polis MA, Haase AT, Feinberg MB, Sullivan JL, Jamieson BD, Zack JA, Picker LJ, Koup RA. 1998. Changes in thymic function with age and during the treatment of HIV infection. Nature 396:690 – 695. http://dx.doi .org/10.1038/25374. 8. Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R, Katlama C, Debre P, Leibowitch J. 1997. Positive effects of combined antiretroviral therapy on CD4⫹ T cell homeostasis and function in advanced HIV disease. Science 277:112–116. http://dx.doi.org/10.1126/science.277 .5322.112. 9. Zhang ZQ, Notermans DW, Sedgewick G, Cavert W, Wietgrefe S, Zupancic M, Gebhard K, Henry K, Boies L, Chen Z, Jenkins M, Mills

9988

jvi.asm.org

14.

15. 16.

17.

18.

19.

20.

21.

22.

23.

R, McDade H, Goodwin C, Schuwirth CM, Danner SA, Haase AT. 1998. Kinetics of CD4⫹ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection. Proc. Natl. Acad. Sci. U. S. A. 95:1154 –1159. http://dx .doi.org/10.1073/pnas.95.3.1154. Hazenberg MD, Otto SA, Cohen Stuart JW, Verschuren MC, Borleffs JC, Boucher CA, Coutinho RA, Lange JM, Rinke de Wit TF, Tsegaye A, van Dongen JJ, Hamann D, de Boer RJ, Miedema F. 2000. Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection. Nat. Med. 6:1036 –1042. http://dx.doi.org/10.1038/79549. Roederer M, Dubs JG, Anderson MT, Raju PA, Herzenberg LA, Herzenberg LA. 1995. CD8 naive T cell counts decrease progressively in HIVinfected adults. J. Clin. Invest. 95:2061–2066. http://dx.doi.org/10.1172 /JCI117892. Allen TM, Altfeld M, Yu XG, O’Sullivan KM, Lichterfeld M, Le Gall S, John M, Mothe BR, Lee PK, Kalife ET, Cohen DE, Freedberg KA, Strick DA, Johnston MN, Sette A, Rosenberg ES, Mallal SA, Goulder PJ, Brander C, Walker BD. 2004. Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection. J. Virol. 78:7069 –7078. http: //dx.doi.org/10.1128/JVI.78.13.7069-7078.2004. Connolly N, Riddler S, Stanson J, Gooding W, Rinaldo CR, Ferrone S, Whiteside TL. 2007. Levels of antigen processing machinery components in dendritic cells generated for vaccination of HIV-1⫹ subjects. AIDS 21:1683–1692. http://dx.doi.org/10.1097/QAD.0b013e32825eabbc. Sapp M, Engelmayer J, Larsson M, Granelli-Piperno A, Steinman R, Bhardwaj N. 1999. Dendritic cells generated from blood monocytes of HIV-1 patients are not infected and act as competent antigen presenting cells eliciting potent T-cell responses. Immunol. Lett. 66:121–128. http: //dx.doi.org/10.1016/S0165-2478(98)00169-2. Huang XL, Fan Z, Borowski L, Rinaldo CR. 2008. Maturation of dendritic cells for enhanced activation of anti-HIV-1 CD8(⫹) T cell immunity. J. Leukoc. Biol. 83:1530 –1540. http://dx.doi.org/10.1189/jlb.1107795. Huang XL, Fan Z, Colleton BA, Buchli R, Li H, Hildebrand WH, Rinaldo CR, Jr. 2005. Processing and presentation of exogenous HLA class I peptides by dendritic cells from human immunodeficiency virus type 1-infected persons. J. Virol. 79:3052–3062. http://dx.doi.org/10.1128 /JVI.79.5.3052-3062.2005. Fan Z, Huang XL, Zheng L, Wilson C, Borowski L, Liebmann J, Gupta P, Margolick J, Rinaldo C. 1997. Cultured blood dendritic cells retain HIV-1 antigen-presenting capacity for memory CTL during progressive HIV-1 infection. J. Immunol. 159:4973– 4982. Newton PJ, Weller IV, Williams IG, Miller RF, Copas A, Tedder RS, Katz DR, Chain BM. 2006. Monocyte derived dendritic cells from HIV-1 infected individuals partially reconstitute CD4 T-cell responses. AIDS 20: 171–180. http://dx.doi.org/10.1097/01.aids.0000202649.95655.8c. Mailliard RB, Wankowicz-Kalinska A, Cai Q, Wesa A, Hilkens CM, Kapsenberg ML, Kirkwood JM, Storkus WJ, Kalinski P. 2004. Alphatype-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 64:5934 –5937. http://dx.doi.org/10 .1158/0008-5472.CAN-04-1261. Mailliard RB, Egawa S, Cai Q, Kalinska A, Bykovskaya SN, Lotze MT, Kapsenberg ML, Storkus WJ, Kalinski P. 2002. Complementary dendritic cell-activating function of CD8⫹ and CD4⫹ T cells: helper role of CD8⫹ T cells in the development of T helper type 1 responses. J. Exp. Med. 195:473– 483. http://dx.doi.org/10.1084/jem.20011662. Huang XL, Fan Z, Borowski L, Mailliard RB, Rolland M, Mullins JI, Day RD, Rinaldo CR. 2010. Dendritic cells reveal a broad range of MHC class I epitopes for HIV-1 in persons with suppressed viral load on antiretroviral therapy. PLoS One 5:e12936. http://dx.doi.org/10.1371/journal .pone.0012936. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, Lederman MM, Benito JM, Goepfert PA, Connors M, Roederer M, Koup RA. 2006. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8⫹ T cells. Blood 107:4781– 4789. http://dx.doi .org/10.1182/blood-2005-12-4818. Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, Johnston MN, Corcoran C, Wurcel AG, Fitzpatrick CA, Feeney ME, Rodriguez WR, Basgoz N, Draenert R, Stone DR, Brander C, Goulder PJ, Rosenberg ES, Altfeld M, Walker BD. 2003. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demon-

Journal of Virology


only do these cells fail to exert cytolytic function on target cells expressing the variant antigen, they also promote DC maturation and viral spread in vitro. This finding suggests that DC immunotherapy that uses an antigen that is similar to but not the exact antigen to which the subject’s T cells were originally primed may not induce an effective CTL response and may actually hinder viral clearance. It may be that the most effective way of inducing a potent CTL response specific for the autologous HIV-1 reservoir is to generate new CTL from naive precursors (78). The effects of antigen-specific DC stimulation on naive and memory T cells and on their ability to facilitate elimination of the HIV-1 reservoir need to be evaluated.


24.

25.

27.

28.

29.

30.

31.

32. 33.

34.

35.

36.

37.

38.


39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49. 50.

51.

52.

53.

54.

subtype B viruses. Virology 374:229 –233. http://dx.doi.org/10.1016/j .virol.2008.01.029. Tenzer S, Peters B, Bulik S, Schoor O, Lemmel C, Schatz MM, Kloetzel PM, Rammensee HG, Schild H, Holzhutter HG. 2005. Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding. Cell. Mol. Life Sci. 62:1025–1037. http://dx.doi.org/10.1007/s00018-005-4528-2. Peters B, Bulik S, Tampe R, Van Endert PM, Holzhutter HG. 2003. Identifying MHC class I epitopes by predicting the TAP transport efficiency of epitope precursors. J. Immunol. 171:1741–1749. http://dx.doi .org/10.4049/jimmunol.171.4.1741. Hoof I, Peters B, Sidney J, Pedersen LE, Sette A, Lund O, Buus S, Nielsen M. 2009. NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61:1–13. http://dx.doi.org/10 .1007/s00251-008-0341-z. Colleton BA, Huang XL, Melhem NM, Fan Z, Borowski L, Rappocciolo G, Rinaldo CR. 2009. Primary human immunodeficiency virus type 1-specific CD8⫹ T-cell responses induced by myeloid dendritic cells. J. Virol. 83:6288 – 6299. http://dx.doi.org/10.1128/JVI.02611-08. Roederer M, Nozzi JL, Nason MC. 2011. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 79:167– 174. http://dx.doi.org/10.1002/cyto.a.21015. Jones NA, Wei X, Flower DR, Wong M, Michor F, Saag MS, Hahn BH, Nowak MA, Shaw GM, Borrow P. 2004. Determinants of human immunodeficiency virus type 1 escape from the primary CD8⫹ cytotoxic T lymphocyte response. J. Exp. Med. 200:1243–1256. http://dx.doi.org/10 .1084/jem.20040511. Anderson JA, Archin NM, Ince W, Parker D, Wiegand A, Coffin JM, Kuruc J, Eron J, Swanstrom R, Margolis DM. 2011. Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4⫹ T cells. J. Virol. 85:5220 –5223. http://dx.doi.org/10.1128/JVI.00284-11. Troyer RM, McNevin J, Liu Y, Zhang SC, Krizan RW, Abraha A, Tebit DM, Zhao H, Avila S, Lobritz MA, McElrath MJ, Le Gall S, Mullins JI, Arts EJ. 2009. Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog. 5:e1000365. http://dx .doi.org/10.1371/journal.ppat.1000365. Huang XL, Fan Z, Borowski L, Rinaldo CR. 2009. Multiple T-cell responses to human immunodeficiency virus type 1 are enhanced by dendritic cells. Clin. Vaccine Immunol. 16:1504 –1516. http://dx.doi.org/10 .1128/CVI.00104-09. Kearney MF, Spindler J, Shao W, Yu S, Anderson EM, O’Shea A, Rehm C, Poethke C, Kovacs N, Mellors JW, Coffin JM, Maldarelli F. 2014. Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy. PLoS Pathog. 10:e1004010. http://dx.doi.org/10.1371 /journal.ppat.1004010. Betts MR, Harari A. 2008. Phenotype and function of protective T cell immune responses in HIV. Curr. Opin. HIV AIDS 3:349 –355. http://dx .doi.org/10.1097/COH.0b013e3282fbaa81. Turk G, Gherardi MM, Laufer N, Saracco M, Luzzi R, Cox JH, Cahn P, Salomon H. 2008. Magnitude, breadth, and functional profile of T-cell responses during human immunodeficiency virus primary infection with B and BF viral variants. J. Virol. 82:2853–2866. http://dx.doi.org/10.1128 /JVI.02260-07. Harari A, Cellerai C, Bellutti Enders F, Kostler J, Codarri L, Tapia G, Boyman O, Castro E, Gaudieri S, James I, John M, Wagner R, Mallal S, Pantaleo G. 2007. Skewed association of polyfunctional antigen-specific CD8 T cell populations with HLA-B genotype. Proc. Natl. Acad. Sci. U. S. A. 104:16233–16238. http://dx.doi.org/10.1073/pnas.0707570104. Almeida JR, Sauce D, Price DA, Papagno L, Shin SY, Moris A, Larsen M, Pancino G, Douek DC, Autran B, Saez-Cirion A, Appay V. 2009. Antigen sensitivity is a major determinant of CD8⫹ T-cell polyfunctionality and HIV-suppressive activity. Blood 113:6351– 6360. http://dx.doi .org/10.1182/blood-2009-02-206557. Saez-Cirion A, Lacabaratz C, Lambotte O, Versmisse P, Urrutia A, Boufassa F, Barre-Sinoussi F, Delfraissy JF, Sinet M, Pancino G, Venet A. 2007. HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc. Natl. Acad. Sci. U. S. A. 104:6776 – 6781. http://dx.doi .org/10.1073/pnas.0611244104. Pandrea I, Gaufin T, Gautam R, Kristoff J, Mandell D, Montefiori D, Keele BF, Ribeiro RM, Veazey RS, Apetrei C. 2011. Functional cure of

jvi.asm.org 9989


26.

strate broadly directed responses, but no correlation to viral load. J. Virol. 77:2081–2092. http://dx.doi.org/10.1128/JVI.77.3.2081-2092.2003. Pereyra F, Addo MM, Kaufmann DE, Liu Y, Miura T, Rathod A, Baker B, Trocha A, Rosenberg R, Mackey E, Ueda P, Lu Z, Cohen D, Wrin T, Petropoulos CJ, Rosenberg ES, Walker BD. 2008. Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J. Infect. Dis. 197:563–571. http://dx.doi.org/10.1086 /526786. Kaslow RA, Ostrow DG, Detels R, Phair JP, Polk BF, Rinaldo CR, Jr. 1987. The Multicenter AIDS Cohort study: rationale, organization, and selected characteristics of the participants. Am. J. Epidemiol. 126:310 – 318. http://dx.doi.org/10.1093/aje/126.2.310. Detels R, Jacobson L, Margolick J, Martinez-Maza O, Munoz A, Phair J, Rinaldo C, Wolinsky S. 2012. The Multicenter AIDS Cohort study, 1983 to. . .. Public Health 126:196 –198. http://dx.doi.org/10.1016/j.puhe .2011.11.013. Shankarappa R, Margolick JB, Gange SJ, Rodrigo AG, Upchurch D, Farzadegan H, Gupta P, Rinaldo CR, Learn GH, He X, Huang XL, Mullins JI. 1999. Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J. Virol. 73:10489 –10502. Schenker EL, Hultin LE, Bauer KD, Ferbas J, Margolick JB, Giorgi JV. 1993. Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program. Cytometry 14:307–317. http://dx.doi.org/10.1002/cyto.990140311. Giorgi JV, Cheng HL, Margolick JB, Bauer KD, Ferbas J, Waxdal M, Schmid I, Hultin LE, Jackson AL, Park L, Taylor JMG, Multicenter AIDS Cohort Study Group. 1990. Quality control in the flow cytometric measurement of T-lymphocyte subsets: the Multicenter AIDS Cohort study experience. Clin. Immunol. Immunopathol. 55:173–186. http://dx .doi.org/10.1016/0090-1229(90)90096-9. Siliciano JD, Siliciano RF. 2005. Enhanced culture assay for detection and quantitation of latently infected, resting CD4⫹ T-cells carrying replication-competent virus in HIV-1-infected individuals. Methods Mol. Biol. 304:3–15. http://dx.doi.org/10.1385/1-59259-907-9:003. Laird GM, Eisele EE, Rabi SA, Lai J, Chioma S, Blankson JN, Siliciano JD, Siliciano RF. 2013. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog. 9:e1003398. http://dx .doi.org/10.1371/journal.ppat.1003398. Chen Y, Rinaldo C, Gupta P. 1997. A semiquantitative assay for CD8⫹ T-cell-mediated suppression of human immunodeficiency virus type 1 infection. Clin. Diagn. Lab. Immunol. 4:4 –10. Frenkel LM, Mullins JI, Learn GH, Manns-Arcuino L, Herring BL, Kalish ML, Steketee RW, Thea DM, Nichols JE, Liu SL, Harmache A, He X, Muthui D, Madan A, Hood L, Haase AT, Zupancic M, Staskus K, Wolinsky S, Krogstad P, Zhao J, Chen I, Koup R, Ho D, Korber B, Apple RJ, Coombs RW, Pahwa S, Roberts NJ, Jr. 1998. Genetic evaluation of suspected cases of transient HIV-1 infection of infants. Science 280:1073–1077. http://dx.doi.org/10.1126/science.280.5366.1073. Liu Y, McNevin J, Cao J, Zhao H, Genowati I, Wong K, McLaughlin S, McSweyn MD, Diem K, Stevens CE, Maenza J, He H, Nickle DC, Shriner D, Holte SE, Collier AC, Corey L, McElrath MJ, Mullins JI. 2006. Selection on the human immunodeficiency virus type 1 proteome following primary infection. J. Virol. 80:9519 –9529. http://dx.doi.org/10 .1128/JVI.00575-06. Rodrigo AG, Goracke PC, Rowhanian K, Mullins JI. 1997. Quantitation of target molecules from polymerase chain reaction-based limiting dilution assays. AIDS Res. Hum. Retroviruses 13:737–742. http://dx.doi.org /10.1089/aid.1997.13.737. Delwart EL, Shpaer EG, Louwagie J, McCutchan FE, Grez M, Rubsamen-Waigmann H, Mullins JI. 1993. Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes. Science 262:1257–1261. http://dx.doi.org/10.1126/science.8235655. Liu Y, Holte S, Rao U, McClure J, Konopa P, Swain JV, LanxonCookson E, Kim M, Chen L, Mullins JI. 2013. A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture. J. Virol. Methods 189:157–166. http://dx.doi.org/10.1016/j .jviromet.2012.10.016. Liu Y, Curlin ME, Diem K, Zhao H, Ghosh AK, Zhu H, Woodward AS, Maenza J, Stevens CE, Stekler J, Collier AC, Genowati I, Deng W, Zioni R, Corey L, Zhu T, Mullins JI. 2008. Env length and N-linked glycosylation following transmission of human immunodeficiency virus type 1

Smith et al.

55. 56. 57.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

9990

jvi.asm.org

69.

70.

71.

72.

73.

74.

75.

76.

77.

78. 79.

Rosenberg ES, Nguyen T, Allen R, Trocha A, Altfeld M, He S, Bunce M, Funkhouser R, Pelton SI, Burchett SK, McIntosh K, Korber BT, Walker BD. 2001. Evolution and transmission of stable CTL escape mutations in HIV infection. Nature 412:334 –338. http://dx.doi.org/10.1038/35085576. Couillin I, Culmann-Penciolelli B, Gomard E, Choppin J, Levy JP, Guillet JG, Saragosti S. 1994. Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein. J. Exp. Med. 180:1129 – 1134. http://dx.doi.org/10.1084/jem.180.3.1129. Meier UC, Klenerman P, Griffin P, James W, Koppe B, Larder B, McMichael A, Phillips R. 1995. Cytotoxic T lymphocyte lysis inhibited by viable HIV mutants. Science 270:1360 –1362. http://dx.doi.org/10.1126 /science.270.5240.1360. Mailliard RB, Smith KN, Fecek RJ, Rappocciolo G, Nascimento EJ, Marques ET, Watkins SC, Mullins JI, Rinaldo CR. 2013. Selective induction of CTL helper rather than killer activity by natural epitope variants promotes dendritic cell-mediated HIV-1 dissemination. J. Immunol. 191:2570 –2580. http://dx.doi.org/10.4049/jimmunol.1300373. Draenert R, Verrill CL, Tang Y, Allen TM, Wurcel AG, Boczanowski M, Lechner A, Kim AY, Suscovich T, Brown NV, Addo MM, Walker BD. 2004. Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection. J. Virol. 78:630 – 641. http://dx.doi.org/10.1128/JVI.78.2.630-641 .2004. Iversen AK, Stewart-Jones G, Learn GH, Christie N, Sylvester-Hviid C, Armitage AE, Kaul R, Beattie T, Lee JK, Li Y, Chotiyarnwong P, Dong T, Xu X, Luscher MA, MacDonald K, Ullum H, Klarlund-Pedersen B, Skinhoj P, Fugger L, Buus S, Mullins JI, Jones EY, van der Merwe PA, McMichael AJ. 2006. Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope. Nat. Immunol. 7:179 –189. http://dx.doi.org/10.1038/ni1298. Hay CM, Ruhl DJ, Basgoz NO, Wilson CC, Billingsley JM, DePasquale MP, D’Aquila RT, Wolinsky SM, Crawford JM, Montefiori DC, Walker BD. 1999. Lack of viral escape and defective in vivo activation of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes in rapidly progressive infection. J. Virol. 73:5509 –5519. Berger CT, Frahm N, Price DA, Mothe B, Ghebremichael M, Hartman KL, Henry LM, Brenchley JM, Ruff LE, Venturi V, Pereyra F, Sidney J, Sette A, Douek DC, Walker BD, Kaufmann DE, Brander C. 2011. High-functional-avidity cytotoxic T lymphocyte responses to HLA-Brestricted Gag-derived epitopes associated with relative HIV control. J. Virol. 85:9334 –9345. http://dx.doi.org/10.1128/JVI.00460-11. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, Reddy S, de Pierres C, Mncube Z, Mkhwanazi N, Bishop K, van der Stok M, Nair K, Khan N, Crawford H, Payne R, Leslie A, Prado J, Prendergast A, Frater J, McCarthy N, Brander C, Learn GH, Nickle D, Rousseau C, Coovadia H, Mullins JI, Heckerman D, Walker BD, Goulder P. 2007. CD8⫹ T-cell responses to different HIV proteins have discordant associations with viral load. Nat. Med. 13:46 –53. http://dx.doi .org/10.1038/nm1520. Rolland M, Heckerman D, Deng W, Rousseau CM, Coovadia H, Bishop K, Goulder PJ, Walker BD, Brander C, Mullins JI. 2008. Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads. PLoS One 3:e1424. http://dx.doi.org/10.1371/journal.pone.0001424. Rinaldo CR. 2009. Dendritic cell-based human immunodeficiency virus vaccine. J. Intern. Med. 265:138 –158. http://dx.doi.org/10.1111/j.1365-2796 .2008.02047.x. Harari A, Cellerai C, Enders FB, Kostler J, Codarri L, Tapia G, Boyman O, Castro E, Gaudieri S, James I, John M, Wagner R, Mallal S, Pantaleo G. 2007. Skewed association of polyfunctional antigen-specific CD8 T cell populations with HLA-B genotype. Proc. Natl. Acad. Sci. U. S. A. 104: 16233–16238. http://dx.doi.org/10.1073/pnas.0707570104.

Journal of Virology


58.

SIVagm infection in rhesus macaques results in complete recovery of CD4⫹ T cells and is reverted by CD8⫹ cell depletion. PLoS Pathog. 7:e1002170. http://dx.doi.org/10.1371/journal.ppat.1002170. Baker BM, Block BL, Rothchild AC, Walker BD. 2009. Elite control of HIV infection: implications for vaccine design. Expert Opin. Biol. Ther. 9:55– 69. http://dx.doi.org/10.1517/14712590802571928. Bangham CR. 2009. CTL quality and the control of human retroviral infections. Eur. J. Immunol. 39:1700 –1712. http://dx.doi.org/10.1002/eji .200939451. Bazykin GA, Dushoff J, Levin SA, Kondrashov AS. 2006. Bursts of nonsynonymous substitutions in HIV-1 evolution reveal instances of positive selection at conservative protein sites. Proc. Natl. Acad. Sci. U. S. A. 103:19396 –19401. http://dx.doi.org/10.1073/pnas.0609484103. Grossman Z, Meier-Schellersheim M, Paul WE, Picker LJ. 2006. Pathogenesis of HIV infection: what the virus spares is as important as what it destroys. Nat. Med. 12:289 –295. http://dx.doi.org/10.1038/nm1380. Grossman Z, Meier-Schellersheim M, Sousa AE, Victorino RM, Paul WE. 2002. CD4⫹ T-cell depletion in HIV infection: are we closer to understanding the cause? Nat. Med. 8:319 –323. http://dx.doi.org/10.1038 /nm0402-319. Gurunathan S, Stobie L, Prussin C, Sacks DL, Glaichenhaus N, Iwasaki A, Fowell DJ, Locksley RM, Chang JT, Wu CY, Seder RA. 2000. Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8⫹ T cells. J. Immunol. 165:915–924. http://dx.doi.org/10.4049/jimmunol.165.2.915. Melhem NM, Smith KN, Huang XL, Colleton BA, Jiang W, Mailliard RB, Mullins JI, Rinaldo CR. 2014. The impact of viral evolution and frequency of variant epitopes on primary and memory human immunodeficiency virus type 1-specific CD8(⫹) T cell responses. Virology 450 – 451:34 – 48. http://dx.doi.org/10.1016/j.virol.2013.10.015. Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, Nowak MA, Giangrande P, Luzzi G, Morgan B, Edwards A, McMichael AJ, Rowland-Jones S. 1997. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat. Med. 3:212–217. http://dx.doi.org/10.1038/nm0297-212. Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, Nelson JA, Gairin JE, Hahn BH, Oldstone MB, Shaw GM. 1997. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat. Med. 3:205–211. http://dx.doi.org/10.1038/nm0297-205. Price DA, Goulder PJ, Klenerman P, Sewell AK, Easterbrook PJ, Troop M, Bangham CR, Phillips RE. 1997. Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection. Proc. Natl. Acad. Sci. U. S. A. 94:1890 –1895. http://dx.doi.org/10.1073/pnas.94.5.1890. Evans DT, O’Connor DH, Jing P, Dzuris JL, Sidney J, da Silva J, Allen TM, Horton H, Venham JE, Rudersdorf RA, Vogel T, Pauza CD, Bontrop RE, DeMars R, Sette A, Hughes AL, Watkins DI. 1999. Virusspecific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef. Nat. Med. 5:1270 –1276. http://dx.doi.org/10.1038/15224. Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, Peyerl FW, Krivulka GR, Beaudry K, Lifton MA, Gorgone DA, Montefiori DC, Lewis MG, Wolinsky SM, Letvin NL. 2002. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 415:335–339. http://dx.doi.org/10.1038/415335a. Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, Workman C, Shaunak S, Olson K, Goulder P, Brander C, Ogg G, Sullivan JS, Dyer W, Jones I, McMichael AJ, Rowland-Jones S, Phillips RE. 2001. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J. Exp. Med. 193:375–386. http://dx.doi.org/10.1084/jem.193.3.375. Goulder PJ, Brander C, Tang Y, Tremblay C, Colbert RA, Addo MM,

Dendritic cells decide CD8(+) T cell fate.

Proliferative reactivity of T cells to autologous, cell-associated antigens.

Human CD8+ T cell clone regulates autologous CD4+ myelin basic protein specific T cells.

Combinatorial immunotherapeutic approaches to restore the function of anergic tumor-reactive cytotoxic CD8+ T cells.

On the Role of Dendritic Cells Versus Other Cells in Inducing Protective CD8+ T Cell Responses.

Combining dendritic cells and B cells for presentation of oxidised tumour antigens to CD8+ T cells.

Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses.

Dendritic cell-secreted lipocalin2 induces CD8+ T-cell apoptosis, contributes to T-cell priming and leads to a TH1 phenotype.

Mycophenolic acid-treated dendritic cells generate regulatory CD4+ T cells that suppress CD8+ T cells' allocytotoxicity.

Dual roles of autologous CD8+ T cells in hematopoietic progenitor cell mobilization and engraftment.

The Role of Invariant Natural Killer T Cells in Dendritic Cell Licensing, Cross-Priming, and Memory CD8(+) T Cell Generation.

Targeting dendritic cell function during systemic autoimmunity to restore tolerance.

Antitumor Efficacy of Radiation plus Immunotherapy Depends upon Dendritic Cell Activation of Effector CD8+ T Cells.

Reprogramming tumor-infiltrating dendritic cells for CD103+ CD8+ mucosal T-cell differentiation and breast cancer rejection.

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity.

Neuroantigen-specific autoregulatory CD8+ T cells inhibit autoimmune demyelination through modulation of dendritic cell function.

CD11b+ migratory dendritic cells mediate CD8 T cell cross-priming and cutaneous imprinting after topical immunization.

Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus.

Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response.

CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in both type 1 and type 2 diabetes.

Plasmacytoid Dendritic Cells Require Direct Infection To Sustain the Pulmonary Influenza A Virus-Specific CD8 T Cell Response.

Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8(+) and CD4(+) T Cells.

Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells.

CD8(+) T activation attenuates CD4(+) T proliferation through dendritic cells modification.