Eur. J. Immunol. 2014. 44: 715–727
DOI: 10.1002/eji.201343775
Immunity to infection
Transcription factor ELF4 promotes development and function of memory CD8+ T cells in Listeria monocytogenes infection Maksim Mamonkin1 , Monica Puppi1 and H. Daniel Lacorazza1,2 1
Department of Pathology & Immunology, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX, USA 2 Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA Most differentiated CD8+ T cells die off at the end of an infection, revealing two main subsets of memory T cells — central and effector memory — which can be found in lymphoid tissues or circulating through nonlymphoid organs, respectively. The cell intrinsic regulation of the differentiation of CD8+ T cells to effector and central memory remains poorly studied. Herein, we describe a novel role of the ETS transcription factor ELF4 in the development and function of memory CD8+ T cells following infection with Listeria monocytogenes. Adoptively transferred Elf4−/− na¨ıve CD8+ T cells produced lower numbers of effector memory CD8+ T cells despite a normal pool of central memory. This was caused by suboptimal priming and decreased survival of CD8+ T cells at the peak of response while enhanced Notch1 signaling and upregulation of eomesodermin correlated with “normal” development of Elf4−/− central memory. Finally, loss of ELF4 impaired the expansion of both central and effector memory CD8+ T cells in a recall response by also activating Notch1 signaling. Altogether, ELF4 emerges as a novel transcriptional regulator of CD8+ T-cell differentiation in response to infection.
Keywords: Bacterial infection
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CD8+ T cells
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ELF4
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Memory
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction The activation and expansion of na¨ıve CD8+ T cells during infection is associated with downregulation of the LN-guiding molecules CD62L and CCR7 and concomitant emigration of effector T cells from the lymphoid tissue in their quest to find and eliminate infected cells [1, 2]. In acute infections, most terminally differentiated CD8+ T cells undergo cell death leaving behind a small population of stable memory T cells [3–5]. The two classic
Correspondence: Dr. H. Daniel Lacorazza e-mail: [email protected]
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
subsets of memory T cells — central and effector memory — have distinct functional features and tissue distribution [6, 7]. Central memory cells display stem cell features such as longevity, proliferative potential, and expression of CD62L, an adhesion molecule that mediates homing into lymphoid organs where they receive prosurvival, quiescence, and activation cues [8]. Conversely, effector memory T cells share many properties with short-lived effector T cells such as the expression of cytotoxic granules, low levels of CD62L on the cell surface, and surveillance of pathogen entry by trafficking through nonlymphoid tissues [7]. Despite having a lower self-renewal capacity and proliferative potential compared to central memory T cells, the circulating effector memory T cells, and CD62Llow tissue-resident memory CD8+ T cells, play a major role in host protection against pathogens such as SIV, HSV,
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vaccinia virus, and Sendai virus [7, 9–11]. The transcriptional machinery regulating differentiation of effector and central memory T cells is largely unknown. The differentiation of CD8+ T cells induced by infection is regulated by a combination of cell extrinsic and intrinsic factors. For example, inflammatory cytokines augment TCR signaling and modulate the balance of key transcription factors such as eomesodermin (Eomes), T-bet, Blimp-1, and Bcl-6 [12–16]. Eomes promotes the development, maintenance, and function of central memory [12, 17], whereas cooperation with T-bet is crucial for acquisition of cytotoxic functions [18]. The expression of Eomes is controlled by the Wnt, Notch1, and IL-12 signaling pathways [19–21]. Recent work suggests that alterations in the intracellular balance of T-bet and Eomes can affect the fate of differentiating CD8+ T cells [12, 22]. The ETS family of transcription factors is involved in multiple cellular processes including development and function of the immune system. We have previously shown that the ETS transcription factor (E74-like factor 4) ELF4 prevents homeostatic and TCR cross-link driven proliferation of naive CD8+ T cells by regulating the expression of KLF4 and raising the threshold of activation. At least in vitro, ELF4 controls the extent of ERK-mediated activation downstream of TCR signaling by maintaining the pool of DUSP [23, 24]. ELF4 restrains both homeostatic and antigen-induced proliferation of supra-physiological numbers of CD8+ T cells in response to the peptide stimulation [24]. However, the role of ELF4 in shaping T-cell response to pathogens in an inflammatory setting and normal frequency of na¨ıve precursors remains unclear. In this work, we show a new role of ELF4 in the in vivo differentiation of na¨ıve CD8+ T cells. Stimulation of physiological numbers of na¨ıve CD8+ T cells by infection with Listeria monocytogenes showed a lower number of effector memory Elf4−/− CD8+ T cells, which was caused at least in part by reduced proliferation within 2.5 days after infection and enhanced cell death at the peak of response. Defective formation of CD62Llow T cells and enhanced CXCR4 expression skewed tissue distribution of Elf4−/− CD8+ T cells to LNs and BM. However, activation of Notch1 signaling and increased expression of Eomes correlated with normal numbers of central memory Elf4−/− CD8+ T cells. Finally, the defect in memory development was both quantitative and qualitative, since the expansion of central and effector memory Elf4−/− CD8+ T cells was severely impaired in recall responses. Collectively, our data show that ELF4 is required for optimal activation and differentiation of CD8+ T cells in response to infection.
Results ELF4 is expressed in proliferating effector CD8+ T cells Infection with Listeria monocytogenes-OVA (Lm-OVA) elicits strong inflammatory immune response with robust activation and differentiation of CD8+ T cells [3]. Therefore, we first assessed the kinetics of ELF4 mRNA and protein expression in OVA-specific CD8+ T cells at different times after infection to investigate the C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2014. 44: 715–727
Figure 1. ELF4 is expressed in proliferating effector CD8+ T cells during Lm-OVA infection. (A) Kinetics of T-cell expansion in the spleen after adoptive transfer of OT-I CD8+ T cells to congenic recipients and subsequent challenge with Lm-OVA (mean ± SD, n = 3). (B) Immunoblot analysis of ELF4 and phospho-Rb expression in OT-I CD8+ T cells purified from the spleen of infected mice. Na¨ıve CD8+ T cells from Elf4−/− mice were used as a negative control of ELF4 detection. (C) Immunoblot analysis of ELF4 expression in na¨ıve (day 0), late effector (day 20), and early memory (day 40) OT-I CD8+ T cells. (A–C) Data are representative of four independent experiments.
role of ELF4 in shaping CD8+ T-cell response to Lm-OVA. Mice adoptively transferred with OT-I CD8+ T cells were systemically infected with Lm-OVA and donor-derived CD8+ T cells were purified at different times post infection from the spleen (Fig. 1A). We found increased levels of ELF4 protein in effector CD8+ T cells at 5 days post infection (dpi), which then gradually decreased in late effector and early memory T cells to the level of na¨ıve CD8+ T cells (Fig. 1B and C). Similar to ELF4, the levels of phosphorylated Rb protein were elevated at 5 dpi indicating that ELF4 is upregulated in proliferating CD8+ T cells (Fig. 1B). Of note, a reduction of ELF4 transcripts at 5 dpi (Supporting Information Fig. 1A), suggested that proliferating effector CD8+ T cells generated by Lm-OVA displayed enhanced stability of the ELF4 protein. This was confirmed in experiments using inhibitors of www.eji-journal.eu
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protein synthesis (cycloheximide, CHX) and proteasomal degradation (MG132) that demonstrated enhanced ELF4 degradation in naive compared to effector CD8+ T cells (Supporting Information Fig. 1B and C).
ELF4 controls formation of memory CD8+ T cells Previous work showed that loss of ELF4 results in profound defects in the development and function of NK and NK-T cells [25]. Hence, we decided to adoptively transfer low numbers of OT-I WT and OT-I Elf4−/− CD8+ T cells into WT congenic recipients to delineate the CD8+ T cell intrinsic role of ELF4 in response to LmOVA. OT-I WT and OT-I Elf4−/− CD8+ T cells were injected either separately (single transfer) or mixed at a 1:1 ratio (cotransfer). Mice were infected 24 h later with Lm-OVA and monitored for the expansion of donor-derived T cells in peripheral blood. In the cotransfer model, loss of ELF4 reduced the expansion of effector T cells and formation of CD8+ T-cell memory after Lm-OVA infection (Fig. 2A). However, Elf4−/− CD8+ T cells displayed normal expression of IFN-γ, TNF-α, granzyme B, perforin, and CD107a (Supporting Information Fig. 1A), suggesting that ELF4 does not regulate acquisition of effector functions. We observed a similar decrease in memory formation in the single transfer model, despite a lower reduction of effector T cells (Fig. 2B). Interestingly, the overall decrease of immunological memory was due to a specific defect in CD62Llow effector memory cells since we observed similar numbers of circulating CD62Lhigh Elf4−/− CD8+ T cells in both models (Fig. 2C and D). Of note, based on the expression of killer cell lectin-like receptor G1 (KLRG1) and CD127 [13], Elf4−/− CD8+ T cells showed normal differentiation to short-lived effector cells (SLEC; KLRG1high ; CD127low ) and memory precursor effector cells (MPECs; KLRG1low ; CD127high ) at the peak of response (Supporting Information Fig. 1B). We also examined the endogenous response of OVA-specific Elf4−/− CD8+ T cells in mixed WT:Elf4−/− BM chimeras. Similar to the adoptive transfer model, Elf4−/− CD8+ T cells produced less OVA-specific memory after Lm-OVA infection while displaying a concomitant increase in the frequency of CD62Lhigh cells (Supporting Information Fig. 2). Hence, data from three different models suggest that ELF4 is required for the formation of CD62Llow effector memory CD8+ T cells.
ELF4 regulates tissue distribution of OVA-specific CD8+ T cells We next analyzed the tissue distribution of WT and Elf4−/− OT-I cells coactivated in the same environment because CD62L (L-selectin) is an important homing molecule. Consistent with a specific defect in the generation of CD62Llow CD8+ T cells, the ratio of WT to Elf4−/− cells was higher in blood, lung, and the spleen, whereas the relative frequencies of both populations were comparable in inguinal LNs (Fig. 3A). Interestingly, we observed increased homing of effector and memory Elf4−/− OT-I CD8+ C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Immunity to infection
T cells to BM (Fig. 3A). Since the chemokine receptor CXCR4 mediates homing of T cells and hematopoietic stem cells to BM [17, 26], we assessed the expression of CXCR4. As expected, circulating Elf4−/− CD8+ T cells expressed relatively higher levels of CXCR4 than controls, both at mRNA and protein levels (Fig. 3B and C). These data indicate that ELF4 modulates tissue distribution of effector and memory CD8+ T cells, generated during infection with Lm-OVA, in a CD62L- and CXCR4-dependent manner.
Ectopic expression of ELF4 augments formation of effector memory CD8+ T cells Deletion of ELF4 can deregulate the expression of genes directly involved in the development of effector memory CD8+ T cells; therefore, we decided to confirm that ELF4 mediates differentiation of effector and memory T cells upon infection by overexpressing ELF4 in OT-I WT CD8+ T cells. We transplanted BM cells from CD45.2+ OT-I mice that were transduced with retrovirus (RV) coexpressing murine ELF4 and GFP (RV-ELF4) into lethally irradiated congenic recipients. Following hematological reconstitution (>2 months), CD44low GFP+ CD8+ T cells were purified from the spleen of transplanted mice by cell sorting and adoptively transferred to congenic CD45.1+ mice, which were then infected with Lm-OVA. The overexpression of ELF4 was confirmed in CD8+ T cells used for adoptive transfer by immunoblots (Fig. 4A). As expected, ectopic ELF4 expression led to increased numbers of CD62Llow memory CD8+ T cells in the spleen with no alterations in the frequency of CD62Lhigh cells (Fig. 4B and C). Therefore, ELF4 specifically promotes formation of effector memory CD8+ T cells.
ELF4 deficiency impairs priming and survival of OVA-specific CD8+ T cells Pathogen-specific CD8+ T cells are activated by splenic macrophages and DCs that have captured circulating L. monocytogenes [27, 28]. Therefore, we examined whether differences in early expansion of co-transferred OT-I WT and OT-I Elf4−/− CD8+ T cells (1:1) could account for the lower expansion of Elf4−/− CD8+ T cells. Despite similar frequencies of WT and Elf4−/− OT-I cells in the spleen at 1.5 dpi, we detected a 2-fold increase in the WT to Elf4−/− ratio at 2.5 dpi (Fig. 5A), which was likely caused by impaired proliferation or survival of activated OT-I Elf4−/− CD8+ T cells. Of note, expression of CD69 and CD25 (1.5 dpi) and CD44 and CD5 (3.5 dpi) revealed a similar activation of Elf4−/− and WT CD8+ T cells (Fig. 5B–D). However, the lower numbers of CD25high Elf4−/− CD8+ T cells observed at 2.5 dpi (Fig. 5C) correlated with decreased proliferation of Elf4−/− CD8+ T cells (Fig. 5E). A defective generation of memory could be due to enhanced cell death of activated Elf4−/− CD8+ T cells. Although no differences in Annexin V-positive cells were initially observed, splenic Elf4−/− effector CD8+ T cells showed increased apoptosis at the peak of response (Fig. 5F). This correlated with decreased mRNA expression of the www.eji-journal.eu
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Figure 2. Impaired development of memory by Elf4−/− CD8+ T cells in response to Lm-OVA. (A) OT-I WT (CD45.1+ ) and OT-I Elf4−/− (CD45.2+ ) CD8+ T cells (500 cells each per mouse) were cotransferred into congenic recipient mice (CD45.1+ CD45.2+ ), which were infected with Lm-OVA 24 h later. Contribution of each donor (WT, CD45.1+ , and Elf4−/− , CD45.2+ ) to circulating CD8+ T cells is shown for the input, 9 dpi, and 70 dpi. The immune response to Lm-OVA is shown on the right for WT and Elf4−/− cells as percentage of total CD8+ T cells. (B) OT-I WT (CD45.2+ ) and OT-I Elf4−/− (CD45.2+ ) CD8+ T cells (1000/mouse) were separately transferred into congenic recipient mice (CD45.1+ ) 24 h before Lm-OVA infection. Contribution of each donor (WT, CD45.2+ , and Elf4−/− , CD45.2+ ) to circulating CD8+ T cells is shown for 7 and 40 dpi. Kinetics of expansion in blood is shown on the right as percentage of total CD8+ T cells. (C) CD62L expression in donor WT and Elf4−/− CD8+ T cells and overall frequency of CD62Lhigh and CD62Llow in total blood CD8+ T cells. (D) Same as (C) for single adoptive transfer. (C, D) Data are shown as mean ± SD (n = 5) and (A–D) are representative of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Immunity to infection
Figure 3. ELF4 modulates tissue distribution of Lm-OVA-specific CD8+ T cells. (A) The contribution of each donor (WT, CD45.1+ , and Elf4−/− , CD45.2+ ) was analyzed by flow cytometry in blood, lung, spleen, LN, and BM at the peak of response (7 dpi) and 60 dpi. Ratios of WT to Elf4−/− OT-I CD8+ T cells in different tissues are shown on the right as mean ± SD (n = 3). Dashed line indicates 1:1 ratio of the input. (B) Expression of CXCR4 transcripts was measured by qPCR in donor WT and Elf4−/− OT-I CD8+ T cells at the peak of response. (C) Cell surface expression of CXCR4 in donor WT and Elf4−/− OT-I CD8+ T cells at the peak of response. MFI is shown for effector T cells from three pairs of WT and Elf4−/− OT-I CD8+ T cells cotransferred in recipient mice (connected by dashed lines). (A–B) Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. *p < 0.05; **p < 0.01 (two-tailed Student’s t-test).
anti-apoptotic gene bcl2a1a in Elf4−/− effector CD8+ T cells at 7 dpi (Fig. 5G). These data indicate that ELF4 is required for optimal priming of na¨ıve CD8+ T cells and survival of Lm-OVA-specific effector CD8+ T cells.
Activated Notch1 pathway in Elf4−/− CD8+ T cells A reduced pool of memory T cells with skewed ratio of centralto-effector memory in Elf4−/− CD8+ T cells may be caused by an imbalance of key intrinsic factors of CD8+ T-cell differentiation, such as T-bet and Eomes [12, 13, 17, 20, 29, 30]. For example, the development of central memory depends on the expression of Eomes in differentiating T cells [17, 21]. Elf4−/− CD8+ T cells expressed higher levels of Eomes, both mRNA and protein, with normal levels of T-bet, lowering the T-bet/Eomes ratio at the peak of response (Fig. 6A and B). This was not caused by deregulation of Tcf-1 and Runx3 [21,31] (Supporting Information Fig. 4). In addition, loss of ELF4 did not affect the expression C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of Blimp-1, Bcl-6, Id2, Id3, and KLF2 (Supporting Information Fig. 4). Since Notch1 signaling drives the expression of Eomes [19], we assessed the activity of Notch1 in WT and Elf4−/− effector CD8+ T cells. The expression of Notch1 target genes Hes1, Hey1 and HeyL was significantly upregulated in Elf4−/− effector CD8+ T cells (Fig. 6C). Moreover, the increased levels of cleaved Notch1 in Elf4−/− effector CD8+ T cells further suggested augmented Notch1 signaling (Fig. 6D). The increased expression of Eomes and Notch1 target genes was not a result of a selective enrichment of CD62L positive cells within effector Elf4−/− CD8+ T cells as we also detected increased expression of these genes in CD62Llow Elf4−/− CD8+ T cells purified at the peak of response (Fig. 6E). Loss of ELF4 did not alter the transcript levels of Notch1 inhibitors such as Numb, Itch and Fbxw7 (Fig. 6F), indicating that ELF4 does not increase Notch1 signaling by modulating expression of Notch1 inhibitors. These data suggest that enhanced Notch1 signaling in Elf4−/− CD8+ T cells increases expression of Eomes and promotes development of central memory. www.eji-journal.eu
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Figure 4. Ectopic expression of ELF4 augments production of effector memory CD8+ T cells. (A) BM cells from OT-I mice were transduced with either empty (RV) or ELF4-expressing (RV-ELF4) RV and transplanted into lethally irradiated recipient mice. GFP+ CD8+ T cells were purified from the spleen of reconstituted mice and analyzed by immunoblot. Numbers indicate fold change in ELF4 expression estimated by densitometry. (B) Purified GFP+ OT-I cells (CD45.2+ ) were injected into B6.SJL (CD45.1+ ) mice that were challenged with Lm-OVA. Percentages of donor-derived memory T cells and CD62L profiles are shown in the spleen at 47 dpi. Data represent mean ± SD for each group. (C) The number of CD62Lhigh and CD62Llow memory CD8+ T cells in the spleen is shown for RV and RV-ELF4 groups. Data are shown as mean ± SD (n = 5) and (A–B) are representative of two independent experiments. *p < 0.05 (two-tailed Student’s t-test).
Loss of ELF4 impairs recall response of memory CD8+ T cells Elf4−/− CD8+ T cells display impaired development of immunological memory in response to Lm-OVA infection due to defective formation of effector memory CD8+ T cells. We confirmed that CD62Lhigh Elf4−/− CD8+ T memory cells also expressed other markers of central memory such as CCR7 and CD27 (Fig. 7A). An important property of memory T cells is their capacity to mount a robust secondary expansion upon encounter with the same antigen. To assess the functional role of ELF4 in memory T cells, purified WT and Elf4−/− CD62Lhigh memory OT-I CD8+ T cells were mixed at a 1:1 ratio, adoptively transferred into congenic recipients and challenged with a lethal dose of Lm-OVA. Elf4−/− CD8+ T cells failed to expand as WT controls (Fig. 7B), suggesting that central memory Elf4−/− CD8+ T cells exhibit a severe functional defect despite an apparent normal differentiation. Strikingly, loss of ELF4 abrogated the expansion of CD62Llow effector memory CD8+ T cells (Fig. 7C). Similar to primary response, the tissue distribution of secondary effector Elf4−/− CD8+ T cells showed reduced distribution in blood and lung (Fig. 7D). These data suggest that ELF4 is required for recall responses of memory CD8+ T cells. To further characterize the defective expansion of Elf4−/− central memory CD8+ T cells, we adoptively transferred WT and Elf4−/− CD62Lhigh memory OT-I CD8+ T cells separately to congenic recipients and assessed their presence in blood and the spleen. Similar to the co-transfer experiment, Elf4−/− central memory cells displayed a severe impairment in their capacity to expand (Fig. 8A). The frequency of Elf4−/− secondary effector cells was significantly reduced in the spleen as early as 4 days after re C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
challenge (Fig. 8B) whereas the bacterial burden in the spleen was controlled nearly as efficiently as controls (Fig. 8C). However, neither proliferation or cell death was altered at least 4 dpi (Fig. 8D and E) and 6 dpi (data not shown) in expanding Elf4−/− CD8+ T cells, suggesting that loss of ELF4 compromised early activation or priming of central memory CD8+ T cells. The expression of Eomes, Hes1 and CXCR4 mRNA in Elf4−/− secondary effector CD8+ T cells was also elevated (Fig. 8F). Collectively, our results demonstrate a previously unknown function of ELF4 in promoting development of functional memory CD8+ T cells in response to infection.
Discussion A better understanding of the molecular circuitry controlling the development and function of effector and memory CD8+ T cells is the first step toward the generation of vaccines and immunotherapy for cancer and autoimmune disorders. In this work, we show that the ETS transcription factor ELF4 is required for the differentiation of effector memory CD8+ T cells and the recall response of effector and central memory CD8+ T cells at least in part by modulating Notch1 signaling. Loss of ELF4 impaired the proliferation and survival of OVAspecific CD8+ T cells upon infection with L. monocytogenes, resulting in a smaller pool of effector memory T cells. We ruled out differential homing of adoptively co-transferred wild type and Elf4−/− CD8+ T cells to the priming site and enhanced activationinduced cell death upon initial antigen encounter. Even though the numbers of central memory CD8+ T cells were not significantly affected in the primary response, the ability to expand www.eji-journal.eu
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Immunity to infection
Figure 5. Loss of ELF4 impairs priming and survival of Lm-OVA-specific CD8+ T cells. (A) Expansion in the spleen of WT and Elf4−/− OT-I CD8+ T cells cotransferred into recipient mice prior to Lm-OVA infection is shown at 1.5 and 2.5 dpi. Ratios of WT to Elf4−/− T cells are shown on the right. (B) Expression of the activation marker CD69 in WT and Elf4−/− OT-I CD8+ T cells at 1.5 dpi. Numbers of CD69high cells (per 104 of total splenic CD8+ T cells) are shown on the right. (C) Numbers of splenic CD25high WT and Elf4−/− CD8+ T cells per 104 of total splenic CD8+ T cells at 1.5 and 2.5 dpi (n = 4, mean ± SD). (D) Expression of CD44 and CD5 in WT and Elf4−/− OT-I CD8+ T cells at 3.5 dpi. (E) Dilution of eFluor670 dye in WT and Elf4−/− OT-I CD8+ T cells labeled before transfer and analyzed at 2.5 dpi. Bar graphs show percent of undivided cells and division index for CD8+ T cells (n = 4, mean ± SD). (F) Annexin V staining of splenic WT and Elf4−/− OT-I CD8+ T cells at different times post infection is shown for three pairs of WT and Elf4−/− OT-I CD8+ T cells cotransferred to recipient mice (connected by dashed lines). (G) mRNA expression of pro- and anti-apoptotic genes in WT and Elf4−/− CD8+ T cells at 7 dpi. Pooled cDNA from three mice in each group was used for the PCR array. (A–F) The data are representative of 2–3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
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Figure 6. Increased Eomes expression and enhanced Notch signaling in Elf4−/− effector CD8+ T cells. (A) The mRNA levels of T-bet and Eomes were measured in WT and Elf4−/− OT-I CD8+ T cells purified from the spleen of infected mice (7 dpi) and normalized to β-actin. (B) Expression of Eomes and T-bet was assessed by intracellular staining in WT (open histogram) and Elf4−/− (shaded histogram) effector CD8+ T cells at the peak of response (7 dpi). (C) Transcript levels of Notch target genes (Hes1, Hey1, and HeyL) normalized to β-actin was measured in splenic WT and Elf4−/− OT-I CD8+ T cells at 7 dpi. (D) Levels of cleaved Notch1 were analyzed by immunoblot using WT and Elf4−/− OT-I CD8+ T cells isolated at 5 and 7 dpi. Numbers indicate relative levels estimated by densitometry normalized to β-actin. (E) Expression of Eomes, Hes1, and Hey1 mRNA in WT and Elf4−/− CD62low CD8+ T cells at 7 dpi. (F) Transcript levels of Notch1 inhibitors in WT and Elf4−/− CD62low CD8+ T cells at 7 dpi. (A–F) Data are shown as mean + SD (n = 3) and are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
in a secondary infection was severely compromised. In contrast, Elf4−/− effector memory CD8+ T cells showed both developmental and functional defects. This impaired formation of immunological memory was not due to competition with WT OT-I cells for antigen and/or cytokines because we observed same outcome in two additional models of infection (single adoptive transfer and mixed BM chimeras). We previously showed that ELF4 controls the proliferation of na¨ıve CD8+ T cells both in vitro and in vivo [23, 24]. The use of supra-physiological numbers of Elf4−/− CD8+ T cells and a low inflammatory model of activation (peptide-pulsed DCs) led to increased numbers of memory T cells [24]. In contrast, the activa C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tion of physiological numbers of CD8+ T cells by bacterial infection resulted in decreased expansion and defective differentiation of effector memory T cells, indicating that the frequency of antigenspecific CD8+ T cells combined with an inflammatory environment altered the fate of activated Elf4−/− CD8+ T cells. Thus, it is possible that the lower threshold of activation in Elf4−/− OT-I CD8+ T cells and the addition of more signals elicited by inflammatory cytokines in a physiological priming overruled the stimulation of proliferation and survival of differentiating CD8+ T cells. In support of T-cell extrinsic mechanisms, a recent study showed that inflammatory cytokines induced by infection with Lm-OVA augment the TCR signaling in OT-I CD8+ T cells [32]. Furthermore,
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Immunity to infection
Figure 7. Loss of ELF4 impairs recall responses of memory CD8+ T cells. (A) Coexpression of CCR7, CD27, and CD62L in WT and Elf4−/− memory CD8+ T cells (110 dpi). Numbers indicate the percentage of double-positive cells (mean ± SD). (B) Expansion of central memory CD8+ T cells in a recall response. Central memory CD8+ T cells (CD62Lhigh ) were purified from infected mice cotransferred with WT and Elf4−/− CD8+ T cells (110 dpi) and adoptively transferred as a 1:1 mixture to secondary recipient mice, that were infected a day after with Lm-OVA (200 × 103 CFU, i.v.). (C) Effector memory CD8+ T cells (CD62Llow ) were purified from infected mice cotransferred with WT and Elf4−/− CD8+ T cells and tested as described in (A). (D) Tissue distribution of secondary effector CD8+ T cells was analyzed 20 days after recall response of cotransferred central memory WT and Elf4−/− OT-I CD8+ T cells (as shown in B). The ratio of WT to Elf4−/− was calculated for lung, blood, spleen, inguinal LNs, and BM. Dashed line indicates input ratio of transferred memory T cells. (A–D) Data are shown as mean ± SD (n = 5) and (A–D) are representative of two independent experiments. ns, not significant; *p < 0.05; ***p < 0.001. (two-tailed Student’s t-test).
the Notch1 pathway was activated in Elf4−/− CD8+ T cells likely in a ligand-independent manner downstream of the TCR signaling [33]. In turn, the enhanced Notch1 signaling probably increased the expression of Eomes — a known positive regulator of central memory development — in Elf4−/− CD8+ T cells resulting in skewed differentiation toward central memory T cells to the detriment of CD62Llow memory precursors. This increased expression of Eomes also correlated with elevated levels of CXCR4 in Elf4−/− OT-I cells and augmented homing to BM, a known site for homeostatic maintenance of central memory T cells [17, 34]. The activation of the Notch1 pathway can also affect proliferation and cytokine production in T cells. IL-2 signaling plays a criti C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cal role for optimal formation of CD62Llow effector and effector memory CD8+ T cells [35, 36]. Therefore, impaired generation of CD25 high Elf4−/− CD8+ T cells at an early stage of expansion may limit the development of effector memory CD8+ T cells. Previous studies have linked enhanced Notch1 signaling with decreased proliferation and IL-2 production in activated T cells [37–39]. Therefore, the enhanced Notch1 activity may be an important factor in the suboptimal formation of effector and effector memory Elf4−/− CD8+ T cells. A model of CD8+ T-cell differentiation postulates that infection triggers a developmental program that simultaneously generate terminal effector and memory precursor T cells [5]. At least three www.eji-journal.eu
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Figure 8. Proliferation and survival of Elf4−/− secondary effector CD8+ T cells. (A) Expansion of WT and Elf4−/− central memory CD8+ T cells in peripheral blood that were transferred separately to secondary recipient mice and re-activated by infection with Lm-OVA (200 × 103 CFU, i.v.) as in Fig. 7A. (B) Frequencies of WT and Elf4−/− secondary effector CD8+ T cells in the spleen of recipient mice at 4 dpi. (C) Bacterial burden in the spleen of infected mice at 4 dpi (n.s., no statistical difference). (D) Proliferation of donor-derived WT and Elf4−/− CD8+ T cells in the spleen at 4 dpi was assessed by BrdU incorporation assay. Open histograms show BrdU incorporated in WT and Elf4−/− CD8+ T cells. Shaded histograms correspond to the BrdU staining profile of total splenic CD8+ T cells. Bar graph indicates total percentage of BrdU-positive cells in both groups. (E) Annexin V staining of splenic donor-derived WT and Elf4−/− CD8+ T cells. Gates indicate Annexin V positive cells, with total percentage of Annexin V positive cells shown in the bar graph. (F) Transcript levels of Eomes, Hes1, CXCR4, and T-bet in WT and Elf4−/− secondary effector CD8+ T cells at 5 dpi. (A–F) Data are shown as mean ± SD (n = 3) and (A–D) are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
distinct populations can be identified at the peak of response by flow cytometric analysis: terminal effector (CD127low CD62Llow ) cells, effector memory precursors (CD127high CD62Llow ), and central memory precursors (CD127high CD62Lhigh ) [5]. Hence, ELF4 may selectively regulate commitment of differentiating T cells toward an effector memory lineage. Even though we detected normal numbers of immunophenotypic SLEC and MPEC populations,
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Elf4−/− precursors of effector memory CD8+ T cells may exhibit functional defects. In addition to a developmental defect, loss of ELF4 severely impaired the expansion of adoptively transferred central and effector memory CD8+ T cells in a recall response. The mechanism of impaired expansion Elf4−/− memory CD8+ T cells in a recall response is similar to the primary response. This defect
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could have been imprinted in memory T cells early in the process during a suboptimal primary response or rather reflects a greater role of ELF4 in regulating the expansion of memory T cells. In addition, a recall response with lethal dose of Lm-OVA entails a greater inflammation compared to the primary response. Therefore, loss of ELF4 did not only decrease the total number of memory cells upon Lm-OVA infection but also compromised their function. Collectively, this study describes a novel function of ELF4 in regulating differentiation and function of memory CD8+ T-cell in response to infection. In the context of inflammation, ELF4 promotes priming and survival of na¨ıve and effector CD8+ T cells and differentiation of effector memory CD8+ T cells. Furthermore, ELF4 has a critical role in recall responses of both effector and central memory CD8+ T cells. Therefore, this work offers a novel molecular target to improve fitness of memory T cells.
Materials and methods Mice C57BL/6 (B6), C57BL/6.SJL (B6.SJL), and OT-I mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). B6 mice (CD45.2+ ) were bred with B6.SJL mice (CD45.1+ ) to generate B6xB6.SJL F1 (CD45.1+ CD45.2+ ) recipients. Elf4−/− mice were obtained from S. Nimer [25], backcrossed for more than 12 generations to B6, and bred with OT-I B6 to generate OT-I Elf4−/− mice. OT-I transgenic mice were crossed to B6 (CD45.2+ ) and to B6.SJL (CD45.1+ ). Mice were maintained in specific pathogenfree conditions at Baylor College of Medicine. All experiments and procedures were done in compliance with the Institutional Animal Care and Usage Committee of Baylor College of Medicine (AN3698).
Adoptive transfer and infection with L. monocytogenes For adoptive transfer experiments, na¨ıve CD44low OT-I CD8+ T cells were either negatively enriched (90–97% purity) with BDImag magnetic separation system (BD Biosciences), with addition of biotinylated anti-CD44 antibody (BD Biosciences), or purified using a MoFlo cell sorter (Cytomation; 97–99% purity). For all cotransfer experiments, naive CD8+ T cells were purified from the spleen of OT-I WT (CD45.1+ ) and OT-I Elf4−/− (CD45.2+ ) mice, mixed at 1:1 ratio, and adoptively transferred to B6xB6.SJL F1 (CD45.1+ CD45.2+ ) recipients (1000 cells/mouse i.v., to approach physiological frequency). For analysis of proliferation at 2.5 dpi, naive purified CD8+ T cells were labeled with 4 μM eFluor670 (eBioscience), mixed at a 1:1 ratio, and adoptively transferred to congenic recipients (2 × 105 cells per mouse, to facilitate detection). Recipient mice were infected 24 h later with recombinant OVA-expressing L. monocytogenes (Lm-OVA) strain (4 × 103 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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CFU, i.v.). For recall responses, donor-derived memory CD62Lhigh and CD62Llow OT-I CD8+ T cells were purified by cell sorting at 110 dpi and adoptively transferred (104 cells/mouse) either separately or mixed (1:1) into congenic unchallenged recipients followed by a challenge with a lethal dose of Lm-OVA (200 × 103 CFU). Bacterial burden was measured in the spleen of infected mice by plating single-cell suspension permeabilized with 0.05% Triton on brain-heart infusion (Fluka) agar plates supplemented with 10 μg/ml erythromycin.
Flow cytometry Longitudinal analysis of T-cell expansion was monitored in blood collected by tail vein bleeding. For tissue distribution analysis, lungs were perfused with 5 mL cold PBS and processed as described before [24]. BM cells were flushed out from femurs and tibias. Cells were incubated with anti-CD16/32 (Biolegend) prior to cell surface staining. All antibodies for surface proteins were purchased from Biolegend (anti-CD45.2, anti-CD107, anti-CD45.1, anti-CD8, anti-CD25, anti-CD62L, and anti-CD27), eBioscience (anti-CD127, streptavidin, anti-CXCR4, and antiCD69) and BD Biosciences (streptavidin and anti-KLRG1). Kb -OVA tetramers were synthesized at the MHC Tetramer Core Laboratory (Baylor College of Medicine). CCR7 was detected using recombinant CCL19-Fc protein (eBioscience) as previously described [24]. For intracellular cytokine staining, cells were incubated with 1 μg/mL OVA(257-264) peptide (AnaSpec) in RPMI + 10% FBS for 5 h at 37◦ C in the presence of 1 μl/mL Golgi Plug and 0.8 μL/mL Golgi Stop (BD Biosciences). Cells were stained on the cell surface for 20 min on ice, followed by intracellular staining with anti-IFNγ, anti-TNFα (BD Biosciences), anti-perforin, or antigranzyme B (eBioscience) in BD Cytofix/Cytoperm buffer according to the manufacturer’s instructions (BD Biosciences). Apoptosis was measured with Annexin V (Biolegend). Detection of Eomes was performed using anti-Eomes (eBioscience) antibody and FoxP3 staining buffer set (eBioscience). For BrdU incorporation assay, mice were injected with BrdU (2 mg, i.p.) and 4 h later splenocytes were stained using BrdU Flow Kit (BD Biosciences). Flow cytometry was performed using FACSCanto instrument (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).
BM transduction and transplantation To generate mixed WT:Elf4−/− BM chimeras, lethally irradiated (960 Rad) CD45.1+ WT recipient mice were transplanted with a mixture (1:1) of WT (CD45.1+ ) and Elf4−/− (CD45.2+ ) BM cells (10 × 106 cells/mouse). Three months later, transplanted mice were challenged with Lm-OVA (4 × 103 CFU, i.v.). To overexpress ELF4, BM cells were flushed out from femur and tibia of OT-I CD45.2+ mice that received 5-FU (150 mg/Kg i.p.) 5 days prior. Cells were cultured in X-VIVO 15 medium (Lonza) in the presence of SCF (100 ng/mL), IL-6 (10 ng/mL), and IL-3 (6 ng/mL) www.eji-journal.eu
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for 48 h before retroviral transduction. Cells were transferred to retronectin-coated plates and transduced by spinoculation (400 g, 1 h at 25◦ C) using viral supernatant containing either MigR1 (empty RV) or MigR1-ELF4 (RV-ELF4) [40] in the presence of polybrene (8 μg/mL). BM cells were cultured for additional 48 h and injected i.v. to irradiated (960 Rad) congenic recipient mice.
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ELF4 protein was detected using IgG-purified serum from immunized rabbits [25]. Rabbit antibodies against phospho-Rb (Cell Signaling), Notch1 (D1E11, Cell Signaling), and β-Actin (A5060, Sigma-Aldrich) were used. HRP-linked goat antirabbit IgG (Cell Signaling) was used as secondary antibodies and membranes were developed using ECL Western Blotting Substrate (Pierce) on X-ray films (Amersham). For the protein stability assay, naive or effector CD8+ OT-I cells from 2 to 3 donors were cultured in X-VIVO 15 medium at 37◦ C in the presence 20 μg/mL CHX or 20 μM MG132. Cells were collected at indicated time points, washed twice with PBS and analyzed by immunoblot.
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Acknowledgments: Authors acknowledge T. Goltsova and C. Threeton for cell sorting, P.-H. Lee for critical reading of the manuscript, Drs. K. Schluns and V. Badovinac for providing Lm-OVA, and Karen Prince for the preparation of figures. This work was supported by National Institutes of Health Grants R01AI077536 and R01-AI077536-02S1 (to H.D.L).
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Conflict of interests: Authors declare no financial or commercial conflict of interests.
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Abbreviations: CHX: cycloheximide · dpi: days post infection · ELF4: E74like factor 4 · Eomes: eomesodermin · KLRG1: killer cell lectin-like receptor G1 · Lm-OVA: Listeria monocytogenes-OVA · RV: retrovirus
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Full correspondence: Dr. H. Daniel Lacorazza, Department of Pathology & Immunology, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX 77030, USA e-mail: [email protected]
ondary effector and memory CD8 T cells. J. Immunol. 2011. 187: 4068–4076. 31 Cruz-Guilloty, F., Pipkin, M. E., Djuretic, I. M., Levanon, D., Lotem, J., Lichtenheld, M. G., Groner, Y. et al., Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs. J. Exp. Med. 2009. 206: 51–59.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 5/6/2013 Revised: 11/10/2013 Accepted: 10/12/2013 Accepted article online: 13/12/2013
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DOI: 10.1002/eji.201343775
Immunity to infection
Transcription factor ELF4 promotes development and function of memory CD8+ T cells in Listeria monocytogenes infection Maksim Mamonkin1 , Monica Puppi1 and H. Daniel Lacorazza1,2 1
Department of Pathology & Immunology, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX, USA 2 Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA Most differentiated CD8+ T cells die off at the end of an infection, revealing two main subsets of memory T cells — central and effector memory — which can be found in lymphoid tissues or circulating through nonlymphoid organs, respectively. The cell intrinsic regulation of the differentiation of CD8+ T cells to effector and central memory remains poorly studied. Herein, we describe a novel role of the ETS transcription factor ELF4 in the development and function of memory CD8+ T cells following infection with Listeria monocytogenes. Adoptively transferred Elf4−/− na¨ıve CD8+ T cells produced lower numbers of effector memory CD8+ T cells despite a normal pool of central memory. This was caused by suboptimal priming and decreased survival of CD8+ T cells at the peak of response while enhanced Notch1 signaling and upregulation of eomesodermin correlated with “normal” development of Elf4−/− central memory. Finally, loss of ELF4 impaired the expansion of both central and effector memory CD8+ T cells in a recall response by also activating Notch1 signaling. Altogether, ELF4 emerges as a novel transcriptional regulator of CD8+ T-cell differentiation in response to infection.
Keywords: Bacterial infection
r
CD8+ T cells
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ELF4
r
Memory
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction The activation and expansion of na¨ıve CD8+ T cells during infection is associated with downregulation of the LN-guiding molecules CD62L and CCR7 and concomitant emigration of effector T cells from the lymphoid tissue in their quest to find and eliminate infected cells [1, 2]. In acute infections, most terminally differentiated CD8+ T cells undergo cell death leaving behind a small population of stable memory T cells [3–5]. The two classic
Correspondence: Dr. H. Daniel Lacorazza e-mail: [email protected]
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subsets of memory T cells — central and effector memory — have distinct functional features and tissue distribution [6, 7]. Central memory cells display stem cell features such as longevity, proliferative potential, and expression of CD62L, an adhesion molecule that mediates homing into lymphoid organs where they receive prosurvival, quiescence, and activation cues [8]. Conversely, effector memory T cells share many properties with short-lived effector T cells such as the expression of cytotoxic granules, low levels of CD62L on the cell surface, and surveillance of pathogen entry by trafficking through nonlymphoid tissues [7]. Despite having a lower self-renewal capacity and proliferative potential compared to central memory T cells, the circulating effector memory T cells, and CD62Llow tissue-resident memory CD8+ T cells, play a major role in host protection against pathogens such as SIV, HSV,
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vaccinia virus, and Sendai virus [7, 9–11]. The transcriptional machinery regulating differentiation of effector and central memory T cells is largely unknown. The differentiation of CD8+ T cells induced by infection is regulated by a combination of cell extrinsic and intrinsic factors. For example, inflammatory cytokines augment TCR signaling and modulate the balance of key transcription factors such as eomesodermin (Eomes), T-bet, Blimp-1, and Bcl-6 [12–16]. Eomes promotes the development, maintenance, and function of central memory [12, 17], whereas cooperation with T-bet is crucial for acquisition of cytotoxic functions [18]. The expression of Eomes is controlled by the Wnt, Notch1, and IL-12 signaling pathways [19–21]. Recent work suggests that alterations in the intracellular balance of T-bet and Eomes can affect the fate of differentiating CD8+ T cells [12, 22]. The ETS family of transcription factors is involved in multiple cellular processes including development and function of the immune system. We have previously shown that the ETS transcription factor (E74-like factor 4) ELF4 prevents homeostatic and TCR cross-link driven proliferation of naive CD8+ T cells by regulating the expression of KLF4 and raising the threshold of activation. At least in vitro, ELF4 controls the extent of ERK-mediated activation downstream of TCR signaling by maintaining the pool of DUSP [23, 24]. ELF4 restrains both homeostatic and antigen-induced proliferation of supra-physiological numbers of CD8+ T cells in response to the peptide stimulation [24]. However, the role of ELF4 in shaping T-cell response to pathogens in an inflammatory setting and normal frequency of na¨ıve precursors remains unclear. In this work, we show a new role of ELF4 in the in vivo differentiation of na¨ıve CD8+ T cells. Stimulation of physiological numbers of na¨ıve CD8+ T cells by infection with Listeria monocytogenes showed a lower number of effector memory Elf4−/− CD8+ T cells, which was caused at least in part by reduced proliferation within 2.5 days after infection and enhanced cell death at the peak of response. Defective formation of CD62Llow T cells and enhanced CXCR4 expression skewed tissue distribution of Elf4−/− CD8+ T cells to LNs and BM. However, activation of Notch1 signaling and increased expression of Eomes correlated with normal numbers of central memory Elf4−/− CD8+ T cells. Finally, the defect in memory development was both quantitative and qualitative, since the expansion of central and effector memory Elf4−/− CD8+ T cells was severely impaired in recall responses. Collectively, our data show that ELF4 is required for optimal activation and differentiation of CD8+ T cells in response to infection.
Results ELF4 is expressed in proliferating effector CD8+ T cells Infection with Listeria monocytogenes-OVA (Lm-OVA) elicits strong inflammatory immune response with robust activation and differentiation of CD8+ T cells [3]. Therefore, we first assessed the kinetics of ELF4 mRNA and protein expression in OVA-specific CD8+ T cells at different times after infection to investigate the C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. ELF4 is expressed in proliferating effector CD8+ T cells during Lm-OVA infection. (A) Kinetics of T-cell expansion in the spleen after adoptive transfer of OT-I CD8+ T cells to congenic recipients and subsequent challenge with Lm-OVA (mean ± SD, n = 3). (B) Immunoblot analysis of ELF4 and phospho-Rb expression in OT-I CD8+ T cells purified from the spleen of infected mice. Na¨ıve CD8+ T cells from Elf4−/− mice were used as a negative control of ELF4 detection. (C) Immunoblot analysis of ELF4 expression in na¨ıve (day 0), late effector (day 20), and early memory (day 40) OT-I CD8+ T cells. (A–C) Data are representative of four independent experiments.
role of ELF4 in shaping CD8+ T-cell response to Lm-OVA. Mice adoptively transferred with OT-I CD8+ T cells were systemically infected with Lm-OVA and donor-derived CD8+ T cells were purified at different times post infection from the spleen (Fig. 1A). We found increased levels of ELF4 protein in effector CD8+ T cells at 5 days post infection (dpi), which then gradually decreased in late effector and early memory T cells to the level of na¨ıve CD8+ T cells (Fig. 1B and C). Similar to ELF4, the levels of phosphorylated Rb protein were elevated at 5 dpi indicating that ELF4 is upregulated in proliferating CD8+ T cells (Fig. 1B). Of note, a reduction of ELF4 transcripts at 5 dpi (Supporting Information Fig. 1A), suggested that proliferating effector CD8+ T cells generated by Lm-OVA displayed enhanced stability of the ELF4 protein. This was confirmed in experiments using inhibitors of www.eji-journal.eu
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protein synthesis (cycloheximide, CHX) and proteasomal degradation (MG132) that demonstrated enhanced ELF4 degradation in naive compared to effector CD8+ T cells (Supporting Information Fig. 1B and C).
ELF4 controls formation of memory CD8+ T cells Previous work showed that loss of ELF4 results in profound defects in the development and function of NK and NK-T cells [25]. Hence, we decided to adoptively transfer low numbers of OT-I WT and OT-I Elf4−/− CD8+ T cells into WT congenic recipients to delineate the CD8+ T cell intrinsic role of ELF4 in response to LmOVA. OT-I WT and OT-I Elf4−/− CD8+ T cells were injected either separately (single transfer) or mixed at a 1:1 ratio (cotransfer). Mice were infected 24 h later with Lm-OVA and monitored for the expansion of donor-derived T cells in peripheral blood. In the cotransfer model, loss of ELF4 reduced the expansion of effector T cells and formation of CD8+ T-cell memory after Lm-OVA infection (Fig. 2A). However, Elf4−/− CD8+ T cells displayed normal expression of IFN-γ, TNF-α, granzyme B, perforin, and CD107a (Supporting Information Fig. 1A), suggesting that ELF4 does not regulate acquisition of effector functions. We observed a similar decrease in memory formation in the single transfer model, despite a lower reduction of effector T cells (Fig. 2B). Interestingly, the overall decrease of immunological memory was due to a specific defect in CD62Llow effector memory cells since we observed similar numbers of circulating CD62Lhigh Elf4−/− CD8+ T cells in both models (Fig. 2C and D). Of note, based on the expression of killer cell lectin-like receptor G1 (KLRG1) and CD127 [13], Elf4−/− CD8+ T cells showed normal differentiation to short-lived effector cells (SLEC; KLRG1high ; CD127low ) and memory precursor effector cells (MPECs; KLRG1low ; CD127high ) at the peak of response (Supporting Information Fig. 1B). We also examined the endogenous response of OVA-specific Elf4−/− CD8+ T cells in mixed WT:Elf4−/− BM chimeras. Similar to the adoptive transfer model, Elf4−/− CD8+ T cells produced less OVA-specific memory after Lm-OVA infection while displaying a concomitant increase in the frequency of CD62Lhigh cells (Supporting Information Fig. 2). Hence, data from three different models suggest that ELF4 is required for the formation of CD62Llow effector memory CD8+ T cells.
ELF4 regulates tissue distribution of OVA-specific CD8+ T cells We next analyzed the tissue distribution of WT and Elf4−/− OT-I cells coactivated in the same environment because CD62L (L-selectin) is an important homing molecule. Consistent with a specific defect in the generation of CD62Llow CD8+ T cells, the ratio of WT to Elf4−/− cells was higher in blood, lung, and the spleen, whereas the relative frequencies of both populations were comparable in inguinal LNs (Fig. 3A). Interestingly, we observed increased homing of effector and memory Elf4−/− OT-I CD8+ C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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T cells to BM (Fig. 3A). Since the chemokine receptor CXCR4 mediates homing of T cells and hematopoietic stem cells to BM [17, 26], we assessed the expression of CXCR4. As expected, circulating Elf4−/− CD8+ T cells expressed relatively higher levels of CXCR4 than controls, both at mRNA and protein levels (Fig. 3B and C). These data indicate that ELF4 modulates tissue distribution of effector and memory CD8+ T cells, generated during infection with Lm-OVA, in a CD62L- and CXCR4-dependent manner.
Ectopic expression of ELF4 augments formation of effector memory CD8+ T cells Deletion of ELF4 can deregulate the expression of genes directly involved in the development of effector memory CD8+ T cells; therefore, we decided to confirm that ELF4 mediates differentiation of effector and memory T cells upon infection by overexpressing ELF4 in OT-I WT CD8+ T cells. We transplanted BM cells from CD45.2+ OT-I mice that were transduced with retrovirus (RV) coexpressing murine ELF4 and GFP (RV-ELF4) into lethally irradiated congenic recipients. Following hematological reconstitution (>2 months), CD44low GFP+ CD8+ T cells were purified from the spleen of transplanted mice by cell sorting and adoptively transferred to congenic CD45.1+ mice, which were then infected with Lm-OVA. The overexpression of ELF4 was confirmed in CD8+ T cells used for adoptive transfer by immunoblots (Fig. 4A). As expected, ectopic ELF4 expression led to increased numbers of CD62Llow memory CD8+ T cells in the spleen with no alterations in the frequency of CD62Lhigh cells (Fig. 4B and C). Therefore, ELF4 specifically promotes formation of effector memory CD8+ T cells.
ELF4 deficiency impairs priming and survival of OVA-specific CD8+ T cells Pathogen-specific CD8+ T cells are activated by splenic macrophages and DCs that have captured circulating L. monocytogenes [27, 28]. Therefore, we examined whether differences in early expansion of co-transferred OT-I WT and OT-I Elf4−/− CD8+ T cells (1:1) could account for the lower expansion of Elf4−/− CD8+ T cells. Despite similar frequencies of WT and Elf4−/− OT-I cells in the spleen at 1.5 dpi, we detected a 2-fold increase in the WT to Elf4−/− ratio at 2.5 dpi (Fig. 5A), which was likely caused by impaired proliferation or survival of activated OT-I Elf4−/− CD8+ T cells. Of note, expression of CD69 and CD25 (1.5 dpi) and CD44 and CD5 (3.5 dpi) revealed a similar activation of Elf4−/− and WT CD8+ T cells (Fig. 5B–D). However, the lower numbers of CD25high Elf4−/− CD8+ T cells observed at 2.5 dpi (Fig. 5C) correlated with decreased proliferation of Elf4−/− CD8+ T cells (Fig. 5E). A defective generation of memory could be due to enhanced cell death of activated Elf4−/− CD8+ T cells. Although no differences in Annexin V-positive cells were initially observed, splenic Elf4−/− effector CD8+ T cells showed increased apoptosis at the peak of response (Fig. 5F). This correlated with decreased mRNA expression of the www.eji-journal.eu
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Figure 2. Impaired development of memory by Elf4−/− CD8+ T cells in response to Lm-OVA. (A) OT-I WT (CD45.1+ ) and OT-I Elf4−/− (CD45.2+ ) CD8+ T cells (500 cells each per mouse) were cotransferred into congenic recipient mice (CD45.1+ CD45.2+ ), which were infected with Lm-OVA 24 h later. Contribution of each donor (WT, CD45.1+ , and Elf4−/− , CD45.2+ ) to circulating CD8+ T cells is shown for the input, 9 dpi, and 70 dpi. The immune response to Lm-OVA is shown on the right for WT and Elf4−/− cells as percentage of total CD8+ T cells. (B) OT-I WT (CD45.2+ ) and OT-I Elf4−/− (CD45.2+ ) CD8+ T cells (1000/mouse) were separately transferred into congenic recipient mice (CD45.1+ ) 24 h before Lm-OVA infection. Contribution of each donor (WT, CD45.2+ , and Elf4−/− , CD45.2+ ) to circulating CD8+ T cells is shown for 7 and 40 dpi. Kinetics of expansion in blood is shown on the right as percentage of total CD8+ T cells. (C) CD62L expression in donor WT and Elf4−/− CD8+ T cells and overall frequency of CD62Lhigh and CD62Llow in total blood CD8+ T cells. (D) Same as (C) for single adoptive transfer. (C, D) Data are shown as mean ± SD (n = 5) and (A–D) are representative of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. ELF4 modulates tissue distribution of Lm-OVA-specific CD8+ T cells. (A) The contribution of each donor (WT, CD45.1+ , and Elf4−/− , CD45.2+ ) was analyzed by flow cytometry in blood, lung, spleen, LN, and BM at the peak of response (7 dpi) and 60 dpi. Ratios of WT to Elf4−/− OT-I CD8+ T cells in different tissues are shown on the right as mean ± SD (n = 3). Dashed line indicates 1:1 ratio of the input. (B) Expression of CXCR4 transcripts was measured by qPCR in donor WT and Elf4−/− OT-I CD8+ T cells at the peak of response. (C) Cell surface expression of CXCR4 in donor WT and Elf4−/− OT-I CD8+ T cells at the peak of response. MFI is shown for effector T cells from three pairs of WT and Elf4−/− OT-I CD8+ T cells cotransferred in recipient mice (connected by dashed lines). (A–B) Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. *p < 0.05; **p < 0.01 (two-tailed Student’s t-test).
anti-apoptotic gene bcl2a1a in Elf4−/− effector CD8+ T cells at 7 dpi (Fig. 5G). These data indicate that ELF4 is required for optimal priming of na¨ıve CD8+ T cells and survival of Lm-OVA-specific effector CD8+ T cells.
Activated Notch1 pathway in Elf4−/− CD8+ T cells A reduced pool of memory T cells with skewed ratio of centralto-effector memory in Elf4−/− CD8+ T cells may be caused by an imbalance of key intrinsic factors of CD8+ T-cell differentiation, such as T-bet and Eomes [12, 13, 17, 20, 29, 30]. For example, the development of central memory depends on the expression of Eomes in differentiating T cells [17, 21]. Elf4−/− CD8+ T cells expressed higher levels of Eomes, both mRNA and protein, with normal levels of T-bet, lowering the T-bet/Eomes ratio at the peak of response (Fig. 6A and B). This was not caused by deregulation of Tcf-1 and Runx3 [21,31] (Supporting Information Fig. 4). In addition, loss of ELF4 did not affect the expression C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of Blimp-1, Bcl-6, Id2, Id3, and KLF2 (Supporting Information Fig. 4). Since Notch1 signaling drives the expression of Eomes [19], we assessed the activity of Notch1 in WT and Elf4−/− effector CD8+ T cells. The expression of Notch1 target genes Hes1, Hey1 and HeyL was significantly upregulated in Elf4−/− effector CD8+ T cells (Fig. 6C). Moreover, the increased levels of cleaved Notch1 in Elf4−/− effector CD8+ T cells further suggested augmented Notch1 signaling (Fig. 6D). The increased expression of Eomes and Notch1 target genes was not a result of a selective enrichment of CD62L positive cells within effector Elf4−/− CD8+ T cells as we also detected increased expression of these genes in CD62Llow Elf4−/− CD8+ T cells purified at the peak of response (Fig. 6E). Loss of ELF4 did not alter the transcript levels of Notch1 inhibitors such as Numb, Itch and Fbxw7 (Fig. 6F), indicating that ELF4 does not increase Notch1 signaling by modulating expression of Notch1 inhibitors. These data suggest that enhanced Notch1 signaling in Elf4−/− CD8+ T cells increases expression of Eomes and promotes development of central memory. www.eji-journal.eu
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Figure 4. Ectopic expression of ELF4 augments production of effector memory CD8+ T cells. (A) BM cells from OT-I mice were transduced with either empty (RV) or ELF4-expressing (RV-ELF4) RV and transplanted into lethally irradiated recipient mice. GFP+ CD8+ T cells were purified from the spleen of reconstituted mice and analyzed by immunoblot. Numbers indicate fold change in ELF4 expression estimated by densitometry. (B) Purified GFP+ OT-I cells (CD45.2+ ) were injected into B6.SJL (CD45.1+ ) mice that were challenged with Lm-OVA. Percentages of donor-derived memory T cells and CD62L profiles are shown in the spleen at 47 dpi. Data represent mean ± SD for each group. (C) The number of CD62Lhigh and CD62Llow memory CD8+ T cells in the spleen is shown for RV and RV-ELF4 groups. Data are shown as mean ± SD (n = 5) and (A–B) are representative of two independent experiments. *p < 0.05 (two-tailed Student’s t-test).
Loss of ELF4 impairs recall response of memory CD8+ T cells Elf4−/− CD8+ T cells display impaired development of immunological memory in response to Lm-OVA infection due to defective formation of effector memory CD8+ T cells. We confirmed that CD62Lhigh Elf4−/− CD8+ T memory cells also expressed other markers of central memory such as CCR7 and CD27 (Fig. 7A). An important property of memory T cells is their capacity to mount a robust secondary expansion upon encounter with the same antigen. To assess the functional role of ELF4 in memory T cells, purified WT and Elf4−/− CD62Lhigh memory OT-I CD8+ T cells were mixed at a 1:1 ratio, adoptively transferred into congenic recipients and challenged with a lethal dose of Lm-OVA. Elf4−/− CD8+ T cells failed to expand as WT controls (Fig. 7B), suggesting that central memory Elf4−/− CD8+ T cells exhibit a severe functional defect despite an apparent normal differentiation. Strikingly, loss of ELF4 abrogated the expansion of CD62Llow effector memory CD8+ T cells (Fig. 7C). Similar to primary response, the tissue distribution of secondary effector Elf4−/− CD8+ T cells showed reduced distribution in blood and lung (Fig. 7D). These data suggest that ELF4 is required for recall responses of memory CD8+ T cells. To further characterize the defective expansion of Elf4−/− central memory CD8+ T cells, we adoptively transferred WT and Elf4−/− CD62Lhigh memory OT-I CD8+ T cells separately to congenic recipients and assessed their presence in blood and the spleen. Similar to the co-transfer experiment, Elf4−/− central memory cells displayed a severe impairment in their capacity to expand (Fig. 8A). The frequency of Elf4−/− secondary effector cells was significantly reduced in the spleen as early as 4 days after re C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
challenge (Fig. 8B) whereas the bacterial burden in the spleen was controlled nearly as efficiently as controls (Fig. 8C). However, neither proliferation or cell death was altered at least 4 dpi (Fig. 8D and E) and 6 dpi (data not shown) in expanding Elf4−/− CD8+ T cells, suggesting that loss of ELF4 compromised early activation or priming of central memory CD8+ T cells. The expression of Eomes, Hes1 and CXCR4 mRNA in Elf4−/− secondary effector CD8+ T cells was also elevated (Fig. 8F). Collectively, our results demonstrate a previously unknown function of ELF4 in promoting development of functional memory CD8+ T cells in response to infection.
Discussion A better understanding of the molecular circuitry controlling the development and function of effector and memory CD8+ T cells is the first step toward the generation of vaccines and immunotherapy for cancer and autoimmune disorders. In this work, we show that the ETS transcription factor ELF4 is required for the differentiation of effector memory CD8+ T cells and the recall response of effector and central memory CD8+ T cells at least in part by modulating Notch1 signaling. Loss of ELF4 impaired the proliferation and survival of OVAspecific CD8+ T cells upon infection with L. monocytogenes, resulting in a smaller pool of effector memory T cells. We ruled out differential homing of adoptively co-transferred wild type and Elf4−/− CD8+ T cells to the priming site and enhanced activationinduced cell death upon initial antigen encounter. Even though the numbers of central memory CD8+ T cells were not significantly affected in the primary response, the ability to expand www.eji-journal.eu
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Figure 5. Loss of ELF4 impairs priming and survival of Lm-OVA-specific CD8+ T cells. (A) Expansion in the spleen of WT and Elf4−/− OT-I CD8+ T cells cotransferred into recipient mice prior to Lm-OVA infection is shown at 1.5 and 2.5 dpi. Ratios of WT to Elf4−/− T cells are shown on the right. (B) Expression of the activation marker CD69 in WT and Elf4−/− OT-I CD8+ T cells at 1.5 dpi. Numbers of CD69high cells (per 104 of total splenic CD8+ T cells) are shown on the right. (C) Numbers of splenic CD25high WT and Elf4−/− CD8+ T cells per 104 of total splenic CD8+ T cells at 1.5 and 2.5 dpi (n = 4, mean ± SD). (D) Expression of CD44 and CD5 in WT and Elf4−/− OT-I CD8+ T cells at 3.5 dpi. (E) Dilution of eFluor670 dye in WT and Elf4−/− OT-I CD8+ T cells labeled before transfer and analyzed at 2.5 dpi. Bar graphs show percent of undivided cells and division index for CD8+ T cells (n = 4, mean ± SD). (F) Annexin V staining of splenic WT and Elf4−/− OT-I CD8+ T cells at different times post infection is shown for three pairs of WT and Elf4−/− OT-I CD8+ T cells cotransferred to recipient mice (connected by dashed lines). (G) mRNA expression of pro- and anti-apoptotic genes in WT and Elf4−/− CD8+ T cells at 7 dpi. Pooled cDNA from three mice in each group was used for the PCR array. (A–F) The data are representative of 2–3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
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Figure 6. Increased Eomes expression and enhanced Notch signaling in Elf4−/− effector CD8+ T cells. (A) The mRNA levels of T-bet and Eomes were measured in WT and Elf4−/− OT-I CD8+ T cells purified from the spleen of infected mice (7 dpi) and normalized to β-actin. (B) Expression of Eomes and T-bet was assessed by intracellular staining in WT (open histogram) and Elf4−/− (shaded histogram) effector CD8+ T cells at the peak of response (7 dpi). (C) Transcript levels of Notch target genes (Hes1, Hey1, and HeyL) normalized to β-actin was measured in splenic WT and Elf4−/− OT-I CD8+ T cells at 7 dpi. (D) Levels of cleaved Notch1 were analyzed by immunoblot using WT and Elf4−/− OT-I CD8+ T cells isolated at 5 and 7 dpi. Numbers indicate relative levels estimated by densitometry normalized to β-actin. (E) Expression of Eomes, Hes1, and Hey1 mRNA in WT and Elf4−/− CD62low CD8+ T cells at 7 dpi. (F) Transcript levels of Notch1 inhibitors in WT and Elf4−/− CD62low CD8+ T cells at 7 dpi. (A–F) Data are shown as mean + SD (n = 3) and are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
in a secondary infection was severely compromised. In contrast, Elf4−/− effector memory CD8+ T cells showed both developmental and functional defects. This impaired formation of immunological memory was not due to competition with WT OT-I cells for antigen and/or cytokines because we observed same outcome in two additional models of infection (single adoptive transfer and mixed BM chimeras). We previously showed that ELF4 controls the proliferation of na¨ıve CD8+ T cells both in vitro and in vivo [23, 24]. The use of supra-physiological numbers of Elf4−/− CD8+ T cells and a low inflammatory model of activation (peptide-pulsed DCs) led to increased numbers of memory T cells [24]. In contrast, the activa C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tion of physiological numbers of CD8+ T cells by bacterial infection resulted in decreased expansion and defective differentiation of effector memory T cells, indicating that the frequency of antigenspecific CD8+ T cells combined with an inflammatory environment altered the fate of activated Elf4−/− CD8+ T cells. Thus, it is possible that the lower threshold of activation in Elf4−/− OT-I CD8+ T cells and the addition of more signals elicited by inflammatory cytokines in a physiological priming overruled the stimulation of proliferation and survival of differentiating CD8+ T cells. In support of T-cell extrinsic mechanisms, a recent study showed that inflammatory cytokines induced by infection with Lm-OVA augment the TCR signaling in OT-I CD8+ T cells [32]. Furthermore,
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Figure 7. Loss of ELF4 impairs recall responses of memory CD8+ T cells. (A) Coexpression of CCR7, CD27, and CD62L in WT and Elf4−/− memory CD8+ T cells (110 dpi). Numbers indicate the percentage of double-positive cells (mean ± SD). (B) Expansion of central memory CD8+ T cells in a recall response. Central memory CD8+ T cells (CD62Lhigh ) were purified from infected mice cotransferred with WT and Elf4−/− CD8+ T cells (110 dpi) and adoptively transferred as a 1:1 mixture to secondary recipient mice, that were infected a day after with Lm-OVA (200 × 103 CFU, i.v.). (C) Effector memory CD8+ T cells (CD62Llow ) were purified from infected mice cotransferred with WT and Elf4−/− CD8+ T cells and tested as described in (A). (D) Tissue distribution of secondary effector CD8+ T cells was analyzed 20 days after recall response of cotransferred central memory WT and Elf4−/− OT-I CD8+ T cells (as shown in B). The ratio of WT to Elf4−/− was calculated for lung, blood, spleen, inguinal LNs, and BM. Dashed line indicates input ratio of transferred memory T cells. (A–D) Data are shown as mean ± SD (n = 5) and (A–D) are representative of two independent experiments. ns, not significant; *p < 0.05; ***p < 0.001. (two-tailed Student’s t-test).
the Notch1 pathway was activated in Elf4−/− CD8+ T cells likely in a ligand-independent manner downstream of the TCR signaling [33]. In turn, the enhanced Notch1 signaling probably increased the expression of Eomes — a known positive regulator of central memory development — in Elf4−/− CD8+ T cells resulting in skewed differentiation toward central memory T cells to the detriment of CD62Llow memory precursors. This increased expression of Eomes also correlated with elevated levels of CXCR4 in Elf4−/− OT-I cells and augmented homing to BM, a known site for homeostatic maintenance of central memory T cells [17, 34]. The activation of the Notch1 pathway can also affect proliferation and cytokine production in T cells. IL-2 signaling plays a criti C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cal role for optimal formation of CD62Llow effector and effector memory CD8+ T cells [35, 36]. Therefore, impaired generation of CD25 high Elf4−/− CD8+ T cells at an early stage of expansion may limit the development of effector memory CD8+ T cells. Previous studies have linked enhanced Notch1 signaling with decreased proliferation and IL-2 production in activated T cells [37–39]. Therefore, the enhanced Notch1 activity may be an important factor in the suboptimal formation of effector and effector memory Elf4−/− CD8+ T cells. A model of CD8+ T-cell differentiation postulates that infection triggers a developmental program that simultaneously generate terminal effector and memory precursor T cells [5]. At least three www.eji-journal.eu
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Figure 8. Proliferation and survival of Elf4−/− secondary effector CD8+ T cells. (A) Expansion of WT and Elf4−/− central memory CD8+ T cells in peripheral blood that were transferred separately to secondary recipient mice and re-activated by infection with Lm-OVA (200 × 103 CFU, i.v.) as in Fig. 7A. (B) Frequencies of WT and Elf4−/− secondary effector CD8+ T cells in the spleen of recipient mice at 4 dpi. (C) Bacterial burden in the spleen of infected mice at 4 dpi (n.s., no statistical difference). (D) Proliferation of donor-derived WT and Elf4−/− CD8+ T cells in the spleen at 4 dpi was assessed by BrdU incorporation assay. Open histograms show BrdU incorporated in WT and Elf4−/− CD8+ T cells. Shaded histograms correspond to the BrdU staining profile of total splenic CD8+ T cells. Bar graph indicates total percentage of BrdU-positive cells in both groups. (E) Annexin V staining of splenic donor-derived WT and Elf4−/− CD8+ T cells. Gates indicate Annexin V positive cells, with total percentage of Annexin V positive cells shown in the bar graph. (F) Transcript levels of Eomes, Hes1, CXCR4, and T-bet in WT and Elf4−/− secondary effector CD8+ T cells at 5 dpi. (A–F) Data are shown as mean ± SD (n = 3) and (A–D) are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t-test).
distinct populations can be identified at the peak of response by flow cytometric analysis: terminal effector (CD127low CD62Llow ) cells, effector memory precursors (CD127high CD62Llow ), and central memory precursors (CD127high CD62Lhigh ) [5]. Hence, ELF4 may selectively regulate commitment of differentiating T cells toward an effector memory lineage. Even though we detected normal numbers of immunophenotypic SLEC and MPEC populations,
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Elf4−/− precursors of effector memory CD8+ T cells may exhibit functional defects. In addition to a developmental defect, loss of ELF4 severely impaired the expansion of adoptively transferred central and effector memory CD8+ T cells in a recall response. The mechanism of impaired expansion Elf4−/− memory CD8+ T cells in a recall response is similar to the primary response. This defect
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could have been imprinted in memory T cells early in the process during a suboptimal primary response or rather reflects a greater role of ELF4 in regulating the expansion of memory T cells. In addition, a recall response with lethal dose of Lm-OVA entails a greater inflammation compared to the primary response. Therefore, loss of ELF4 did not only decrease the total number of memory cells upon Lm-OVA infection but also compromised their function. Collectively, this study describes a novel function of ELF4 in regulating differentiation and function of memory CD8+ T-cell in response to infection. In the context of inflammation, ELF4 promotes priming and survival of na¨ıve and effector CD8+ T cells and differentiation of effector memory CD8+ T cells. Furthermore, ELF4 has a critical role in recall responses of both effector and central memory CD8+ T cells. Therefore, this work offers a novel molecular target to improve fitness of memory T cells.
Materials and methods Mice C57BL/6 (B6), C57BL/6.SJL (B6.SJL), and OT-I mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). B6 mice (CD45.2+ ) were bred with B6.SJL mice (CD45.1+ ) to generate B6xB6.SJL F1 (CD45.1+ CD45.2+ ) recipients. Elf4−/− mice were obtained from S. Nimer [25], backcrossed for more than 12 generations to B6, and bred with OT-I B6 to generate OT-I Elf4−/− mice. OT-I transgenic mice were crossed to B6 (CD45.2+ ) and to B6.SJL (CD45.1+ ). Mice were maintained in specific pathogenfree conditions at Baylor College of Medicine. All experiments and procedures were done in compliance with the Institutional Animal Care and Usage Committee of Baylor College of Medicine (AN3698).
Adoptive transfer and infection with L. monocytogenes For adoptive transfer experiments, na¨ıve CD44low OT-I CD8+ T cells were either negatively enriched (90–97% purity) with BDImag magnetic separation system (BD Biosciences), with addition of biotinylated anti-CD44 antibody (BD Biosciences), or purified using a MoFlo cell sorter (Cytomation; 97–99% purity). For all cotransfer experiments, naive CD8+ T cells were purified from the spleen of OT-I WT (CD45.1+ ) and OT-I Elf4−/− (CD45.2+ ) mice, mixed at 1:1 ratio, and adoptively transferred to B6xB6.SJL F1 (CD45.1+ CD45.2+ ) recipients (1000 cells/mouse i.v., to approach physiological frequency). For analysis of proliferation at 2.5 dpi, naive purified CD8+ T cells were labeled with 4 μM eFluor670 (eBioscience), mixed at a 1:1 ratio, and adoptively transferred to congenic recipients (2 × 105 cells per mouse, to facilitate detection). Recipient mice were infected 24 h later with recombinant OVA-expressing L. monocytogenes (Lm-OVA) strain (4 × 103 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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CFU, i.v.). For recall responses, donor-derived memory CD62Lhigh and CD62Llow OT-I CD8+ T cells were purified by cell sorting at 110 dpi and adoptively transferred (104 cells/mouse) either separately or mixed (1:1) into congenic unchallenged recipients followed by a challenge with a lethal dose of Lm-OVA (200 × 103 CFU). Bacterial burden was measured in the spleen of infected mice by plating single-cell suspension permeabilized with 0.05% Triton on brain-heart infusion (Fluka) agar plates supplemented with 10 μg/ml erythromycin.
Flow cytometry Longitudinal analysis of T-cell expansion was monitored in blood collected by tail vein bleeding. For tissue distribution analysis, lungs were perfused with 5 mL cold PBS and processed as described before [24]. BM cells were flushed out from femurs and tibias. Cells were incubated with anti-CD16/32 (Biolegend) prior to cell surface staining. All antibodies for surface proteins were purchased from Biolegend (anti-CD45.2, anti-CD107, anti-CD45.1, anti-CD8, anti-CD25, anti-CD62L, and anti-CD27), eBioscience (anti-CD127, streptavidin, anti-CXCR4, and antiCD69) and BD Biosciences (streptavidin and anti-KLRG1). Kb -OVA tetramers were synthesized at the MHC Tetramer Core Laboratory (Baylor College of Medicine). CCR7 was detected using recombinant CCL19-Fc protein (eBioscience) as previously described [24]. For intracellular cytokine staining, cells were incubated with 1 μg/mL OVA(257-264) peptide (AnaSpec) in RPMI + 10% FBS for 5 h at 37◦ C in the presence of 1 μl/mL Golgi Plug and 0.8 μL/mL Golgi Stop (BD Biosciences). Cells were stained on the cell surface for 20 min on ice, followed by intracellular staining with anti-IFNγ, anti-TNFα (BD Biosciences), anti-perforin, or antigranzyme B (eBioscience) in BD Cytofix/Cytoperm buffer according to the manufacturer’s instructions (BD Biosciences). Apoptosis was measured with Annexin V (Biolegend). Detection of Eomes was performed using anti-Eomes (eBioscience) antibody and FoxP3 staining buffer set (eBioscience). For BrdU incorporation assay, mice were injected with BrdU (2 mg, i.p.) and 4 h later splenocytes were stained using BrdU Flow Kit (BD Biosciences). Flow cytometry was performed using FACSCanto instrument (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).
BM transduction and transplantation To generate mixed WT:Elf4−/− BM chimeras, lethally irradiated (960 Rad) CD45.1+ WT recipient mice were transplanted with a mixture (1:1) of WT (CD45.1+ ) and Elf4−/− (CD45.2+ ) BM cells (10 × 106 cells/mouse). Three months later, transplanted mice were challenged with Lm-OVA (4 × 103 CFU, i.v.). To overexpress ELF4, BM cells were flushed out from femur and tibia of OT-I CD45.2+ mice that received 5-FU (150 mg/Kg i.p.) 5 days prior. Cells were cultured in X-VIVO 15 medium (Lonza) in the presence of SCF (100 ng/mL), IL-6 (10 ng/mL), and IL-3 (6 ng/mL) www.eji-journal.eu
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for 48 h before retroviral transduction. Cells were transferred to retronectin-coated plates and transduced by spinoculation (400 g, 1 h at 25◦ C) using viral supernatant containing either MigR1 (empty RV) or MigR1-ELF4 (RV-ELF4) [40] in the presence of polybrene (8 μg/mL). BM cells were cultured for additional 48 h and injected i.v. to irradiated (960 Rad) congenic recipient mice.
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Conflict of interests: Authors declare no financial or commercial conflict of interests.
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Abbreviations: CHX: cycloheximide · dpi: days post infection · ELF4: E74like factor 4 · Eomes: eomesodermin · KLRG1: killer cell lectin-like receptor G1 · Lm-OVA: Listeria monocytogenes-OVA · RV: retrovirus
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C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 5/6/2013 Revised: 11/10/2013 Accepted: 10/12/2013 Accepted article online: 13/12/2013
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