Cytokine xxx (2014) xxx–xxx

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Cytokine journal homepage: www.journals.elsevier.com/cytokine

Review Article

T cell exhaustion and Interleukin 2 downregulation Mumtaz Y. Balkhi a,⇑, Qiangzhong Ma a, Shazia Ahmad b, Richard P. Junghans a a b

Department of Medicine, Tufts Medical Center and Tufts University School of Medicine, Boston, MA, United States Boston University Medical Center, Boston, MA, United States

a r t i c l e

i n f o

Article history: Received 2 October 2014 Received in revised form 13 November 2014 Accepted 14 November 2014 Available online xxxx Keywords: T cells Exhaustion Interleukin 2 Chromatin PD1

a b s t r a c t T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigenrich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion. Ó 2014 Elsevier Ltd. All rights reserved.

1. T cell exhaustion T cells are a central component of an adaptive immune system that is indispensable for immune system maintenance and human health. A principal function of T cells is immune surveillance to eliminate transformed cells and suppress virus infection. There are two processes, correlated, by which cognate T cells may lose their ability to react to antigens, anergy and exhaustion. Anergy is defined in terms of T cells receiving insufficient antigen activation and co-stimulation [1,2]. Exhaustion is a process in which T cells reactive to tumor antigens or viral antigens lose their ability to kill cancer cells or virus infected cells upon chronic antigen exposure. Antigen reactive T cells may be particularly vulnerable to exhaustion during immune surveillance, as reactive T cells may enter antigen rich-environment and be repeatedly stimulated triggering exhaustion. Exhaustion affects both long lived central memory and short lived effector memory T cells with debilitating consequences over immune functions. As, upon recurrent exposure to viruses and tumor antigens, the memory T cell pool loses its ability to reactivate. T cell exhaustion is characterized by a downregulation of effector functions including diminished cytolytic activity and type I cytokine secretion [3–6] and an upregulation in the expression of inhibitory molecules such as programmed death receptor 1 ⇑ Corresponding author at: Tufts Medical Center, 800 Washington, St. #817 Boston, MA 02111, United States. Tel.: +1 617 636 8247; fax: +1 617 636 8516. E-mail address: [email protected] (M.Y. Balkhi).

(PD1), cytotoxic T-lymphocyte antigen 4 (CTLA4), T cell immunoglobulin and mucin domain-containing molecule 3 (TIM3), lymphocyte activation gene 3 (LAG3), CD244 (2B4) and shift in the cytokine expression to IL10 and TGFb production that mark a suppressed state [7–9]. The gene profiling studies have demonstrated that the molecular profile of exhausted T cells in malignancy is similar to the one observed in chronic viral infections [10]. For example, tumor infiltrating CD8+ T cells have been shown to express inhibitory receptors such as PD1, BTLA, Tim3, Lag3, CTLA4 and shift in their cytokines expression, that is similar to molecular profiles in T cells exposed to chronic viral infections [2]. The T cell exhaustion to tumor cells is exacerbated by the presence of inhibitory ligands on tumor cells, regulatory T cells and immunomodulatory enzymes in tumor microenvironment [11]. The molecular details of exhaustion are not fully characterized yet, thus, making its distinction difficult from the other forms of T cell dysfunction such as anergy. However, kinetics of calcium signaling, constitutive MAPK and ERK signaling pathways could be distinctive features of exhaustion. The gene transcription profiles of exhausted T cells are distinct from anergic, memory and terminally differentiated T cells [2,12,13]. A core set of transcription factors such as Blimp1, Eomes, T-bet, IKZF, and Foxo can distinguish these T cell subsets (Table 1). Crawford et al. demonstrated recently that the pattern of expression of these transcription factors along with expression of inhibitory molecules and type I cytokine expression could distinguish exhausted CD4+ T cells from the effector and memory T cells [14].

http://dx.doi.org/10.1016/j.cyto.2014.11.024 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Balkhi MY et al. T cell exhaustion and Interleukin 2 downregulation. Cytokine (2014), http://dx.doi.org/10.1016/ j.cyto.2014.11.024

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Table 1 Distinct transcription factor profile of T cell subtypes. Characteristics

Terminally differentiated T cells

Exhausted T cells

Anergic T cells

Memory T cells

Transcription factors

EOMES, T-bet, BLIMP1, THPOK, KLRG1

BATF, Eomesodermin, BLIMP1, IKZF2, Eomes

GRAIL, CBL-B, ITCH, NEDD4

Foxo1, BCL-2

Table 2 Unanswered questions about T cell exhaustion. No.

Unanswered questions about T cell exhaustion

1 2 3 4 5 6

Whether exhausted T cells represent a distinct differentiation state or exhaustion represent dysfunctional CD8+ and CD4+ subtypes Whether T cell exhaustion arises to certain types of viruses and tumor-antigens only or a process is more common to viruses and tumor-antigens Signaling pathways contributing to the transcriptional changes associated with T cell exhaustion Apart from T cells of Th1/Th2 lineage, can other cell types such as Treg, Th17, Tfh or antigen presenting cells be exhausted Mechanism leading to loss of the IL2 production and other cytokines in exhausted T cells Whether T cell exhaustion is genetically regulated or only acquired through chronic infections and in malignancy

T cell exhaustion is a reversible state. Several studies have demonstrated that exhausted tumor reactive T cells can be recovered functionally in metastatic melanomas during immunotherapy [15]. Such T cells begin to produce IL2, IFNc and TNFa cytokines and the activation of STAT1, STAT5 and ERK1/2 signaling pathway which are crucial for cell survival and cytokine synthesis [16,17]. An antibody mediated blockade of Tim3 and PD1 partially reverses T cell exhaustion to colon carcinoma tumors in mice [18]. Likewise, an antibody mediated blockade of PD1 receptor or ligand alone or in combination with inhibition of IL10 in early clinical trials has shown promise in reversing T cell exhaustion and promoting anti-viral responses against LCMV infection [19–22]. Despite progress, much remains to be elucidated about the mechanism of T cell exhaustion (Table 2). In the following sections we will discuss various aspects of exhaustion. 1.1. T cell exhaustion in virus infections The phenomenon of T cell exhaustion was first observed in CD8+ T cells of immune-competent mice upon chronic infection with noncytopathic lymphocytic choriomeningitis virus (LCMV) [5,23]. Two separate studies by Richter et al., and Ahmed et al., showed that antigen presenting cells play central role in T cell exhaustion. Antigen presentation and the relative amount of antigen presented through MHC class I play critical role in the process of T cell exhaustion [24,25]. Whether high viral load through continuing replication of virus within the cells is sufficient to induce T cell exhaustion or additional mechanisms such as impairment of CD4 and NK cell activities are needed is still unclear [26]. Apart from LCMV, T cell exhaustion is also observed for many different chronic viruses including hepatitis C virus, hepatitis B Virus, HIV, and HTLV, etc. [27,28]. However, no such T cell exhaustion was observed for gamma herpes viruses such as cytomegalovirus and Epstein-Barr virus which persist in infected host indefinitely [29], confirming that T cell exhaustion does not arise to all viruses. The viral load is consistently high in settings with exhaustion [24,25], and this along with differences in antigen presentation, may distinguish those viral infections that induce exhaustion and those that do not [24,30]. Robust T cell activity can be recovered in exhausted T cells upon depletion of virus or antigens from the body, indicating that viral load play major role in the process of exhaustion [31]. 1.2. T cell exhaustion in parasitic and bacterial infections T cell exhaustion is reported in parasitic infections. Malaria is caused by zoonotic parasite plasmodium that during blood stage infection induces exhaustion of T cells [32,33]. Enhanced expression of PD1 in CD4+ and CD8+ T cells is reported in patients

infected with malaria and in experimental rodent malaria model [33,34]. In murine model of blood stage malaria, combined blockade of PDL-1 and LAG3 has shown enhanced clearance of Plasmodium yoelii and Plasmodium chabaudi [35]. Likewise, exhaustion related phenotype is also reported in murine and canine leishmaniasis. Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL). In canine leishmaniasis and murines infected with Leishmania major strain, CD4+ and CD8+ T cell express PD1 exhaustion markers and show impaired IFNc synthesis [36,37]. Likewise, CD8+ T cells undergo exhaustion in diffuse cutaneous leishmaniasis caused by Leishmania mexicana [38]. Toxoplasmosis is another case in which T cell undergoes exhaustion. It is caused by a protozoan Toxoplasma gondii (T. gondii). Infection of mice with T. gondii has revealed exhaustion of CD8+ T cells [39]. These T cells express PD1 in later phase of chronic toxoplasmosis and blockade of PD1/PDL-1 control Toxoplasma reactivation [40]. Exhaustion in bacterial infections is also reported. For example, higher expression of PD1 and its ligand have been reported in PBMCs of patients infected with active Mycobacterium tuberculosis antigens. While as blockade of PD1 in vitro in T cells derived from M.tb infected patients resist apoptosis and decline in IL2 and IFNc synthesis [41]. However, these results are inconsistent with the findings made in mice model of M.tb infection. A study by Reiley et al., has revealed in mice M.tb infection model that a fraction of antigen specific CD4+ T though express PD1 and KLRG1 markers but does not undergo exhaustion as evidenced by the continued production of IFNc and TNFa [42]. T cell exhaustion is also reported during gut bacterial infections of patients suffering from rare genetic disease, common variable immunodeficiency (CVID). The PD-1-PD-L1/2 blockade in vitro reactivated the T cells of CVID patients [43]. 1.3. T cell exhaustion in malignancy Tumor antigens are broadly divided into tumor associated antigens (TAA) and tumor specific antigens (TSA). TAA are self-antigens expressed on tumor cells and normal cells. Three means have been proposed by which self-antigens can become tumor associated antigen; (1) through overexpression of peptides because of mutations or epigenetic changes in the regulatory elements of the genes, (2) due to mutations or recombination in the genes itself leading to overexpression of proteins, (3) due to posttranslational modification of a peptides such as phosphorylation, glycosylation, and acetylation, leading to abnormal processing and presentation by the MHC class I [44]. Examples include, CEA, Her2/Neu, Mucin, vimentin, alpha-catenin and melanoma antigens, tyrosinaserelated protein-1 and tyrosinase-related protein-2, Survivin, and NY-ESO-1 etc [45–48]. Failure of the immune system to suppress tumors expressing TAA is attributed to the phenomenon of

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Table 3 Distinct features of tolerance and exhaustion. No.

T cell exhaustion

T cell tolerance

1

Arises against chronic tumor and viral antigens

2

Exhaustion does not require any tissue specific antigen presentation. Phenotypically exhausted T cells could be T-bethi PD1int or Emoes hiPD1hi [2] Inhibitory molecules such as PD1, TIM3, LAG3, CTLA4, B- and T-lymphocyte attenuator (BTLA) [134], BLIMP1 are commonly expressed Cytokines such as IL2, TNFa, IFNc are lost in hierarchical order IL10 and TGFb cytokines are synthesized Immunosuppressive microenvironment such as the presence of Treg, MDSCs, galectin 3, and iNOS may play role in inducing T cell exhaustion

Arises against self-antigens and is an important mechanism to prevent autoimmunity (central tolerance). Commonly observed for tumor antigens that are self-antigens Lymph nodes and spleen are the primary sites of tumor antigen presentation for T cell tolerance induction [131,132]. APCs play a major role in T cell tolerance. Such T cells may be CD44hi, CD69int, CD25, CD62Lint, CD49dlo [133] Inhibitory molecules such as PD1 and LAG3 are commonly expressed

3 4 5 6

tolerance. Two principal mechanisms of tolerance operate in T cells exposed to TAAs. In one mechanism, if T cells are strongly activated (i.e., with high avidity TCRs) against tumor associated antigens presented by the MHC, there is a chance that T cells will attack the normal tissue which will be detrimental to the host. These T cells are typically deleted in the thymus (negative selection) [49,50]. In a second mechanism, weakly activated T cells (i.e., with low avidity TCR to tumor associated antigens) will survive in the host if they escape deletion (positive selection). These T cells under persistent tumor antigens will become anergic due to lack of co-stimulation. These anergic T cells express inhibitory molecules such PD1, Lag3 and secretion of IFNc declines contributing to the overall decline in anti-tumor immunity [50]. In malignancy, both tumor cells and immature dendritic cells fail to provide optimal co-stimulations to the T cells [51]. Tumor specific antigens (TSA) are the antigens that are uniquely expressed by tumors. TSA are more likely to be immunogenic generating tumor-antigen specific T cells. Some examples of TSA include viral oncoproteins, HTLV virus Tax oncoprotein, HPV oncogenes E6 and E7, SV40 tumor (T) antigen, mutated p53, oncogenic mutant Ras, etc. [50,52]. Immunogenic fusion oncoproteins, thyroid tumor specific antigen rearranged during transfection/papillary thyroid carcinoma (RET/PTC) fusion protein and Chronic myelogenous leukemia (CML) specific BCR-ABL [53–55]. Cancer germline genes, melanoma antigens (MAGE-1) and somatic tumor mutations also termed as neo-antigens such as BRAFV600E, and CDKR24C [56,57], are some other examples of tumor specific antigens [58]. Exhaustion is likely to arise in T cells reactive to immunogenic tumor specific antigens under the settings of antigen-rich environment [56]. For example, exhaustion of the leukemia antigen specific cytotoxic T lymphocytes was observed in the murine models of retroviral induced chronic phase CML and blast crisis

Cytokines such as IL2, TNFa, IFNc are lost. IFNc loss is critical for tolerance induction Expresses IL15 receptor alpha chain [135] Immunosuppressive tumor microenvironment such as the presence of CD11b+Gr-1+ MDSc play prominent role in T cell tolerance [136]

CML [59]. Exhaustion is also reported to occur to metastases melanoma antigen, Melan-A/MART-1 [16], suggesting that the process of exhaustion could be more related to presentation and antigenicity of tumor antigens [57,60]. Several algorithms are now available that can be used to predict the antigenicity of tumor antigens [61–63]. This information can be used to examine the possible exhaustion of T cells in patients presented with immunogenic tumor antigens. Important distinctions between tolerance and exhaustion are provided (Table 3).

2. Interleukin 2 downregulation during T cell exhaustion Changes in the cytokines synthesis constitute a major component of the exhaustion phenotype. During T cell exhaustion, loss of secretion of IL2, IFNc and TNFa occurs in hierarchical manner [9]. One of the most important cytokines whose production is lost during T cell exhaustion is IL2. IL2 is a pivotal cytokine for T cell survival and for activating a robust cellular immune response against infections and transformation. The role of IL2 in activating T cell response against tumor cells is depicted schematically (Fig. 1A). The schematics describe if IL2 synthesis is inhibited under antigen persistence, the overall antitumor response declines, resulting in tumor progression (Fig. 1B). It is reported extensively that exogenous IL2 infusion can enhance destructive immune responses against tumors, both in adoptively transferred tumor reactive T cell and gene modified T cells [15,64–66]. However, addition of exogenous IL2 does not by itself support long term antigenic response and survival [67,68]. In adoptive T cell transfers, T cells capable of sustaining IL2 production after coming in contact with reactive-antigens may produce stronger antigenic responses, thus, helping to break T cell exhaustion.

Fig. 1. (A) Schematics illustrate the crucial role of IL2 in antitumor response. (B) Illustrates negative impact of IL2 shutdown on overall antitumor response.

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The signaling pathways, both stimulatory and inhibitory, to the IL2 transcription have been extensively studied and new knowledge is constantly being added. The knowledge about these signaling pathways could provide vital clues about dysregulation of the IL2 synthesis during exhaustion. T cell receptor (TCR) mediated signaling pathway and CD28 costimulatory signaling are important for the IL2 transcription. These two signaling pathways are briefly described here. TCR signaling pathways: Upon ligation of MHC molecules loaded with antigenic peptides with TCR-CD3 complex, the first event to occur within T cells is the phosphorylation of tyrosine residues within cytoplasmic side of immunoreceptor tyrosine-based transactivation motifs (ITAMs) of TCR associated CD3 molecules by Src family tyrosine-protein kinases, lymphocyte protein tyrosine kinase (LCK) and Fyn [69]. Both LCK and Fyn undergo phosphorylation in the process, the mechanism of which is not completely understood. However, LCK is also activated by the interaction of CD4 and CD8 molecules with MHC class II [70,71]. Phosphorylated tyrosine residues of ITAM of CD3 zeta chains serve as a docking site for the zeta-chain associated protein tyrosine kinase (ZAP70). Phosphorylation in the tyrosine residues Tyr319/Tyr352 of ZAP70 amplifies the signal by promoting the recruitment of membrane proximal SH2 domain-containing leukocyte protein-76 (SLP76) scaffold protein complex, membrane integral protein linker for activation of T cells (LAT) and IL2 inducible T-cell kinase (ITK) [72]. Phosphorylation of LAT at tyrosine residues Tyr171 and Tyr191 by ZAP70 enables the binding and recruitment of SLP76, PLCc1 and GRB2 adaptor protein complex that also includes Gads/SOS/VAV. Phosphorylation of Vav1 stimulates guanine nucleotide exchange factors (GEFs) for CDC42-Rac molecules activating MEKK [73–75]. MEKK phosphorylates MKK3/6 and MKK4/7. Phoshpho-MKK3(Ser189)/MKK6(Ser207) and phospho-MKK4(Ser257/Thr261) activates p38MAPK and cJunN-terminal kinase (JNK) pathways, respectively [76,77]. Phosphop38MAPK(Thr180/Tyr182) in turn activates cJun transcription factor. Phospho-JNK (Thr183/Tyr185) activates transcription factor Fos and ATF2 [78,79]. The PLCc1 hydrolysis Phosphotidylinositol-4, 5 Biphosphate (PIP2) into inositol-1, 4, 5 triphosphatase (IP3) and diacyl glycerol (DAG). Second messenger DAG activates and recruits RAS-GRP and PKCh to the plasma membrane. Ras-GRP activates RAS, RAF, MEK1/2 and ERK kinase cascade activating transcription factor Fos. Fos heterodimerizes with cJun to form AP1 transcription factor that activates IL2 transcription by binding to the AP1 consensus binding site, ATGAGTCAT, of IL2 gene [3,80–83]. The IP3 binds to the IP3 receptor on the endoplasmic reticulum triggering calcium release from the ER. The two protein families, the stromal interacting molecules-(STIM) 1 and 2, functions as sensors of calcium ion levels in the ER. The depletion of calcium stores in the ER allow STIM proteins to migrate to the plasma membrane where they interact with Orai family proteins. This interaction is essential for the store-operated calcium entry through activating CRAC channels [84]. Influx of extracellular calcium ions into the T cells is sensed by calmodulin (CaM). CaM activates calcineurin phosphatase (CaN) by binding to its regulatory domain (RD) and displacing an autoinhibitory domain (AID) from the active site [85,86]. Activated calcineurin subsequently activates NF-AT transcription factor by dephosphorylating serine residues, allowing NF-AT to translocate to nucleus to initiate the IL2 gene transcription. NF-AT cooperates with AP1 to form dimers to bind to the NF-AT consensus sites GGAGGAAAAA or on several composite NF-AT-AP1 sites on IL2 promoter [87,88]. CD28 costimulatory signaling pathway: CD28 generated signaling pathway engages molecules that are partially distinct from TCR-CD3 receptor complex. NF-jB is the main transcription factor that is activated upon CD28 costimulation through the signaling

pathway that is yet to be completely defined. NF-jB binds to a sequence on the IL2 promoter, the CD28 response element, to activate IL2 transcription. Evidences also exist that support the integration of CD28 and TCR-CD3 signaling pathway by PDK1 molecule and Grb2 [89,90]. Ligation of CD28 extracellular domain motif comprised of a conserved sequence, MYPPPY, with B7 costimulatory molecules of the antigen presenting cells initiates CD28 costimulatory signaling cascade [91]. Phosphorylation of tyrosine residues within the short cytoplasmic domain of CD28 by the Src family kinases LCK and Fyn act as a docking site for p85 SH2 domain of phosphatidylinositol 3-kinase (PI3K) and Grb2 [92]. CD28 binds with PI3K through YMNM motif. As reported in mice the phosphorylation of PI3K on Tyr173 during CD28/B7.1 ligation is crucial for IL2 synthesis [93]. T cell expresses four different isoforms of PI3K, each capable of catalyzing PIP2 to PIP3 (PtdIns (3,4,5) P3) [94]. The requirement of second messenger PIP3 for further downstream signaling for the IL2 transcription is still unclear. Grb2-CD28 binding is required for the phosphorylation and activation of Vav1 for relaying and integrating signaling to the Rac1-CDC42 [95]. Another molecule that has gained much attention recently for providing distinct signals to the NF-jB during CD28 costimulation is Akt. Several studies have reported the activation of Akt during CD28 costimulation and its requirement for the IL2 synthesis [96,97]. CD28 family signaling pathways inhibitory to IL2 transcription: The signaling pathways generated by CD28 family molecules also block T cell activation. There are several CD28 family members chief among them CTLA4 and PD1 that have proven inhibitory to the T cell activation. The mechanism by which these molecules deliver coinhibitory signals to limit T cell response is not completely understood. However, the activation of phosphatases such as SHP2 and PP2A have been reported and those are believed to generate inhibitory signals [98]. CTLA4 uses YVKM motif to bind with PI3K, SHP2 and PP2A [90,91]. Likewise, PD1 ligation is reported to block PI3K activity and Akt response [99]. How exactly these varied signals integrate and influence IL2 synthesis and contribute to the exhaustion is yet to be discovered. 2.1. Epigenetic and posttranscriptional regulation of IL2 during exhaustion One of the reasons for gene repression or cellular inactivation is the epigenetic changes associated with the chromatin such as phosphorylation, methylation and acetylation. Several studies have shown that specific epigenetic modifications of chromatin represent distinct differentiation states of T cell subtypes such as naïve, effector and memory T cells [100–102]. However, it remains to be investigated whether exhausted T cells are presented with specific epigenetic signature and whether such epigenetic marks are conserved in T cells encountering malignancy and chronic viral infections. Along with the epigenetic regulation, posttranscriptional gene regulation could represent another mechanism of regulating IL2 mRNA metabolism during T cell exhaustion (Fig. 2). Mature microRNAs are class of small naturally occurring non-coding RNA molecules that extensively regulate protein coding genes by targeting mRNA 30 UTRs. More than 1000 microRNAs have been discovered. A single microRNA can regulate multiple genes due to their ability to pair with mRNAs with partial complimentary in Watson–Crick base pairing mechanism. MicroRNA targeting of mRNA can result in either mRNA decay, deadenylation or translational repression in RNA-induced silencing complex (RISC) [103]. The role of microRNAs in T cell activity and lineage differentiation has been extensively reported [104,105]. In a bioinformatics screen, Targetscan and miRANDA databases predicted a perfect seed match for human IL2 30 UTR with miR-181a (Fig. 3). The data suggest that miR 181a could be regulating IL2 mRNA stability

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Fig. 2. Schematics illustrate the possible involvement of chromatin remodeling complex in IL2 gene transcription (Upper panel). Lower panel demonstrates the posttranscriptional regulation of IL2 mRNA through microRNAs and RNA binding proteins during T cell exhaustion.

Fig. 3. Bioinformatics Targetscan analysis predicted miR-181a as a potential microRNA targeting IL2 30 UTR.

during T cell activation. This mechanism is supported by findings that show miR 181a expression in mature T cells is required for antigen sensitivity that regulates the positive and negative selection process for T cell tolerance to self antigens [104]. RNA binding proteins represent a class of proteins that specifically bind to mRNAs and play a prominent role in the posttranscriptional gene regulation. In a bioinformatics screen, RNA binding protein PUM2 turns out to be a predicted target for IL2 mRNA (Table 4). Human Pumilio Proteins have been reported to recruit multiple deadenylases to efficiently repress messenger RNAs [106]. PUM1 and PUM2 also known as PUMILIO proteins have been shown to cause repression of p27 mRNA through microRNA dependent gene silencing in rapidly proliferating cells [107]. Several other genes including hunchback (mat) mRNA have also been shown to be repressed by binding of PUM2. The signaling pathways regulating IL2 shutdown during T cell exhaustion are unknown. One interesting link could be the association of PD1/PDL-1 signaling pathway with IL2 transcription. The rationale for this model is derived from the studies that have demonstrated elevated expression of PD1 in tumor infiltrating T lymphocytes and the higher levels of PD1 ligand (PDL-1) on tumors. The PD1/PDL-1 interaction is associated with exhaustion phenotype and impaired cytokine production including that of IL2 [108,109]. Experimental evidence shows that PD1 activation through its ligand PDL-1/2 inhibits PI3K activation by activating src-homology phosphatases (SHP2) [110]. A relevant question to ask is whether PI3K inhibition could block IL2 transcription. It is possible, due to the PI3K inhibition; T cells do not receive sufficient CD28 costimulatory signals resulting in decline of the IL2 production. Lack of IL2 production in turn could render PI3K inactivation persistent because PI3Ks are strongly activated not only through IL2 receptor signaling but also by cytokines such as IL15, IL3, IL6, and interferons (IFNs), creating a cumulative effect contributing to T cell exhaustion [111,112]. A model is proposed that describes the relation between PD1 signaling and IL2 transcription (Fig. 4). Table 4 Pum2 scored 100% coverage for IL2 30 UTR binding. Score

Relative score

RBP name

Start

End

Matching sequence

7.2294196 7.2294196 7.2294196 7.2294196

100% 100% 100% 100%

Pum2 Pum2 Pum2 Pum2

132 113 188 272

135 116 191 275

UGUA UGUA UGUA UGUA

Fig. 4. Model depicting the signaling pathway involving PD1 mediated regulation of IL2 transcription.

The observation of development of spontaneous autoimmune diseases in PD1, PDL-1 and IL2 knockout mice provides a rationale that PD1 signaling could be regulating the IL2 transcription [113–116]. Thus, PD1 signaling may be contributing significantly to decline of IL2 production and to the process of T cell exhaustion [113]. Further research into this direction will reveal in-depth of understanding of the mechanism of T cell exhaustion. 3. Prevention of T cell exhaustion and its therapeutic applications As T cells play critical role in virus clearance and tumor rejection, there has been considerable interest in preventing or reversing T cell exhaustion during chronic viral infection and malignancy. For example, PD1/PDL-1 blockade reverses CD8+ T cell exhaustion to chronic virus infections [19]. An antibody mediated blockade of PD1 or PDL-1 molecules restored T cell responses against tumor-antigens and demonstrated promising clinical benefits for melanomas [117,118]. Doedens et al., showed recently

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that genetically inactivating the tumor suppressor VHL (von Hippel-Lindau), which is a negative regulator of hypoxia-inducible factor (HIF) signaling pathway, is able to prevent exhaustion. Moreover, CD8+ T cells were able to maintain potent effector functions while encountering persistent melanoma tumor antigens and chronic LCMV Armstrong virus [119]. Our lab is currently investigating whether rescuing IL2 production or opposing IL2 decline in T cells exposed to chronic tumor antigens could reverse the T cell exhaustion (Balkhi et al.; unpublished data). T cell adoptive immunotherapy is a promising tool for cancer cure. It has been reported that T cell exhaustion not only affects T cells encountering persistent tumor antigens in a body but also ex vivo expanded therapeutic T cells such as tumor infiltrating T cells (TILs). TILs are tumor infiltrated T cells that are isolated from tumors. TILs are extensively expanded ex vivo and infused back into cancer patients to generate anti-tumor reactivity. However, exhaustion limits the potential of TILs to suppress tumors. Exhaustion in TILs has been demonstrated in human prostate cancer patients and during metastases and in mice bearing solid tumors [16,18,109,120]. Exhaustion is also reported in chimeric antigen receptor modified T cells (CAR-T) [121,122]. CAR-T cells are generated by genetically modifying T cell receptor with immunoglobulin fusion products of an antibody binding domain (immunoglobulin, Ig) with the zeta signaling chain of the TCR and CD28 costimulatory molecule sequences, to form Immunoglobulin chimeric antigen receptor (IgCAR), which redirects T cells for cancer or viral antigen specific cytotoxicity [123–125]. The single chain variable fragment (scFV) antibody binding domain of CAR is specific of tumor associated antigens. On tumor contact T cell chimeric antigen receptor ‘‘IgCAR with the fusion of CD28’’ yields signal 1 + 2. Signal 1 + 2 is important for the full activation of T cells and IL2 synthesis. The design of CAR-T cells is represented sche-

matically in Fig. 5. The design of CAR-T cells, apart from CD28 molecule, has also included several other costimulatory molecules to improve antitumor activity. For example, CAR-T cells modified with B cell specific CD19 antigen and costimulatory molecule CD137 (4-1BB) have shown success in clearing chronic lymphocytic leukemia [64]. However, one factor that could limit designer T cell efficacy is their failure to sustain IL2 production in vivo and in patients as reported by Emtage et al. [121,122]. IL2 shutdown is consistent feature in antigen-specific T cells and gene modified T cells. In-depth understanding of the mechanism of IL2 shutdown in T cells exposed to chronic antigens will significantly help improving the design of CAR-T cells in a way that T cells maintain long term antigenic responses and promote powerful antitumor or antiviral T cell responses. 4. Future directions A global approach to identify epigenetic modifications associated with the chromatin in T cells at various stages of differentiation including exhaustion could be very informative. Such an approach has already been tested successfully in activating and resting T cells [126,127]. A global approach to identify repressive histone marks on IL2 region using ChIP-Seq technique could be useful in understanding the mechanism leading to T cell exhaustion to persistent infections or in malignancy. This technique could be particularly useful in determining whether histones around IL2 genomic region ‘‘4q26-q27 Chromosome: 4; NC_000004.11 (123372625 to 123377650’’ undergoes chromatin modifications [128]. The binding of high mobility group proteins (HMGA1) to the CD28 response element has been reported to promote interaction of NF-jB, NF-AT, and AP1 at this region [129]. The HMGA1 putative binding site on CD28 response element lies in close proximity to TATA binding protein, TFIID. Proximity to these sites makes all these transcription factors suitable candidates to influence gene expression [130]. It could be potentially interesting to investigate how these factors influence IL2 shutdown in T cells exposed to persistent antigens. The role of microRNAs and RNA binding proteins in the regulation of IL2 and T cell exhaustion in malignancy and in chronic virus infections is not commonly described. It could be interesting to determine the expression levels miR-181 in T cells infiltrating tumor antigens. This will allow exploring in vitro whether modulating miR-181 expression produces effect on the IL2 production. Likewise, investigating the expression levels of ‘‘PUMA’’ RNA binding protein in T cells infiltrating tumors or exploring its significance for T cell exhaustion is potentially interesting. Likewise, investigating whether PUMA and miR-181a cooperate to influence the stability of IL2 mRNA could be vital for understanding the mechanism of IL2 downregualtion in exhaustion. 5. Conclusions The in-depth understanding of the mechanism of IL2 decline in T cells exposed to chronic antigens will help genetically modify T cells, so that when they are exposed back to an antigen-rich environment could resist IL2 decline. Maintaining constant production of the IL2 in antigen-rich environment will allow T cell to harness powerful antigenic responses. Thus, the identification of the signaling pathways involved in the IL2 downregulation and posttranscriptional regulation of the IL2 mRNA could prove vital in making breakthrough improvements in T cell immunotherapy.

Fig. 5. Structure of T cell chimeric antigen receptor (IgCAR). Upper panel: Tumor antigen specific Ab binding domain is cloned together with TCR zeta chain and co-stimulatory CD28 molecule and packaged in retroviral vector and subsequently expressed in T cells. Lower: Designer T cells expressing IgCAR.

Acknowledgement This work is supported by Breast Cancer Research Program, Department of Defense.

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