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. Author manuscript; available in PMC: 2014 Sep 15.
Published in final edited form as: J Immunol. 2013 Aug 9;191(6):3471–3477. doi: 10.4049/jimmunol.1202350

Death Receptor Independent Activation Induced Cell Death (AICD) in Human Melanoma Antigen Specific MHC class I-Restricted TCR Engineered CD4 T Cells

Arvind Chhabra 1,*, Bijay Mukherji 1
PMCID: PMC3779684  NIHMSID: NIHMS509480  PMID: 23935194

Abstract

Engaging CD4 T cells in anti-tumor immunity has been quite challenging, especially in an antigen specific manner, since most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma associated antigenic epitope, MART-127-35, specific MHC class I restricted transgenic T cell receptor (TCR) function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope specific and MHC class I restricted manner (Chhabra et al., JI, 2008, Ray et al., J. Clin. Immunol., 2010). TCR engineered (TCReng) CD4 T cells therefore have translational potential and clinical trials with MHC class I TCR engineered CD4 T cells are underway. We here show that while TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope specific AICD. We also show that the AICD in TCReng CD4 T cells is a death receptor (DR)-independent process, and that JNK andp53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53 mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights towards AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

Introduction

Generation of a protective CD8+ cytolytic T lymphocyte (CTL) response has been a major focus of most T cell based cancer immunotherapy approaches. Since CD4 T cells play an important role in the generation of a long-lived antigen specific CD8+ CTL response (1, 2), a simultaneous engagement of CD4 and CD8 T cells in cancer immunotherapy could significantly improve the clinical outcome of T cell based cancer immunotherapy. However, engaging CD4 T cells in anti-tumor immunity is a challenging proposition, especially in an antigen specific manner, since natural CD4 T cells function in a MHC class II-restricted manner and as a large fraction of non-lymphoid human tumor cells usually do not express MHC class II molecules (3). However, it should be pointed out that some non-lymphoid tumors can express MHC class II molecules, and IFN-γ exposure can further induce the expression of MHC class II molecules on tumor cells (4, 5). Interestingly, increased HLA-DR expression on tumor cells has been associated with poor prognosis in melanoma and osteosarcoma, and improved prognosis in squamous cell carcinoma, breast carcinoma, colorectal carcinoma, cervical carcinoma and laryngeal carcinoma (3, 6). Engagement of natural CD4 T cells in tumor immunity in general and adoptive cancer immunotherapy in particular, in an antigen specific manner, will require the identification and characterization of HLA allele matched MHC class II restricted tumor antigenic epitopes and isolation of TCRs against these epitopes. However, compared to a large number of well characterized MHC class I restricted antigenic epitopes available for generating CD8+ CTL responses and against tumor associated antigens, very few allele matched MHC class II-restricted tumor antigenic epitopes have been identified to date. In this context, we have recently shown that a high avidity MHC Class I restricted transgenic T cell receptor (TCR) can be utilized to effectively program human CD4 T cells to function as MHC class I directed anti-tumor effectors (7-9). These MHC class I restricted CD4 T cells exhibit an eptope specific Th1 biased effector cytokine response, help the expansion of CD8+ CTLs, and also exhibit a potent MHC class I restricted and granule exocytosis-mediated cytolytic function of their own (7, 8). However, MHC class I–restricted epitope specific TCR engineered (TCReng) CD4 T cells are non-physiologic effector T cells. Hence, their biology needs to be fully understood to effectively utilize them in cancer immunotherapy.

Just as signaling through a TCR leads to effector function such signaling, including signaling through transgenic TCR, can also lead to epitope specific activation induced cell death (AICD). While program cell death (PCD) in T cells following an immune response, is essential to maintain homeostasis, AICD, especially premature AICD, could be a limiting factor in T cell-based cancer immunotherapy. Presently, nothing is known on AICD in MHC class I restricted CD4 T cells. Therefore, we examined the susceptibility as well as the mechanism underlying AICD in TCReng CD4 T cells. We here show that the cognate antigen stimulated and in-vitro expanded (antigen experienced) but not the freshly transduced (antigen inexperienced) TCReng CD4 T cells are susceptible to AICD in an epitope specific manner. We further show that AICD in TCReng CD4 T cells is a death receptor (DR)-independent, JNK activation-driven, intrinsic process, similar to the MHC class I TCR driven AICD we have recently shown in melanoma epitope specific primary human CD8+ cytolytic T lymphocytes (CTL) (10). We also show that the p53 mediated non-transcription dependent mitochondria-centric pathway also plays a critical role in this process, and that the interference with this pathway prevents AICD in a significant fraction of these cells. Our findings offer novel insights on AICD in MHC class I-restricted TCReng CD4 T cells that could have implications for cancer immunotherapy with MHC class I restricted tumor epitope specific TCReng CD4 T cells.

Materials and Methods

Study population, cell lines, culture medium and reagents

The study population was comprised of HLA-A2-positive melanoma patients and healthy donors, enrolled with an informed consent. Culture medium, cytokines and medium supplements used to differentiate peripheral blood derived monocytes into dendritic cells (DC), and for the generation and culture of antigen specific T cells have also been described before (10-13). T2-A2 cell line used in the study to present peptide epitopes has been described before (11). Antibodies and tetramer reagents to track antigen specific CTL and annexin-V reagents to quantify CTL undergoing AICD were was purchased from BD Pharmingen (USA), Beckman Coulter (USA), and R & D Systems Inc. (USA), and have also been described before (10, 13). Pharmacological inhibitors targeting specific kinases, SB203580 (p38 kinase), SP600125 (JNK), PD98059 (ERK) were purchased from EMD Biosciences, USA. Reagents blocking the death-receptor engagement with their ligands, human Fas/Fc chimera, human TNF-RI/Fc chimera, human TRAIL-RI/Fc chimera, human TRAIL-RII/Fc chimera, and human IFN-γ RI/Fc chimeric proteins were purchased from R&D Systems, USA. P53 inhibitor, p-fithrin-μ, was purchased from EMD Biosciences, USA. Antibodies for western blot analyses of JNK, c-Jun and phospho-c-Jun were purchased from the Santa Cruz Biosciences, USA.

Transduction of CD4 and CD8 T cells with transgenic TCR

CD4+CD25− and CD8+ T cells were isolated from the Ficoll-Hypaque density gradient purified peripheral blood lymphocytes (PBL) by Dynal magnetic beads purification method (Invitrogen Inc., USA) as reported before (10, 11). Purified CD4+CD25− and CD8+ T cells were activated with plate-bound anti-CD3 (5μg for CD4 and 10μg for CD8) & CD28 antibodies (5μg for both CD4 and CD8) and cultured in the presence of 100 u/ml IL-2 for 3-5 days. Transduction of activated CD4 T cells (1-2 ×106 cells) with MART-127–35 (M1) epitope specific transgenic TCR was performed as described before (8). In brief, activated CD4 T cells were cultured on retronectin (Fisher Scientific, USA) coated recombinant virus bound cell culture plates in the presence of IL-2 for 3 days. Transduction efficiency was quantified by staining the transduced cells with M1 epitope specific tetramer (Beckman Coulter, USA). TCR transduced CD4 and CD8 T cell populations were expanded by co-culturing them with the M1 peptide pulsed mature DC, according to our published methods (10, 11).

Assay for induction of AICD in MHC class I restricted CD4 and CD8 T cells

To test whether or not the MHC class I TCR engineered CD4 T cells undergo AICD, primary transduced as well as in-vitro expanded MHC class I restricted CD4 T cells were exposed to peptide (1 μg/ml) loaded T2 cells (E:T = 100). Apoptosis was determined by flow cytometry with triple color staining (CD8, MART-127–35 epitope specific tetramer, and annexin V) at different time points (4–18 h). Annexin staining was also confirmed by mitochondrial membrane integrity breakdown assay, discussed below. To evaluate the effect of various agents in modulating AICD, the MHC class I restricted CD4 T cells were pre-incubated with various compounds at optimal concentration for 45 min at 37°C and then exposed to T2 cells alone or loaded with peptide. The optimal dose used in the experiments shown in this paper was determined by using these compounds at different concentrations.

Death-Receptor-Mediated Cell Death

To examine the functional activity of DR-blocking antibodies, DR-mediated cell death was induced in Jurkar cells exposed to respective DR-ligands and cells undergoing death were quantified 4 hr post DR-ligand exposure by FACS-mediated staining for annexin. To examine the activity of DR-blocking reagents, Jurkat cells exposed to DR-ligands we pre-incubated with respective DR-blocking antibodies (1μg/ml) and cells undergoing death were examined by quantifying annexin +ve cells by FACS, 4 hr post DR-ligand exposure.

Assay for mitochondria membrane integrity during AICD

Mitochondrial membrane integrity of MART-127–35 epitope specific TCR engineered CD4 and CD8 T cells was done by staining with mitotracker dye (Invitrogen Inc. USA). In brief, untreated TCReng CD4 T cells were exposed to control MAGE-3271–279 and the cognate MART-127–35 peptide co-incubated with mitotracker dye (25nM) 2 hour post peptide exposure. Status of mitochondria membrane integrity was quantified 4 hour post peptide exposure, following staining with MART-127–35 epitope specific tetramer by FACS.

Western Blot Analysis

Western blot analysis was done as described previously (13). In brief, MHC class I TCR engineered CD4 T cells co-incubated with the T2 cells alone or the T2 cells pulsed with the control peptide, MAGE-3271–279, or the cognate peptide, MART-127–35, with or without pre-incubation with pharmacological inhibitors for 4 hours. Cells undergoing AICD were quantified by annexin staining as discussed above, and the cell lysates were prepared in RIPA buffer to examine the status of different proteins by western blot analysis, as described previously (13).

Results

TCR engineered anti-tumor CD4 T cells were generated by transducing human peripheral blood derived CD4+25− T cells with the MART-127-35 epitope specific transgenic TCR, according to our published methods (8). Fig.1A shows generation and in-vitro expansion of MART-127-35 epitope specific TCR engineered CD4 T cell populations. While the initial transduction efficiency varied from a 30-60% in different experiments, the number of epitope specific cells could be substantially expanded to 70-80% MART-127-35 upon re-stimulation with the MART-127-35 peptide pulsed DC. Fig. 1B reconfirms our published findings that the TCReng CD4 T cells exhibit a Th1 biased functional profile (7, 8). Fig. 1C shows that the MART-127–35 TCR engineered CD4 T cells are also susceptible to epitope specific AICD, in a dose dependent manner. Interestingly, we have found that the TCReng CD4 T cells show a different form of susceptibility to AICD than the CD8+ T cells. For example, while the TCReng CD8 T cells exhibit considerable susceptibility to AICD following transduction (i.e. antigen inexperienced TCReng CD8 T cells) (Fig. 2B-ii), the antigen inexperienced TCReng CD4 T cells show remarkable resistance to AICD (Fig. 2B-i). Of further interest, following stimulation with the cognate epitope pulsed DC (i.e. antigen experienced) TCReng CD4 T cells exhibit significant susceptibility to AICD (Fig. 2A & 2B).

Figure. 1.

Figure. 1

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Epitope specific AICD in MART-127-35 epitope specific TCR engineered CD4 T cells. (A). MART-127-35 epitope specific TCR engineered CD4 T cells were generated by engineering the activated human peripheral blood derived CD4 T cells with a MART-127-35 epitope specific transgenic TCR, and transduced cells were tracked by MART-127-35 epitope specific tetramer staining, 3 days post transduction. As shown, we can generate ~60% MART-127-35 epitope specific TCReng CD4 T cells 3 days post TCR transduction that could be expanded to a ~80% epitope specific CTL population 7-10 days upon re-stimulation with the MART-127-35 peptide pulsed DC. (B). Effector function profile of TCR engineered CD4 T cells. TCReng CD4 T cells were exposed to T 2 cells pulsed with cognate or control peptide (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-3271-279 control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide), and the cytokines released were measured by ELISA. As shown, the TCReng CD4 T cells exhibit a Th1 biased cytokine profile, in accord with our published findings (7, 8). (C). Peptide dose kinetics of AICD in in-vitro expanded MART-127-35 epitope specific TCR engineered CD4 T cells. MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the different doses of cognate peptide, MART-127-35, or control peptide, MAGE-3271-279, pulsed T2 cells and 4h post co-culture, CD4 T cells undergoing AICD were quantified by following the exposure of annexin-V on their cell surface (upper panel) and mitochondria membrane hypo-polarization (lower panel) (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-s control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide). Statistical analysis is shown on top of respective panels, and boxes on top of respective panels shows percent M1 tetramer +ve annexin +ve TCReng CD4 T cells. As shown, the TCReng CD4 T cells undergo AICD in a dose dependent manner. The data shown is representative of more than five independent experiments.

Figure. 2.

Figure. 2

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Comparative analysis of AICD in freshly transduced (antigen inexperienced) and cognate peptide pulsed DC re-stimulated (antigen experienced) TCReng CD4 and CD8 T cells. (A). AICD in antigen experienced TCR engineered CD4 and CD8 T cells. In-vitro expanded CD4 (i) and CD8 (ii) T ce lls were exposed to the peptide pulsed T2 cells. T cells undergoing AICD were quantified by following the exposure of annexin-V on their cell surface (upper panel), and mitochondria membrane hypo-polarization (middle and lower panels), 4h post antigen encounter. Statistical analysis is shown on top of respective panels. As shown, T cells exposed to peptide pulsed T2 cells undergo AICD in an epitope specific manner (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-3271-279 control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide). (B). Comparative analysis of AICD in freshly transduced (antigen inexperienced) and cognate peptide pulsed DC re-stimulated (antigen experienced) TCReng CD4 and CD8 T cells. AICD in primary transduced CD4 and CD8 T cells was performed by exposing them to T2 cells pulsed with cognate peptide and annexin positive MART-1 tetramer positive cells were quantified by FACS. As shown while antigen inexperienced as well as antigen experienced CD8 T cells significantly undergo epitope specific AICD, antigen inexperienced TCReng CD4 T cells do not undergo AICD but antigen experienced TCReng CD4 T cells do (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-3 control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide). The data shown is representative of more than five independent experiments.

We have recently shown that the MHC class I TCR driven epitope specific AICD in MART-127-35 epitope specific CD8+ CTL is a death receptor (DR)-independent, caspase-independent, JNK activation driven process (10, 13, 14). Therefore, we next examined whether AICD in these TCReng CD4 T cells also follows a mitochondria-centric death cascade. As shown in Fig. 3A, blocking several cell surface death receptors (Fas/Fc, TRAIL-R1 & R2, IFNg-R1, TNF-sRI/Fc) by DR neutralizing antibodies had no major effect on the AICD susceptibility of the TCR engineered CD4 T cells, suggesting that AICD in MHC class I restricted CD4 T cells is a DR-independent process. Fig. 3B shows a positive control for the ability of DR blocking antibodies to interfere with the DR-mediated cell death. As shown in Fig. 4, AICD in TCR engineered CD4 T cells involves JNK activation, evident from the phosphorylation of JNK target gene c-Jun (Fig. 4B), and blocking JNK activation by pharmacological inhibitor, SP600125, significantly prevented AICD, evident by the annexin-V staining as well as mitochondria hypo-polarization readouts (Fig. 4A). The dose kinetics studies of JNK inhibitor found 25uM to be an optimal dose for preventing AICD without affecting the effector function of TCReng CD4 T cells (data not shown), as previously shown in natural CTL (10). Fig. 4C shows data that the pretreatment of TCReng CD4 T cells with JNK inhibitor did not affect their effector function, evident from their TNF-α and IL-2 cytokine production profiles.

Figure. 3.

Figure. 3

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AICD in MART-127-35 epitope specific TCR engineered CD4 T cells is death receptor (DR)-independent process. (A). MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the different doses of cognate peptide, MART-127-35, or control peptide, MAGE-3271-279, pulsed T2 cells, in the presence or absence of DR-blocking antibodies (1μg/ml), and 4h post co-culture T cells undergoing AICD were quantified by following the exposure of the annexin-V on their cell surface (upper panel), and mitochondria membrane hypo-polarization (middle and lower panels). The box on top of respective panels shows percent M1 tetramer +ve annexin +ve TCReng CD4 T cells. As shown DR-blockade did not prevent AICD. (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-3271-279 control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide). The data shown is representative of three independent experiments. (B). Data showing activity of DR-blocking antibodies. DR-mediated death was induced in Jurkat cells by TRAIL (100ug/ml) (upper panel) or FAS agonistic antibody (100ug/ml) (lower panel), w/wo pre-incubation with the TRAIL receptor blocking antibodies (1μg/ml) or the FAS blocking antibody (1μg/ml). As shown DR-blocking antibodies effectively blocked the DR-mediated death in Jurkat cells.

Figure. 4.

Figure. 4

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AICD in MART-127-35 epitope specific TCR engineered CD4 T cells involves JNK. (A). JNK activation is involved in AICD of TCReng CD4 T cells and blocking JNK activation rescues TCReng CD4 T cells from undergoing AICD. (i). MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the control peptide, MAGE-3271-279, or the cognate peptide, MART-127-35, pulsed T2 cells. T cells undergoing AICD were quantified 4h post antigen encounter by exposure of the annexin-V on their cell surface (upper panel), and mitochondria membrane hypo-polarization (middle and lower panels). To examine the effect of inhibitors targeting JNK, SP600125 (SP) or ERK, PD098059 (PD) on AICD in TCReng CD4 T cells, cells were exposed to the cognate epitope following pre-treatment with respective kinase inhibitors. As shown, SP600125 pre-treatment (25μM) rescued TCReng CD4 T cells from AICD, but PD098059 pre-treatment could not. The statistical analysis is shown on top of respective panels, and the boxes on top of respective panels shows percent M1 tetramer +ve annexin −ve TCReng CD4 T cells. (ii). A bar plot showing the percent M1 tetramer +ve annexin −ve TCReng CD4 T cells (from Fig. 4A-i) and the effect of JNK activation blockade. The p-value (0.0049) was calculated by two tailed student t-test. (B). Western blot data showing JNK activation during AICD in TCReng CD4 T cells and its blockade by SP600125. As shown JNK expression level was not affected during AICD; JNK activation during AICD resulted in phosphorylation of JNK target gene, c-Jun, and the JNK inhibitor (SP) blocked c-Jun phosphorylation while ERK inhibitor (PD) did not. (C). Effect of JNK blockade on effector function of TCR engineered CD4 T cells. To examine the effect of JNK inhibition on effector function of TCR engineered CD4 T cells, in-vitro expanded TCR engineered CD4 T cells were pre-treated with inhibitors targeting JNK, SP600125 (SP) or ERK, PD098059 (PD), and cytokines released in the supernatant were measured. As shown, SP600125 pre-treatment did not significantly affect their effector function. The data shown is representative of more than three independent experiments.

P53 has recently been shown to have a transcriptional independent role in mitochondria-centric cell death (15, 16). Since our findings suggest that the AICD in TCR engineered CD4 T cells might be an intrinsic mitochondria-centric process, we next examined the involvement of p53 in MHC class I TCR driven AICD of engineered CD4 T cells. As shown in Fig. 5, pretreatment of TCR engineered CD4 T cells with the p53 inhibitor (p-pifithrin-μ), that blocks binding of p53 to mitochondria, rescued these cells from undergoing AICD without affecting their effector function. As shown in Fig. 5D, p53 inhibitor had no effect on p53 target gene expression.

Figure. 5.

Figure. 5

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AICD in MART-127-35 epitope specific TCR engineered CD4 T cells involves p53. (A). MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the cognate peptide, MART-127-35, or control peptide, MAGE-3271-279, pulsed T2 cells, in the presence or absence of p-53 inhibitor, p-fithrin-μ, and 4h post co-culture T cells undergoing AICD were quantified by following the exposure of the annexin-V on their cell surface. The box on top of respective panels shows percent M1 tetramer +ve annexin +ve TCReng CD4 T cells. As shown, p-53 inhibitor, p-fithrin-μ interfered with the AICD in a dose dependent manner. (T2: cells exposed to T2 cells alone, T2+M3: cells exposed to T2 pulsed with MAGE-s control peptide, T2+M1: cells exposed to T2 cells pulsed with the MART-127-35 cognate peptide). (B). MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the different doses of cognate peptide, MART-127-35, or control peptide, MAGE-3271-279, pulsed T2 cells, in the presence or absence of 50uM p-53 inhibitor, p-fithrin-μ, and 4h post co-culture CTL undergoing AICD were quantified by following the exposure of the annexin-V on their cell surface (upper panel), and mitochondria membrane hypo-polarization (middle and lower panels). Statistical analysis is shown on top of respective panels, and box on top of respective panels shows percent M1 tetramer +ve annexin +ve TCReng CD4 T cells. As shown, p-fithrin-μ rescued TCReng CD4 T cells from undergoing AICD. (T2: T2 cells alone, T2+M3: T2 pulsed with MAGE-s control peptide, T2+M1: T2 cells pulsed with the MART-127-35cognate peptide). (C). Effect of p53 inhibitor on effector function of TCR engineered CD4 T cells. To examine the effect of JNK inhibition on effector function of TCR engineered CD4 T cells, in-vitro expanded TCR engineered CD4 T cells were pre-treated with inhibitors targeting p53-mediated mitochondria-centric death pathway, p-fithrin-μ (p53μ-20μM) or ERK inhibitor, PD098059 (PD), and cytokines released in the supernatant were measured. As shown, p53μ pre-treatment did not significantly affect their effector function. The data shown is representative of more than three independent experiments. (D). Effect of p53 inhibitor did not affect the gene expression levels of p53 target genes. MART-127-35 epitope specific TCR engineered CD4 T cells were exposed to the p53-inhibitor and expression of p53 gene as well as p53 target genes involved in cell death, Bax, PUMA, was examined by intracellular FACS. The data shows overlay plots of respective protein expression levels in control cells (c) or cells pre-incubated with the p53 inhibitor (p53u). As shown no change in expression was observed.

Discussion

CD4+ T cells play important roles in cell-mediated immunity by providing “help” towards the generation of a robust and long-lived CTL response, and also towards the generation of an antigen specific memory response (1, 2, 17-20). As such engaging CD4 T cells in tumor immunotherapies is highly desirable, considering that T cell based cancer immunotherapies--whether it is active specific immunization or the adoptive immunotherapy--have generated limited success (21-24). However, engaging natural CD4 T cells in cancer immunotherapy has been a challenging proposition, especially in an antigen specific manner, as CD4 T cells recognize their target epitopes on MHC class II molecules and many non-lymphoid tumors are usually MHC class II negative. It should however be pointed out that some non-lymphoid tumors can express MHC class II molecules, IFN-γ can increase MHC class II expression on tumor cells. Interestingly, increased MHC class II expression has been associated with poor prognosis in melanoma and osteosarcoma, and improved prognosis in squamous cell carcinoma, breast carcinoma, colorectal carcinoma, cervical carcinoma and laryngeal carcinoma (3, 6).

Utilizing a TCR-based T cell engineering approach of engrafting human peripheral blood derived T cells with a desired antigen specific transgenic TCR (23, 25-30), we have recently shown that human CD4 T cells, when engineered to express a MHC class I restricted melanoma epitope specific TCR, can be programmed to function as MHC class I restricted “multi-functional” anti-melanoma effectors capable of simultaneously functioning as lytic effectors as well as helper cells (7-9). Understandably, the availability of a single MHC class I restricted TCR that can program both CD4 as well as CD8 T cells to function as potent anti-tumor effectors makes it easier to engage CD4 T cells in tumor immunity. However, it should be emphasized here that natural CD4 T cells do not express MHC class I restricted TCRs. Therefore, systematic studies are needed to fully understand their physiology under the control of a MHC class I TCR to effectively utilize them in cancer immunotherapy. Since, MHC class I TCR triggered signal cascades, besides leading to the generation of effector function, can also lead to AICD, we here examined the susceptibility to and the mechanism of epitope specific AICD in TCReng CD4 T cells. Our data show that the antigen experienced MHC class I restricted CD4+ anti-tumor effector T cells are quite susceptible to epitope specific AICD (Figs. 1C & 2). Interestingly, we have found that while antigen inexperienced freshly transduced CD8 T cells show susceptibility to AICD even upon encountering the cognate epitope for the very first time, antigen inexperienced freshly transduced CD4 T cells do not undergo epitope specific AICD (Fig. 2). This adds a new dimension to the functional profile of these MHC class I restricted CD4 T cells, epitope specific AICD. Although the basis for the AICD resistance in antigen inexperienced TCReng CD4 T cell presently remains unclear, the lack of co-receptor engagement in these cells might be an explanation. Further studies will be needed to clarify this interesting difference in susceptibility to AICD by antigen inexperienced CD4 and CD8 T cells. In addition, our findings also highlight the need for a systematic characterization of the molecular mechanism of AICD in antigen experienced TCReng CD4 T cells to sustain them longer.

Epitope specific AICD is one of the salient features of an antigen specific T cell response that involves activation of the TCR triggered death cascade leading to the elimination of a significant fraction of antigen specific T cells. Although, AICD and PCD are essential immune homeostasis mechanisms, premature AICD of anti-tumor T cells could be a limitation in T cell based cancer immunotherapy, especially in adoptive immunotherapy. Since CD4+ and CD8+ T cells follow distinct developmental programs, perform distinctly different effector functions in the physiology, and as CD4+ helper T cells have been shown to rescue CTL from AICD (31), understanding the MHC class I TCR triggered molecular cascade leading to epitope specific AICD in TCReng CD4 T cells is critical to effectively utilize them in cancer immunotherapy. Understandably, availability of methods to sustain these cells long enough in the physiology would allow them to orchestrate their full multifunctional anti-tumor repertoire for generating a productive clinical outcome.

AICD in T cells has long been considered to be initiated by the engagement of their surface death receptors with the corresponding death ligands and executed by caspases (32-35). Most of these early studies, however, were done in T cell lines or T cell clones and not in human primary T cells (32-35). Interestingly, T cells have been shown to also die in a DR-independent and caspase independent manner (36-38). Towards understanding the mechanism of epitope specific AICD in CD8+ human primary CTL, we have also recently shown that the AICD in human melanoma associated antigen specific CD8+ primary CTL is a JNK activation driven, DR-independent, caspase-independent, mitochondria-centric process (10, 13, 14). We have now found that the AICD in MHC class I restricted epitope specific transgenic TCR triggered AICD in CD4 T cells is also an intrinsic death process that is not initiated by DR engagement (Fig. 3), and JNK activation plays a critical role in this death cascade as blocking JNK activation rescued significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function (Fig. 4).

Mitochondria play an essential role in cell death pathways, as they harbor death mediators that can execute cell death through caspase-dependent as well as casepase-independent manner (39, 40). We have shown that the MHC class I epitope specific TCR driven AICD in CD8+ human primary CTL is a mitochondrion-centric process that involves mitochondria resident caspase-independent death executioner, apoptosis-inducing factor (AIF) (10, 13). We have also found that the mitochondria-centric p53-mediated cell death pathway (15, 16) is involved in AICD in CD8+ CTL, as p-pifithrin-μ, a p53 inhibitor that blocks the interaction of p53 with the mitochondrial and interferes with its non-transcription-dependent death cascade (41), blocks this AICD (unpublished data). We have now found that the epitope specific AICD in MHC class I restricted CD4 T cells also involves p53-mediated non-transcription-dependent mitochondria-centric death cascade and that the p53 inhibitor, p-pifithrin-μ, could prevent this AICD, without affecting their effector function (Fig. 5). Interestingly, p53 inhibitor had no effect on p53 target gene expression levels (Fig. 5D). While further studies will be needed to systematically examine the role JNK-p53-cross-talk in regulation of AICD in MHC class I restricted CD4 and CD8 T cells, taken together, our findings offer novel insights towards the biology of MHC class I restricted CD4 T cells by showing that the AICD in antigen experienced TCReng CD4 T cells is a DR-independent, mitochondria-centric process. In addition, our findings provide potential targets (JNK and p53) to interfere with this death cascade for sustaining these multifunctional effectors longer for an effective cancer immunotherapy.

Acknowledgments

The work was supported by PHS grants CA83130 (BM), CA88059 (BM), CA132681 (BM), Breast Cancer Alliance, Greenwich, Connecticut (AC), the State of Connecticut Stem Cell Research Initiative (AC), and the MO 1RR06192 grant, GCRC, UCHC.

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