CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.     

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.     

Th17 cells undergo Fas-mediated activation-induced cell death independent of IFN-gamma

IL-17-secreting CD4+ T cells (Th17 cells) play a critical role in immune responses to certain infections and in the development of many autoimmune disorders. The mechanisms controlling homeostasis in this cell population are largely unknown. In this study, we show that murine Th17 cells undergo rapid apoptosis in vitro upon restimulation through the TCR. This activation-induced cell death (AICD), a common mechanism for elimination of activated T cells, required the Fas and FasL interaction: Fas was stably expressed, while FasL was up-regulated upon TCR reactivation of Th17 cells; Ab ligation of Fas induced Th17 cell death; and AICD was completely absent in Th17 cells differentiated from gld/gld CD4+ T cells. Thus, the Fas/FasL pathway is essential in regulating the AICD of Th17 cells. Interestingly, IFN-gamma, a cytokine previously found to be important for the AICD of T cells, did not affect Th17 cell apoptosis. Furthermore, Th17 cells derived from mice deficient in IFN-gamma receptor 1 (IFN-gammaR1-/-) underwent AICD similar to wild-type cells. Thus, AICD of Th17 cells occurs via the Fas pathway, but is independent of IFN-gamma.     

Fas-dependent, activation-induced cell death of gammadelta cells.

Activated gammadelta T cells undergo apoptosis upon restimulation of their T cell receptor (TCR)/CD3 complex. We demonstrate that in these cells, the activation-induced cell death (AICD) is mediated by Fas and Fas ligand (FasL) interaction. The activated gammadelta T cells are prone to AICD initiated by exposure to mitogens, anti-TCR/CD3 antibodies, as well as specific antigens such as Daudi cells or ethylpyrophosphate (Etpp). Cells that have been activated twice, and consequently more susceptible to AICD than primary cells, display augmented tyrosine phosphorylation in comparison with control cells. These studies outline a mechanism that may regulate gammadelta T cell activities in immune responses and limit the expansion of activated T cells repeatedly exposed to antigens.     

T helper lymphocytes rescue CTL from activation-induced cell death

T cell activation is characterized by a vast expansion of Ag-specific T cells followed by an equally extensive reduction in T cell numbers. This decline is due, in part, to activation-induced apoptosis of the responding T cells during repeated encounter with Ag. In the current study, we used solid-phase MHC class I/peptide monomers to cause activation-induced cell death (AICD) of previously activated CD8 T cells in an Ag-specific manner. AICD occurred rapidly and was mediated primarily by Fas-FasL interactions. Most interestingly, we observed that Th cells could provide survival signals to CTL significantly reducing the level of AICD. Both Th1 and Th2 subsets were capable of protecting CTL from AICD, and a major role for soluble factors in this protection was ruled out, as cell-to-cell contact was an essential component of this Th-mediated protection. Upon encounter with Ag-expressing tumor cells, CTL underwent significant apoptosis. However, in the presence of Th cells, the CTL not only were protected against death, but also had significantly greater lytic ability. In vivo tumor protection studies using peptide immunization showed that the activation of Ag-specific Th cells was crucial for optimal protection, but did not affect the magnitude of the CTL response in the lymphoid tissues. In this study, we examine the type of help that CD4 T cells may provide and propose a model of Th cell-CTL interaction that reduces CTL death. Our results show a novel role for Th cells in the maintenance of CTL responses.     

Regulation of activation-induced cell death of mature T-lymphocyte populations

Resting mature T lymphocytes are activated when triggered via their antigen-specific T-cell receptor (TCR) to elicit an appropriate immune response. In contrast, preactivated T cells may undergo activation-induced cell death (AICD) in response to the same signals. along with cell death induced by growth factor deprivation, AICD followed by the elimination of useless or potentially harmful cells preserves homeostasis, leads to the termination of cellular immune responses and ensures peripheral tolerance. T-cell apoptosis and AICD are controlled by survival cytokines such as interleukin-2 (IL-2) and by death factors such as tumor necrosis factor (TNF) and CD95 ligand (CD95L). In AICD-sensitive T cells, stimulation upregulates expression of one or several death factors, which in turn engage specific death receptors on the same or a neighboring cell. Death receptors are activated by oligomerization to rapidly assemble a number of adapter proteins and enzymes to result in an irreversible activation of proteases and nucleases that culminates in cell death by apoptosis. Increased knowledge of the molecular mechanisms that regulate AICD of lymphocytes opens new immunotherapeutic perspectives for the treatment of certain autoimmune diseases, and has implications in other areas such as transplantation medicine and AIDS research.     

CD133表位联合热休克蛋白佐剂疫苗引发抗白血病的免疫应答

肿瘤干细胞具有肿瘤形成、自我更新和抗化疗的特性,因而受到广泛关注和研究。CD133作为重要的肿瘤干细胞标志物与肿瘤细胞的干性与恶性密切相关。本研究筛选并且鉴定了CD133的3个H2-Kd限制性的细胞毒性T细胞 (CTL) 的表位,分别是CD133419–428、CD133702–710和CD133760–769。将重组热休克蛋白gp96为佐剂联合CD133表位制备表位疫苗,将疫苗免疫CD133+白血病移植的小鼠后引发抗肿瘤的特异性T细胞免疫应答。转输CD133表位特异的T细胞同样可以抑制小鼠淋巴瘤的生长。该研究为设计抗CD133+的白血病和其他肿瘤的表位疫苗提供了依据。     

Fas-mediated suicide of tumor-reactive T cells following activation by specific tumor: selective rescue by caspase inhibition

CD8+ T lymphocytes that specifically recognize tumor cells can be isolated and expanded ex vivo. While the lytic properties of these cells have been well described, their fate upon encounter with cognate tumor is not known. We performed reverse 51Cr release assays in which the lymphocyte effectors rather than the tumor cell targets were radioactively labeled. We found that melanoma tumor cells caused the apoptotic death of tumor-specific T cells only upon specific MHC class I-restricted recognition. This death was entirely blockable by the addition of an Ab directed against the Fas death receptor (APO-1, CD95). Contrary to the prevailing view that tumor cells cause the death of anti-tumor T cells by expressing Fas ligand (FasL), our data suggested that FasL was instead expressed by T lymphocytes upon activation. While the tumor cells did not express FasL by any measure (including RT-PCR), functional FasL (as well as FasL mRNA) was consistently found on activated anti-tumor T cells. We could successfully block the activation-induced cell death with z-VAD-fmk, a tripeptide inhibitor of IL-1 beta-converting enzyme homologues, or with anti-Fas mAbs. Most importantly, these interventions did not inhibit T cell recognition as measured by IFN-gamma release, nor did they adversely affect the specific lysis of tumor cell targets. These results imply that Fas-mediated activation-induced cell death could be a limiting factor in the in vivo efficacy of adoptive transfer of class I-restricted CD8+ T cells and provide a means of potentially enhancing their growth in vitro as well as their function in vivo.     

An HLA-A2.1-transgenic rabbit model to study immunity to papillomavirus infection

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.     

Synergistic effect of adoptive T-cell therapy and intratumoral interferon gamma-inducible protein-10 transgene expression in treatment of established tumors

The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have previously shown that transplanted SP2/0 myeloma tumors engineered to express lymphotactin invariably induced tumor regress mediated by SP2/0 tumor-specific T cells. Herein, we further systemically characterize these activated T cells and investigate their therapeutic efficacy, either alone or with the chemokine interferon gamma (IFN-gamma)-inducible protein-10 (IP-10) gene therapy. Following stimulation with SP2/0 cells, these activated T cells were CD25(+)FasL(+) L-selectin(low), expressed CXCR3 receptor and were chemoattracted by IP-10 in vitro. They comprised 64% CD4(+) Th1 and 36% CD8(+) Tc1 cells, both of which expressed IFN-gamma, perforin, and TNF-alpha, but not IL-4. The activated T cells were strongly cytotoxic for SP2/0 tumor cells (79% specific killing; E:T ratio, 50), mainly via perforin-mediated pathway. Cell tracking using labeled T cells confirmed that these T cells infiltrated better into the IP-10-expressing tumors than non-IP-10-expressing ones. In vivo, combined intratumoral IP-10 gene transfer and adoptive T-cell immunotherapy for well-established SP2/0 tumors eradicated the tumors in 7 of the 8 mice. Control or IP-10 adenoviral treatments by themselves neither alter the lethal outcome for tumor-bearing mice nor did T-cell therapy by itself, although the latter two treatments did slow its time-frame. Taken together, our data provide solid evidence of a potent synergy between adoptive T-cell therapy and IP-10 gene transfer into tumor tissues, which culminated in the eradication of well-established tumor masses.     

Activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs) by human dendritic cells infected with an attenuated influenza A virus expressing a CTL epitope derived from the HER-2/neu proto-oncogene

The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-gamma) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75(+) CD45RO(+) cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-alpha in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.     

Enhanced priming of antigen-specific CTLs in vivo by embryonic stem cell-derived dendritic cells expressing chemokine along with antigenic protein: application to antitumor vaccination

Dendritic cell (DC)-based immunotherapy is regarded as a promising means for anti-cancer therapy. The efficiency of T cell-priming in vivo by transferred DCs should depend on their encounter with T cells. In the present study, we attempted to improve the capacity of DCs to prime T cells in vivo by genetic modification to express chemokine with a T cell-attracting property. For genetic modification of DCs, we used a recently established method to generate DCs from mouse embryonic stem cells. We generated double-transfectant DCs expressing a chemokine along with a model Ag (OVA) by sequential transfection of embryonic stem cells, and then induced differentiation to DCs. We comparatively evaluated the effect of three kinds of chemokines; secondary lymphoid tissue chemokine (SLC), monokine induced by IFN-gamma (Mig), and lymphotactin (Lptn). All three types of double transfectant DCs primed OVA-specific CTLs in vivo more efficiently than did DCs expressing only OVA, and the coexpression of SLC or Lptn was more effective than that of Mig. Immunization with DCs expressing OVA plus SLC or Mig provided protection from OVA-expressing tumor cells more potently than did immunization with OVA alone, and SLC was more effective than Mig. In contrast, coexpression of Lptn gave no additive effect on protection from the tumor. Collectively, among the three chemokines, expression of SLC was the most effective in enhancing antitumor immunity by transferred DCs in vivo. The findings provide useful information for the development of a potent DC-based cellular immunotherapy.     

Generation of CD8(+) T cell memory in response to low, high, and excessive levels of epitope

The magnitude of a virus-specific memory CTL population can dramatically influence the outcome of secondary infections, yet little is known about the determinants of memory size. We investigated the impact of epitope levels on CTL memory generation by using a recombinant vaccinia virus system that allows for a broad range of epitope expression with the same infectious dose of virus. The size of the memory pool was examined using MHC class I/peptide tetramer staining and IFN-gamma ELISPOT analysis following priming with viruses expressing low, high, or excessive epitope levels. The size of the epitope-specific CD8(+) T cell memory population correlates with Ag dose at the low and high levels of epitope expression. However, at excessive epitope levels, the number of functional, IFN-gamma-producing, epitope-specific memory cells is significantly reduced compared with the number of tetramer(+) cells. These results demonstrate that the level of epitope expressed during an acute viral infection in vivo can dramatically influence CTL memory size. Furthermore, when epitope is overexpressed, the quality of the response can be adversely affected. Therefore, epitope expression level is an important consideration when developing approaches to optimize CTL memory induction.     

Human macrophages and dendritic cells can equally present MART-1 antigen to CD8(+) T cells after phagocytosis of gamma-irradiated melanoma cells

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8⁺ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8⁺ T cell clone. Confocal microscopy with Alexa Fluor®⁶⁴⁷-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8⁺ T cell cross-presentation thereafter.     

Activation of antigen-specific CD8 T cells results in minimal killing of bystander bacteria

Memory CD8 T cells play a critical role in protective immunity against intracellular pathogens. In addition to their ability to specifically recognize and lyse infected targets, activated CD8 T cells secrete cytokines that induce phagocytic cells to engulf and kill bacterial pathogens. In this study, we asked whether activation of Ag-specific CD8 T cells results in nonspecific killing of bystander bacteria during a mixed infection. Mice with epitope-specific memory CD8 T cells were coinfected with two isogenic strains of recombinant Listeria monocytogenes that differ in the cognate epitope. Recall responses by epitope-specific CD8 T cells rapidly inhibited the growth of epitope-bearing bacteria, impeding the course of infection within 6 h after challenge. This rapid inhibition was highly specific and did not affect the growth of coinfecting bacteria without the epitope. CTL recall did not enhance activation of innate immune cells, as evidenced by the absence of inducible NO synthase production in infectious foci. Our observations demonstrate the remarkable specificity of the bactericidal mechanisms of CTL and reveal the possibility for escape mutants to prevail in the hostile environment of a specific immune response. This implication has a bearing on subunit vaccine design strategies and understanding failure of immunization against bacterial infection.     

FADD deficiency sensitises Jurkat T cells to TNF-alpha-dependent necrosis during activation-induced cell death

Activation-induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas-associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD(-/-)) Jurkat T cells undergo AICD to a similar extent as wild-type cells. AICD in wild-type Jurkat T cells is via apoptosis, whereas it is non-apoptotic in FADD(-/-) cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase-8 or -3 activation, suggesting that these cells die by necrosis. Wild-type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF-alpha. In contrast, FADD(-/-) Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF-alpha. We showed that neutralising anti-TNF-alpha blocked AICD as well as TNF-alpha-induced necrosis in FADD(-/-) Jurkat T cells. Furthermore, down regulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF-alpha signalling, markedly inhibited AICD in FADD(-/-) Jurkat T cells. In addition, caspase-8-deficient Jurkat T cells are resistant to Fas- and TNF-alpha-induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase-8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF-alpha-dependent necrosis during AICD.     

CD8+ cytotoxic T lymphocytes in cancer immunotherapy: A review

CD8⁺ cytotoxic T lymphocytes (CTLs) are preferred immune cells for targeting cancer. During cancer progression, CTLs encounter dysfunction and exhaustion due to immunerelated tolerance and immunosuppression within the tumor microenvironment (TME), with all favor adaptive immune-resistance. Cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and regulatory T cells (Tregs) could make immunologic barriers against CD8 ⁺ T cell-mediated antitumor immune responses. Thus, CD8 ⁺ T cells are needed to be primed and activated toward effector CTLs in a process called tumor immunity cycle for making durable and efficient antitumor immune responses. The CD8 ⁺ T cell priming is directed essentially as a corroboration work between cells of innate immunity including dendritic cells (DCs) and natural killer (NK) cells with CD4 ⁺ T cells in adoptive immunity. Upon activation, effector CTLs infiltrate to the core or invading site of the tumor (so-called infiltrated–inflamed [I–I] TME) and take essential roles for killing cancer cells. Exogenous reactivation and/or priming of CD8 ⁺ T cells can be possible using rational immunotherapy strategies. The increase of the ratio for costimulatory to coinhibitory mediators using immune checkpoint blockade (ICB) approach. Programmed death-1 receptor (PD-1)–ligand (PD-L1) and CTL-associated antigen 4 (CTLA-4) are checkpoint receptors that can be targeted for relieving exhaustion of CD8 ⁺ T cells and renewing their priming, respectively, and thereby eliminating antigen-expressing cancer cells. Due to a diverse relation between CTLs with Tregs, the Treg activity could be dampened for increasing the number and rescuing the functional potential of CTLs to induce immunosensitivity of cancer cells.     

A HLA-DQ5 restricted Melan-A/MART-1 epitope presented by melanoma tumor cells to CD4+ T lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.