CD4+ T cells are able to promote tumor growth through inhibition of tumor-specific CD8+ T-cell responses in tumor-bearing hosts

Modulation of the immune response by established tumors may contribute to the limited success of therapeutic vaccination for the treatment of cancer compared with vaccination in a preventive setting. We analyzed the contribution of the CD4+ T-cell population to the induction or suppression of tumor-specific CD8+ T cells in a tumor model in which eradication of tumors crucially depends on CD8+ T cell-mediated immunity. Vaccine-mediated induction of protective antitumor immunity in the preventive setting (i.e., before tumor challenge) was CD4+ T cell dependent because depletion of this T-cell subset prevented CD8+ T-cell induction. In contrast, depletion of CD4+ cells in mice bearing established E1A+ tumors empowered the mice to raise strong CD8+ T-cell immunity capable of tumor eradication without the need for tumor-specific vaccination. Spontaneous eradication of tumors, which had initially grown out, was similarly observed in MHC class II-deficient mice, supporting the notion that the tumor-bearing mice harbor a class II MHC-restricted CD4+ T-cell subset capable of suppressing a tumor-specific CD8+ T-cell immune response. The deleterious effects of the presence of CD4+ T cells in tumor-bearing hosts could be overcome by CD40-triggering or injection of CpG. Together these results show that CD4+ T cells with a suppressive activity are rapidly induced following tumor development and that their suppressive effect can be overcome by agents that activate professional antigen-presenting cells. These observations are important for the development of immune interventions aiming at treatment of cancer.     

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8⁺ T-cell responses against transgene expressing cells, we created WAP-T_NP mice, in which the transgene additionally codes for the NP_118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-T_NP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-Ag_NP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-Ag_NP in WAP-T_NP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-T_NP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-T_NP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-T_NP mice. LCMV infection of WAP-T_NP mice induced a strong, LCMV NP-epitope specific CD8⁺ T-cell response, which was able to specifically eliminate T-Ag_NP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-T_NP tumor mice contained LCMV NP-epitope specific CD8⁺ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-T_NP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-T_NP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.     

Immunization with tumor-derived ER chaperone grp170 elicits tumor-specific CD8+ T-cell responses and reduces pulmonary metastatic disease

Glucose-regulated protein (grp) 170 is a molecular chaperone localized in endoplasmic reticulum (ER), which has been demonstrated to interact with the peptides translocated by transporter associated with antigen presentation (TAP). In our study, we have evaluated the therapeutic efficacy of tumor-derived grp170 against the highly metastatic and poorly immunogenic murine melanoma B16F10. Immunization of mice with grp170 preparations from autologous tumor significantly delayed progression of the primary cancer and reduced established pulmonary metastases, which was associated with the prolonged survival of metastases-bearing mice. However, grp170 from normal liver or antigenically distinct tumor failed to exhibit therapeutic effect. In addition, tumor-derived grp170 elicited a potent cytotoxic T-lymphocyte response specific for B16F10 tumor, which correlates with in vivo protective effects. Adoptive transfer of splenocytes obtained from B16F10-grp170-primed animals remarkably suppressed pulmonary metastases. Depletion of either CD4(+) or CD8(+) T cells in priming phase significantly abrogated the tumor immunity induced by the B16F10-grp170. However, the vaccine activity was intact when CD4(+), not CD8(+), T cells were depleted in effector phase. It suggests that CD4(+) T helper cells play an important role in the generation of effective antitumor response, whereas CD8(+) T cells are predominantly involved in direct killing of tumor cells. These observations have strong clinical implications for using tumor-derived grp170 as a therapeutic vaccine against metastatic diseases.     

Tumor-induced interleukin 10 suppresses the ability of splenic dendritic cells to stimulate CD4 and CD8 T-cell responses

Dendritic cell (DC) maturation and function are influenced by the surrounding cytokine milieu. We demonstrate tumor-associated suppression of DCs in stimulating allogeneic and tumor-specific CTL and type 1 (IFN-gamma-producing) responses in both CD4- and CD8-positive T cells. DCs from MB49-bearing female mice fail to stimulate proliferative and IFN-gamma-producing responses in allogeneic mixed lymphocyte cultures. MB49 also inhibited DC function in stimulating type 1 responses against our tumor-specific antigen, the male antigen, HY. DCs from MB49-bearing male mice were unable to restimulate effective HY-specific CTLs or IFN-gamma. Tumor-induced interleukin (IL) 10 was found to be specifically responsible for DC dysfunction in response to antigenic driven maturation. This was demonstrated by restoration of DC function in splenic DCs from MB49-bearing female IL-10 knockout mice (HY disparity), whereas not in MB49-bearing male IL-10 knockout mice (no HY disparity). Finally, any tumor-induced systemic inhibitory effect on bone marrow precursors could be overcome by generation of bone marrow-derived DCs ex vivo. These bone marrow-derived DCs derived from MB49-bearing B6 mice were capable of inducing control levels of proliferation in allogeneic mixed lymphocyte reactions and a type 1 (IFN-gamma) cytokine profile. The BM-DCs were also capable of restimulating HY-specific CTL and IFN-gamma production. These studies reveal the tumor-associated in vivo effects of IL-10 inhibition on DC function in eliciting a type 1 immune response in both allogeneic and tumor-specific responses.     

Co-immunization with L-Myc enhances CD8+ or CD103+ DCs mediated tumor-specific multi-functional CD8+ T cell responses

Renal carcinoma shows a high risk of invasion and metastasis without effective treatment. Herein, we developed a chitosan (CS) nanoparticle-mediated DNA vaccine containing an activated factor L-Myc and a tumor-specific antigen CAIX for renal carcinoma treatment. The subcutaneous tumor models were intramuscularly immunized with CS-pL-Myc/pCAIX or control vaccine, respectively. Compared with single immunization group, the tumor growth was significantly suppressed in CS-pL-Myc/pCAIX co-immunization group. The increased proportion and mature of CD11c⁺ DCs, CD8⁺CD11c⁺ DCs and CD103⁺CD11c⁺ DCs were observed in the splenocytes from CS-pL-Myc/pCAIX co-immunized mice. Furthermore, the enhanced antigen-specific CD8⁺ T lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and multi-functional CD8⁺ T cell induction were detected in CS-pL-Myc/pCAIX co-immunization group compared with CS-pCAIX immunization group. Of note, the depletion of CD8 T cells resulted in the reduction of CD8⁺ T cells or CD8⁺CD11c⁺ DCs and the loss of anti-tumor efficacy induced by CS-pL-Myc/pCAIX vaccine, suggesting the therapeutic efficacy of the vaccine was required for CD8⁺ DCs and CD103⁺ DCs mediated CD8⁺ T cells responses. Likewise, CS-pL-Myc/pCAIX co-immunization also significantly inhibited the lung metastasis of renal carcinoma models accompanied with the increased induction of multi-functional CD8⁺ T cell responses. Therefore, these results indicated that CS-pL-Myc/pCAIX vaccine could effectively induce CD8⁺ DCs and CD103⁺ DCs mediated tumor-specific multi-functional CD8⁺ T cell responses and exert the anti-tumor efficacy. This vaccine strategy offers a potential and promising approach for solid or metastatic tumor treatment.     

Local administration of PF-3512676 CpG-B instigates tumor-specific CD8+ T-cell reactivity in melanoma patients

PURPOSE: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. EXPERIMENTAL DESIGN: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. RESULTS: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. CONCLUSIONS: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.     

三级淋巴结构在肿瘤免疫治疗中的研究进展

三级淋巴结构（tertiary lymphoid structures,TLS）是指在病理生理环境下,淋巴细胞在非淋巴器官内聚集形成的有组织的异位淋巴结构。成熟的TLS包括被T细胞包围的B细胞区域和生发中心,普遍存在于多个瘤种中,是肿瘤细胞与免疫细胞相互作用的重要场所,与患者预后密切相关。因此,诱导形成TLS,使T细胞和B细胞发育分化为能够识别杀伤肿瘤细胞的效应细胞和记忆细胞,可以介导抗肿瘤免疫反应。本综述主要介绍了TLS的组成成分和形成过程,总结归纳现有的检测手段,强调了TLS不同成熟度、位置和内部组成等特征对肿瘤免疫疗效的影响和预测价值,并探讨诱导TLS作为肿瘤治疗手段的应用潜能。     

Ex vivo detectable human CD8 T-cell responses to cancer-testis antigens

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.     

Simultaneous generation of CD8+ and CD4+ melanoma-reactive T cells by retroviral-mediated transfer of a single T-cell receptor

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen-reactive T cells for transfer into cancer patients. Genes encoding tumor antigen-specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor-specific for a class I MHC (HLA-A2)-restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4(+) tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor-transduced T cells secreted various cytokines when cocultured with tyrosinase peptide-loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8(+) and CD4(+) transduced T cells could recognize HLA-A2(+) melanoma cells, giving us the possibility of engineering class I MHC-restricted effector and T helper cells against melanoma. The ability to confer class I MHC-restricted tumor cell recognition to CD4(+) T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer-based immunotherapy.     

CD4+ T cells inhibit the neu-specific CD8+ T-cell exhaustion during the priming phase of immune responses against breast cancer

Studies conducted in animal model of infectious diseases or H-Y antigen model suggest a crucial role for CD4+ T cells in providing help for CD8+ T-cell memory responses. This concept suggests that inclusion of T helper epitopes in vaccine formulation will result in improved CD8+ T-cell responses. Although this concept has been applied to cancer vaccine design, the role of CD4+ T cells in the memory differentiation of CD8+ T cells and retention of their anti-tumor function have never been tested in breast cancer model. Using the FVB mouse model of neu-positive breast carcinoma we report for the first time that helpless T cells showed cytostatic or tumor inhibitory effects during primary tumor challenge whereas, helped T cells showed cytotoxic effects and resulted in complete tumor rejection. Such differential effects, in vivo, were associated with higher frequency of CD8+PD-L1+ and CD8+PD-1+ T cells in animals harboring helpless T cells as well as higher titer of IL-2 in the sera of animals harboring helped T cells. However, depletion of CD4+ T cells did not alter the ability of neu-specific CD8+ T cells to differentiate into memory cells and to retain their effector function against the tumor during recall challenge. These results suggest the inhibitory role of CD4+ T cells on CD8+ T-cell exhaustion without substantial effects on the differentiation of memory T cells during priming phase of the immune responses against breast cancer.     

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor-associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte-derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA-derived CEA and MUC1 antigens and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4(+) and CD8(+) T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN-gamma. More importantly, coculture of fusion cells with patient-derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen-specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1-positive and HLA partially matched target cells. Antigen-specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA- and MUC1-specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen-specific CTL responses. Our report demonstrates the simultaneous induction of CRCA-specific CTL responses restricted by HLA-A2 and -A24.     

Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity

Immunization of mice with plasmids encoding xenogeneic orthologues of tumor differentiation antigens can break immune ignorance and tolerance to self and induce protective tumor immunity. We sought to improve on this strategy by combining xenogeneic DNA vaccination with an agonist anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) monoclonal antibody (mAb), DTA-1, which has been shown previously both to costimulate activated effector CD4(+) and CD8(+) T cells and to inhibit the suppressive activity of CD4(+)CD25(+) regulatory T cells. We found that ligation of GITR with DTA-1 just before the second, but not the first, of 3 weekly DNA immunizations enhanced primary CD8(+) T-cell responses against the melanoma differentiation antigens gp100 and tyrosinase-related protein 2/dopachrome tautomerase and increased protection from a lethal challenge with B16 melanoma. This improved tumor immunity was associated with a modest increase in focal autoimmunity, manifested as autoimmune hypopigmentation. DTA-1 administration on this schedule also led to prolonged persistence of the antigen-specific CD8(+) T cells as well as to an enhanced recall CD8(+) T-cell response to a booster vaccination given 4 weeks after the primary immunization series. Giving the anti-GITR mAb both during primary immunization and at the time of booster vaccination increased the recall response even further. Finally, this effect on vaccine-induced CD8(+) T-cell responses was partially independent of CD4(+) T cells (both helper and regulatory), consistent with a direct costimulatory effect on the effector CD8(+) cells themselves.     

Importance of tumor-specific cytotoxic CD8+ T-cells in eradication of a large subcutaneous MOPC-315 tumor following low-dose melphalan therapy

We have previously demonstrated that depletion of CD8+ T-cells by the use of a monoclonal anti-Lyt-2.2 antibody abolishes the curative effectiveness of low-dose melphalan (L-phenylalanine mustard; L-PAM) therapy for BALB/c mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor and extensive metastases (Mokyr et al., Cancer Res., 49: 4597-4606, 1989). Here we show that as a consequence of low-dose L-PAM therapy, CD8+ T-cells accumulate in the s.c. tumor nodules of MOPC-315 tumor bearers. Specifically, an 80-fold increase in the number of CD8+ T-cells was seen within 5 days after the chemotherapy. Treatment of MOPC-315 tumor bearers with low-dose L-PAM in conjunction with monoclonal anti-Thy-1.2 or anti-Lyt-2.2 antibody, in contrast to treatment with monoclonal anti-L3T4 antibody, prevented the appearance of the massive CD8+ T-cell infiltrate in the s.c. tumor nodules. Fresh CD8+ T-cells derived from s.c. MOPC-315 tumor nodules that were regressing as a consequence of low-dose L-PAM therapy exhibited a potent direct lytic activity against the MOPC-315 plasmacytoma in a short-term in vitro assay. The specificity of the lytic activity exhibited by the CD8+ T-cells was illustrated not only by the inability of the CD8+ T-cells to lyse two antigenically unrelated thymomas (the WEHI 22.1 and the EL-4) and a natural killer-sensitive lymphoma (the YAC-1), but also by their relatively weak lytic activity against an antigenically related plasmacytoma (the MOPC-104E). Thus, CD8+ T-cells that infiltrate the s.c. tumor nodules of MOPC-315 tumor bearers following low-dose L-PAM therapy most likely exploit a CTL-type lytic mechanism to eradicate at least part of the large tumor burden not eliminated by the direct antitumor effects of the drug.     

Correlated ultrastructural and immunological findings in CD8+ and CD4+ peripheral T-cell lymphomas

Ultrastructural features of CD8+ and CD4+ peripheral T-cell lymphomas were studied and the results revealed distinct traits correlated with phenotype. CD8+ peripheral T-cell lymphomas were characterized by a rich organular pattern with a well-developed Golgi apparatus, dense granules, numerous and atypical giant mitochondria. CD4+ peripheral T-cell lymphomas were characterized by wide cytoplasmic areas devoid of organelles. Since similar ultrastructural features differentiated normal and pathological CD8+ and CD4+ peripheral T-lymphocytes, we conclude that under both normal and pathological conditions the expression on the part of the peripheral T-lymphocytes of CD8+ and CD4+ phenotypes is associated to morphological features which distinguish the two subsets.     

T-cell prolymphocytic leukemia with an unusual phenotype CD4+ CD8+

A patient with T-cell prolymphocytic leukemia (T-PLL) is described. The outcome was poor, with death 8 months after diagnosis, despite several therapeutic interventions. The cells carried both CD4 and CD8 epitopes, but other thymocytic markers were absent. The spleen showed infiltration of CD4+ CD8+ prolymphocytes in the red pulp and in T-cell-dependent areas of the white pulp. Immunologic function studies revealed proliferation after stimulation with mitogens and even several antigens. However, in the mixed lymphocyte culture the T-PLL cells did not proliferate. Cytotoxic T-cells could not be induced. In T-non-T recombination experiments neither helper nor suppressor cell function was found for pokeweed mitogen-dependent plasmablast generation of normal B-cells. Cytogenetically, many abnormalities were found. Among them, 14q+; absence of chromosomes 8, 11, and 22; and the presence of large marker chromosomes and fragments.     

CD8 T-cell recognition of human 5T4 oncofetal antigen

The 5T4 oncofetal antigen is expressed by a wide variety of human carcinomas, including colorectal, ovarian and gastric carcinomas. The restricted expression of 5T4 on tumor tissues as well as its implication in tumor progression and bad prognosis makes 5T4 a promising new candidate for immunotherapy. An MVA vaccine encoding 5T4 antigen has been successfully evaluated in preclinical studies in a murine tumor model. Here, we report the generation of human CD8 T cells specific for the 5T4 antigen by stimulation with autologous monocyte derived DC infected with a replication defective adenovirus encoding the 5T4 cDNA (Ad5T4). Analysis of several donors confirms a repertoire of such CD8 responses. In a parallel approach, incorporating the results of proteasome-mediated digestion of 5T4 derived 35-mer peptides and the potential high affinity epitopes predicted by a computer-based algorithm, we identified 8 putative HLA-A*0201-presented CD8 MHC class I epitopes of 5T4 antigen. Two of these generated specific CD8 T cells after restimulation with peptide loaded autologous DC and assay by cytotoxicity and IFN gamma ELISPOT. Moreover these particular peptide generated T cells recognized naturally 5T4 positive tumor cells only if they expressed HLA-A*0201 as judged by IFN gamma ELISPOT or ELISA. Also, HLA-A*0201 CD8 T cells recognized these peptides in a DC-Ad5T4 polyclonal response. In conclusion, there is a repertoire of CD8 T cell recognition of 5T4 in normal human donors and some candidate HLA-A*0201 epitopes have been identified.     

Spontaneous regression of grade 3 vulvar intraepithelial neoplasia associated with human papillomavirus-16-specific CD4(+) and CD8(+) T-cell responses

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in six patients affected with grade 3 vulvar intraepithelial neoplasia (VIN3, also known as bowenoid papulosis). Five of the patients presented with a chronic and persistent disease that relapsed after destructive treatments. They showed no detectable anti-HPV blood T-cell responses and no T-cell intraepidermal vulvar infiltrate containing both CD4+ and CD8+ lymphocytes. The last patient had a complete clearance of viral lesions, 8 months after disease onset and 2 months after electrocoagulation of <50% of the VIN3 lesions. She showed high frequency anti-E6 and anti-E7 effector blood T cells by ex vivo ELISpot-IFNgamma assay before clinical regression. Immunohistochemical study of her vulvar biopsy revealed a marked dermal infiltrate containing a majority of CD4+ T lymphocytes and an epidermal infiltrate made up of both CD4(+) and CD8(+) T cells. This seems to be the first evidence of an association between spontaneous regression of VIN3 lesions and HPV-specific T-cell responses detectable in the blood. Hence, an increase of HPV-specific effector T lymphocyte responses by vaccine-based therapeutic strategies might be useful to clear the lesions in bowenoid papulosis disease.     

The apoptosis inhibitor protein survivin induces tumor-specific CD8+ and CD4+ T cells in colorectal cancer patients

The identification of tumor-associated antigens expressed by colorectal carcinoma remains one of the major goals for designing novel immunological treatments for this tumor. By using a reverse-immunology approach, we show here that the inhibitor of apoptosis protein, survivin, is immunogenic in colorectal cancer patients. In particular, we found that survivin elicited CD8(+) T cell-mediated responses in peripheral blood or in tumor-associated lymphocytes from patients at different disease stage. Colorectal carcinoma cells were recognized by survivin-specific T lymphocytes, and the survivin-specific, class-I HLA-restricted T lymphocytes were fully activated and released interleukin-2 in response to HLA/survivin-peptide complexes expressed by tumor cells. In addition to CD8-mediated responses, survivin specifically stimulated CD4+ T-cell reactivity in peripheral blood lymphocytes from the same patients, thus suggesting that a complete activation of the immune system may occur in response to this antiapoptotic protein. These findings indicate that survivin could be considered a valuable tumor-associated antigen for immune-based clinical approaches in colorectal cancer.