MHC II lung cancer vaccines prime and boost tumor‐specific CD4+ T cells that cross‐react with multiple histologic subtypes of nonsmall cell lung cancer cells

Nonsmall cell lung cancer (NSCLC) is the major cause of lung cancer‐related deaths in the United States. We are developing cell‐based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into 3 histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens, and vaccines that induce immune reactivity against 1 subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor‐specific CD4⁺ T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8⁺ T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous or large cell carcinomas each activate CD4⁺ T cells that cross‐react with the other NSCLC subtypes and do not react with HLA‐DR‐matched normal lung fibroblasts or other HLA‐DR‐matched nonlung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7 or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA‐DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA‐DR allele activate NSCLC‐specific CD4⁺ T cells that react with the 3 major classes of NSCLC, and the antigens recognized by the activated T cells are presented by several common HLA‐DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients.     

Cyclophosphamide treatment induces rejection of established P815 mastocytoma by enhancing CD4 priming and intratumoral infiltration of P1E/H‐2Kd‐specific CD8+ T cells

There is increasing evidence that the effect of chemotherapy on tumor growth is not cell autonomous but relies on the immune system. The objective of this study was therefore to decipher the cellular and molecular mechanisms underlying the role of innate and adaptive immunity in chemotherapy‐induced tumor rejection. Treatment of DBA/2 mice bearing P815 mastocytoma with cyclophosphamide induced rejection and long‐term protection in a CD4‐ and CD8‐dependent manner. A population of inflammatory‐type dendritic cells was dramatically expanded in the lymph nodes of mice that rejected the tumor and correlated with CD4‐dependent infiltration, in tumor bed, of tumor‐specific CD8⁺ T lymphocytes. Our data point to a major role of CD4⁺ T cells in inducing chemokine expression in the tumor, provoking migration of tumor‐specific CXCR3⁺ CD8⁺ T lymphocytes. Importantly, the analysis of CD8⁺ T cells specific to P1A/H‐2L^d and P1E/H‐2K^d revealed that cyclophosphamide altered the P815‐specific CD8 T repertoire by amplifying the response specific to the mutated P1E antigen.     

Blood monocytes sample MelanA/MART1 antigen for long‐lasting cross‐presentation to CD8+ T cells after differentiation into dendritic cells

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross‐presenting antigens to CD8⁺ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen‐specific CD8⁺ T cell clones after 5 days of differentiation into monocytes‐derived dendritic cells (MoDCs). Tagged‐long peptides can be visualized in electron‐dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14⁺ monocytes from two patients effectively activate a MelanA‐specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor‐specific immune response and should be taken into account for cancer immunotherapy.     

CD8+Foxp3+ tumor infiltrating lymphocytes accumulate in the context of an effective anti‐tumor response

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3‐expressing CD8⁺ T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti‐tumor response. CD8⁺Foxp3⁺ T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM‐CSF secreting HER‐2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen‐specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3⁺ and Foxp3^? CD8⁺ T cells have similar IFN‐γ production and antigen‐specific degranulation after stimulation with RNEU_420–429, the immunodominant HER‐2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU_(420–429)‐specific effector T cells into neu‐N mice (a model that results in immune tolerance to neu), confirm that CD8⁺Foxp3⁺ T cells are present in tumors only if there is an existing pool of tumor‐rejecting effector T cells. CD8⁺Foxp3⁺ TILs mark the presence of tumor‐rejecting antigen‐specific T cells and their accumulation serves as a marker for an effective T cell response.     

Regulatory T cells in tumor immunity

Recent studies have revealed that Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self‐tolerance, play critical roles for the control of antitumor immune responses. For example, a large number of Foxp3⁺Tregs infiltrate into tumors, and systemic removal of Foxp3⁺Tregs enhances natural as well as vaccine‐induced antitumor T‐cell responses. Tregs are recruited to tumor tissues via chemokines, such as CCL22 binding to CCR4 expressed by Tregs. They appear to expand and become activated in tumor tissues and in the draining lymph nodes by recognizing tumor‐associated antigens as well as normal self‐antigen expressed by tumor cells. These results indicate that cancer vaccines targeting tumor‐associated self‐antigens may potentially expand/activate Tregs and hamper effective antitumor immune responses, and that tumor immunity can therefore be enhanced by depleting Tregs, attenuating Treg suppressive function, or rendering effector T cells refractory to Treg‐mediated suppression. Recent attempts have indeed demonstrated that combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity and are more effective than a single monoclonal antibody therapy. Combination therapy targeting a variety of molecules expressed in antigen‐presenting cells, effector T cells and Tregs is envisaged to be a promising anticancer immunotherapy.     

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high‐grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19^?/? mice inoculated with CD19⁺ B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19^?/? mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19‐derived peptides. The majority of mice exhibited a CD4⁺ T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27‐specific CD4⁺ T cell line protected CD19^?/? mice against challenge with CD19⁺ lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19‐specific CD4⁺ T cells for adoptive T cell therapy of B cell lymphomas.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

In vivo antitumor function of tumor antigen‐specific CTLs generated in the presence of OX40 co‐stimulation in vitro

Adoptive cell transfer (ACT) is an emerging and promising cancer immunotherapy that has been improved through various approaches. Here, we described the distinctive characteristics and functions of tumor Ag‐specific effector CD8⁺ T‐cells, co‐cultured with a tumor‐specific peptide and a stimulatory anti‐OX40 antibody, before being used for ACT therapy in tumor‐bearing mouse recipients. Splenic T‐cells were obtained from wild‐type FVB/N mice that had been injected with a HER2/neu (neu)‐expressing tumor and a neu‐vaccine. The cells were then incubated for 7 days in vitro with a major histocompatibility complex (MHC) class I peptide derived from neu, in the presence or absence of an agonistic anti‐OX40 monoclonal antibody, before CD8⁺ T cells were isolated for use in ACT therapy. The proliferative ability of OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells in vitro was less than that of non‐OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells, but they expressed significantly more early T‐cell differentiation markers, such as CD27, CD62L and CCR7, and significantly higher levels of Bcl‐2, an anti‐apoptotic protein. These OX40‐driven tumor Ag‐specific effector CD8⁺ T‐cells, when transferred into tumor‐bearing recipients, demonstrated potent proliferation capability and successfully eradicated the established tumor. In addition, these cells exhibited long‐term antitumor function, and appeared to be established as memory T‐cells. Our findings suggest a possible in vitro approach for improving the efficacy of ACT, which is simple, requires only a small amount of modulator, and can potentially avoid several toxicities associated with co‐stimulation in vivo.     

Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

CD169‐positive sinus macrophages in the lymph nodes determine bladder cancer prognosis

CD169⁺ macrophages are suggested to play a pivotal role in establishing anti‐tumor immunity. They capture dead tumor cell‐associated antigens and transfer their information to lymphocsytes, including CD8⁺ T cells, which is important for successful tumor suppression. This study aimed to determine the prognostic significance of CD169⁺ macrophages residing in the tumor‐draining lymph nodes from cases of bladder cancer. In this retrospective study, 44 bladder cancer patients who received radical cystectomy were examined. The abundance of CD169⁺ macrophages in the regional lymph nodes and the number of CD8⁺ T cells in the primary tumor were investigated by immunohistochemistry. A CD169 score was calculated based on the intensity of CD169 staining and the proportion of CD169⁺ macrophages, and the scores were compared to the patients’ clinicopathological parameters. A high CD169 score was significantly associated with low T stage and with a high number of CD8⁺ T cells infiltrating into the tumor. The group with high CD169 expression had significantly longer cancer‐specific survival than the group with low CD169 expression (5‐year cancer‐specific survival rate: 83.3% vs 31.3%). In a multivariate analysis, the CD169 score was identified as a strong and independent favorable prognostic factor for cancer‐specific survival. Our findings suggest that CD169⁺ macrophages in the lymph nodes enhance anti‐tumor immunity by expanding CD8⁺ T cells in bladder cancer. The CD169 score may serve as a novel marker for the evaluation of bladder cancer prognosis.     

NY‐CO‐58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer

NY‐CO‐58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY‐CO‐58/KIF2C by RT‐PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full‐length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY‐CO‐58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki‐67 expression (r = 0.69; p = 0.0003). Investigating NY‐CO‐58/KIF2C‐specific T cell responses, CD8⁺ T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4⁺ T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN‐γ and TNF‐α. Depletion of CD4⁺CD25⁺ T cells before stimulation significantly increased the intensity of the preexisting response. NY‐CO‐58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY‐CO‐58/KIF2C was able to induce spontaneous CD4⁺ T cell responses of the Th1‐type, which were tightly controlled by peripheral T regulatory cells.     

Immune landscape of vulvar cancer patients treated with surgery and adjuvant radiotherapy revealed restricted T cell functionality and increased IL-17 expression associated with cancer relapse

For vulvar cancers, radiotherapy is targeting cancer cells, but also affects the host immune system. As this may affect treatment outcome, in this prospective study, we characterized the individual T cell immune milieu induced by surgery and adjuvant radio +/− chemotherapy (aRT) systemically in the blood of vulvar cancer patients and found increased frequencies of Interleukin (IL)-17-producing CD4⁺ and CD8⁺ T cells after aRT while frequencies of Th1 and perforin-producing CD8⁺ killer cells were strongly diminished. Phenotypic characterization revealed enhanced expression of the ectonucleotidase CD39 on Th17 and Tc17 cells as well as CD8⁺ perforin⁺ cells after aRT. Furthermore, the aRT cohort exhibited increased proportions of Programmed Cell Death Protein (PD-1) expressing cells among Th1 and CD8⁺ perforin⁺ cells, but not among Th17 and Tc17 cells. High post-therapeutic levels of Th17 and Tc17 cells and low proportions of Th1 and CD8⁺ perforin⁺ cells expressing PD-1 was associated with reduced recurrence free survival on follow-up. In conclusion, our study defines individual therapy-induced changes in the cellular immune milieu of patients and their association with cancer relapse. Our results may help to explain differences in the individual courses of disease of vulvar cancer patients and suggest PD-1 and IL-17 as targets for immunotherapy in vulvar cancer.     

Chemotherapy‐induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy‐induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high‐mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC‐1, or PSA‐specific CD8⁺ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen‐processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT‐blocking peptide. A docetaxel‐resistant cell line was selected (MDR‐1⁺, CD133⁺) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.     

Adenovirus vector‐based prime‐boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV‐infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus‐specific antigens for vaccine‐induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV‐specific CD8⁺ T cells producing IFN‐γ and TNF‐α to the cervicovaginal tract. Importantly, the cervicovaginal CD8⁺ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue‐resident memory CD8⁺ T cells. This prime‐boost strategy targeting heterologous locations also induced circulating HPV‐specific CD8⁺ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.     

CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome

The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4⁺ and CD8⁺ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4⁺ and CD8⁺ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4⁺ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4⁺ and CD8⁺ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8⁺ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4⁺ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4⁺ and CD8⁺ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Trypanosoma cruzi extracts elicit protective immune response against chemically induced colon and mammary cancers

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, has anticancer effects mediated, at least in part, by parasite‐derived products which inhibit growth of tumor cells. We investigated whether immunity to T. cruzi antigens could induce antitumor activity, using two rat models which reproduce human carcinogenesis: colon cancer induced by 1,2‐dimethylhydrazine (DMH), and mammary cancer induced by N‐nitroso‐N‐methylurea (NMU). We found that vaccination with T. cruzi epimastigote lysates strongly inhibits tumor development in both animal models. Rats immunized with T. cruzi antigens induce activation of both CD4⁺ and CD8⁺ T cells and splenocytes from these animals showed higher cytotoxic responses against tumors as compared to rats receiving adjuvant alone. Tumor‐associated immune responses included increasing number of CD11b/c⁺ His48^? MHC II⁺ cells corresponding to macrophages and/or dendritic cells, which exhibited augmented NADPH‐oxidase activity. We also found that T. cruzi lysate vaccination developed antibodies specific for colon and mammary rat cancer cells, which were capable of mediating antibody‐dependent cellular cytotoxicity (ADCC) in vitro. Anti‐T. cruzi antibodies cross‐reacted with human colon and breast cancer cell lines and recognized 41/60 (68%) colon cancer and 38/63 (60%) breast cancer samples in a series of 123 human tumors. Our results suggest that T. cruzi antigens can evoke an integrated antitumor response involving both the cellular and humoral components of the immune response and provide novel insights into the understanding of the intricate relationship between parasite infection and tumor growth.     

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3⁺ T cells, and relatively high levels of BDCA2⁺ and FOXP3⁺ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3⁺ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c⁺ myeloid dendritic cells, and high numbers of FOXP3⁺ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.     

Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin

BACKGROUND:

Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44⁺ ovarian cancer stem cells, we found a high expression of the genes encoding for claudin‐4. Because this tight junction protein is the natural high‐affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo.

METHODS:

Real‐time polymerase chain reaction and flow cytometry were used to evaluate claudin‐3/‐4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE‐induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B‐17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo.

RESULTS:

CD44⁺ ovarian cancer stem cells expressed claudin‐4 gene at significantly higher levels than matched autologous CD44^? ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 μg/mL of CPE in vitro. Conversely, small‐interfering RNA‐mediated knockdown of claudin‐3/‐4 expression in CD44⁺ cancer stem cells significantly protected cancer stem cells from CPE‐induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy‐resistant CD44⁺ ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long‐term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001).

CONCLUSIONS:

CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy‐resistant cancer stem cells. Cancer 2011;. © 2011 American Cancer Society.