Tumor expression of 4-1BB ligand sustains tumor lytic T cells

Inadequate costimulation by solid tumors is generally believed to induce immune tolerance during primary tumor growth. We looked for tumor-specific immunity vs. tolerance in patients with Ewing's sarcoma. Circulating T cells from patients with progressively growing Ewing's tumors displayed MHC restricted tumor-induced proliferation and robust tumor lysis. Tumor-reactive T cells reside within the memory CD3+CD8+ subset and are CD28-/4-1BB+. Autologous Ewing's tumors expressed 4-1BBL, and tumor-induced T cell proliferation and activation required costimulation by 4-1BBL. Stimulation of PBL with anti-CD3/4-1BBL, but not anti-CD3/anti-CD28 induced tumor lytic effectors. Similarly, in a xenograft model, anti-CD3/4-1BBL expanded T cells controlled primary growth and prevented metastasis of autologous tumors while nonactivated and anti-CD3/anti-CD28 activated CD8+ cells did not. These results question prevailing models of tumor induced tolerance accompanying progressive tumor growth; rather, we show coexistence of progressive tumor growth and anti-tumor immunity, with costimulation provided by the tumor itself. They further demonstrate a potential new therapeutic role for 4-1BBL mediated costimulation in expanding tumor reactive CTLs for use in the adoptive immunotherapy of cancer.     

IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.     

Adoptively transferred tumor-specific T cells stimulated ex vivo using herpes simplex virus amplicons encoding 4-1BBL persist in the host and show antitumor activity in vivo

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.     

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.     

A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL

4-1BB Ligand (4-1BBL), a transmembrane molecule, member of the tumor necrosis factor ligand superfamily, is an important costimulatory molecule in the immune response. In this study a functional anti-human 4-1BBL MAb 1F1 was obtained and the specificity of this MAb was verified by flow cytometry and Western blotting. This MAb effectively recognized the 4-1BBL molecule expressed on a series of malignant cell lines as well as on DC and monocytes and it inhibited the proliferation of T lymphocytes, costimulated by soluble 4-1BBL and agonist anti-human CD3 MAb. Furthermore, we demonstrated that MAb 1F1 induced an impressive proliferation of monocytes from peripheral blood by triggering the reverse signal through 4-1BBL. This functional anti-human 4-1BBL MAb provides a valuable tool for further study of biological functions as well as signal transduction of 4-1BBL/4-1BB.     

Melanoma‐associated chondroitin sulphate proteoglycan as a new target antigen for CD4+ T cells in melanoma patients

Melanoma‐associated chondroitin sulfate proteoglycan (MCSP) (also known as high molecular weight‐melanoma‐associated antigen) represents an interesting target antigen for cancer immunotherapy which is expressed on human melanomas and other tumors such as breast carcinomas, gliomas, neuroblastomas and acute leukemias. MCSP seems to play an important functional role in melanoma as it is involved in tumor cell migration, invasion and angiogenesis. In this study, we isolated CD4⁺ T helper cells from the blood of a healthy donor, recognizing a peptide from the MCSP core protein presented by HLA‐DBR1*1101 molecules. T cell reactivity against the identified peptide could be detected in the blood of healthy donors and melanoma patients. MCSP specific T cells from the blood of a patient could be readily expanded by repeated peptide stimulation and recognized MCSP and HLA‐DR expressing tumor cells. Our findings suggest that vaccination against MCSP helper T cell epitopes might be a promising approach to fight melanoma. © 2009 Wiley‐Liss, Inc.     

Interferon‐γ down‐regulates NKG2D ligand expression and impairs the NKG2D‐mediated cytolysis of MHC class I‐deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of αβ CD8⁺ T cells. Ligands of NKG2D, the MHC class I chain‐related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon‐γ (IFN‐γ) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I‐negative and ‐positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN‐γ treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down‐regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT‐PCR revealed a decrease of MICA‐specific mRNA levels upon IFN‐γ treatment and siRNA experiments pointed to an involvement of STAT‐1 in this process. Importantly, IFN‐γ‐treated MHC class I‐negative melanoma cells were less susceptible to NKG2D‐mediated NK cell cytotoxicity. Our study suggests that IFN‐γ, by down‐regulating ligand expression, might facilitate escape of MHC class I‐negative melanoma cells from NKG2D‐mediated killing by NK cells. © 2008 Wiley‐Liss, Inc.     

Enhancement of DNA vaccine potency by antigen linkage to IFN‐γ‐inducible protein‐10

DNA vaccines have emerged as an attractive approach to generate antigen‐specific T‐cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1‐polarizing chemokine, IP‐10, to a model tumor antigen, human papillomavirus type 16 (HPV‐16) E7, enhanced DNA vaccine potency. IP‐10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP‐10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP‐10/E7 DNA exhibited a dramatic increase in the number of E7‐specific CD4⁺ Th1 T‐cells and CD8⁺ T‐cells and, consequently, were strongly resistant over the long term to E7‐expressing tumors compared to mice vaccinated with wild‐type E7 DNA. Thus, because of the increase in tumor antigen‐specific T‐cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP‐10 linked to a tumor antigen holds great promise for treating tumors.     

Synergistic effects of CTLA‐4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo

Anti‐CTLA‐4 monoclonal antibodies (mAb) that block the interaction of CTLA‐4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de‐repress tumor‐specific cellular immunity. Addition of the fully human anti‐CTLA‐4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead‐mediated elimination of CD4⁺ CD25⁺ regulatory T cells (T_reg) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4⁺ CD25⁺ T_reg and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T‐cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA‐4 expression in effector cells as a result of T_reg elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2^?/? IL‐2Rγ^?/? mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T_reg cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T‐cell compartment. In addition, T_reg depletion and CTLA‐4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV‐transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T_reg depletion.     

Activated invariant natural killer T cells directly recognize leukemia cells in a CD1d‐independent manner

Invariant natural killer T (iNKT) cells are innate‐like CD1d‐restricted T cells that express the invariant T cell receptor (TCR) composed of Vα24 and Vβ11 in humans. iNKT cells specifically recognize glycolipid antigens such as α‐galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d‐positive tumor cells, especially when CD1d presents glycolipid antigens. However, iNKT cell recognition of CD1d‐negative tumor cells is unknown, and direct cytotoxicity of iNKT cells toward CD1d‐negative tumor cells remains controversial. Here, we demonstrate that activated iNKT cells recognize leukemia cells in a CD1d‐independent manner, however still in a TCR‐mediated way. iNKT cells degranulated and released Th1 cytokines toward CD1d‐negative leukemia cells (K562, HL‐60, REH) as well as αGalCer‐loaded CD1d‐positive Jurkat cells. The CD1d‐independent cytotoxicity was enhanced by natural killer cell‐activating receptors such as NKG2D, 2B4, DNAM‐1, LFA‐1 and CD2, but iNKT cells did not depend on these receptors for the recognition of CD1d‐negative leukemia cells. In contrast, TCR was essential for CD1d‐independent recognition and cytotoxicity. iNKT cells degranulated toward patient‐derived leukemia cells independently of CD1d expression. iNKT cells targeted myeloid malignancies more than acute lymphoblastic leukemia. These findings reveal a novel anti–tumor mechanism of iNKT cells in targeting CD1d‐negative tumor cells and indicate the potential of iNKT cells for clinical application to treat leukemia independently of CD1d.     

Combination immunotherapy with interleukin‐2 surface‐modified tumor cell vaccine and programmed death receptor‐1 blockade against renal cell carcinoma

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4⁺, CD8⁺ and CD8⁺interferon‐γ⁺ but not CD4⁺Foxp3⁺ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.     

Allorestricted T lymphocytes with a high avidity T‐cell receptor towards NY‐ESO‐1 have potent anti‐tumor activity

The cancer‐testis antigen NY‐ESO‐1 has been targeted as a tumor‐associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T‐cell‐based therapy is the induction of T cells capable of recognizing the NY‐ESO‐1‐expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY‐ESO‐1_157–165 epitope known to be naturally presented with HLA‐A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY‐ESO‐1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA‐A2‐positive, NY‐ESO‐1‐expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY‐ESO‐1‐specific T lymphocytes displayed TCRs with the highest avidity and best anti‐tumor recognition activity. TCRs derived from allorestricted, NY‐ESO‐1‐specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR‐transduced T cells for the treatment of NY‐ESO‐1‐expressing hematological malignancies and solid tumors. © 2009 UICC     

CD155, an onco‐immunologic molecule in human tumors

CD155 is the fifth member in the nectin‐like molecule family, and functions as the receptor of poliovirus; therefore, CD155 is also referred to as necl‐5, or PVR. As an immunoglobulin‐like adhesion molecule, CD155 is involved in cell motility, and natural killer and T cell‐mediated immunity. CD155 is barely or weakly expressed in various normal human tissues, but frequently overexpressed in human malignant tumors. CD155 overexpression promotes tumor cell invasion and migration, and is associated with tumor progression and poor prognosis. As the ligand for both costimulatory receptor CD226 and coinhibitory receptor TIGIT and CD96 on natural killer and T cells, CD155 seems to play a dual role in oncoimmunity. However, some recent studies indicate that CD155 overexpression may induce tumor immune escape. Taken together, CD155 may be considered as a target for the treatment of tumors with CD155 overexpression.     

Tumor‐reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor‐reactive T‐cell subset in metastatic colorectal cancers

High level of T‐cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor‐reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor‐reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4⁺CD8αβ⁺ (DP) αβ T cells. Interestingly, a high proportion (16–20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class‐I, proliferated poorly and secreted higher amounts of IL‐4 and IL‐13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor‐reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL‐4 and IL‐13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.     

Leukemia-specific T-cell reactivity induced by leukemic dendritic cells is augmented by 4-1BB targeting.

PURPOSE: Acute myelogenous leukemia (AML) blasts are able to differentiate into leukemia-derived dendritic cells (AML-DC), thereby enabling efficient presentation of known and unknown leukemic antigens. Advances in culture techniques and AML-DC characterization justify clinical application. However, additional measures are likely needed to potentiate vaccines and overcome the intrinsic tolerant state of the patients' immune system. Engagement of the costimulatory molecule 4-1BB can break immunologic tolerance and increase CTL responses. In this study, we examined the role of the 4-1BB ligand (4-1BBL) on T-cell responses induced by AML-DC. EXPERIMENTAL DESIGN: In allogeneic and autologous cocultures of T cells and AML-DC, the effect of the addition of 4-1BBL on T-cell proliferation, T-cell subpopulations, and T-cell function was determined. RESULTS: Addition of 4-1BBL to cocultures of AML-DC and T cells induced a preferential increase in the proliferation of CD8(+) T cells. Increased differentiation into effector and central memory populations was observed in both CD4(+) and CD8(+) T cells in the presence of 4-1BBL. AML-DC induce a T helper 1 response, characterized by high IFN-gamma production, which is significantly increased by targeting 4-1BB. T cells primed in the presence of 4-1BBL show specificity for the leukemia-associated antigen Wilms' tumor 1, whereas cytotoxicity assays with leukemic blast targets showed the cytolytic potential of T cells primed in the presence of 4-1BBL. CONCLUSION: We conclude that 4-1BBL is an effective adjuvant to enhance T-cell responses elicited by AML-DC.     

A multimeric carcinoembryonic antigen signal inhibits the activation of human T cells by a SHP‐independent mechanism: A potential mechanism for tumor‐mediated suppression of T‐cell immunity

Carcinoembryonic antigen (CEA) is a well‐known tumor antigen that is found in the serum of patients with various cancers and is correlated with an increased risk of cancer recurrence and metastasis. To understand the tumor environment and to develop antitumor therapies, CEA has been studied as an antigen to activate/tolerate specific T cells. In this study, we show that CEA can function as a coinhibitory molecule and can inhibit the activation of human peripheral blood mononucleated cell‐derived T cells. The addition of CEA‐overexpressing tumor cells or immobilized CEA dampened both cell proliferation and the expression of IL‐2 and CD69 expression in T cells after TCR stimulation. The phosphorylation of ERK and translocation of NFAT were hampered in these cells, whereas the phosphorylation of proximal TCR signaling molecules such as ZAP70 and phospholipase Cγ was not affected by immobilized CEA. To determine the relevance of carcinoembryonic antigen‐related cell adhesion molecule‐1 and Src homology region 2 domain‐containing phosphatase (SHP) molecules to CEA‐mediated suppression, we tested the effect of the SHP inhibitor, NSC‐87877, on CEA‐mediated suppression of T cells; however, it did not reverse the effect of CEA. Collectively, these results indicate that CEA can function as a modulator of T‐cell responses suggesting a novel mechanism of tumor evasion.     

Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity

Co-stimulatory molecules play an important role in initiating antitumor immune responses. Engineered tumor cells expressing co-stimulatory molecules have been used as cancer vaccines in both experimental tumor models and clinical trials. In this study, we cloned a cDNA gene coding for the mouse co-stimulatory molecule 4-1BBL by RTPCR. The expression vector pCI-4-1BBL was constructed by DNA recombinant technology and further transfected into a moderately immunogenic EL4 and a poorly immunogenic BL6-10 tumor cell line. Expression of the co-stimulatory molecule 4-1BBL is able to induce tumor regression of EL4/4-1BBL but not BL6-10/4-1BBL tumor cell line in syngeneic BALB/c mice. The tumor regression which is mainly mediated by CD8+ T cells further leads to protective immunity against the parental EL4 tumor. Our results thus indicate the potential utility of engineered tumor cells expressing co-stimulatory molecule 4-1BBL, especially in combination with other co-stimulatory molecules such as B7-1 in cancer vaccine.     

T cell recognition of HLA‐A2 restricted tumor antigens is impaired by the oncogene HER2

The HER2 oncogene is frequently over‐expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over‐expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor‐antigen specific cytotoxic T lymphocytes (CTLs) in HLA‐A2.1⁺ melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA‐A2. A significant reduction of HLA‐A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling‐competent HER2 molecule was crucial for the observed HLA‐A2 down‐regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA‐A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen‐specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN‐γ mediated HLA‐A2 up‐regulation and improved recognition by HLA‐A2‐restricted CTLs in treated cells. Moreover, key APM molecules were down‐regulated by HER2. These findings implicate that HER2 over‐expressing tumors may be more prone to escape from HLA‐A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.