PD-L1 (CD274) contributes to functional exhaustion of T cells and limits immune responses in patients with cancer. In this study, we report the identification of an human leukocyte antigen (HLA)-A2-restricted epitope from PD-L1, and we describe natural, cytolytic T-cell reactivity against PD-L1 in the peripheral blood of patients with cancer and healthy individuals. Notably, PD-L1-specific T cells were able not only to recognize and kill tumor cells but also PD-L1-expressing dendritic cells in a PD-L1-dependent manner, insofar as PD-L1 ablation rescued dendritic cells from killing. Furthermore, by incubating nonprofessional antigen-presenting cells with long peptides from PD-L1, we found that PD-L1 was rapidly internalized, processed, and cross-presented by HLA-A2 on the cell surface. Apparently, this cross-presentation was TAP-independent, as it was conducted not only by B cells but in addition by TAP-deficient T2-cells. This is intriguing, as soluble PD-L1 has been detected in the sera from patients with cancer. PD-L1-specific CTL may boost immunity by the killing of immunosuppressive tumor cells as well as regulatory cells. However, PD-L1-specific CTLs may as well suppress immunity by the elimination of normal immune cells especially PD-L1 expressing mature dendritic cells. Cancer Res; 73(6);

Purpose: To investigate targeting of indoleamine 2,3 dioxygenase (IDO) enzyme using a synthetic peptide vaccine administered to patients with metastatic non-small cell lung cancer (NSCLC).Experimental Design: In a clinical phase I study, we treated 15 HLA-A2-positive patients with stage III-IV NSCLC in disease stabilization after standard chemotherapy. Patients were treated with imiquimod ointment and subcutaneous vaccinations (100 mg IDO5 peptide, sequence ALLEIASCL, formulated in 900 mL Montanide). Primary endpoint was toxicity. Clinical benefit and immunity were assessed as secondary endpoints.Results: No severe toxicity was observed. One patient developed a partial response (PR) after one year of vaccine treatment, whereas long-lasting stable disease (SD) ! 8.5 months was demonstrated in another six patients. The median overall survival (OS) was 25.9 months. Patients demonstrated significant improved OS (P ¼ 0.03) when compared with the group of patients excluded because of HLA-A2 negativity. IDOspecific CD8 þ T-cell immunity was demonstrated by IFN-g Elispot and Tetramer staining. Fluorescenceactivated cell sorting analyses demonstrated a significant reduction of the Treg population (P ¼ 0.03) after the sixth vaccine (2.5 months) compared with pretreatment levels. Furthermore, expression of IDO was detected in nine of ten tumor biopsies by immunohistochemistry. High-performance liquid chromatography analyses of kynurenine/tryptophan (Kyn/Trp) ratio in sera were performed. In long-term analyses of two clinical responding patients, the ratio of Kyn/Trp remained stable. Conclusions:The vaccine was well tolerated with no severe toxicity occurring. A median OS of 25.9 months was demonstrated and long-lasting PRþSD was seen in 47% of the patients.

The presence of natural killer (NK) cells in the tumor microenvironment correlates with outcome in a variety of cancers. However, the role of intratumoral NK cells is unclear. Preclinical studies have shown that, while NK cells efficiently kill circulating tumor cells of almost any origin, they seem to have very little effect against the same type of tumor cells when these have extravasated. The ability to kill extravasated tumor cells is, however, is dependent of the level of activation of the NK cells, as more recent published and unpublished studies, discussed below, have demonstrated that interleukin-2–activated NK cells are able to attack well-established solid tumors.

Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2-(HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using the Enzyme-Linked ImmunoSPOT assay, we examined peripheral blood mononuclear cells from HLA-A2+ cancer patients and healthy volunteers for reactivity against the CCL22-derived T-cell epitope. This revealed spontaneous T-cell responses against the CCL22-derived epitope in cancer patients and in healthy donors. Finally, we performed tetramer enrichment/depletion experiments to examine the impact of HLA-A2-restricted CCL22-specific T cells on CCL22 levels among PMBCs. The addition or activation of CCL22-specific T cells decreased the CCL22 level in the microenvironment. Activating CCL22-specific T cells (e.g., by vaccination) may directly target cancer cells and tumor-associated macrophages, thereby modulating Treg recruitment into the tumor environment and augmenting anticancer immunity.

BackgroundThe enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tumor antigens. Recently, we described cytotoxic CD8+ T-cell reactivity towards IDO-derived peptides.Methods and FindingsIn the present study, we show that CD4+ helper T cells additionally spontaneously recognize IDO. Hence, we scrutinized the vicinity of the previously described HLA-A*0201-restricted IDO-epitope for CD4+ T-cell epitopes. We demonstrated the presence of naturally occurring IDO-specific CD4+ T cells in cancer patients and to a lesser extent in healthy donors by cytokine release ELISPOT. IDO-reactive CD4+ T cells released IFN-γ, TNF-α, as well as IL-17. We confirm HLA class II-restriction by the addition of HLA class II specific blocking antibodies. In addition, we detected a trend between class I- and class II-restricted IDO responses and detected an association between IDO-specific CD4+ T cells and CD8+ CMV-responses. Finally, we could detect IL-10 releasing IDO-reactive CD4+ T cells.ConclusionIDO is spontaneously recognized by HLA class II-restricted, CD4+ T cells in cancer patients and in healthy individuals. IDO-specific T cells may participate in immune-regulatory networks where the activation of pro-inflammatory IDO-specific CD4+ responses may well overcome or delay the immune suppressive actions of the IDO-protein, which are otherwise a consequence of the early expression of IDO in maturing antigen presenting cells. In contrast, IDO-specific regulatory T cells may enhance IDO-mediated immune suppression.

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Tryptophan-2,3-dioxygenase (TDO) physiologically regulates systemic tryptophan levels in the liver. However, numerous studies have linked cancer with activation of local and systemic tryptophan metabolism. Indeed, similar to other heme dioxygenases TDO is constitutively expressed in many cancers. In the present study, we detected the presence of both CD8 and CD4 T-cell reactivity toward TDO in peripheral blood of patients with malignant melanoma (MM) or breast cancer (BC) as well as healthy subjects. However, TDO-reactive CD4 T cells constituted distinct functional phenotypes in health and disease. In healthy subjects these cells predominately comprised interferon (IFN)γ and tumor necrosis factor (TNF)-α producing Th1 cells, while in cancer patients TDO-reactive CD4 T-cells were more differentiated with release of not only IFNγ and TNFα, but also interleukin (IL)-17 and IL-10 in response to TDO-derived MHC-class II restricted peptides. Hence, in healthy donors (HD) a Th1 helper response was predominant, whereas in cancer patients CD4 T-cell responses were skewed toward a regulatory T cell (Treg) response. Furthermore, MM patients hosting a TDO-specific IL-17 response showed a trend toward an improved overall survival (OS) compared to MM patients with IL-10 producing, TDO-reactive CD4 T cells. For further characterization, we isolated and expanded both CD8 and CD4 TDO-reactive T cells . TDO-reactive CD8 T cells were able to kill HLA-matched tumor cells of different origin. Interestingly, the processed and presented TDO-derived epitopes varied between different cancer cells. With respect to CD4 TDO-reactive T cells, expanded T-cell cultures comprised a Th1 and/or a Treg phenotype. In summary, our data demonstrate that the immune modulating enzyme TDO is a target for CD8 and CD4 T cell responses both in healthy subjects as well as patients with cancer; notably, however, the functional phenotype of these T-cell responses differ depending on the respective conditions of the host.

Tumor-infiltrating CD4+CD25+ regulatory T cells (Tregs) are associated with an impaired prognosis in several cancers. The transcription factor forkhead box P3 (Foxp3) is generally expressed in Tregs. Here, we identify and characterize spontaneous cytotoxic immune responses to Foxp3-expressing cells in peripheral blood of healthy volunteers and cancer patients. These immune responses were directed against a HLA-A2-restricted peptide epitope derived from Foxp3. Foxp3-reactive T cells were characterized as cytotoxic CD8+ T cells. These cells recognized dendritic cells incubated with recombinant Foxp3 protein indicating that this protein was indeed internalized, processed and cross-presented in the context of HLA-A2. More importantly, however, Foxp3-specific T cells were able to specifically recognize Tregs. Similarly, Foxp3+ malignant T cells established from a Cutaneous T-cell lymphomas (CTCL) patient were readily killed by the Foxp3-specific cytotoxic T lymphocytes. The spontaneous presence of Foxp3-specific cytotoxic T-cell responses suggest a general role of such T cells in the complex network of immune regulation as such responses may eliminate Tregs, that is, suppression of the suppressors. Consequently, induction of Foxp3-specific cytotoxic T-cell responses appears as an attractive tool to boost spontaneous or therapeutically provoked immune responses, for example, for the therapy of cancer.