CCL22-specific T Cells: Modulating the immunosuppressive tumor microenvironment.

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.

Cellular immune responses towards regulatory cells.

This thesis describes the results from two published papers identifying spontaneous cellular immune responses against the transcription factors Foxp3 and Foxo3. The tumor microenvironment is infiltrated by cells that hinder effective tumor immunity from developing. Two of these cell types, which have been linked to a bad prognosis for patients, are regulatory T cells (Treg) and tolerogenic dendritic cells (DC). Tregs inhibit effector T cells from attacking the tumor through various mechanisms, including secreted factors and cell-to-cell contact. Tregs express the transcription factor Foxp3, which is necessary for their development and suppressive activities. Tolerogenic DCs participate in creating an environment in the tumor where effector T cells become tolerant towards the tumor instead of attacking it. The transcription factor Foxo3 was recently described to be highly expressed by tolerogenic DCs and to programme their tolerogenic influence. This thesis describes for the first time the existence of spontaneous cellular immune responses against peptides derived from Foxp3 and Foxo3. We have detected the presence of cytotoxic T cells that recognise these peptides in an HLA-A2 restricted manner in cancer patients and for Foxp3 in healthy donors as well. In addition, we have demonstrated that the Foxp3- and Foxo3-specific CTLs recognize Foxp3- and Foxo3-expressing cancer cell lines and importantly, suppressive immune cells, namely Tregs and in vitro generated DCs. Cancer immunotherapy is recently emerging as an important treatment modality improving the survival of selected patients. The current progress is largely owing to targeting of the immune suppressive milieu that is dominating the tumor microenvironment. This is being done through immune checkpoint blockade with CTLA-4 and PD-1/PD-L1 antibodies and through lymphodepleting conditioning of patients and ex vivo activation of TILs in adoptive cell transfer. Several strategies are being explored for depletion of Tregs and for modulation of Treg and DC suppressive activity, including CD25 antibodies and chemotherapy. The research presented in this thesis offer an alternative approach to targeting suppressive cells subsets, by activating the immune system against proteins expressed by these cell types.

Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer.

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 in vitro. 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, in vitro 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.

Broadening the repertoire of melanoma-associated T-cell epitopes.

Immune therapy has provided a significant breakthrough in the treatment of metastatic melanoma. Despite the remarkable clinical efficacy and established involvement of effector CD8 T cells, the knowledge of the exact peptide-MHC complexes recognized by T cells on the tumor cell surface is limited. Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer-based enrichment of peripheral blood from 39 melanoma patients and 10 healthy donors. To dissect the T-cell reactivity against this large peptide library, we used combinatorial-encoded MHC multimers and observed the T-cell responses against 17 different peptide-MHC complexes in the patient group and four in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing the appropriate antigen and HLA molecule. We further found T-cell reactivity against two of the identified sequences among tumor-infiltrating lymphocytes from melanoma patients, suggesting a potential clinical relevance of these sequences.

Long-lasting disease stabilization in the absence of toxicity in metastatic lung cancer patients vaccinated with an epitope derived from indoleamine 2,3 dioxygenase.

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 µg IDO5 peptide, sequence ALLEIASCL, formulated in 900 µL 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 = 0.03) when compared with the group of patients excluded because of HLA-A2 negativity. IDO-specific CD8(+) T-cell immunity was demonstrated by IFN-γ Elispot and Tetramer staining. Fluorescence-activated cell sorting analyses demonstrated a significant reduction of the Treg population (P = 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 PR+SD was seen in 47% of the patients.

Spontaneous presence of FOXO3-specific T cells in cancer patients.

In the present study, we describe forkhead box O3 (FOXO3)-specific, cytotoxic CD8+ T cells existent among peripheral-blood mononuclear cells (PBMCs) of cancer patients. FOXO3 immunogenicity appears specific, as we did not detect reactivity toward FOXO3 among T cells in healthy individuals. FOXO3 may naturally serve as a target antigen for tumor-reactive T cells as it is frequently over-expressed in cancer cells. In addition, expression of FOXO3 plays a critical role in immunosuppression mediated by tumor-associated dendritic cells (TADCs). Indeed, FOXO3-specific cytotoxic T lymphocytes (CTLs) were able to specifically recognize and kill both FOXO3-expressing cancer cells as well as dendritic cells. Thus, FOXO3 was processed and presented by HLA-A2 on the cell surface of both immune cells and cancer cells. As FOXO3 programs TADCs to become tolerogenic, FOXO3 signaling thereby comprises a significant immunosuppressive mechanism, such that FOXO3 targeting by means of specific T cells is an attractive clinical therapy to boost anticancer immunity. In addition, the natural occurrence of FOXO3-specific CTLs in the periphery suggests that these T cells hold a function in the complex network of immune regulation in cancer patients.

HLA-restricted CTL that are specific for the immune checkpoint ligand PD-L1 occur with high frequency in cancer patients.

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.

Natural CD4+ T-cell responses against indoleamine 2,3-dioxygenase.

BACKGROUND: The 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 FINDINGS: In 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. CONCLUSION: IDO 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.

Characterization of Spontaneous Immune Responses against Long Peptides Derived from Bcl-X(L) in Cancer Patients Using Elispot.

In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9-10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126-188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20-23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides.