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);

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.

Treatment with the demethylating agent 5-Azacytidine leads to prolonged survival for patients with myelodysplastic syndrome, and the demethylation induces upregulation of cancer-testis antigens. Cancer-testis antigens are well-known targets for immune recognition in cancer, and the immune system may have a role in this treatment regimen. We show here that 5-Azacytidine treatment leads to increased T-cell recognition of tumor cells. T-cell responses against a large panel of cancer-testis antigens were detected before treatment, and these responses were further induced upon initiation of treatment. These characteristics point to an ideal combination of 5-Azacytidine and immune therapy to preferentially boost T-cell responses against cancer-testis antigens. To initiate such combination therapy, essential knowledge is required about the general immune modulatory effect of 5-Azacytidine. We therefore examined potential treatment effects on both immune stimulatory (CD8 and CD4 T cells and Natural Killer (NK) cells) and immune inhibitory cell subsets (myeloid-derived suppressor cells and regulatory T cells). We observed a minor decrease and modulation of NK cells, but for all other populations no effects could be detected. Together, these data support a strategy for combining 5-Azacytidine treatment with immune therapy for potential clinical benefit.

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry

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.

We designed conditional ligands restricted to HLA-B*08:01, 2B*35:01, and 2B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context. V C 2015 International Society for Advancement of Cytometry Key terms CD8 T cells; MHC multimer; HLA-B molecules; flow cytometry; conditional ligands THE tracking of specific T cells by use of fluorochrome-conjugated major histocompatibility complex (MHC) multimers for flow cytometry has increased our understanding in all areas of T-cell immunology (1-3). Staining of cells for binding to MHC multimers, combined with a set of antibodies to identify the CD8 T-cell population, represents an attractive way to quantitatively and phenotypically measure the CD8 T cells with certain specificities in a complex cell sample. It is thus possible to directly investigate the frequency and phenotype of these peptide-MHC (pMHC) multimer-specific T cells. The design and use of conditional ligands for the generation of MHC monomers was introduced in 2006 and revolutionized our ability to generate MHC reagents in a high-throughput fashion (4). These are HLA-binding peptides containing an UV-labile bond next to a 2-nitrophenylglycine or 3-amino-3-(2-nitrophenyl)-propionic acid residue (denoted "J") in their amino acid sequence, and can be integrated into the standard refolding procedure. Upon short-term UV light (366 nm) exposure, the amide bond next to the "J" residue is cleaved, and the fragmented peptide can be substituted with another ligand. The rescue of the MHC Class I complex after UV light exposure is dependent on the binding affinity between the ligand and the MHC molecule. Thus, this technique allows high-throughput affinity estimation using an MHC ELISA (5,6) and the generation of large libraries of pMHC complexes for the determinations of antigen-specific T cells using MHC Class I multimers in flow (7,8) and mass cytometry (9). The UV exchange technology has been extended to several MHC Class I variants (2,6,10), but conditional ligands has previously not been defined for some of the most frequently expressed HLA-B alleles.

Immunotherapy has increased overall survival of metastatic cancer patients, and cancer antigens are promising vaccine targets. To fulfill the promise, appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice, and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart, we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC, both important in human cancer development and progression, were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09, CASAC, Montanide ISA 51 VG, or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1*04:01, −1*07:02, −2*04:01, −2*05:02, and/or −3*04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer.