In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

SummaryCharacterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential. It is now well established that human tumor cells, notably cutaneous melanoma, may express multiple CTL-defined antigens that are shared among tumors, providing the prospect for generic vaccines applicable to large subsets of cancer patients (1, 2). Hence, numerous clinical trials are underway aimed at inducing vigorous CTL responses against defined tumor antigens. However, further progress in understanding natural or vaccine-induced CTL responses to tumor antigens has been prevented by the lack of direct assays of CTL activity. Previous assays for detection of antigen-specific CTLs have depended on their ability to proliferate extensively and acquire lytic activity, or to release relatively large amounts of cytokine (3), such that accurate quantification of these cells has not been possible. The necessity of stimulating CTLs with antigen in order to detect them has also prevented characterization of their phenotype in vivo. Recently, tetrameric arrays of soluble class I MHC-peptide complexes ("tetramers") have been used to identify antigen-specific CTLs (4), and techniques for detecting and isolating low-frequency CTLs using these reagents have been developed and validated (5). We report here the first analyses of patient samples using tetramers incorporating melanoma-derived antigenic peptides. These reagents were used to directly enumerate and phenotype melanoma-specific CTLs ex vivo from both metastatic lesions and peripheral blood, using multiparameter flow cytometry. Materials and MethodsTissues and Cells. Melanoma patients subjected to therapeutic surgical LN resection were selected for this study on the basis of P. Romero and P.R. Dunbar contributed equally to this work. T on

SummaryVitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer ϩ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer ϩ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer ϩ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer ϩ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR–peptide–MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)–A2 tumor epitope NY–ESO-1157–165–SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine–tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA–A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

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CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipidspecific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone-and foldase-assisted refolding of denatured CD1d molecules and ␤2-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-␣-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

NP105–113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105–113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105–113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105–113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105–113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2Kb H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.