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Cytotoxic CD8+ T lymphocyte (CTL) recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells and also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes and long-term survival of patients with melanoma. Using MART1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior co-stimulatory effects of DNAM1 over CD28 involved enhanced TCR signaling, CTL killer function, and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in antigen specificity and selectivity of killer function. In addition, the elevation of NKR ligand expression on cancer cells due to chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAAs. Our data help explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T-cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells., (©2024 American Association for Cancer Research.)

The Melan-A (melanocyte antigen) protein, also termed 'melanoma antigen recognized by T cells 1' (MART-1) is a protein with unknown function whose expression is specific for the melanocyte lineage. Antibodies against Melan-A are thus used for identifying melanocytic tumors, but some Melan-A antibodies show an additional - diagnostically useful - cross-reactivity against an unspecified protein involved in corticosteroid hormone synthesis. To comprehensively compare the staining patterns of a specific and a cross-reactive Melan-A antibody in normal and neoplastic tissues, tissue microarrays containing 15,840 samples from 133 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. For the Melan-A-specific antibody 'Melan-A specific' (MSVA-900M), Melan-A positivity was seen in 96.0% of 25 benign nevi, 93.0% of 40 primary and 86.7% of 75 metastatic melanomas, 82.4% of 85 renal angiomyolipomas as well as 96.4% of 84 neurofibromas, 2.2% of 46 granular cell tumors, 1.0% of 104 schwannomas, and 1.1% of 87 leiomyosarcomas. The cross-reactive antibody 'Melan-A+' (MSVA-901M+) stained 98.1% of the tumors stained by 'Melan-A specific'. In addition, high positivity rates were seen in sex-cord-stroma tumors of the ovary (35.3%-100%) and the testis (86.7%) as well as for adrenocortical neoplasms (76.3%-83.0%). Only nine further tumor groups showed Melan-A+ staining, including five different categories of urothelial carcinomas. Our data provide a comprehensive overview on the staining patterns of specific and cross-reactive Melan-A antibodies. The data demonstrate that both antibodies are highly useful for their specific purpose. It is important for pathologists to distinguish these two Melan-A antibody subtypes for their daily work., (© 2024 The Author(s). APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Introduction: Naked DNA vaccination could be a powerful and safe strategy to mount antigen-specific cellular immunity. We designed a phase I clinical trial to investigate the toxicity of naked DNA vaccines encoding CD8+ T-cell epitope from tumor-associated antigen MART-1 in patients with advanced melanoma., Methods: This dose escalating phase Ia clinical trial investigates the toxicity and immunological response upon naked DNA vaccines encoding a CD8+ T-cell epitope from the tumor-associated antigen MART-1, genetically linked to the gene encoding domain 1 of subunit-tetanus toxin fragment C in patients with advanced melanoma (inoperable stage IIIC-IV, AJCC 7th edition). The vaccine was administrated via intradermal application using a permanent make-up or tattoo device. Safety was monitored according to CTCAE v.3.0 and skin biopsies and blood samples were obtained for immunologic monitoring., Results: Nine pretreated, HLA-A*0201-positive patients with advanced melanoma expressing MART-1 and MHC class I, with a good performance status, and adequate organ function, were included. With a median follow-up of 5.9 months, DNA vaccination was safe, without treatment-related deaths. Common treatment-emergent adverse events of any grade were dermatologic reactions at the vaccination site (100%) and pain (56%). One patient experienced grade 4 toxicity, most likely related to tumor progression. One patient (11%) achieved stable disease, lasting 353 days. Immune analysis showed no increase in vaccine-induced T cell response in peripheral blood of 5 patients, but did show a MART-1 specific CD8+ T cell response at the tattoo administration site. The maximum dose administered was 2 mg due to lack of clinical activity., Conclusion: We showed that the developed DNA vaccine, applied using a novel intradermal application strategy, can be administered safely. Further research with improved vaccine formats is required to show possible clinical benefit of DNA vaccination., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8 + T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8 + T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background: Anti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better understanding of the modes of action and predictive biomarkers for clinical outcome is urgently required. Cancer rejection is mostly T cell-mediated. We previously showed that the presence of NY-ESO-1-reactive and/or Melan-A-reactive T cells in the blood correlated with prolonged overall survival (OS) of patients with melanoma with a heterogeneous treatment background. Here, we investigated whether such reactive T cells can also be informative for clinical outcomes in metastatic melanoma under PD-1 immune-checkpoint blockade (ICB)., Methods: Peripheral blood T cell stimulation by NY-ESO-1 and Melan-A overlapping peptide libraries was assessed before and during ICB in two independent cohorts of a total of 111 patients with stage IV melanoma. In certain cases, tumor-infiltrating lymphocytes could also be assessed for such responses. These were characterized using intracellular cytokine staining for interferon gamma (IFN-γ), tumor negrosis factor (TNF) and CD107a. Digital pathology analysis was performed to quantify NY-ESO-1 and Melan-A expression by tumors. Endpoints were OS and progression-free survival (PFS)., Results: The initial presence in the circulation of NY-ESO-1- or Melan-A-reactive T cells which became no longer detectable during ICB correlated with validated, prolonged PFS (HR:0.1; p>0.0001) and OS (HR:0.2; p=0.021). An evaluation of melanoma tissue from selected cases suggested a correlation between tumor-resident NY-ESO-1- and Melan-A-reactive T cells and disease control, supporting the notion of a therapy-associated sequestration of cells from the periphery to the tumor predominantly in those patients benefitting from ICB., Conclusions: Our findings suggest a PD-1 blockade-dependent infiltration of melanoma-reactive T cells from the periphery into the tumor and imply that this seminally contributes to effective treatment., Competing Interests: Competing interests: CG reports receiving commercial research grants from Bristol-Myers Squibb, Novartis, and Roche; and is a consultant/advisory board member for Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, and Roche. BW reports receiving commercial research grants from, is a consultant/advisory board member for, and reports receiving travel reimbursement from Bristol-Myers Squibb and Merck Sharp & Dohme. F. Meier reports receiving commercial research grants from Novartis and Roche; and has received travel support or/and speaker’s fees or/and advisor’s honoraria by Novartis, Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, and Pierre Fabre. LF reported grants from the Swiss National Science Foundation, Swiss Cancer League, Hookipa Pharma, and Novartis Foundation as well as an advisory role for Novartis, Sanofi, Philogen and Bristol-Myers Squibb. PT has received speaker’s honoraria from Bristol-Myers Squibb, Novartis, Merck Sharp & Dohme, Pierre-Fabre, CureVac, Roche, Kyowa Kirin, Biofrontera, consultant’s honoraria from Bristol-Myers Squibb, Merck Serono, Novartis, Sanofi, Kirin Kyowa, and Roche and travel support from Bristol-Myers Squibb and Pierre-Fabre. SF reports receiving speaker’s fees by TAKEDA Pharmaceutical. TE has received speaker’s honoraria from Bristol-Myers Squibb, Novartis, Almiral Hermal, and Roche, consultant’s honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Sanofi, Pierre Fabre and Roche. TS and HN received a research grant from Novartis. GP has received speaker’s honoraria from Novartis, Roche, GlaxoSmithKline and Astellas. MWL is a co-inventor of several patents owned by Immatics Biotechnologies, and has acted as a consultant and/or advisory board member for Boehringer Ingelheim Pharma Gmbh & Co. KG. HZ is employed by CeGaT GmbH. KW-H received commercial research grants from the CatalYm GmbH and travel support from SITC (Society for Immunotherapy of Cancer). No potential conflicts of interest were disclosed by the other authors. KW-H is the guarantor for this manuscript., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201 + MART-1 + tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These "off-the-shelf" AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hamieh, Chatillon, Dupel, Bayeux, Fauquembergue, Maby, Drouet, Duval-Modeste, Adriouch, Boyer and Latouche.)

The efficacy of adoptive cell therapy (ACT) relies on the abilities of T cells in self-expansion, survival and the secretion of effector molecules. Here, we presented an optimized method to generate T cells with improved functions by supplementing the culture medium with p38 inhibitor and the combination of IL-7 and IL-15 or IL-2 alone. The addition of p38 inhibitor, Doramapimod or SB202190, to IL-7 and IL-15 culture largely increased the capacity of T cells in the proliferation with enrichment of the naïve-like subsets and expression of CD62L. Importantly, we found this regimen has generated complete T cell resistance to TGF-β-induced functional suppression, with sustained levels of the IFN-γ and Granzyme-B productions. Such findings were also validated in the melanoma-associated antigen recognized by T cells (MART-1) specific T cell receptor (TCR) engineered T cells, which were expanded in Doramapimod and IL-7 + IL-15 added media. In conclusion, we have established and optimized a protocol with the combination of p38 inhibitor, IL-7 and IL-15, rather than IL-2, for the generation of functionally enhanced T cells applicable for ACT., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen-loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized that the combined transfer of TIL, containing a melanoma antigen recognized by T cells 1 (MART-1) specific population, with MART-1-pulsed DC will result in enhanced proliferation and prolonged survival of transferred MART-1 specific T cells in vivo ultimately leading to improved clinical responses., Design: We tested the combination of TIL and DC in a phase II clinical trial of patients with advanced stage IV melanoma. HLA-A0201 patients whose early TIL cultures demonstrated reactivity to MART-1 peptide were randomly assigned to receive TIL alone or TIL +DC pulsed with MART-1 peptide. The primary endpoint was to evaluate the persistence of MART-1 TIL in the two arms. Secondary endpoints were to evaluate clinical response and survival., Results: Ten patients were given TIL alone while eight patients received TIL+DC vaccine. Infused MART-1 reactive CD8 + TIL were tracked in the blood over time by flow cytometry and results show good persistence in both arms, with no difference in the persistence of MART-1 between the two arms. The objective response rate was 30% (3/10) in the TIL arm and 50% (4/8) in the TIL+DC arm. All treatments were well tolerated., Conclusions: The combination of TIL +DC showed no difference in the persistence of MART-1 TIL compared with TIL therapy alone. Although more patients showed a clinical response to TIL+DC therapy, this study was not powered to resolve differences between groups., Trial Registration Number: NCT00338377., Competing Interests: Competing interests: CLH is on the SAB for Briacell. PH consulting agreement with Immatics, Dragonfly, Sanofi, GSK. CB is on the SAB of Myst therapeutics and received research funding from Iovance biotherapeutics., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Rationale: TCR-T cell therapy plays a critical role in the treatment of malignant cancers. However, it is unclear how TCR-T cells are affected by PD-L1 molecule in the tumor environment. We performed an in-depth evaluation on how differential expressions of tumor PD-L1 can affect the functionality of T cells. Methods: We used MART-1-specific TCR-T cells (TCR-T MART-1 ), stimulated with MART-1 27-35 peptide-loaded MEL-526 tumor cells, expressing different proportions of PD-L1, to perform cellular assays and high-throughput single-cell RNA sequencing. Results: Different clusters of activated or cytotoxic TCR-T MART-1 responded divergently when stimulated with tumor cells expressing different percentages of PD-L1 expression. Compared to control T cells, TCR-T MART-1 were more sensitive to exhaustion, and secreted not only pro-inflammatory cytokines but also anti-inflammatory cytokines with increasing proportions of PD-L1 + tumor cells. The gene profiles of chemokines were modified by increased expression of tumor PD-L1, which concurrently downregulated pro-inflammatory and anti-inflammatory transcription factors. Furthermore, increased expression of tumor PD-L1 showed distinct effects on different inhibitory checkpoint molecules (ICMs). In addition, there was a limited correlation between the enrichment of cell death signaling in tumor cells and T cells and increased tumor PD-L1 expression. Conclusion: Overall, though the effector functionality of TCR-T cells was suppressed by increased expression percentages of tumor PD-L1 in vitro , scRNA-seq profiles revealed that both the anti-inflammatory and pro-inflammatory responses were triggered by a higher expression of tumor PD-L1. This suggests that the sole blockade of tumor PD-L1 might inhibit not only the anti-inflammatory response but also the pro-inflammatory response in the complicated tumor microenvironment. Thus, the outcome of PD-L1 intervention may depend on the final balance among the highly dynamic and heterogeneous immune regulatory circuits., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Metastatic melanoma in sentinel lymph nodes is often elusive to detect with morphology alone. Per American Joint Committee on Cancer staging guidelines, a single atypical melanocyte in lymph node qualifies as metastasis, whether identified by morphology or immunohistochemistry, but single cell staining must be convincing. We propose that the use of a second immunohistochemical run performed on a single slide will allow for more confident diagnosis of micrometastases., Materials and Methods: We designed a technical study to determine whether a second antibody application on previously stained slides can successfully detect the same population of cells. Melanocytic neoplasms were stained with SOX-10 using Ventana Benchmark Ultra stainers, coverslipped, and examined, followed by coverslip removal and application of MART-1 (Ventana A103). The order of antibody application and chromagen detection kit (AP-RED vs. DAB) was reversed to establish reliability and robustness of the protocol., Results: All melanocytes marked with SOX-10 and MART-1, and produced a range of staining quality that varied based on order of stain application and chromagen kit were used. The optimal combination was red MART-1 applied first followed by brown SOX-10 applied second., Conclusions: Consecutive staining of melanocytes with SOX-10 and MART-1 may improve diagnostic confidence of melanocyte identification, particularly in detection of single cell, micrometastases in sentinel lymph nodes or in situations where dual immunohistochemical stains may be unavailable.

Neoantigen-based cancer immunotherapies hold the promise of being a truly personalized, effective treatment for diverse cancer types. ELISPOT assays, as a powerful experimental technique, can verify the existence of antigen specific T cells to support basic clinical research and monitor clinical trials. However, despite the high sensitivity of ELISPOT assays, detecting immune responses of neoantigen specific T cells in a patient or healthy donor's PBMCs is still extremely difficult, since the frequency of these T cells can be very low. We developed a novel experimental method, by co-stimulation of T cells with anti-CD28 and IL-2 at the beginning of ELISPOT, to further increase the sensitivity of ELISPOT and mitigate the challenge introduced by low frequency T cells. Under the optimal concentration of 1 μg/ml for anti-CD28 and 1 U/ml for IL-2, an 11.7-fold increase of T cell response against CMV peptide was observed by using our method, and it outperforms other cytokine stimulation alternatives (5-10 folds). We also showed that this method can be effectively applied to detect neoantigen-specific T cells in healthy donors' and a melanoma patient's PBMCs. To the best of our knowledge, this is the first report that the co-stimulation of anti-CD28 and IL-2 is able to significantly improve the sensitivity of ELISPOT assays, indicating that anti-CD28 and IL-2 signaling can act in synergy to lower the T cell activation threshold and trigger more neoantigen-specific T cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Long-lasting CD8 + T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-1 26(27L)-35 antigenic peptide ELAGIGILTV. The Cu I -catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-1 26-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function., Methods: To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus., Results: Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2 - or swine thymus compared with an HLA-A2 + thymus. Additionally, CD8 MART1 TCR bright T cells were detected in the spleens of mice transplanted with HLA-A2 + thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2 - thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses.

A vaccine that could expand melanoma-specific T cells might reduce the risk of recurrence of resected melanoma and could provide an alternative or adjunct to standard immunotherapy options. We tested the safety and immunogenicity of a vaccine coupling a melanoma-associated peptide with a xenogenic peptide (to promote epitope spreading) and/or resiquimod (to activate antigen-presenting cells). HLA-A2-positive patients with resected stage II, III, and IV melanoma were assigned to treatment on one of three schedules. All patients received three subcutaneous doses of the peptide MART-1a mixed with Montanide. In addition, patients on schedule 1 received the xenoantigen peptide Gag267-274, patients on schedule 2 received topical resiquimod, and patients on schedule 3 received both Gag267-274 and resiquimod. Blood samples were tested for the frequency of antigen-specific T cells by tetramer assay, as well as immune cell subtypes and plasma cytokine levels. Patients enrolled from October 2012 to December 2014, with 10 patients enrolling to each schedule. The most common adverse events were injection site reaction (26 patients) and fatigue (15 patients). Tetramer analysis revealed antigen-specific responses (defined as doubling of MART-1a-specific T cells from pretreatment to post-treatment) in 20, 60, and 40% of patients treated on schedules 1, 2, and 3, respectively. Vaccine treatment consisting of MART-1a peptide, Gag267-274, Montanide, and topical resiquimod was well-tolerated. The addition of the Gag267-274 xenoantigen was not associated with an increase in the response to MART-1a, whereas use of topical resiquimod was associated with a higher frequency of MART-1a-specific T-cell responses that did not meet statistical significance.

Objective: To illustrate a case of sensorineural hearing loss (SNHL) after immunotherapy based on T cell receptor (TCR) gene therapy using modified T cells recognizing melanoma antigen recognized by T cells 1 for disseminated melanoma., Patient: We present a 59-year-old woman with profound subacute bilateral SNHL including unilateral deafness after immunotherapy based on TCR gene therapy using modified T cells recognizing melanoma antigen recognized by T cells 1 for disseminated melanoma. Ten days after treatment, the patient developed hearing loss of 57 dB hearing loss air conduction at pure-tone average 0.5-1-2-4 kHz in the right ear, and >100 dB hearing loss air conduction at pure-tone average 0.5-1-2-4 in the left ear. The right ear recovered partially, while the left ear remained deaf, despite oral prednisolone (1.0 mg/kg) and salvage treatment with three transtympanic injections of 0.5 ml dexamethasone (4.0 mg/ml)., Conclusion: Based on our presented case and a vast amount of literature there is circumstantial evidence that TCR gene therapy for melanoma targets the perivascular macrophage-like melanocytes in the stria vascularis, resulting in SNHL. We suggest that SNHL after TCR gene therapy may be caused by a disruption of the blood-labyrinth-barrier and the endolymphatic potential and/or a sterile inflammation of the stria vascularis. In severe cases like our subject, we posit that endolymphatic hydrops or hair cell loss may cause irreversible and asymmetrical deafness. Steroid prophylaxis via transtympanic application is debatable.

We used four-color ImmunoSpot® assays, in conjunction with peptide pools that cover the sequence of tyrosinase (Tyr), melanoma-associated antigen A3 (MAGE-A3), melanocyte antigen/melanoma antigen recognized by T cells 1 (Melan-A/MART-1), glycoprotein 100 (gp100), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) to characterize the melanoma antigen (MA)-specific CD8 + cell repertoire in PBMC of 40 healthy human donors (HD). Tyr triggered interferon gamma (IFN-γ)-secreting CD8 + T cells in 25% of HD within 24 h of antigen stimulation ex vivo. MAGE-A3, Melan-A/MART-1, and gp100 also induced recall responses in 10%, 7.5%, and 2.5% of HD, respectively. At this time point, these CD8 + T cells did not yet produce GzB (granzyme B). However, they engaged in GzB production after 72 h of antigen stimulation. By this 72-h time point, 57.5% of the HD responded to at least one, and typically several, of the MA. A closer characterization of the Tyr-specific CD8 + T cell repertoire indicated that it was low-affinity, and to primarily entail a stem cell-like subpopulation. Collectively, our data reveal pre-existing endogenous T cell immunity against melanoma antigens in healthy donors, and analogous to natural autoantibodies, we have termed this "natural T cell autoreactivity".

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved in vitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated in vitro. To test this notion, we compared the TCR-Vα/Vβ (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated in vitro to that from ex vivo naïve T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naïve T cells and TILs, which showed the expected narrow TRAV repertoire, in vitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated in vitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells., (© 2019 Australasian Society for Immunology Inc.)

The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.

T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. However, though antigen specificity is a hallmark of adaptive immunity, TCRs themselves do not possess the high specificity of monoclonal antibodies. Although a necessary function of T cell biology, the resulting cross-reactivity presents a significant challenge for TCR-based therapeutic development, as it creates the potential for off-target recognition and immune toxicity. Efforts to enhance TCR specificity by mimicking the antibody maturation process and enhancing affinity can inadvertently exacerbate TCR cross-reactivity. Here we demonstrate this concern by showing that even peptide-targeted mutations in the TCR can introduce new reactivities against peptides that bear similarity to the original target. To counteract this, we explored a novel structure-guided approach for enhancing TCR specificity independent of affinity. Tested with the MART-1-specific TCR DMF5, our approach had a small but discernible impact on cross-reactivity toward MART-1 homologs yet was able to eliminate DMF5 cross-recognition of more divergent, unrelated epitopes. Our study provides a proof of principle for the use of advanced structure-guided design techniques for improving TCR specificity, and it suggests new ways forward for enhancing TCRs for therapeutic use., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Thymic selection constitutes the first checkpoint in T-cell development to purge autoreactive T cells. Most of our understanding of this process comes from animal models because of the challenges of studying thymopoiesis and how T cell receptor (TCR) specificity impacts thymocyte phenotype in humans. We developed a humanized mouse model involving the introduction of autoreactive TCRs and cognate autoantigens that enables the analysis of selection of human T cells in human thymic tissue in vivo . Here, we describe the thymic development of MART1-specific autoreactive CD8 + T cells that normally escape deletion and how their phenotype and survival are affected by introduction of the missing epitope in the hematopoietic lineage. Expression of the epitope in a fraction of hematopoietic cells, including all major types of antigen-presenting cells (APCs), led to profound yet incomplete deletion of these T cells. Upregulation of PD-1 upon antigen encounter occurred through the different stages of thymocyte development. PD-1 and CCR7 expression were mutually exclusive in both transgenic and non-transgenic thymocytes, challenging the view that CCR7 is necessary for negative selection in humans. In the presence of antigen, MART1-reactive T cells down-regulated TCR, CD3, CD8, and CD4 in the thymus and periphery. Moreover, expression of secondary TCRs influences MHC class I-restricted T cells to develop as CD4 + , particularly regulatory T cells. This new model constitutes a valuable tool to better understand the development of autoreactive T cells identified in different human autoimmune diseases and the role of different APC subsets in their selection.

Antigen recognition by T cells bearing αβ T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αβ TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Immunodeficient mice engrafted with human peripheral blood cells are promising tools for in vivo analysis of human patient individual immune responses. However, when human peripheral blood mononuclear cells (PBMCs) are transferred into NOG (NOD/Shi-scid, IL-2rg null ) mice, severe graft versus host disease (GVHD) hinders long term detailed analysis. Administration of human PBMCs into newly developed murine MHC class I- and class II-deficient NOG (NOG-dKO; NOG- Iab, B2m-double-knockout) mice showed sufficient engraftment of human immune cells with little sign of GVHD. Immunization with influenza vaccine resulted in an increase in influenza-specific human IgG Ab, indicating induction of antigen-specific B cells in the NOG-dKO mice. Immunization with human dendritic cells pulsed with HLA-A2 restricted cytomegalovirus peptide induced specific cytotoxic T cells, indicating the induction of antigen-specific T cells in the NOG-dKO mice. Adoptive cell therapies (ACTs) using melanoma antigen recognized by T cells (MART-1)-specific TCR-transduced activated T cells showed strong tumor growth inhibition in NOG-dKO mice without any sign of GVHD accompanied by preferential expansion of the transferred MART-1-specific T cells. ACTs using cultured human melanoma infiltrating T cells also showed anti-tumor effects against autologous melanoma cells in NOG-dKO mice, in which changes in human cancer phenotypes by immune intervention, such as increased CD271 expression, could be evaluated. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both the induction and effector phases of T-cell and B-cell responses for a longer period than regular NOG mice.

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.

A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor-expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation. Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935-43. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 899 ., (©2018 American Association for Cancer Research.)

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vβ) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vβ interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vβ association or direct contact with the ligand. More surprisingly, many Vα/Vβ interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45βY) was most prevalent. Further analysis of F45βY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vβ interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Increased numbers of CD8 + CD28 neg. T cells have been detected in the peripheral blood of patients with several types of malignancies. However, the role of these cells in anticancer immunity are not yet clear and CD8 + CD28 neg. T cells are a controversially discussed subpopulation reported both as immunosuppressive and cytotoxic. In this study, we examined the T cell receptor (TCR) repertoire and complementarity-determining region 3 sequences of CD28 neg. T cells in a melanoma patient with recurrent disease who achieved long-term disease-free status. As a result, the patient's oligoclonal CD8 + CD28 neg. T cell compartment holds TCRs that are public and specific for Melan-A as well as several public TCRs reported for common viral antigens. While over 80% of his CD8 + CD28 neg. T cells expressed a cytotoxicity marker, CD57, only 0.01% of CD8 + CD28 neg. T cells were positive for Foxp3. In conclusion, our results demonstrate that besides virus-specific also tumor-associated self-antigen targeting T cells accumulate in the CD28 neg. compartment of the immunological memory. Since the patient is in ongoing complete remission for more than 9 years, CD8 + CD28 neg. T cells with the Melan-A-specific TCR might contribute to antitumor immunity in this patient.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αβ heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (K D values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross-presenting antigens to CD8 + T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen-specific CD8 + T cell clones after 5 days of differentiation into monocytes-derived dendritic cells (MoDCs). Tagged-long peptides can be visualized in electron-dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14 + monocytes from two patients effectively activate a MelanA-specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor-specific immune response and should be taken into account for cancer immunotherapy., (© 2017 UICC.)

Background: Transplantation of hematopoietic stem cells (HSCs) from peripheral blood (PB) or cord blood (CB) is well established. HSCs from CB are associated with a lower risk of graft-versus-host disease (GVHD), but antigen-independent expanded CB- and PB-derived T cells can induce GVHD in allo-HSC recipients. CB-derived cells might be more suitable for adoptive immunotherapy as they have unique T-cell characteristics. Here, we describe functional differences between CB and PB T cells stimulated with different cytokine combinations involved in central T-cell activation., Study Design and Methods: Isolated CD8+ T cells from CB and PB were stimulated antigen independently with anti-CD3/CD28 stimulator beads or in an antigen-dependent manner with artificial antigen-presenting cells loaded with the HLA-A*02:01-restricted peptide of tumor-associated melanoma antigen recognized by T cells 1 (MART1). CB and PB T cells cultured in the presence of interleukin (IL)-7, IL-15, IL-12, and IL-21 were characterized for T-cell phenotype and specificity, that is, by CD107a, interferon-γ, tumor necrosis factor-α, and IL-2 expression., Results: After antigen-independent stimulation, activated CD8+ CB T cells exhibited stronger proliferation and function than those from PB. After antigenic stimulation, MART1-reactive CB T cells were naïve (CD45RA+CCR7+), cytotoxic, and highly variable in expressing homing marker CD62L. Addition of IL-21 resulted in increased T-cell proliferation, whereas supplementation with IL-12 decreased IL-21-induced expansion, but increased the functionality and cytotoxicity of CB and PB T cells., Conclusion: MART1-reactive CB T cells with a more naïve phenotype and improved properties for homing can be generated. The results contribute to better understanding the effects on GVHD and graft versus tumor., (© 2017 AABB.)

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy. Cancer Res; 77(24); 7083-93. ©2017 AACR ., (©2017 American Association for Cancer Research.)

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials., (© 2017 John Wiley & Sons Ltd.)

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Patients with cancer benefit increasingly from T-cell-based therapies, such as adoptive T-cell transfer, checkpoint blockade, or vaccination. We have previously shown that serial vaccinations with Melan-A MART-1 26-35 peptide, CpG-B, and incomplete Freund adjuvant (IFA) generated robust tumor-specific CD8 T-cell responses in patients with melanoma. Here, we describe the detailed kinetics of early- and long-term establishment of T-cell frequency, differentiation (into memory and effector cells), polyfunctionality, and clonotype repertoire induced by vaccination. Experimental Design: Twenty-nine patients with melanoma were treated with multiple monthly subcutaneous vaccinations consisting of CpG-B, and either the native/EAA ( n = 13) or the analogue/ELA ( n = 16) Melan-A MART-1 26-35 peptide emulsified in IFA. Phenotypes and functionality of circulating Melan-A-specific CD8 T cells were assessed directly ex vivo by multiparameter flow cytometry, and TCR clonotypes were determined ex vivo by mRNA transcript analyses of individually sorted cells. Results: Our results highlight the determining impact of the initial vaccine injections on the rapid and strong induction of differentiated effector T cells in both patient cohorts. Moreover, long-term polyfunctional effector T-cell responses were associated with expansion of stem cell-like memory T cells over time along vaccination. Dominant TCR clonotypes emerged early and persisted throughout the entire period of observation. Interestingly, one highly dominant clonotype was found shared between memory and effector subsets. Conclusions: Peptide/CpG-B/IFA vaccination induced powerful long-term T-cell responses with robust effector cells and stem cell-like memory cells. These results support the further development of CpG-B-based cancer vaccines, either alone or as specific component of combination therapies. Clin Cancer Res; 23(13); 3285-96. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.

Background: Indications to treat invasive melanoma with Mohs micrographic surgery (MMS) or analogous techniques with exhaustive microscopic margin assessment have not been defined., Objective: Identify clinical and histologic factors associated with subclinical spread of invasive melanoma., Methods: This retrospective, cross-sectional study evaluated 216 invasive melanomas treated with MMS and melanoma antigen recognized by T cells 1 immunostaining. Logistic regression models were used to correlate clinicopathologic risk factors with subclinical spread and construct a count prediction model., Results: Risk factors associated with subclinical spread by multivariate analysis included tumor localization on the head and neck (OR 3.28, 95% confidence interval [CI] 1.16-9.32), history of previous treatment (OR 4.18, 95% CI 1.42-12.32), age ≥65 (OR 4.45, 95% CI 1.29-15.39), and ≥1 mitoses/mm 2 (OR 2.63, 95% CI 1.01-6.83). Tumor thickness and histologic subtype were not associated with subclinical spread. The probability of subclinical spread increased per number of risk factors, ranging from 9.22% (95% CI 2.57%-15.86%) with 1 factor to 80.32% (95% CI 68.13%-92.51%) with 5 factors., Limitations: This study was conducted at a single academic institution with a small study population using a retrospective study design that was subject to potential referral bias., Conclusion: Clinical and histologic factors identify invasive melanomas that are at increased risk for subclinical spread and might benefit from MMS or analogous techniques prior to reconstruction., (Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Mohs micrographic surgery (MMS) with melanoma antigen recognized by T-cell (MART-1) immunostaining is an effective treatment of cutaneous melanoma., Objective: To determine the efficacy of MMS with MART-1 immunostain in the management of invasive and in situ melanoma., Methods and Materials: A retrospective cohort study evaluated 2,114 melanomas in 1,982 patients excised using MMS and MART-1 immunostain. The margins required for excision were calculated based on Breslow thickness, location, and size. Survival and local recurrence rates were calculated and compared with those of historical controls., Results: The mean follow-up period was 3.73 years. Local recurrence was identified in 0.49% (7/1,419) of primary melanomas. Approximately 82% of melanomas were excised with ≤6-mm margins. The surgical margin was significantly related to tumor location and size but not to Breslow thickness. The five-year Kaplan-Meier local recurrence and disease-specific survival rates were 0.59 ± 0.30 and 98.53 ± 0.42, respectively. Mohs micrographic surgery with MART-1 immunostain achieved lower local recurrence rates and equivalent or higher Kaplan-Meier survival rates than conventional wide local excision., Conclusion: Mohs micrographic surgery with MART-1 immunostain is an effective treatment of melanoma as evidenced by low local recurrence rates. It offers the advantage of more tissue-conserving margins than those recommended for conventional excision.

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

Recent high throughput sequencing analysis has revealed that the TCRβ repertoire is largely different between CD8(+) and CD4(+) T cells. Here, we show that the transduction of SIG35α, the public chain-centric HLA-A*02:01(A2)/MART127-35 TCRα hemichain, conferred A2/MART127-35 reactivity to a substantial subset of both CD8(+) and CD4(+) T cells regardless of their HLA-A2 positivity. T cells individually reconstituted with SIG35α and different A2/MART127-35 TCRβ genes isolated from CD4(+) or CD8(+) T cells exhibited a wide range of avidity. Surprisingly, approximately half of the A2/MART127-35 TCRs derived from CD4(+) T cells, but none from CD8(+) T cells, were stained by A2/MART127-35 monomer and possessed broader cross-reactivity. Our results suggest that the differences in the primary structure of peripheral CD4(+) and CD8(+) TCRβ repertoire indeed result in the differences in their ability to form extraordinarily high avidity T cells which would otherwise have been deleted by central tolerance.

Purpose: Cancer vaccines aim to generate and maintain antitumor immune responses. We designed a phase I/IIa clinical trial to test a vaccine formulation composed of Montanide ISA-51 (Incomplete Freund's Adjuvant), LAG-3Ig (IMP321, a non-Toll like Receptor agonist with adjuvant properties), and five synthetic peptides derived from tumor-associated antigens (four short 9/10-mers targeting CD8 T-cells, and one longer 15-mer targeting CD4 T-cells). Primary endpoints were safety and T-cell responses., Experimental Design: Sixteen metastatic melanoma patients received serial vaccinations. Up to nine injections were subcutaneously administered in three cycles, each with three vaccinations every 3 weeks, with 6 to 14 weeks interval between cycles. Blood samples were collected at baseline, 1-week after the third, sixth and ninth vaccination, and 6 months after the last vaccination. Circulating T-cells were monitored by tetramer staining directly ex vivo, and by combinatorial tetramer and cytokine staining on in vitro stimulated cells., Results: Side effects were mild to moderate, comparable to vaccines with Montanide alone. Specific CD8 T-cell responses to at least one peptide formulated in the vaccine preparation were found in 13 of 16 patients. However, two of the four short peptides of the vaccine formulation did not elicit CD8 T-cell responses. Specific CD4 T-cell responses were found in all 16 patients., Conclusions: We conclude that vaccination with IMP321 is a promising and safe strategy for inducing sustained immune responses, encouraging further development for cancer vaccines as components of combination therapies., (©2015 American Association for Cancer Research.)

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy.

In 1985, John Monaco--the discoverer of LMP-2 and -7, the inducible components of the immunoproteasome--asked his advanced immunology class as to why the MHC region contained not only structural genes, but several others as well, whose functions were then unknown. As we drew a blank, he quipped: perchance because many of the MHC genes are induced by IFN-γ! The ensuing three decades have witnessed the unveiling of the profound fundamental and clinical implications of that classroom tête-à-tête. Amongst its multitudinous effects, IFN-γ induces genes enhancing antigen processing and presentation to T cells; such as those encoding cellular proteases and activators of proteases. In this issue, Keller et al. [Eur. J. Immunol. 2015. 45: 3257-3268] demonstrate that the limited success of MART-1/Melan-A-targeted immunotherapy in melanoma patients could be due to inefficient MART-1(26-35) presentation, owing to the proteolytic activities of IFN-γ-inducible β2i/MECL-1, proteasome activator 28 (PA28), and endoplasmic reticulum-associated aminopeptidase-associated with antigen processing (ERAP). Specifically, whilst β2i and PA28 impede MART-1(26-35) liberation from its precursor protein, ERAP-1 degrades this epitope. Hence, critical to effective cancer immunotherapy is deep knowledge of T-cell-targeted tumor antigens and how cellular proteases generate protective epitope(s) from them, or destroy them., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Endogenous tumor-specific T cells are detectable in patients with different tumor types including malignant melanoma (MM). They can control tumor growth, have impact on patient survival and correlate with improved clinical response to immune checkpoint therapy. Thus, they may represent a potent biomarker for respective treatment decisions. So far, major target antigens of endogenous MM-reactive T cells have not been determined systematically. Instead, autoantibodies are discussed as surrogate parameter for MM-specific T cells. Throughout a period of more than 60 days after tumor resection, we therefore determined in 38 non-metastasized primary MM patients and in healthy individuals by IFNγ ELISpot and bead-based fluorescent multiplex assay major target antigens of spontaneous T cell and humoral responses using a broad panel of MM antigens and assessed the presence and suppressive impact of MM-reactive regulatory T cells (Tregs). We show that MM-reactive T cells are frequent in MM patients, transiently increase after tumor removal and are mostly directed against Melan-A/MART-1, Tyrosinase, NA17-A and p53. MM-specific Tregs were only detected in few patients and inhibited MM-reactive T cells particularly early after tumor resection. Tumor-specific autoantibodies occurred in most patients, but did not correlate with T cell responses. Thus, endogenous antibodies may not be reliable surrogate parameters of MM-reactive T cells.

Dendritic cells (DC) vaccination is a potent therapeutic approach for inducing tumor-directed immunity, but challenges remain. One of the particular interest is the induction of an immune response targeting multiple (unknown) tumor-associated antigens (TAA), which requires a polyvalent source of TAA. Previously, we described the preferred use of apoptotic cell-derived blebs over the larger apoptotic cell remnants, as a source of TAA for both in situ loading of skin-resident DC and in vitro loading of monocyte-derived DC (MoDC). Recent reports suggest that MoDC cultured in the presence of GM-CSF supplemented with IFNα (IFNα MoDC), as compared to IL-4 (IL-4 MoDC), have an increased capacity to cross-present antigen to CD8(+) T cells. As culture conditions, maturation methods and antigen sources differ between the conducted studies, we analyzed the functional differences between IL-4 MoDC and IFNα MoDC, loaded with blebs, in a head-to-head comparison using commonly used protocols. Our data show that both MoDC types are potent (cross-) primers of CD8(+) T cells. Whereas IFNα MoDC were more potent in their capacity to cross-present a 25-mer MART-1 synthetic long peptide (SLP) to a MART-1aa26-35 recognizing CD8(+) T cell line, IL-4 MoDC proved more potent cross-primers of antigen-specific CD8(+) T cells when loaded with blebs. The latter is likely due to the observed greater capacity of IL-4 MoDC to ingest apoptotic blebs. In conclusion, our data indicate the use of IFNα MoDC over IL-4 MoDC in the context of DC vaccination with SLP, whereas IL-4 MoDC are preferred for vaccination with bleb-derived antigens.

Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs.

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.