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Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advanced solid tumors; however, many patients experience treatment resistance. In preclinical models 4-1BB co-stimulation synergizes with ICI by activating cytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize the mechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecific antibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhanced anti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional 4-1BB agonist activity that was dependent on simultaneous binding to PD-L1. mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of 4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB strongly enhanced T-cell proliferation, cytokine production and antigen-specific cytotoxicity using primary mouse cells in vitro. Furthermore, mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38 models in vivo, leading to the rejection of CT26 tumors that were unresponsive to PD-L1 blockade alone. Anti-tumor activity was associated with increased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumor microenvironment and tumor-draining lymph nodes. In immunocompetent tumor-free mice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into the liver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and 4-1BB with a bispecific antibody may therefore address key limitations of first generation 4-1BB-agonistic antibodies, and may provide a novel approach to improve PD-1/PD-L1 checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) represents a challenge in oncology, with limited treatment options for advanced-stage patients. Chimeric antigen receptor T cell (CAR T) therapy targeting mesothelin (MSLN) shows promise, but challenges such as the hostile immunosuppressive tumor microenvironment (TME) hinder its efficacy. This study explores the synergistic potential of combining proton radiation therapy (RT) with MSLN-targeting CAR T therapy in a syngeneic PDAC model. Proton RT significantly increased MSLN expression in tumor cells and caused a significant increase in CAR T cell infiltration into tumors. The combination therapy reshaped the immunosuppressive TME, promoting antitumorigenic M1 polarized macrophages and reducing myeloid-derived suppressor cells (MDSC). In a flank PDAC model, the combination therapy demonstrated superior attenuation of tumor growth and improved survival compared to individual treatments alone. In an orthotopic PDAC model treated with image-guided proton RT, tumor growth was significantly reduced in the combination group compared to the RT treatment alone. Further, the combination therapy induced an abscopal effect in a dual-flank tumor model, with increased serum interferon-γ levels and enhanced proliferation of extratumoral CAR T cells. In conclusion, combining proton RT with MSLN-targeting CAR T therapy proves effective in modulating the TME, enhancing CAR T cell trafficking, and exerting systemic antitumor effects. Thus, this combinatorial approach could present a promising strategy for improving outcomes in unresectable PDAC. [ABSTRACT FROM AUTHOR]

As a strategy to improve the therapeutic success of chimeric antigen receptor T cells (CART) directed against solid tumors, we here test the combinatorial use of CART and IMSA101, a newly developed stimulator of interferon genes (STING) agonist. In two syngeneic tumor models, improved overall survival is observed when mice are treated with intratumorally administered IMSA101 in addition to intravenous CART infusion. Transcriptomic analyses of CART isolated from tumors show elevated T cell activation, as well as upregulated cytokine pathway signatures, in particular IL-18, in the combination treatment group. Also, higher levels of IL-18 in serum and tumor are detected with IMSA101 treatment. Consistent with this, the use of IL-18 receptor negative CART impair anti-tumor responses in mice receiving combination treatment. In summary, we find that IMSA101 enhances CART function which is facilitated through STING agonist-induced IL-18 secretion. It has been previously suggested that STING agonists can improve response to CAR-T therapy. Here the authors report the characterization of the STING agonist IMSA101, showing that STING-induced IL18 secretion enhances CAR-T activity in preclinical cancer models. [ABSTRACT FROM AUTHOR]

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

The article presents a study which explores how chimeric antigen receptor T cells (CARTs) act as adjuvant therapy for unresectable adenocarcinoma. It mentions that study finds that local delivery of CARTs in a fibrin glue–based carrier was effective in clearing residual cancer cells following incomplete surgery.

Integrin beta 7 (β7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell–cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific β7 expression in immune cells after T-cell-enriched mixed lymphocyte culture—finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients' whole blood (WB) was treated with Kit-M (granulocyte–macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of β7-expressing T-cells, degranulating and intracellular cytokine-producing β7-expressing immune cells and, in patients' samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of β7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) β7-expressing immune cells. Furthermore, β7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide due to high recurrence rates after curative treatment and being frequently diagnosed at an advanced stage. Immune-checkpoint inhibition (ICPI) has yielded impressive clinical successes in a variety of solid cancers, however results in treatment of HCC have been modest. Vaccination could be a promising treatment to synergize with ICPI and enhance response rates. Cancer testis antigens (CTAs) were recently discovered to be widely expressed in HCC and expression in macroscopically tumor-free tissues correlated with recurrence, implying the presence of micro-satellites. To determine whether CTAs are immunogenic in HCC patients, we analyzed systemic T-cell and humoral responses against seven CTAs in 38 HCC patients using a multitude of techniques; flowcytometry, ELISA and whole antigen and peptide stimulation assays. CTA-specific T-cells were detected in all (25/25) analyzed patients, of which most had a memory phenotype but did not exhibit unequivocal signs of chronic stimulation or recent antigen encounter. Proliferative CD4+ and CD8+ T-cell responses against these CTAs were found in 14/16 analyzed HCC patients. CTA-peptide stimulation-induced granzyme B, IL2, and TNFa in 8/8 analyzed patients, including two MAGEA1 peptides included based on in silico prediction. Finally, IgG responses were observed in 13/32 patients, albeit with low titers. The presence of CD4+ and CD8+ T-cells and IgG responses shows the immunogenicity of these CTAs in HCC-patients. We hypothesize that vaccines based on these tumorspecific antigens may boost preexisting CTA-specific immunity and could enhance therapeutic efficacy of ICPI in advanced HCC. [ABSTRACT FROM AUTHOR]

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies. [ABSTRACT FROM AUTHOR]

This study shows that melanoma-associated fibroblasts (MAFs) suppress cytotoxic T lymphocyte (CTL) activity and reveals a pivotal role played by arginase in this phenomenon. MAFs and normal dermal fibroblasts (DFs) were isolated from surgically resected melanomas and identified as Melan-A-/gp100-/FAP+ cells. CTLs of healthy blood donors were activated in the presence of MAF- and DF-conditioned media (CM). Markers of successful CTL activation, cytotoxic degranulation, killing activity and immune checkpoint regulation were evaluated by flow cytometry, ELISPOT, and redirected killing assays. Soluble mediators responsible for MAF-mediated effects were identified by ELISA, flow cytometry, inhibitor assays, and knock-in experiments. In the presence of MAF-CM, activated/non-naïve CTLs displayed dysregulated ERK1/2 and NF-κB signaling, impeded CD69 and granzyme B production, impaired killing activity, and upregulated expression of the negative immune checkpoint receptors TIGIT and BTLA. Compared to DFs, MAFs displayed increased amounts of VISTA and HVEM, a known ligand of BTLA on T cells, increased l-arginase activity and CXCL12 release. Transgenic arginase over-expression further increased, while selective arginase inhibition neutralized MAF-induced TIGIT and BTLA expression on CTLs. Our data indicate that MAF interfere with intracellular CTL signaling via soluble mediators leading to CTL anergy and modify immune checkpoint receptor availability via l-arginine depletion. [ABSTRACT FROM AUTHOR]

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8+ T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema. [ABSTRACT FROM AUTHOR]

HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade with anti-PD-1 antibodies is showing great promise for patients with metastatic melanoma and other malignancies, but despite good responses by some patients who achieve partial or complete regression, many others still do not respond. Here, we sought peripheral blood T-cell biomarker candidates predicting treatment outcome in 75 stage IV melanoma patients treated with anti-PD-1 antibodies. We investigated associations with clinical response, progression-free survival (PFS) and overall survival (OS). Univariate analysis of potential biological confounders and known biomarkers, and a multivariate model, was used to determine statistical independence of associations between candidate biomarkers and clinical outcomes. We found that a lower than median frequency of peripheral PD-1+CD56+ T-cells was associated with longer OS (p = 0.004), PFS (p = 0.041) and superior clinical benefit (p = 0.009). However, neither frequencies of CD56-CD4+ nor CD56-CD8+ T-cells, nor of the PD-1+ fraction within the CD4 or CD8 subsets was associated with clinical outcome. In a multivariate model with known confounders and biomarkers only the M-category (HR, 3.11; p = 0.007) and the frequency of PD-1+CD56+ T-cells (HR, 2.39; p = 0.028) were identified as independent predictive factors for clinical outcome under PD-1 blockade. Thus, a lower than median frequency of peripheral blood PD-1+CD56+ T-cells prior to starting anti-PD-1 checkpoint blockade is associated with superior clinical response, longer PFS and OS of stage IV melanoma patients. [ABSTRACT FROM AUTHOR]

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8 T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells. [ABSTRACT FROM AUTHOR]

Thymomas with immunodeficiency (formerly Good's syndrome) are a rare acquired disease of combined T- and B-cell immunodeficiency accompanying a thymoma. Recurrent opportunistic infections associated with disorders of both humoral and cell-mediated immunity frequently accompany this rare primary, adult-onset immunodeficiency. This is a report of a case of a thymoma with immunodeficiency in a 65-year-old male patient who developed recurrent oral herpetic infections. He consulted us about recurrent vesiculo-ulcerative lesions on his tongue, lower lip, and buccal mucosa. Results of laboratory examinations indicated hypogammaglobulinemia accompanied by diminished B cells in the peripheral blood, which is consistent with the characteristic features of a thymoma with immunodeficiency. After a diagnosis confirming herpes simplex virus infection, systemic antiviral therapy was administered, which was effective for his vesiculo-ulcerative lesions at follow-up. When an intractable infection accompanied by a thymoma is encountered, increased awareness about the clinical and immunological profiles of this primary immunodeficiency may help in its early diagnosis, thereby preventing mortality. [ABSTRACT FROM AUTHOR]

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models. [ABSTRACT FROM AUTHOR]

Introduction: Myeloid leukaemic blasts can be converted into leukaemia-derived dendritic cells (DCleu), characterised by the simultaneous expression of dendritic- and leukaemia-associated antigens, which have the competence to prime and enhance (leukaemia-specific) immune responses with the whole leukaemic antigen repertoire. To display and further specify dendritic cell (DC)- and DCleu-mediated immune responses, we analysed the interferon gamma (IFNy) secretion of innate and adaptive immune cells. Methods: DC/DCleu were generated from leukaemic whole blood (WB) with (blast)modulatory Kit-I (granulocyte-macrophage colony-stimulating factor [GM-CSF] + Picibanil [OK-432]) and Kit-M (GM-CSF + prostaglandin E1) and were used to stimulate T cell-enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay. Initiated IFNy secretion of T, NK, CIK, and iNKT cells was investigated with a cytokine secretion assay (CSA). IFNy positivity was additionally evaluated with an intracellular cytokine assay (ICA). Recent activation of leukaemia-specific cells was verified through addition of leukaemia-associated antigens (LAA; WT-1 and Prame) Results: We found Kit-I and Kit-M competent to generate mature DC and DCleu from leukaemic WB without induction of blast proliferation. Stimulation of immunoreactive cells with DC/DCleu regularly resulted in an increased anti-leukaemic cytotoxicity and increased IFNy secretion of T, NK, and CIK cells, pointing to the significant role of DC/DCleu in leukaemia-specific alongside anti-leukaemic reactions. Interestingly, an addition of LAA did not further increase IFNy secretion, suggesting an efficient activation of leukaemia-specific cells. Here, both the CSA and ICA yielded comparable frequencies of IFNy-positive cells. Remarkably, the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in TCD3+, TCD4+, TCD8+, and NKCD56+ cells. Conclusion: Ultimately, the IFNy secretion of innate and adaptive immune cells appeared to be a suitable parameter to assess and monitor the efficacy of in vitro and potentially in vivo acute myeloid leukaemia immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy-secreting cells. In respect to our studies on DC-based immunomodulation, we were able to display the potential of DC/DCleu to induce or improve leukaemia-specific and anti-leukaemic activity. [ABSTRACT FROM AUTHOR]

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8+ T cells and CD11b+ conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8+ T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound. [ABSTRACT FROM AUTHOR]

Tumor cells can develop immune escape mechanisms to bypass T cell recognition, e.g., antigen loss or downregulation of the antigen presenting machinery, which represents a major challenge in adoptive T cell therapy. To counteract these mechanisms, we transferred not only one, but two receptors into the same T cell to generate T cells expressing two additional receptors (TETARs). We generated these TETARs by lentiviral transduction of a gp100-specific T cell receptor (TCR) and subsequent electroporation of mRNA encoding a second-generation CSPG4-specific chimeric antigen receptor (CAR). Following pilot experiments to optimize the combined DNA- and RNA-based receptor transfer, the functionality of TETARs was compared to T cells either transfected with the TCR only or the CAR only. After transfection, TETARs clearly expressed both introduced receptors on their cell surface. When stimulated with tumor cells expressing either one of the antigens or both, TETARs were able to secrete cytokines and showed cytotoxicity. The confirmation that two antigen-specific receptors can be functionally combined using two different methods to introduce each receptor into the same T cell opens new possibilities and opportunities in cancer immunotherapy. For further evaluation, the use of these TETARs in appropriate animal models will be the next step towards a potential clinical use in cancer patients. [ABSTRACT FROM AUTHOR]

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8+ T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8+ T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8+ T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant. [ABSTRACT FROM AUTHOR]

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cellsin vitrowith EC50values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+T cells and TEMtype cells but not Tregsand display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. [ABSTRACT FROM PUBLISHER]

We have developed a highly versatile platform for the systematic retrieval of T-cell receptors (TCRs) from single-antigen-reactive T cells and for characterization of their function and specificity. This approach enables rapid extraction of multiple TCRs from repertoires in individuals and not only broadens the diversity of TCRs suitable for clinical use, but also sets the stage for actively personalized immunotherapeutic strategies. [ABSTRACT FROM PUBLISHER]

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches. [Display omitted] • Lymph nodes contain non-circulating, tissue-derived UTCs that immigrate via lymphatics • Heterogeneity of tissue-derived UTCs imprints immune responses in lymph nodes • UTCs form functional units across TCR-based lineages • UTCs within functional units share location, function, and homeostatic niche Lymphatic vessels are immunological highways that connect lymph nodes to peripheral tissues. Ataide et al. found that tissue-derived UTCs also continuously migrate via this route. This migration pattern leads to characteristic innate and adaptive immune responses that differ between lymph nodes, mirrors the drained tissue, and is based on the immediate effector function of UTCs. [ABSTRACT FROM AUTHOR]

Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology. [Display omitted] • Determined differentiation trajectory and location of Tex cells and their precursors • cDC1s are essential for viral control but not for Tpex cell expansion • cDC1s maintain Tpex cell niches in an MHC-I-dependent manner • cDC1s limit activation-driven T cell exhaustion and immunopathology Dähling et al. establish the role of cDCs for immunotherapy and find that cDC1s are required to mediate viral control. cDC1s provide a niche to maintain the precursors of exhausted T (Tpex) cell population and prevent their overactivation and subsequent immunopathology. These findings reveal the importance of cDC1s in maintaining Tpex cells to balance viral control, exhaustion, and immunopathology. [ABSTRACT FROM AUTHOR]