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Abstract After activation, some invariant natural killer T (iNKT) cells are differentiated into Klrg1+ long-lived effector NKT1 cells. However, the regulation from the effector phase to the memory phase has not been elucidated. Zeb2 is a zinc finger E homeobox-binding transcription factor and is expressed in a variety of immune cells, but its function in iNKT cell differentiation remains also unknown. Here, we show that Zeb2 is dispensable for development of iNKT cells in the thymus and their maintenance in steady state peripheral tissues. After ligand stimulation, Zeb2 plays essential roles in the differentiation to and maintenance of Klrg1+ Cx3cr1+GzmA+ iNKT cell population derived from the NKT1 subset. Our results including single-cell-RNA-seq analysis indicate that Zeb2 regulates Klrg1+ long-lived iNKT cell differentiation by preventing apoptosis. Collectively, this study reveals the crucial transcriptional regulation by Zeb2 in establishment of the memory iNKT phase through driving differentiation of Klrg1+ Cx3cr1+GzmA+ iNKT population.

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific T cell receptor (TCR)-engineered T (TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.

IntroductionCancer is categorized into two types based on the microenvironment: cold and hot tumors. The former is challenging to stimulate through immunity. The immunogenicity of cancer relies on the quality and quantity of cancer antigens, whether recognized by T cells or not. Successful cancer immunotherapy hinges on the cancer cell type, antigenicity and subsequent immune reactions. The T cell response is particularly crucial for secondary epitope spreading, although the factors affecting these mechanisms remain unknown. Prostate cancer often becomes resistant to standard therapy despite identifying several antigens, placing it among immunologically cold tumors. We aim to leverage prostate cancer antigens to investigate the potential induction of epitope spreading in cold tumors. This study specifically focuses on identifying factors involved in secondary epitope spreading based on artificial adjuvant vector cell (aAVC) therapy, a method established as invariant natural killer T (iNKT) -licensed DC therapy.MethodsWe concentrated on three prostate cancer antigens (prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP)). By introducing allogeneic cells with the antigen and murine CD1d mRNA, followed by α-galactosylceramide (α-GalCer) loading, we generated five types of aAVCs, i.e, monovalent, divalent and trivalent antigen-expressing aAVCs and four types of prostate antigen-expressing cold tumors. We evaluated iNKT activation and antigen-specific CD8+ T cell responses against tumor cells prompted by the aAVCs.ResultsOur study revealed that monovalent aAVCs, expressing a single prostate antigen, primed T cells for primary tumor antigens and also induced T cells targeting additional tumor antigens by triggering a tumor antigen-spreading response. When we investigated the immune response by trivalent aAVC (aAVC-PROS), aAVC-PROS therapy elicited multiple antigen-specific CD8+ T cells simultaneously. These CD8+ T cells exhibited both preventive and therapeutic effects against tumor progression.ConclusionsThe findings from this study highlight the promising role of tumor antigen-expressing aAVCs, in inducing efficient epitope spreading and generating robust immune responses against cancer. Our results also propose that multivalent antigen-expressing aAVCs present a promising therapeutic option and could be a more comprehensive therapy for treating cold tumors like prostate cancer.

Abstract Coronaviruses regularly cause outbreaks of zoonotic diseases characterized by severe pneumonia. The new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the global pandemic disease COVID-19 that began at the end of 2019 and spread rapidly owing to its infectious nature and rapidly progressing pneumonia. Although the infectivity of SARS-CoV-2 is high, indicated by the worldwide spread of the disease in a very short period, many individuals displayed only subclinical infection, and some of them transmitted the disease to individuals who then developed a severe symptomatic infection. Furthermore, there are differences in the severity of infection across countries, which can be attributed to factors such as the emergence of viral mutations in a short period of time as well as to the immune responses to viral factors. Anti-viral immunity generally consists of neutralizing antibodies that block viral infection and cytotoxic CD8+ T cells that eliminate the virus-infected cells. There is compelling evidence for the role of neutralizing antibodies in protective immunity in SARS-CoV-2 infection. However, the role of CD4+ and CD8+ T cells after the viral entry is complex and warrants a comprehensive discussion. Here, we discuss the protection afforded by cellular immunity against initial infection and development of severe disease. The initial failure of cellular immunity to control the infection worsens the clinical outcomes and functional profiles that inflict tissue damage without effectively eliminating viral reservoirs, while robust T cell responses are associated with mild outcomes. We also discuss persistent long-lasting memory T cell-mediated protection after infection or vaccination, which is rather complicated as it may involve SARS-CoV-2-specific cytotoxic T lymphocytes or cross-reactivity with previously infected seasonal coronaviruses, which are largely related to HLA genotypes. In addition, cross-reactivity with mutant strains is also discussed. Lastly, we discuss appropriate measures to be taken against the disease for immunocompromised patients. In conclusion, we provide evidence and discuss the causal relationship between natural infection- or vaccine-mediated memory T cell immunity and severity of COVID-19. This review is expected to provide a basis to develop strategies for the next generation of T cell-focused vaccines and aid in ending the current pandemic.

Background A key to success of cancer immunotherapy is the amplification and sustenance of various effector cells. The hallmark of prominent antitumor T cells is their long-term effector function. Although interleukin (IL)-2 is an attractive cytokine, several attempts have been made towards developing IL-2 modalities with improved effectiveness and safety that enhance natural killer (NK) cells or T cells in cancer models. However, whether such IL-2 modalities can simultaneously support long-term innate and adaptive immunity, particularly stem-like memory, has not been shown. To resolve this issue, we compared the antitumor cellular mechanism with two IL-2/anti-IL-2 complexes (IL-2Cxs) administered in combination with a therapeutic cancer vaccine, which we had previously established as an in vivo dendritic cell-targeting therapy.Methods Two types of IL-2Cxs, CD25-biased IL-2Cx and CD122-biased IL-2Cx, together with a Wilms’ tumor 1-expressing vaccine, were evaluated in a leukemic model. The immunological response and synergistic antitumor efficacy of these IL-2Cxs were then evaluated.Results When CD25-biased or CD122-biased IL-2Cxs in combination with the vaccine were assessed in an advanced-leukemia model, the CD122-biased IL-2Cx combination showed 100% survival, but the CD25-biased IL-2Cx did not. We first showed that invariant natural killer T (NKT) 1 cells are predominantly activated by CD122-biased IL-2Cx. In addition, in-depth analysis of immune responses by CD122-biased IL-2Cx in lymphoid tissues and the tumor microenvironment revealed a dramatic increase in the distinct subsets of NK and CD8+ T cells with stem-like phenotype (CD27+Sca-1hi, CXCR3hi, CD127+TCF-1+T-bet+ Eomes+). Moreover, CD122-biased IL-2Cx combination therapy maintained long-term memory CD8+ T cells capable of potent antitumor protection. After the high dimensional profiling analysis of NK and CD8+T cells, principal component analysis revealed that the stem-like-NK cell and stem-like-CD8+T cell state in the combination were integrated in the same group.Conclusions CD122-biased IL-2Cx combined with the vaccine can induce a series of reactions in the immune cascade, including activation of not only NKT1 cells, but also NK and CD8+ T cells with a stem-like memory phenotype. Since it can also lead to a long-term, strong antitumor response, the combination of CD122-biased IL-2Cx with a vaccine may serve as a potential and competent strategy for patients with advanced cancer.

Kanako Shimizu et al. identify HLA-A24 high-binding epitopes in the SARS-CoV-2 spike region and investigate their cross-reactivity with CD8+ T cell receptors. These results provide further insight into CD8+ T cell cross-reactivity toward SARS-CoV-2 and may be useful in future strategies for vaccine development.

CCL5 is an important chemokine for modulation of inflammatory responses, but how CCL5 expression is regulated is still unclear. Here the authors show that the CCL5 locus contains two enhancers, with the proximal enhancer being responsible for homeostatic expression and the distal enhancer enforcing inducibility, while both enhancers are modulated by RUNX3.

Both natural killer T (NKT) and natural killer (NK) cells are innate cytotoxic lymphoid cells that produce inflammatory cytokines and chemokines, and their role in the innate immune response to tumors and microorganisms has been investigated. Especially, emerging evidence has revealed their status and function in the tumor microenvironment (TME) of tumor cells. Some bacteria producing NKT cell ligands have been identified to exert antitumor effects, even in the TME. By contrast, tumor-derived lipids or metabolites may reportedly suppress NKT and NK cells in situ. Since NKT and NK cells recognize stress-inducible molecules or inhibitory molecules on cancer cells, their status or function depends on the balance between inhibitory and activating receptor signals. As a recent strategy in cancer immunotherapy, the mobilization or restoration of endogenous NKT or NK cells by novel vaccines or therapies has become a focus of research. As a new biological evidence, after activation, effector memory-type NKT cells lasted in tumor-bearing models, and NK cell-based immune checkpoint inhibition potentiated the enhancement of NK cell cytotoxicity against cancer cells in preclinical and clinical trials. Furthermore, several new modalities based on the characteristics of NKT and NK cells, including artificial adjuvant vector cells, chimeric antigen receptor-expressing NK or NKT cell therapy, or their combination with immune checkpoint blockade have been developed. This review examines challenges and future directions for improving these therapies.

Summary: Although PD-L1 expression on tumor is related to the prognosis of immune checkpoint blockade (ICB) therapy, a recent study also demonstrated clinical benefits even in patients without PD-L1 expression. To understand the relationship between innate resistance and antitumor cytotoxic T lymphocyte (CTL) responses especially against neoantigens, the interaction between PD-L1+ or genetically PD-L1-deleted colorectal tumors and CTLs was assessed under an ICB therapy, finding the robust CTL activation in PD-L1-deleted tumor-bearing mice. Using antigen libraries based on immunogenomics, we identified three H2-Kb-restricted, somatic-mutated immunogenic neoantigens by utilizing enhanced CTLs responses due to PD-L1 deficiency. Furthermore, we identified three T cell receptor (TCR) repertoires relevant to the neoantigens, confirming the response of TCR-gene-transduced CTLs to parental tumor cells. Notably, neoantigen-pulsed dendritic cell (DC) therapy reversed the tumor tolerance. Thus, innate resistance of tumors determines their responsiveness to neoantigens and mixed neoantigen peptides may be useful in DC therapy against innate resistance type tumor.

Background: Oxatomide, a H1-receptor antagonist, exerts besides its well-known anti-allergic potential an array of anti-inflammatory activities. We wondered whether oxatomide might influence the release of IL-8 from human epithelial cells activated with agonists of TLR2, which mainly expresses on airway epithelial cells. Methodology: We used the human lung epithelial cell line A549 and primary Human nasal epithelial cell line for our in vitro studies. IL-8 releases from these cell lines were determined by IL-8 enzyme immunoassay. NF-kB was analysed by Luciferase reporter assay. Confluent epithelial cell monolayer were pre-incubated with oxatomide for 30 min and afterwards activated with lipoprotein as a TLR2 agonist for 24 h. Results: Epithelial cells stimulated with lipoprotein showed a significantly increased release of IL-8. Pre-incubation with oxatomide diminished the IL-8 release from cells activated with lipoprotein in a significant manner. Furthermore, activity of the NF-kB was determined by luciferase reporter assy. Besides, oxatomide inhibits expression of MIP2, a homologue of human IL-8, and neutrophilic infiltration in nasal membrane of mice intranasally exposed with lipoprotein. These results suggest that oxatomide reduced the release of IL-8 from respiratory epithelial cells stimulated with lipoprotein. Conclusion: Therefore, oxatomide might exert anti-inflammatory effects beyond its H1-receptor antagonistic activity in the course of inflammatory respiratory tract disorders such as acute bacterial rhinitis.

Abstract Background Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. Methods PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. Results Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. Conclusion We present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).

Granzyme A (GzmA), together with perforin, are well-known for their cytotoxic activity against tumor or virus-infected cells. In addition to this cytotoxic function, GzmA stimulates several immune cell types and induces inflammation in the absence of perforin, however, its effect on the dendritic cell (DC) is unknown. In the current study, we showed that recombinant GzmA induced the phenotypic maturation of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), but not their apoptosis. Particularly, GzmA made pDCs more functional, thus leading to production of type I interferon (IFN) via the TLR9-MyD88 pathway. We also demonstrated that GzmA binds TLR9 and co-localizes with it in endosomes. When co-administered with antigen, GzmA acted as a powerful adjuvant for eliciting antigen-specific cytotoxic CD8+ T lymphocytes (CTLs) that protected mice from tumor challenge. The induction of CTL was completely abolished in XCR1+ DC-depleted mice, whereas it was reduced to less than half in pDC-depleted or IFN-α/β receptor knockout mice. Thus, CTL cross-priming was dependent on XCR1+cDC and also type I IFN, which was produced by GzmA-activated pDCs. These results indicate that GzmA -stimulated pDCs enhance the cross-priming activity of cDCs in situ. We also showed that the adjuvant effect of GzmA is superior to CpG-ODN and LPS. Our findings highlight the ability of GzmA to bridge innate and adaptive immune responses via pDC help and suggest that GzmA may be useful as a vaccine adjuvant.

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4+ T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8+ T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.

Immune checkpoint blockade therapy has prevailed for several types of cancer; however, its effectiveness as a single therapy is still limited. In principle, dendritic cells (DCs) should be able to control the post-therapy immune response, in particular since they can link the two major arms of the immune system: innate and adaptive immunity. Therefore, DCs would be a logical and ideal target for the development of immunotherapies. Since DCs are not activated in the steady state, an adjuvant to convert their function from tolerogenic to immunogenic would be desirable. Upon ligand activation, invariant natural killer T (iNKT) cells simultaneously activate NK cells and also energize the DCs, resulting in their full maturation. To utilize such iNKT-licensed “fully” matured DCs as adjuvants, mechanisms of both intercellular communication between DC subsets and iNKT cells and intracellular molecular signaling in DCs have to be clarified and optimized. To generate both innate and adaptive immunity against cancer, a variety of strategies with the potential to target iNKT-licensed DCs in situ have been studied. The benchmark of success in these studies, each with distinct approaches, will be the development of functional NK cells and cytotoxic T cells (CTLs) as well as generation of long-term, memory CTL. In this review, we provide a framework for NKT-mediated immunotherapy through selective DC targeting in situ, describe progress in the design of licensed therapies for iNKT cell targeting of DCs, and highlight the challenge to provide maximal benefit to patients.

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

Introduction: Cancer is categorized into two types based on the microenvironment: cold and hot tumors. The former is challenging to stimulate through immunity. The immunogenicity of cancer relies on the quality and quantity of cancer antigens, whether recognized by T cells or not. Successful cancer immunotherapy hinges on the cancer cell type, antigenicity and subsequent immune reactions. The T cell response is particularly crucial for secondary epitope spreading, although the factors affecting these mechanisms remain unknown. Prostate cancer often becomes resistant to standard therapy despite identifying several antigens, placing it among immunologically cold tumors. We aim to leverage prostate cancer antigens to investigate the potential induction of epitope spreading in cold tumors. This study specifically focuses on identifying factors involved in secondary epitope spreading based on artificial adjuvant vector cell (aAVC) therapy, a method established as invariant natural killer T (iNKT) -licensed DC therapy. Methods: We concentrated on three prostate cancer antigens (prostatespecific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP)). By introducing allogeneic cells with the antigen and murine CD1d mRNA, followed by a-galactosylceramide (a-GalCer) loading, we generated five types of aAVCs, i.e, monovalent, divalent and trivalent antigen-expressing aAVCs and four types of prostate antigenexpressing cold tumors. We evaluated iNKT activation and antigen-specific CD8+ T cell responses against tumor cells prompted by the aAVCs. Results: Our study revealed that monovalent aAVCs, expressing a single prostate antigen, primed T cells for primary tumor antigens and also induced T cells targeting additional tumor antigens by triggering a tumor antigen-spreading response. When we investigated the immune response by trivalent aAVC (aAVC-PROS), aAVC-PROS therapy elicited multiple antigenspecific CD8+ T cells simultaneously. These CD8+ T cells exhibited both preventive and therapeutic effects against tumor progression. Conclusions: The findings from this study highlight the promising role of tumor antigen-expressing aAVCs, in inducing efficient epitope spreading and generating robust immune responses against cancer. Our results also propose that multivalent antigen-expressing aAVCs present a promising therapeutic option and could be a more comprehensive therapy for treating cold tumors like prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA.Methods: PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed.Results: Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.Conclusion: We present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016). [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8+ T cells and play critical roles in cytotoxic T-cell-mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8+ T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4+ T-cell activation by CD11b+ DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103hi) DCs, which immigrate from the skin, and resident (CD8αhi) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8+ T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8+ T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8+ T-cell activation. [ABSTRACT FROM AUTHOR]

A key goal of vaccine immunotherapy is the generation of long-term memory CD8+ T cells capable of mediating immune surveillance. We discovered a novel intercellular pathway governing the development of potent memory CD8+ T cell responses against cell-associated Ags that is mediated through cross-presentation by XCR1+ dendritic cells (DCs). Generation of CD8+ memory T cells against tumor cells pulsed with an invariant NKT cell ligand depended on cross-talk between XCR1+ and plasmacytoid DCs that was regulated by IFN-α/IFN-αR signals. IFN-α production by plasmacytoid DCs was stimulated by an OX40 signal from the invariant NKT cells, as well as an HMGB1 signal from the dying tumor cells. These findings reveal a previously unknown pathway of intercellular collaboration for the generation of tumor-specific CD8+ memory T cells that can be exploited for strategic vaccination in the setting of tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Both innate and adaptive immunity underpin cancer immunosurveillance. To stimulate both these arms of the immune system, we used allogeneic cells loaded with natural killer T (NKT) cell ligands and expressing tumor-associated antigens, resulting in NKT cell activation, dendritic-cell maturation and ultimately in the elicitation of adaptive T-cell responses. This approach holds great promise for the development of novel anticancer immunotherapies. [ABSTRACT FROM AUTHOR]