Cytotoxicity as a form of immunogenic cell death leading to efficient tumor antigen cross-priming.

Antigen cross-priming of CD8+ T cells is a critical process necessary for the effective expansion and activation of CD8+ T cells endowed with the ability to recognize and destroy tumor cells. The cross-presentation of tumor antigens to cross-prime CD8+ T cells is mainly mediated, if not only, by a subset of professional antigen-presenting cells termed type-1 conventional dendritic cells (cDC1). The demise of malignant cells can be immunogenic if it occurs in the context of premortem stress. These ways of dying are termed immunogenic cell death (ICD) and are associated with biochemical features favoring cDC1 for the efficient cross-priming of tumor antigens. Immunosurveillance and the success of immunotherapies heavily rely on the ability of cytotoxic immune cells, primarily CD8+ T cells and NK cells, to detect and eliminate tumor cells through mechanisms collectively known as cytotoxicity. Recent studies have revealed the significance of NK- and CTL-mediated cytotoxicity as a prominent form of immunogenic cell death, resulting in mechanisms that promote and sustain antigen-specific immune responses. This review focuses on the mechanisms underlying the cross-presentation of antigens released during tumor cell killing by cytotoxic immune cells, with an emphasis on the role of cDC1 cells. Indeed, cDC1s are instrumental in the effectiveness of most immunotherapies, underscoring the significance of tumor antigen cross-priming in contexts of immunogenic cell death. The notion of the potent immunogenicity of cell death resulting from NK or cytotoxic T lymphocyte (CTL)-mediated cytotoxicity has far-reaching implications for cancer immunotherapy.

Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network.

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

Recreation of an antigen-driven germinal center in vitro by providing B cells with phagocytic antigen.

Successful vaccines rely on activating a functional humoral immune response through the generation of class-switched high affinity immunoglobulins (Igs). The germinal center (GC) reaction is crucial for this process, in which B cells are selected in their search for antigen and T cell help. A major hurdle to understand the mechanisms of B cell:T cell cooperation has been the lack of an antigen-specific in vitro GC system. Here we report the generation of antigen-specific, high-affinity, class-switched Igs in simple 2-cell type cultures of naive B and T cells. B cell antigen uptake by phagocytosis is key to generate these Igs. We have used the method to interrogate if T cells confer directional help to cognate B cells that present antigen and to bystander B cells. We find that bystander B cells do not generate class-switched antibodies due to a defective formation of T-B conjugates and an early conversion into memory B cells.

Endophilin A2 regulates B-cell endocytosis and is required for germinal center and humoral responses.

Antigen-specific B-cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B-cell endosomal trafficking pathways and their specific roles in B-cell responses have not been systematically investigated. Here, we report high-throughput identification of genes regulating B-cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on constitutive, clathrin-mediated endocytosis and on antigen-induced, clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2-mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B-cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2-deficient mice show defects in GC B-cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B-cell clonal expansion and antibody responses.

Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake-related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.

RRAS2 shapes the TCR repertoire by setting the threshold for negative selection.

Signal strength controls the outcome of αß T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2-/- mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2-/- mice have reduced affinity for MOG/I-Ab tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences that are absent in Rras2-/- mice, suggesting their involvement in autoimmunity.

Antigen phagocytosis by B cells is required for a potent humoral response.

Successful vaccines rely on activating a functional humoral response that results from promoting a proper germinal center (GC) reaction. Key in this process is the activation of follicular B cells that need to acquire antigens and to present them to cognate CD4 T cells. Here, we report that follicular B cells can phagocytose large antigen-coated particles, a process thought to be exclusive of specialized antigen-presenting cells such as macrophages and dendritic cells. We show that antigen phagocytosis by B cells is BCR-driven and mechanistically dependent on the GTPase RhoG. Using Rhog-/- mice, we show that phagocytosis of antigen by B cells is important for the development of a strong GC response and the generation of high-affinity class-switched antibodies. Importantly, we show that the potentiation effect of alum, a common vaccine adjuvant, requires direct phagocytosis of alum-antigen complexes by B cells. These data suggest a new avenue for vaccination approaches by aiming to deliver 1-3 µm size antigen particles to follicular B cells.

Author Correction: Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

The original version of this Article contained an error in the spelling of the author José María González-Granado, which was incorrectly given as José María Gozález-Granado. This has now been corrected in both the PDF and HTML versions of the Article.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

Bacterial phagocytosis and antigen cross-presentation to activate CD8+ T cells are principal functions of professional antigen presenting cells. However, conventional CD4+ T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8+ T cells, which proliferate and become cytotoxic in response. CD4+ T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8+ T cells with low PD-1 expression. Moreover, transphagocytic CD4+ T cells induce protective anti-tumour immune responses by priming CD8+ T cells, highlighting the potential of CD4+ T cells as a tool for cancer immunotherapy.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte development.

Thymocyte-expressed molecule involved in selection (THEMIS) is a recently identified regulator of thymocyte positive selection. THEMIS's mechanism of action is unknown, and whether it has a role in TCR-proximal signaling is controversial. In this article, we show that THEMIS and the adapter molecule growth factor receptor-bound protein 2 (GRB2) associate constitutively through binding of a conserved PxRPxK motif within the proline-rich region 1 of THEMIS to the C-terminal SH3-domain of GRB2. This association is indispensable for THEMIS recruitment to the immunological synapse via the transmembrane adapter linker for activation of T cells (LAT) and for THEMIS phosphorylation by Lck and ZAP-70. Two major sites of tyrosine phosphorylation were mapped to a YY-motif close to proline-rich region 1. The YY-motif was crucial for GRB2 binding, suggesting that this region of THEMIS might control local phosphorylation-dependent conformational changes important for THEMIS function. Finally, THEMIS binding to GRB2 was required for thymocyte development. Our data firmly assign THEMIS to the TCR-proximal signaling cascade as a participant in the LAT signalosome and suggest that the THEMIS-GRB2 complex might be involved in shaping the nature of Ras signaling, thereby governing thymic selection.