Federica Sallusto

Genome-wide association studies investigating autoimmune disorders may reveal previously unrecognized regulators of immune cell functions. A Crohn’s disease risk allele was associated with increased expression in T lymphocytes of the adapter protein Src kinase–associated phosphoprotein 2 (SKAP2), whose functional role in these cells is unknown. By analyzing T cells from individuals homozygous for either allelic variant or engineered to modulate SKAP2 endogenous levels, we found that SKAP2 promotes T cell adhesion while restraining migration, acting nonredundantly with its homologue SKAP1. Consistent with such functional differences, SKAP1 and SKAP2 displayed distinct activation-induced regulation as well as unique protein interactomes. Prolonged TCR activation triggered caspase-mediated SKAP2 cleavage, leading to the disassembly of its interactome and enabling a migratory program. Together, these findings identify SKAP2 as a genetically regulated modulator of T cell function whose expression and activity control the balance between adhesion and migration.

The possibility of enhancing T cell function by deleting specific genes represents a long‐sought goal in preclinical studies and ultimately for clinical applications. Using CRISPR/Cas9 genome editing, we report that, in human cytotoxic CD8 T cell clones, the cell cycle checkpoint gene CDKN2A , encoding p16 ^INK4A , plays a nonredundant role in controlling T cell receptor (TCR)‐dependent and independent cell expansion. Deletion of CDKN2A dramatically enhanced antigen‐driven and homeostatic proliferation, while preserving effector functions. In contrast, the deletion of other cell cycle inhibitors ( CDKN1B , CDKN2C , and CDKN2D ), alone or in combination, had no impact on T cell proliferation. We also report that mediator complex subunit 12 (MED12) and the E3 ubiquitin ligase CBL‐B deletions did not affect proliferative capacity of CD8 T cell clones. Interestingly, deletion of the negative regulator of Ras signaling, RASA2, increased antigen sensitivity and cytotoxic activity, while not improving in vitro expansion. Collectively, these findings reveal a unique and critical nonredundant role for p16 ^INK4A in regulating CD8 T cells. Deletion of CDKN2A offers a promising strategy to enhance CD8 T cell expansion ex vivo, thereby improving TCR discovery pipelines and, potentially, therapeutic applications.

Advances in T cell biology have revealed heterogeneity among T cell populations that is not captured by existing general nomenclature. This issue has caused an ad hoc broadening of core T cell subset definitions and the invention of new subset designations that have not been uniformly delineated. To address this issue, in this Consensus Statement, we propose guidelines that serve three goals. First, they advocate that primary research reports define the experimental basis by which relevant subsets are designated in the methods section of each study. Second, they provide standardized definitions for existing subset designations in popular use, and common experimental criteria for defining each subset are noted. Last, they present an alternative ‘modular nomenclature’ paradigm. The newly proposed modular nomenclature eschews conceptualization of antigen-experienced T cells as belonging to a few idealized subsets, and the nomenclature instead simply indicates individual biological properties present in a T cell population with brief descriptors. Collectively, these guidelines intend to enhance transparency in the literature while facilitating clearer communication of findings and concepts to researchers, students and clinicians.

Genetic variants in the FCRL3 gene are linked to autoimmune disorders. However, the functional properties of FCRL3-expressing T lymphocytes, and the regulation and functional impact of FCRL3 expression remain understudied. Here, we performed a multiomic and functional analysis of human T lymphocytes expressing FCRL3. FCRL3 expression correlated with reduced capacity of T cells to undergo activation and was accompanied by functional specialization toward a cytotoxic phenotype, resembling cytotoxic CD4+ T lymphocytes and CD8+ effector memory TEMRA cells. FCRL3 expression was induced upon repetitive TCR engagement, and sufficed to attenuate T cell responses, indicating a role as a negative regulator of the activation of differentiated T cell subsets with high cytotoxic capacity. Mechanistically, the cytoplasmic domain of FCRL3 engaged inhibitory molecules, suggesting a direct role in limiting activating signals. Overall, our study establishes FCRL3 as a functional immunoregulatory receptor that restrains the activation of highly specialized human memory T cells.

T helper cell subsets—Th1, Th2, and Th17—coordinate pathogen‐specific immune responses. Candida albicans ‐specific T cells include protective Th17 cells alongside other Th subsets. However, the role of alternative Th subsets remains unclear, particularly in individuals with impaired Th17 responses and recurrent candidiasis. Here, we show that patients with STAT1 gain‐of‐function mutations and chronic mucocutaneous candidiasis have a numerically normal but functionally altered pool of C. albicans ‐specific Th cells, skewed toward Th1 and Th2. This imbalance persisted even when assessing responses to the known and the newly identified immunodominant C. albicans antigens MP65 (65‐kilodalton mannoprotein), HYR1 (hyphally regulated cell wall protein 1), and SAP4‐6 (secreted aspartic proteinases 4–6), suggesting that antigen recognition and priming remain intact despite qualitative defects in T cell polarization. Using mucosal infection mouse models, we demonstrate that C. albicans ‐specific transgenic Th17 cells are sufficient to control infection, whereas Th1 and Th2 cells fail to protect, even in high numbers. Moreover, co‐transfer of Th2 cells with Th17 cells impaired fungal control via an IL‐4‐dependent mechanism. These findings highlight the essential role of Th17 cells in protective immunity against C. albicans and reveal that non‐Th17 responses are ineffective and may contribute to susceptibility in both humans and mice.

CD4 ⁺ T cells are indispensable for optimal immunity to Mycobacterium tuberculosis ( M.tb ), a pathogen that triggers tuberculosis (TB) in humans. M.tb -specific human CD4 ⁺ T cells are known to polarize toward an interferon-γ (IFN-γ)–producing, CCR4 ⁻ CCR6 ⁺ CXCR3 ⁺ T-bet ⁺ RORγT ⁺ T helper 1* cell (T _H 1*cell) memory phenotype. We report that autosomal recessive deficiency of the human lymphocytic surface receptor LY9 (SLAMF3 and CD229), which is found in less than 10 ⁻⁵ individuals in the general population, underlies TB in three unrelated patients due to selective impairment in IFN-γ production by T _H 1* cells. T _H 1* cells express higher levels of LY9 than other CD4 ⁺ T cells. Mechanistically, LY9 polarizes naïve CD4 ⁺ T cells toward memory T _H 1* cells by inducing T-bet via signaling lymphocytic activation molecule (SLAM)–associated protein (SAP) and RORγT (thymus-specific retinoid-related orphan receptor γ) without SAP. LY9 costimulation enhances TCR-driven IFN-γ production of memory T _H 1*, but not T _H 1, cells in a T cell–intrinsic manner via NFAT1 (nuclear factor of activated T cells 1) and RORγT. LY9 is likely to govern an optimal T _H 1* cell– and IFN-γ–dependent protective immunity to M.tb in humans.

Autoimmune hepatitis (AIH) is a rare chronic inflammatory liver disease characterized by the presence of autoantibodies, including those targeting O-phosphoseryl-tRNA:selenocysteine-tRNA synthase (SepSecS), also known as soluble liver antigen (SLA). Anti-SepSecS antibodies have been associated with a more severe phenotype, suggesting a key role for the SepSecS autoantigen in AIH. To analyze the immune response to SepSecS in patients with AIH at the clonal level, we combined sensitive high-throughput screening assays with the isolation of monoclonal antibodies (mAbs) and T cell clones. The anti-SepSecS mAbs isolated were primarily IgG1, affinity-matured compared with their germline versions, and recognized at least 3 nonoverlapping epitopes. SepSecS-specific CD4 ⁺ T cell clones were found in patients with AIH who were anti-SLA-positive and anti-SLA-negative,and, to a lesser extent, in patients with non-AIH liver diseases and in healthy individuals. SepSecS-specific T cell clones from patients with AIH produced IFN-γ, IL-4, and IL-10, targeted multiple SepSecS epitopes, and, in one patient, were clonally expanded in both blood and liver biopsy. Finally, SepSecS-specific B cell clones, but not those of unrelated specificities, were able to present soluble SepSecS to specific T cells. Collectively, our study provides the first detailed analysis of B and T cell repertoires targeting SepSecS in patients with AIH, offering a rationale for improved targeted therapies.

Identifying activated T lymphocytes and differentiating antigen‐specific from bystander T cells is crucial for understanding adaptive immune responses. This study investigates the efficacy of activation‐induced markers (AIMs) in distinguishing these cell populations. We measured the expression of commonly used AIMs (CD25, CD38, CD40L, CD69, CD137, HLA‐DR, ICOS, and OX40) in an in vitro T‐cell activation system and evaluated their sensitivity, specificity, and positive predictive value.We demonstrated that individual AIMs, while specific in detecting activated CD4⁺ T cells, poorly discriminate between antigen‐specific and bystander activation, as assessed by a discriminative capacity (DC) score we developed. Our analysis revealed that dual AIM combinations significantly enhanced the ability to distinguish antigen‐specific from bystander‐activated T cells, achieving DC scores above 90%. These combinations also improved positive predictive value and specificity with a modest reduction in sensitivity. The CD25^hi/ICOS^hi combination emerged as the most efficient, with an average sensitivity of 84.35%, specificity of 99.7%, and DC score of 90.12%. Validation through T‐cell cloning and antigen re‐stimulation confirmed the robustness of our predictions.This study provides a practical framework for researchers to optimize strategies for identifying and isolating antigen‐specific human CD4⁺ T lymphocytes and studying their phenotype, function, and T‐cell receptor repertoire.

Open-access immunosequencing data | Sallusto, Federica | Journal of Clinical Investigation | Adaptive Biotechnologies | TBD

Efficient identification of human monoclonal antibodies targeting specific antigenic sites is pivotal for advancing vaccines and immunotherapies against infectious diseases and cancer. Existing screening techniques, however, limit our ability to discover monoclonal antibodies with desired specificity. In this study, we introduce a novel method, blocking of binding (BoB) enzyme‐linked immunoassay (ELISA), enabling the detection of high‐avidity human antibodies directed to defined epitopes. Leveraging BoB‐ELISA, we analyzed the antibody response to known epitopes of influenza A hemagglutinin (HA) in the serum of vaccinated donors. Our findings revealed that serum antibodies targeting head epitopes were immunodominant, whereas antibodies against the stem epitope, although subdominant, were highly prevalent. Extending our analysis across multiple HA strains, we examined the cross‐reactive antibody response targeting the stem epitope. Importantly, employing BoB‐ELISA we identified donors harboring potent heterosubtypic antibodies targeting the HA stem. B‐cell clonal analysis of these donors revealed three novel, genealogically independent monoclonal antibodies with broad cross‐reactivity to multiple HAs. In summary, we demonstrated that BoB‐ELISA is a sensitive technique for measuring B‐cell epitope immunogenicity, enabling the identification of novel monoclonal antibodies with implications for enhanced vaccine development and immunotherapies.