Some naïve T cell fates are sealed: Tissue-resident memory T (T _RM ) cells constitute a subpopulation of memory cells that reside in tissues instead of recirculating. CD8 ⁺ epithelial TRM (eT _RM ) cells, which occupy the epithelium of sites like the skin, require transforming growth factor–β (TGF-β) for their development. Mani et al. found that α _V integrin–expressing dendritic cells, which activate and present TGF-β, are key (see the Perspective by Farber). Surprisingly, this interplay did not occur in the skin or draining lymph nodes during T cell priming. Rather, resting naïve CD8 ⁺ T cells interacted with α _V integrin–expressing migratory dendritic cells during immune homeostasis, reversibly preconditioning them to become eT _RM cells upon activation. A potent cytokine is thus controlled in a context-dependent manner and preimmune T cell repertoires may be less uniform than previously presumed. Science , this issue p. eaav5728 ; see also p. 188

T cell receptor (TCR) profiling using next-generation sequencing (NGS) enables high-throughput, in-depth analysis of repertoire diversity, offering numerous clinical applications. We developed a DNA-based strategy to analyse the TCRβ-chain using NGS and established reference values for T cell repertoire characteristics in 74 healthy donors, including 44 adults, 20 paediatrics, and 10 cord blood units (CBUs). Additionally, four paediatric patients with combined immunodeficiency (CID) or severe CID (SCID) due to deleterious mutations in recombination activating genes (RAG) were analysed. The developed strategy demonstrated high specificity, reproducibility, and sensitivity, and all functional variable and joining genes were detected with minimal PCR bias. All donors had a Gaussian-like distribution of complementary-determining region 3 length, with lower presence of non-templated nucleotides and higher proportion of non-functional clonotypes in CBUs. Both CBUs and paediatrics showed greater convergence and TCRβ diversity was significantly lower in adults and donors with cytomegalovirus-positive serostatus. Finally, an analysis of paediatric patients with RAG-SCID/CID showed significantly shorter CDR3 region length and lower repertoire diversity compared to healthy paediatrics. In summary, we developed a reliable and feasible TCRβ sequencing strategy for application in the clinical setting, and established reference values that could assist in the diagnosis and monitoring of pathological conditions affecting the T cell repertoire.

Purpose The increasing application of virus-specific T cell therapy for treating BK virus infections in immunocompromised patients highlights the necessity for rapid identification of compatible cell donors with optimal BK-specific T cell response. This study aims to characterize the BK virus-specific T cell response in relation to demographic factors, blood group, serological status, and HLA genotypes using samples from a cell donor registry. Methods Peripheral blood mononuclear cells from cell donors were stimulated with peptide pools derived from VP1 and LTA proteins, and the IFN-γ production was analyzed using ELISpot and validated by flow cytometry. Results Our findings provide an overview of the T cell response to BK virus proteins in healthy donors, revealing associations with demographic characteristics, RhD status, CMV or EBV serological status, and HLA alleles. Remarkably, RhD-negative, CMV-seronegative, and EBV-seronegative donors showed a major T cell response against BK virus proteins. Notably, certain HLA alleles were associated with either enhanced or diminished T cell response. Furthermore, our results suggest that HLA-B leader dimorphism, specifically the presence of threonine at position 2, influences the VP1-specific immune response, resulting in enhanced T cell activation. Conclusion This study, beyond advancing our understanding of the relationship between donor characteristics and BK virus-specific T cell response, has significant implications for improving the selection of optimal cell donors for patient-specific adoptive therapy.

During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. Considering the importance of the T cell response in controlling viral infections, adoptive cell therapy with virus-specific T cells has been used as a safe and effective anti-viral prophylaxis and treatment for immunocompromised patients. The main objective of this study was to stablish an effective and save methodology to cryostore whole blood as starting material, and to adapt a T cell activation and expansion protocol to generate an off-the-shelf anti-viral therapeutic option. Additionally, we studied how memory T cell phenotype, clonality based on T cell receptor, and antigen specificity could condition characteristics of final expanded T cell product. Twenty-nine healthy blood donors were selected from a database of convalescent plasma donors with a confirmed history of SARS-CoV-2 infection. Blood was processed using a fully automated, clinical grade, and two-step closed system. Eight cryopreserved bags were advanced to the second phase of the protocol to obtain purified mononucleated cells. We adapted the T cell activation and expansion protocol, without specialized antigen-presenting cells or presenting molecular structures, in a G-Rex culture system with IL-2, IL-7, and IL-15 cytokine stimulation. The adapted protocol successfully activated and expanded virus-specific T cells to generate a T cell therapeutic product. We observed no major impact of post-symptom onset time of donation on the initial memory T cell phenotype or clonotypes resulting in minor differences in the final expanded T cell product. We showed that antigen competition in the expansion of T cell clones affected the T cell clonality based on the TCRβ repertoire. We demonstrated that GMP blood preprocessing and cryopreserving is a successful procedure to obtain an initial cell source able to activate and expand without a specialized antigen presenting agent. Our two-step blood processing allowed recruitment of the cell donors independently of the expansion cell protocol timing, facilitating donor, staff and facility needs. Moreover, the resulting VSTs could be also banked for further use, notably maintaining viability and antigen specificity after cryopreservation. During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. The recent pandemic generated by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been a model to test current knowledge and methods to develop vaccines and treatments. In this study, we propose an alternative methodology to cryopreserve whole blood cells under good manufacturing practice conditions from well-characterized SARS-CoV-2 infected cell donors, to create a cryopreserved cell bank as starting material to develop advanced therapy medicinal products. We adapted a protocol to successfully thaw, activate, and expand virus-specific T cells to generate an off-the-shelf therapeutic option. Additionally, we studied how memory T cell phenotype, clonality based on T cell receptor, and antigen specificity could condition characteristics of final expanded T cell product.

Introduction: Reactivation of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) is a major complication in immunocompromised patients, and understanding donor variability in antiviral immunity is essential for optimizing adoptive T-cell therapy. This study aimed to identify immunogenetic determinants of virus-specific T-cell responses in healthy donors to guide donor selection. Methods: CMV- and EBV-specific T-cell responses were characterized using flow cytometry and IFN-g ELISpot after stimulation with peptide pools, followed by integrated analyses correlating the response magnitude with human leukocyte antigen (HLA) class I and II alleles, HLA haplotypes, and HLA-G 3′UTR polymorphisms. Results: CMV 65-kDa phosphoprotein (pp65) induced stronger CD8 ⁺ and CD4 ⁺ T-cell responses than immediate early 1 (IE1), whereas the EBV consensus peptides preferentially activated CD8 ⁺ T cells. Several HLA alleles and linked haplotypic backgrounds were associated with enhanced or reduced CMV-specific T-cell responses, including an A*01:01~C*07:01~B*08:01~DRB1*03:01~DQB1*02:01 haplotype enriched among low responders. For EBV, several HLA-restricted alleles were associated with stronger peptide-specific activation, supporting the broad immunogenicity of the consensus peptide pool. Moreover, HLA-G 3′UTR polymorphisms selectively modulated the CMV-specific IFN-γ ⁺ CD8 ⁺ T-cell frequencies. Discussion: Together, these results indicate that classical and non-classical HLA variations are associated with inter-donor differences in antiviral T-cell response in an antigen- and virus-dependent manner, providing a translational framework to refine donor selection and improve the design of T-cell-based immunotherapies.