The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The extreme diversity of the TCR repertoire represents a major analytical challenge; this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire. Here, we report on the latest methodological advancements in the field by describing and comparing the available tools; from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis. Finally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared. Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods.

Objective Vedolizumab, a monoclonal antibody directed against the integrin heterodimer α4β7, is approved for the treatment of Crohn’s disease and ulcerative colitis. The efficacy of vedolizumab has been suggested to result from inhibition of intestinal T cell trafficking although human data to support this conclusion are scarce. We therefore performed a comprehensive analysis of vedolizumab-induced alterations in mucosal and systemic immunity in patients with inflammatory bowel disease (IBD), using anti-inflammatory therapy with the TNFα antibody infliximab as control. Design Immunophenotyping, immunohistochemistry, T cell receptor profiling and RNA sequencing were performed using blood and colonic biopsies from patients with IBD before and during treatment with vedolizumab (n=18) or, as control, the anti-TNFα antibody infliximab (n=20). Leucocyte trafficking in vivo was assessed using single photon emission computed tomography and endomicroscopy. Results Vedolizumab was not associated with alterations in the abundance or phenotype of lamina propria T cells and did not affect the mucosal T cell repertoire or leucocyte trafficking in vivo. Surprisingly, however, α4β7 antibody treatment was associated with substantial effects on innate immunity including changes in macrophage populations and pronounced alterations in the expression of molecules involved in microbial sensing, chemoattraction and regulation of the innate effector response. These effects were specific to vedolizumab, not observed in response to the TNFα antibody infliximab, and associated with inhibition of intestinal inflammation. Conclusion Our findings suggest that modulation of innate immunity contributes to the therapeutic efficacy of vedolizumab in IBD. Trial registration number NCT02694588

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However, neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T-cell response nor the diversity of resulting immune memory is well understood. In this study, we use longitudinal high-throughput T-cell receptor (TCR) sequencing to track changes in the T-cell repertoire following two mild cases of COVID-19. In both donors, we identified CD4⁺ and CD8⁺ T-cell clones with transient clonal expansion after infection. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurrence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors, the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T-cell clones were detected in the memory fraction at the pre-infection time point, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.

Human aging is associated with a profound loss of thymus productivity, yet naïve T lymphocytes still maintain their numbers by division in the periphery for many years. The extent of such proliferation may depend on the cytokine environment, including IL-7 and T-cell receptor (TCR) “tonic” signaling mediated by self pMHCs recognition. Additionally, intrinsic properties of distinct subpopulations of naïve T cells could influence the overall dynamics of aging-related changes within the naïve T cell compartment. Here, we investigated the differences in the architecture of TCR beta repertoires for naïve CD4, naïve CD8, naïve CD4⁺CD25⁻CD31⁺ (enriched with recent thymic emigrants, RTE), and mature naïve CD4⁺CD25⁻CD31⁻ peripheral blood subsets between young and middle-age/old healthy individuals. In addition to observing the accumulation of clonal expansions (as was shown previously), we reveal several notable changes in the characteristics of T cell repertoire. We observed significant decrease of CDR3 length, NDN insert, and number of non-template added N nucleotides within TCR beta CDR3 with aging, together with a prominent change of physicochemical properties of the central part of CDR3 loop. These changes were similar across CD4, CD8, RTE-enriched, and mature CD4 subsets of naïve T cells, with minimal or no difference observed between the latter two subsets for individuals of the same age group. We also observed an increase in “publicity” (fraction of shared clonotypes) of CD4, but not CD8 naïve T cell repertoires. We propose several explanations for these phenomena built upon previous studies of naïve T-cell homeostasis, and call for further studies of the mechanisms causing the observed changes and of consequences of these changes in respect of the possible holes formed in the landscape of naïve T cell TCR repertoire.