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Key Points Intestinal regulatory T (T reg ) cells consist of at least three subpopulations: IL-10 + RORγt + microbiota-stimulated peripherally derived T reg (pT reg ) cells, RORγt − NRP1 − dietary antigen-stimulated pT reg cells and GATA3 + thymus-derived T reg (tT reg ) cells. T reg cell subpopulations have complementary functions, including the maintenance of homeostasis against the microbiota and dietary components in the steady state and the suppression of inflammatory responses. The gut microbiota influences the differentiation, accumulation, function and T cell receptor (TCR) repertoire of colonic T reg cells. In turn, the host–microbiota symbiotic relationship in the gut relies on T reg cells that control antigen-specific responses directed to gut microorganisms. Microbial and dietary metabolites, such as short-chain fatty acids, vitamins and amino acids affect the differentiation and the survival of T reg cells. T reg cell generation involves several mechanisms that function in a cell-intrinsic and cell-extrinsic manner. Macrophages, innate lymphoid cells and dendritic cells are strategically positioned beneath the intestinal epithelial cells to sense the types and the features of the intraluminal microorganisms and dietary components and to coordinately promote T cell homeostasis in the intestines. Intestinal regulatory T cells (T reg cells) are distinct from those in other organs and function to maintain tolerance to harmless dietary antigens and commensal microorganisms. The unique features of these cells, as well as the signals involved in their development and maintenance, are discussed in this Review. Gut-resident forkhead box P3 (FOXP3) + CD4 + regulatory T cells (T reg cells) are distinct from those in other organs and have gut-specific phenotypes and functions. Whereas T reg cells in other organs have T cell receptors (TCRs) specific for self antigens, intestinal T reg cells have a distinct set of TCRs that are specific for intestinal antigens, and these cells have pivotal roles in the suppression of immune responses against harmless dietary antigens and commensal microorganisms. The differentiation, migration and maintenance of intestinal T reg cells are controlled by specific signals from the local environment. In particular, certain members of the microbiota continuously provide antigens and immunoregulatory small molecules that modulate intestinal T reg cells. Understanding the development and the maintenance of intestinal T reg cells provides important insights into disease-relevant host–microorganism interactions.

γδ T cells are generally understood to be innate lymphocytes. Prinz and colleagues show that human γδ T cells reconstituted after bone-marrow transplantation have a distinct repertoire that can be shaped by infection with cytomegalovirus, which suggests features of adaptive immunity. To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs ( TRG and TRD ) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer. Single cell RNA sequencing generates short reads from one end of a template, providing incomplete transcript coverage and limiting identification of diverse sequences such as antigen receptors. Here the authors combine long read nanopore sequencing with short read profiling of barcoded libraries to generate full-length antigen receptor sequences.

Secondary mutations can drive the transformation of pre-leukemic clones that carry ETV6 - RUNX1 translocations. Müschen and colleagues show that repeated inflammatory episodes induce aberrant coexpression of the DNA recombinases RAG and AID to promote the development of acute lymphoblastic leukemia. Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure, clonal evolution and leukemic progression, but the pathophysiologic processes driving these events remain incompletely understood. Here, by establishing a comprehensive single-cell transcriptional taxonomy of human MDS, we reveal that inflammatory remodeling of bone marrow stromal niches is a common early feature, irrespective of the genetic driver landscape. We identify an activated CD8-T-cell subset as a source of stromal inflammation via TNF-receptor signaling, which prompts the inflammatory rewiring and loss of repopulating ability of residual normal hematopoietic stem/progenitor cells (HSPC). Mutant HSPCs display relative resistance to this inflammatory stress and reside predominantly in a transcriptional ‘high output’ state, providing a biological framework to their competitive advantage in an inflammatory microenvironment. Consistent with this, stromal inflammation associates with leukemic progression and reduced survival. Our data thus support a model of immune-stromal inflammatory signaling driving tissue failure and clonal evolution in the hematopoietic system. Mechanisms of clonal evolution in myeloid neoplasms remain incompletely understood. Darwinian theory predicts that the (micro)environment of clone-propagating stem cells may contribute to clonal selection. Here, we provide data fitting this model, establishing a relationship between stromal niche inflammation, inflammatory stress in HSPCs, clonal resistance and leukemic evolution in human MDS. Mechanisms of clonal evolution in myeloid neoplasms remain incompletely understood. Darwinian theory predicts that the (micro)environment of clone-propagating stem cells may contribute to clonal selection. Here, authors provide data fitting this model, establishing a relationship between stromal niche inflammation, inflammatory stress in HSPCs, clonal resistance and leukemic evolution in human myelodysplastic syndrome.

Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called ‘memory inflation’ is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8 + T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family’s capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family’s long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation. T cell memory formation is often described as occurring during the chronic phases of infection. Buchholz and colleagues use the phenomenon of ‘memory inflation’ following cytomegalovirus infection to show that a tiny subset of self-renewing T cells branch off early from the bulk population to generate memory.

Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1-/- mice survived significantly longer than immunodeficient Pfp-/- Rag2-/- mice. While tumors in Pfp-/- Rag2-/- mice were highly polyclonal, immunoedited tumors in WT and Pd-1-/- mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ-mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.

Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT. Applying this technology we have shown that the vast majority of shared antigen-specific clonotypes identified in different individuals display the same specificity. We also showed that shared antigen-specific clonotypes are simpler sequences and are present at higher frequencies compared to non-shared clonotypes specific to the same antigen. In conclusion this technology enables sensitive and quantitative monitoring of T cells specific for hundreds or thousands of antigens simultaneously allowing the study of T cell responses with an unprecedented resolution and scale.

Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.

In southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis. 31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR. All 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background. Infections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.