Using T Cell Receptor Repertoires to Understand the Principles of Adaptive Immune Recognition.

Adaptive immune recognition is mediated by antigen receptors on B and T cells generated by somatic recombination during lineage development. The high level of diversity resulting from this process posed technical limitations that previously limited the comprehensive analysis of adaptive immune recognition. Advances over the last ten years have produced data and approaches allowing insights into how T cells develop, evolutionary signatures of recombination and selection, and the features of T cell receptors that mediate epitope-specific binding and T cell activation. The size and complexity of these data have necessitated the generation of novel computational and analytical approaches, which are transforming how T cell immunology is conducted. Here we review the development and application of novel biological, theoretical, and computational methods for understanding T cell recognition and discuss the potential for improved models of receptor:antigen interactions.

The spike-specific TCRß repertoire shows distinct features in unvaccinated or vaccinated patients with SARS-CoV-2 infection.

BACKGROUND: The evolving variants of SARS-CoV-2 may escape immunity from prior infections or vaccinations. It's vital to understand how immunity adapts to these changes. Both infection and mRNA vaccination induce T cells that target the Spike protein. These T cells can recognize multiple variants, such as Delta and Omicron, even if neutralizing antibodies are weakened. However, the degree of recognition can vary among people, affecting vaccine efficacy. Previous studies demonstrated the capability of T-cell receptor (TCR) repertoire analysis to identify conserved and immunodominant peptides with cross-reactive potential among variant of concerns. However, there is a need to extend the analysis of the TCR repertoire to different clinical scenarios. The aim of this study was to examine the Spike-specific TCR repertoire profiles in natural infections and those with combined natural and vaccine immunity. METHODS: A T-cell enrichment approach and bioinformatic tools were used to investigate the Spike-specific TCRß repertoire in peripheral blood mononuclear cells of previously vaccinated (n = 8) or unvaccinated (n = 6) COVID-19 patients. RESULTS: Diversity and clonality of the TCRß repertoire showed no significant differences between vaccinated and unvaccinated groups. When comparing the TCRß data to public databases, 692 unique TCRß sequences linked to S epitopes were found in the vaccinated group and 670 in the unvaccinated group. TCRß clonotypes related to spike regions S135-177, S264-276, S319-350, and S448-472 appear notably more prevalent in the vaccinated group. In contrast, the S673-699 epitope, believed to have super antigenic properties, is observed more frequently in the unvaccinated group. In-silico analyses suggest that mutations in epitopes, relative to the main SARS-CoV-2 variants of concern, don't hinder their cross-reactive recognition by associated TCRß clonotypes. CONCLUSIONS: Our findings reveal distinct TCRß signatures in vaccinated and unvaccinated individuals with COVID-19. These differences might be associated with disease severity and could influence clinical outcomes. TRIAL REGISTRATION: FESR/FSE 2014-2020 DDRC n. 585, Action 10.5.12, noCOVID19@UMG.

Exhaustion of tumour-infiltrating T-cell receptor repertoire diversity is an age-dependent indicator of immunological fitness independently predictive of clinical outcome in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive B-cell-malignancy derived from germinal-centre B-cells. Curative therapy traditionally requires intensive immunochemotherapy. Recently, immuno-oncological approaches, modulating the T-cell tumour response, were approved for the treatment of a variety of malignancies. The architecture of the tumour-infiltrating T-cell receptor (TCR) repertoire in BL remains insufficiently characterized. We therefore performed a large-scale, next-generation sequencing study of the complimentary-determining region (CDR)-3 region of the TCRß chain repertoire in a large cohort of all epidemiological subtypes of BL (n = 82) and diffuse large B-cell lymphoma (DLBCL; n = 34). Molecular data were subsequently assessed for correlation with clinical outcome. Our investigations revealed an age-dependent immunoprofile in BL as in DLBCL. Moreover, we found several public clonotypes in numerous patients suggestive of shared tumour neoantigen selection exclusive to BL and distinct from DLBCL regardless of Epstein-Barr virus and/or human immunodeficiency virus status. Compared with baseline, longitudinal analysis unveiled significant repertoire restrictions upon relapse (P = 0·0437) while productive TCR repertoire clonality proved to be a useful indicator of both overall and progression-free-survival [OS: P = 0·0001; hazard ratio (HR): 6·220; confidence interval (CI): 2·263-11·78; PFS: P = 0·0025; HR: 3·086; CI: 1·555-7·030]. Multivariate analysis confirmed its independence from established prognosticators, including age at diagnosis and comorbidities. Our findings establish the clinical relevance of the architecture and clonality of the TCR repertoire and its age-determined dynamics in BL.

Expansion of the CD4+ effector T-cell repertoire characterizes peanut-allergic patients with heightened clinical sensitivity.

BACKGROUND: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. OBJECTIVE: We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. METHODS: We studied the T-cell receptor ß-chain (TCRß) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRß sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. RESULTS: TCRß analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. CONCLUSIONS: Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.

T Cell Clonal Dynamics Determined by High-Resolution TCR-ß Sequencing in Recipients after Allogeneic Hematopoietic Cell Transplantation.

Delayed reconstitution of the immune system is a long-recognized complication after allogeneic hematopoietic cell transplantation (HCT). Specifically, loss of T cell diversity has been thought to contribute to infectious complications, graft-versus-host disease (GVHD), and disease relapse. We performed serial high-resolution next-generation sequencing of T cell receptor (TCR)-ß in 99 related or unrelated donor (57 unrelated, 42 related) allogeneic HCT recipients (55 with reduced-intensity conditioning, 44 with myeloablative conditioning) during the first 3 months after HCT using the immunoSEQ Assay. We measured T cell fraction, clonality (1- Peilou's evenness) and Daley-Smith richness from recipient samples at multiple time points. In agreement with previous studies, we found that although absolute T cell numbers recover relatively quickly after HCT, T cell repertoire diversity remains diminished. Restricted diversity was associated with conditioning intensity, use of antithymocyte globulin, and donor type. Increased number of expanded clones compared to donor T cell clones at day +30 was associated with the incidence of acute GVHD (hazard ratio [HR], 1.11; P = .00005). Even after exclusion of the 12 patients who developed acute GVHD before day +30, the association between acute GVHD and increased clonal expansion at day +30 remained (HR, 1.098; P = .041), indicating that increased clonal T cell expansion preceded the development of acute GVHD. Our results highlight T cell clonal expansion as a potential novel biomarker for acute GVHD that warrants further study.

Evaluation of the polymerase chain reaction-based T-cell receptor ß clonality test in the diagnosis of early mycosis fungoides.

BACKGROUND: T-cell receptor (TCR) clonality may help establish a diagnosis of mycosis fungoides (MF). Routine clonality analysis is performed by using a polymerase chain reaction TCR- gamma assay, yet with this method, 10% to 50% of T-cell lymphomas escape detection. TCR- beta gene rearrangement is an additional assay. Data about its efficacy are controversial. OBJECTIVE: To evaluate the role of TCR-ß assay in the diagnosis of early MF. METHODS: A retrospective study of 61 skin biopsies, 20 from patients with MF, 30 from patients suspected to have early MF, and 11 from patients with chronic inflammatory skin disease. RESULTS: Monoclonality was detected in 16 of 20 (80%) MF cases: 15 (75%) with TCR-ß and 12 (60%) with TCR-γ assay. Of the 30 suspected cases of early MF, 14 showed monoclonality with TCR-ß, and only 5 of 14 showed monoclonality with TCR-γ assay. None of the chronic inflammatory condition samples showed monoclonality. Therefore, TCR-ß clonality assay was more sensitive than TCR-γ in early MF (83% vs 43%; P = .002). LIMITATIONS: This was a retrospective, relatively small study. CONCLUSION: TCR-ß showed a higher sensitivity rate compared with TCR-γ in early-stage MF. The combined use of the TCR-ß and TCR-γ clonality tests can significantly improve the diagnosis rate of early-stage MF.

Nuclear transfer nTreg model reveals fate-determining TCR-ß and novel peripheral nTreg precursors.

To study the development and function of "natural-arising" T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3(+)CD4(+)Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) ß-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR ß-chain was able to provide stronger TCR signals. This TCR-ß-driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3(-)CD4(+)T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells.

TCRß Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection.

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαß combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRß expressing CD11b+ Ly6G+ neutrophils in the spleen during the early phase of infection. Measurement of TCRß transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRß and peripheral blood parasite burden. In addition, TCRß+ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRß- neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

Comprehensive assessment of peripheral blood TCRß repertoire in infectious mononucleosis and chronic active EBV infection patients.

Epstein-Barr virus (EBV) primary infection is usually asymptomatic, but it sometimes progresses to infectious mononucleosis (IM). Occasionally, some people develop chronic active EBV infection (CAEBV) with underlying immunodeficiency, which belongs to a continuous spectrum of EBV-associated lymphoproliferative disorders (EBV+ LPD) with heterogeneous clinical presentations and high mortality. It has been well established that T cell-mediated immune response plays a critical role in the disease evolution of EBV infection. Recently, high-throughput sequencing of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (T cell receptor ß (TCRß)) has emerged as a sensitive approach to assess the T cell repertoire. In this study, we fully characterized the diversity of peripheral blood TCRß repertoire in IM (n = 6) and CAEBV patients (n = 5) and EBV-seropositive controls (n = 5). Compared with the healthy EBV-seropositive controls, both IM and CAEBV patients demonstrate a significant decrease in peripheral blood TCRß repertoire diversity, basically, including narrowed repertoire breadth, highly expanded clones, and skewed CDR3 length distribution. However, there is no significant difference between IM and CAEBV patients. Furthermore, we observed some disease-related preferences in TRBV/TRBJ usage and combinations, as well as lots of T cell clones shared by different groups (unique or overlapped) involved in public T cell responses, which provide more detailed insights into the divergent disease evolution.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.

A clinical-information-free method for early diagnosis of lung cancer from the patients with pulmonary nodules based on backpropagation neural network model.

Lung cancer is the main cause of cancer-related deaths worldwide. Due to lack of obvious clinical symptoms in the early stage of the lung cancer, it is hard to distinguish between malignancy and pulmonary nodules. Understanding the immune responses in the early stage of malignant lung cancer patients may provide new insights for diagnosis. Here, using high-through-put sequencing, we obtained the TCRß repertoires in the peripheral blood of 100 patients with Stage I lung cancer and 99 patients with benign pulmonary nodules. Our analysis revealed that the usage frequencies of TRBV, TRBJ genes, and V-J pairs and TCR diversities indicated by D50s, Shannon indexes, Simpson indexes, and the frequencies of the largest TCR clone in the malignant samples were significantly different from those in the benign samples. Furthermore, reduced TCR diversities were correlated with the size of pulmonary nodules. Moreover, we built a backpropagation neural network model with no clinical information to identify lung cancer cases from patients with pulmonary nodules using 15 characteristic TCR clones. Based on the model, we have created a web server named "Lung Cancer Prediction" (LCP), which can be accessed at http://i.uestc.edu.cn/LCP/index.html.

Dynamic establishment of recipient resident memory T cell repertoire after human intestinal transplantation.

BACKGROUND: Understanding formation of the human tissue resident memory T cell (TRM) repertoire requires longitudinal access to human non-lymphoid tissues. METHODS: By applying flow cytometry and next generation sequencing to serial blood, lymphoid tissue, and gut samples from 16 intestinal transplantation (ITx) patients, we assessed the origin, distribution, and specificity of human TRMs at phenotypic and clonal levels. FINDINGS: Donor age ≥1 year and blood T cell macrochimerism (peak level ≥4%) were associated with delayed establishment of stable recipient TRM repertoires in the transplanted ileum. T cell receptor (TCR) overlap between paired gut and blood repertoires from ITx patients was significantly greater than that in healthy controls, demonstrating increased gut-blood crosstalk after ITx. Crosstalk with the circulating pool remained high for years of follow-up. TCR sequences identifiable in pre-Tx recipient gut but not those in lymphoid tissues alone were more likely to populate post-Tx ileal allografts. Clones detected in both pre-Tx gut and lymphoid tissue had distinct transcriptional profiles from those identifiable in only one tissue. Recipient T cells were distributed widely throughout the gut, including allograft and native colon, which had substantial repertoire overlap. Both alloreactive and microbe-reactive recipient T cells persisted in transplanted ileum, contributing to the TRM repertoire. INTERPRETATION: Our studies reveal human intestinal TRM repertoire establishment from the circulation, preferentially involving lymphoid tissue counterparts of recipient intestinal T cell clones, including TRMs. We have described the temporal and spatial dynamics of this active crosstalk between the circulating pool and the intestinal TRM pool. FUNDING: This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID) P01 grant AI106697.

Altered T-cell receptor ß repertoire in adults with SARS CoV-2 inactivated vaccine of BBIBP-CorV.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the prolonged and widespread epidemic of coronavirus disease 2019 (COVID-19). The inactivated BBIBP-CorV vaccine has shown safety, efficacy and immunogenicity against COVID-19 in in-vitro studies and clinical trials. However, the characteristics changes of the TCRß repertoire in patients receiving BBIBP-CorV remain unclear. METHODS: TCRß repertoire difference were analyzed between 54 uninfected subjects who received a third dose of the enhanced BBIBP-CorV vaccine and the 16 healthy donors who did not receive the vaccine and 44 COVID-19 patients with different courses of disease (asymptomatic, symptomatic and convalescent). Furthermore, antibody response, anti-inflammatory and pro-inflammatory cytokines also were examined. RESULTS: We found that the third dose inactivated coronavirus vaccine induced widespread changes including the increased TCRß repertoire diversity, a much shorter CDR3 length and high usage of V-J genes segments. Meanwhile, the vaccine-responding clones were also predicted. The results of the antibody response showed that 90.7 % of the vaccinated individuals were positive for NAb seroconversion and 88.9 % for IgG antibody about 60 days after the third dose. The concentration of IL-2 increased significantly compared to baseline inoculation. CONCLUSION: Altered TCRß repertoire in adults with SARS CoV-2 inactivated vaccine of BBIBP-CorV clarified the specific immunity induced by inactivated vaccines. Our research provides insights into the adaptive immune response induced by the new inactivated SARS-CoV-2 vaccine and strengthens the development of immunotherapy.

New insights into the germline genes and CDR3 repertoire of the TCRß chain in Chiroptera.

Introduction: Bats are recognized as natural reservoirs for many viruses, and their unique immune system enables them to coexist with these viruses without frequently exhibiting disease symptoms. However, the current understanding of the bat adaptive immune system is limited due to the lack of a database or tool capable of processing T-cell receptor (TCR) sequences for bats. Methods: We performed germline gene annotation in three bat species using homologous genes and RSSs (Recombinational Signal Sequences) scanning method. Then we used the conserved C gene to construct the TCRß chain receptor library of the Intermediate Horseshoe Bat. Bats' TCRß data will be analyzed using MiXCR and constructed reference library. Results: Regarding the annotation results, we found that the Pale Spear-nosed Bat has 37 members in the TRBV12 family, which is more than the total number of TRBV genes in the Greater Horseshoe Bat. The average number of unique TCRß chain receptor sequences in each Intermediate Horseshoe Bat sample reached 24,904. Discussion: The distinct variations in the distribution of TRBV genes among the three types of bats could have a direct impact on the diversity of the TCR repertoire, as evidenced by the presence of conserved amino acids that indicate the T-cell recognition of antigens in bats is MHC-restricted. The bats' TCRß repertoire is formed through the rearrangement of the V-D-J-C genes, with D-J/V-D deletions and insertions resulting in high diversity.

Cholesterol Binds in a Reversed Orientation to TCRß-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer.

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRß subunit (TCRß-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRß. However, the atomic interaction of cholesterol or CS with TCRß remains elusive. Here, we determined the cholesterol and CS binding site of TCRß-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRß-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRß-TM, validating the NMR and MD results.

Trilineage Sequencing Reveals Complex TCRß Transcriptomes in Neutrophils and Monocytes Alongside T Cells.

Recent findings indicate the presence of T cell receptor (TCR)-based combinatorial immune receptors beyond T cells in neutrophils and monocytes/macrophages. In this study, using a semiquantitative trilineage immune repertoire sequencing approach as well as under rigorous bioinformatic conditions, we identify highly complex TCRß transcriptomes in human circulating monocytes and neutrophils that separately encode repertoire diversities one and two orders of magnitude smaller than that of T cells. Intraindividual transcriptomic analyses reveal that neutrophils, monocytes, and T cells express distinct TCRß repertoires with less than 0.1% overall trilineage repertoire sharing. Interindividual comparison shows that in all three leukocyte lineages, the vast majority of the expressed TCRß variants are private. We also find that differentiation of monocytes into macrophages induces dramatic individual-specific repertoire shifts, revealing a surprising degree of immune repertoire plasticity in the monocyte lineage. These results uncover the remarkable complexity of the two phagocyte-based flexible immune systems which until now has been hidden in the shadow of T cells.

Evaluation of the International Society for Cutaneous Lymphoma Algorithm for the Diagnosis of Early Mycosis Fungoides.

The International Society for Cutaneous Lymphoma (ISCL) proposes a diagnostic algorithm for early mycosis fungoides (MF) that includes clinical, histological, immunophenotypical, and molecular criteria. Here, we analyzed the immunologic markers and features of T-cell clonality in 38 early MF cases and 22 non-MF cases to validate the ISCL algorithm. We found that CD5 and CD7 expression differed significantly between early MF and non-MF cases, with epidermal discordance of CD7 expression more frequently identified in early MF. Notably, increasing the cut-off value for CD7 expression from 10% to 22.5% improved its sensitivity. Furthermore, TCR-γ and ß chain rearrangements were more frequently detected in early MF than in non-MF cases. Based on these findings, we propose CD5 and CD7 deficiency as mandatory immunopathologic criteria and PCR-based testing for TCR-γ and ß chains as required molecular/biologic criteria to improve the efficiency of early MF diagnosis using the ISCL algorithm.

TCRß Sequencing Reveals Spatial and Temporal Evolution of Clonal CD4 T Cell Responses in a Breach of Tolerance Model of Inflammatory Arthritis.

Effective tolerogenic intervention in Rheumatoid Arthritis (RA) will rely upon understanding the evolution of articular antigen specific CD4 T cell responses. TCR clonality of endogenous CD4 T cell infiltrates in early inflammatory arthritis was assessed to monitor evolution of the TCR repertoire in the inflamed joint and associated lymph node (LN). Mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis were used to recapitulate early and late phases of RA. The infiltrating endogenous, antigen experienced CD4 T cells in inflamed joints and LNs were analysed using flow cytometry and TCRß sequencing. TCR repertoires from inflamed late phase LNs displayed increased clonality and diversity compared to early phase LNs, while inflamed joints remained similar with time. Repertoires from late phase LNs accumulated clones with a diverse range of TRBV genes, while inflamed joints at both phases contained clones expressing similar TRBV genes. Repertoires from LNs and joints at the late phase displayed reduced CDR3ß sequence overlap compared to the early disease phase, however the most abundant clones in LNs accumulate in the joint at the later phase. The results indicate CD4 T cell repertoire clonality and diversity broadens with progression of inflammatory arthritis and is first reflected in LNs before mirroring in the joint. These observations imply that antigen specific tolerogenic therapies could be more effective if targeted at earlier phases of disease when CD4 T cell clonality is least diverse.

Resident memory T cells in tumor-distant tissues fortify against metastasis formation.

As a critical machinery for rapid pathogen removal, resident memory T cells (TRMs) are locally generated after the initial encounter. However, their development accompanying tumorigenesis remains elusive. Using a murine breast cancer model, we show that TRMs develop in the tumor, the contralateral mammary mucosa, and the pre-metastatic lung. Single-cell RNA sequencing of TRMs reveals two phenotypically distinct populations representing their active versus quiescent phases. These TRMs in different tissue compartments share the same TCR clonotypes and transcriptomes with a subset of intratumoral effector/effector memory T cells (TEff/EMs), indicating their developmental ontogeny. Furthermore, CXCL16 is highly produced by tumor cells and CXCR6- TEff/EMs are the major subset preferentially egressing the tumor to form distant TRMs. Functionally, releasing CXCR6 retention in the primary tumor amplifies tumor-derived TRMs in the lung and leads to superior protection against metastases. This immunologic fortification suggests a potential strategy to prevent metastasis in clinical oncology.

Using CRISPR to enhance T cell effector function for therapeutic applications.

T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.