Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics.

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

Transcriptomic profile of TNFhigh MAIT cells is linked to B cell response following SARS-CoV-2 vaccination.

Introduction: Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. Methods: To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). Results: We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Discussion: Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.

Human γδ T cell identification from single-cell RNA sequencing datasets by modular TCR expression.

Accurately identifying γδ T cells in large single-cell RNA sequencing (scRNA-seq) datasets without additional single-cell γδ T cell receptor sequencing (sc-γδTCR-seq) or CITE-seq (cellular indexing of transcriptomes and epitopes sequencing) data remains challenging. In this study, we developed a TCR module scoring strategy for human γδ T cell identification (i.e. based on modular gene expression of constant and variable TRA/TRB and TRD genes). We evaluated our method using 5' scRNA-seq datasets comprising both sc-αßTCR-seq and sc-γδTCR-seq as references and demonstrated that it can identify γδ T cells in scRNA-seq datasets with high sensitivity and accuracy. We observed a stable performance of this strategy across datasets from different tissues and different subtypes of γδ T cells. Thus, we propose this analysis method, based on TCR gene module scores, as a standardized tool for identifying and reanalyzing γδ T cells from 5'-end scRNA-seq datasets.

A monoclonal Trd chain supports the development of the complete set of functional γδ T cell lineages.

The clonal selection theory describes key features of adaptive immune responses of B and T cells. For αß T cells and B cells, antigen recognition and selection principles are known at a detailed molecular level. The precise role of the antigen receptor in γδ T cells remains less well understood. To better understand the role of the γδ T cell receptor (TCR), we generate an orthotopic TCRδ transgenic mouse model. We demonstrate a multi-layered functionality of γδ TCRs and diverse roles of CDR3δ-mediated selection during γδ T cell development. Whereas epithelial populations using Vγ5 or Vγ7 chains are almost unaffected in their biology in the presence of the transgenic TCRδ chain, pairing with Vγ1 positively selects γδ T cell subpopulations with distinct programs in several organs, thereby distorting the repertoire. In conclusion, our data support dictation of developmental tropism together with adaptive-like recognition principles in a single antigen receptor.

Corrigendum: Systematic pattern analyses of Vδ2+ TCRs reveal that shared "public" Vδ2+ γδ T cell clones are a consequence of rearrangement bias and a higher expansion status.

[This corrects the article DOI: 10.3389/fimmu.2022.960920.].

Systematic pattern analyses of Vδ2+ TCRs reveal that shared "public" Vδ2+ γδ T cell clones are a consequence of rearrangement bias and a higher expansion status.

Background: Vγ9Vδ2+ T cells are a major innate T cell subset in human peripheral blood. Their Vδ2+ VDJ-rearrangements are short and simple in the fetal thymus and gradually increase in diversity and CDR3 length along with development. So-called "public" versions of Vδ2+ TCRs are shared among individuals of all ages. However, it is unclear whether such frequently occurring "public" Vγ9Vδ2+ T cell clones are derived from the fetal thymus and whether they are fitter to proliferate and persist than infrequent "private" clones. Methods: Shared "public" Vδ2+ TCRs were identified from Vδ2+ TCR-repertoires collected from 89 individuals, including newborns (cord blood), infants, and adults (peripheral blood). Distance matrices of Vδ2+ CDR3 were generated by TCRdist3 and then embedded into a UMAP for visualizing the heterogeneity of Vδ2+ TCRs. Results: Vδ2+ CDR3 distance matrix embedded by UMAP revealed that the heterogeneity of Vδ2+ TCRs is primarily determined by the J-usage and CDR3aa length, while age or publicity-specific motifs were not found. The most prevalent public Vδ2+ TCRs showed germline-like rearrangement with low N-insertions. Age-related features were also identified. Public Vδ2+ TRDJ1 TCRs from cord blood showed higher N-insertions and longer CDR3 lengths. Synonymous codons resulting from VDJ rearrangement also contribute to the generation of public Vδ2+ TCRs. Each public TCR was always produced by multiple different transcripts, even with different D gene usage, and the publicity of Vδ2+ TCRs was positively associated with expansion status. Conclusion: To conclude, the heterogeneity of Vδ2+ TCRs is mainly determined by TRDJ-usage and the length of CDR3aa sequences. Public Vδ2+ TCRs result from germline-like rearrangement and synonymous codons, associated with a higher expansion status.

A CMV-induced adaptive human Vδ1+ γδ T cell clone recognizes HLA-DR.

The innate and adaptive roles of γδ T cells and their clonal γδ T cell receptors (TCRs) in immune responses are still unclear. Recent studies of γδ TCR repertoire dynamics showed massive expansion of individual Vδ1+ γδ T cell clones during viral infection. To judge whether such expansion is random or actually represents TCR-dependent adaptive immune responses, information about their cognate TCR ligands is required. Here, we used CRISPR/Cas9-mediated screening to identify HLA-DRA, RFXAP, RFX5, and CIITA as required for target cell recognition of a CMV-induced Vγ3Vδ1+ TCR, and further characterization revealed a direct interaction of this Vδ1+ TCR with the MHC II complex HLA-DR. Since MHC II is strongly upregulated by interferon-γ, these results suggest an inflammation-induced MHC-dependent immune response of γδ T cells.

Dopamine Signaling Promotes Tissue-Resident Memory Differentiation of CD8+ T Cells and Antitumor Immunity.

Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.

γδ T cells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ T cells show that direct interaction between γδ T cells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ T cells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after in vivo depletion of γδ T cells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ T cells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ T cells an attractive target to treat autoimmunity.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

NKG2A expression identifies a subset of human Vδ2 T cells exerting the highest antitumor effector functions.

Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A+ and NKG2A- cells characterize two distinct "intralineages" of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A+ Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E+ tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients' overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies.

Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI.

Donor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αß T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αß T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor ß (TRB ) sequencing of sorted CD8+ αß T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

A fetal wave of human type 3 effector γδ cells with restricted TCR diversity persists into adulthood.

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process occurs similarly in humans and whether it comprises a dedicated subset of innate-like type 3 effector γδ T cells. Here, we present a protocol for high-throughput sequencing of TRG and TRD pairs that comprise the clonal γδTCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of γδ T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature γδ T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual Vδ1+ TCRs or moderately expanded semi-invariant Vγ9Vδ2+ TCRs. The Vγ9Vδ2+ T cells shared expression of genes that mark innate-like T cells, including ZBTB16 (encoding PLZF), KLRB1, and KLRC1, but consisted of distinct clusters with unrelated Vγ9Vδ2+ TCR clones characterized either by TBX21, FCGR3A, and cytotoxicity-associated gene expression (type 1) or by CCR6, RORC, IL23R, and DPP4 expression (type 3). Effector γδ T cells with type 1 and type 3 innate T cell signatures were detected in a public dataset of early embryonic thymus organogenesis. Together, this study suggests that functionally distinct waves of human innate-like effector γδ T cells with semi-invariant Vγ9Vδ2+ TCR develop in the early fetal thymus and persist into adulthood.

Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity.

BACKGROUND: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear. METHOD: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs. RESULTS: We discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo. CONCLUSION: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.

TCR repertoire analysis reveals phosphoantigen-induced polyclonal proliferation of Vγ9Vδ2 T cells in neonates and adults.

The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.

B cell hyperactivation in an Ackr4-deficient mouse strain is not caused by lack of ACKR4 expression.

The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4-/- and Ackr4GFP/GFP ) show profoundly different phenotypes: Compared to wild-type and Ackr4GFP/GFP mice, Ackr4-/- mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4+/- animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4-/- and Ackr4GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4-/- mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4-/- mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs.

Single-Cell Transcriptomics Identifies the Adaptation of Scart1+ Vγ6+ T Cells to Skin Residency as Activated Effector Cells.

IL-17-producing γδ T cells express oligoclonal Vγ4+ and Vγ6+ TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6+ T cells as a highly homogeneous Scart1+ population in contrast to their Scart2+ IL-17-producing Vγ4+ T cell counterparts. Parabiosis demonstrates that Vγ6+ T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1+ Vγ6+ T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1+ Vγ6+ T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.

Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient Tcrd -/- mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd -/- mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.