T Cell Responses to the Microbiota.

The immune system employs recognition tools to communicate with its microbial evolutionary partner. Among all the methods of microbial perception, T cells enable the widest spectrum of microbial recognition resolution, ranging from the crudest detection of whole groups of microbes to the finest detection of specific antigens. The application of this recognition capability to the crucial task of combatting infections has been the focus of classical immunology. We now appreciate that the coevolution of the immune system and the microbiota has led to development of a lush immunological decision tree downstream of microbial recognition, of which an inflammatory response is but one branch. In this review we discuss known T cell-microbe interactions in the gut and place them in the context of an algorithmic framework of recognition, context-dependent interpretation, and response circuits across multiple levels of microbial recognition resolution. The malleability of T cells in response to the microbiota presents an opportunity to edit immune response cellularity, identity, and functionality by utilizing microbiota-controlled pathways to promote human health.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

An Immunologic Mode of Multigenerational Transmission Governs a Gut Treg Setpoint.

At the species level, immunity depends on the selection and transmission of protective components of the immune system. A microbe-induced population of RORγ-expressing regulatory T cells (Tregs) is essential in controlling gut inflammation. We uncovered a non-genetic, non-epigenetic, non-microbial mode of transmission of their homeostatic setpoint. RORγ+ Treg proportions varied between inbred mouse strains, a trait transmitted by the mother during a tight age window after birth but stable for life, resistant to many microbial or cellular perturbations, then further transferred by females for multiple generations. RORγ+ Treg proportions negatively correlated with IgA production and coating of gut commensals, traits also subject to maternal transmission, in an immunoglobulin- and RORγ+ Treg-dependent manner. We propose a model based on a double-negative feedback loop, vertically transmitted via the entero-mammary axis. This immunologic mode of multi-generational transmission may provide adaptability and modulate the genetic tuning of gut immune responses and inflammatory disease susceptibility.

Single-Cell Mapping of Human Brain Cancer Reveals Tumor-Specific Instruction of Tissue-Invading Leukocytes.

Brain malignancies can either originate from within the CNS (gliomas) or invade from other locations in the body (metastases). A highly immunosuppressive tumor microenvironment (TME) influences brain tumor outgrowth. Whether the TME is predominantly shaped by the CNS micromilieu or by the malignancy itself is unknown, as is the diversity, origin, and function of CNS tumor-associated macrophages (TAMs). Here, we have mapped the leukocyte landscape of brain tumors using high-dimensional single-cell profiling (CyTOF). The heterogeneous composition of tissue-resident and invading immune cells within the TME alone permitted a clear distinction between gliomas and brain metastases (BrM). The glioma TME presented predominantly with tissue-resident, reactive microglia, whereas tissue-invading leukocytes accumulated in BrM. Tissue-invading TAMs showed a distinctive signature trajectory, revealing tumor-driven instruction along with contrasting lymphocyte activation and exhaustion. Defining the specific immunological signature of brain tumors can facilitate the rational design of targeted immunotherapy strategies.

Amphiregulin from regulatory T cells promotes liver fibrosis and insulin resistance in non-alcoholic steatohepatitis.

Production of amphiregulin (Areg) by regulatory T (Treg) cells promotes repair after acute tissue injury. Here, we examined the function of Treg cells in non-alcoholic steatohepatitis (NASH), a setting of chronic liver injury. Areg-producing Treg cells were enriched in the livers of mice and humans with NASH. Deletion of Areg in Treg cells, but not in myeloid cells, reduced NASH-induced liver fibrosis. Chronic liver damage induced transcriptional changes associated with Treg cell activation. Mechanistically, Treg cell-derived Areg activated pro-fibrotic transcriptional programs in hepatic stellate cells via epidermal growth factor receptor (EGFR) signaling. Deletion of Areg in Treg cells protected mice from NASH-dependent glucose intolerance, which also was dependent on EGFR signaling on hepatic stellate cells. Areg from Treg cells promoted hepatocyte gluconeogenesis through hepatocyte detection of hepatic stellate cell-derived interleukin-6. Our findings reveal a maladaptive role for Treg cell-mediated tissue repair functions in chronic liver disease and link liver damage to NASH-dependent glucose intolerance.

Immunomodulatory leptin receptor+ sympathetic perineurial barrier cells protect against obesity by facilitating brown adipose tissue thermogenesis.

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR+) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPCΔIl33) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPCΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR+ SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+IL-33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research.

CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8+ T cell antitumor immunity.

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4+ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3+ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3+ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8+ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8+ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.

Oral tolerance to dietary antigens and Foxp3+ regulatory T cells.

Immune tolerance to foods develops in the intestine upon food ingestion and is essential to prevent IgE-mediated food allergy and gut inflammation. In homeostasis, the intestine is a tolerogenic environment that favors the formation of food-specific Foxp3+ regulatory T cells. A tolerogenic intestinal environment depends on colonization by diverse microbiota and exposure to solid foods at a critical period in early life. These early immune responses lead to the induction of antigen-specific Foxp3+ regulatory T cells in draining mesenteric lymph nodes. These peripherally induced regulatory cells circulate and seed the lamina propria of the gut, exerting suppressive function systemically and locally in the intestine. Successful establishment of a tolerogenic intestinal environment in early life sets the stage for oral tolerance to new antigens in adult life.

YTHDF2/m6 A/NF-κB axis controls anti-tumor immunity by regulating intratumoral Tregs.

N6 -methyladenosine (m6 A) in messenger RNA (mRNA) regulates immune cells in homeostasis and in response to infection and inflammation. The function of the m6 A reader YTHDF2 in the tumor microenvironment (TME) in these contexts has not been explored. We discovered that the loss of YTHDF2 in regulatory T (Treg) cells reduces tumor growth in mice. Deletion of Ythdf2 in Tregs does not affect peripheral immune homeostasis but leads to increased apoptosis and impaired suppressive function of Treg cells in the TME. Elevated tumor necrosis factor (TNF) signaling in the TME promotes YTHDF2 expression, which in turn regulates NF-κB signaling by accelerating the degradation of m6 A-modified transcripts that encode NF-κB-negative regulators. This TME-specific regulation of Treg by YTHDF2 points to YTHDF2 as a potential target for anti-cancer immunotherapy, where intratumoral Treg cells can be targeted to enhance anti-tumor immune response while avoiding Treg cells in the periphery to minimize undesired inflammations.

Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies.

Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy-but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism.

Neuroinflammation: Extinguishing a blaze of T cells.

Inflammation is a biological process that dynamically alters the surrounding microenvironment, including participating immune cells. As a well-protected organ surrounded by specialized barriers and with immune privilege properties, the central nervous system (CNS) tightly regulates immune responses. Yet in neuroinflammatory conditions, pathogenic immunity can disrupt CNS structure and function. T cells in particular play a key role in promoting and restricting neuroinflammatory responses, while the inflamed CNS microenvironment can influence and reshape T cell function and identity. Still, the contraction of aberrant T cell responses within the CNS is not well understood. Using autoimmunity as a model, here we address the contribution of CD4 T helper (Th) cell subsets in promoting neuropathology and disease. To address the mechanisms antagonizing neuroinflammation, we focus on the control of the immune response by regulatory T cells (Tregs) and describe the counteracting processes that preserve their identity under inflammatory challenges. Finally, given the influence of the local microenvironment on immune regulation, we address how CNS-intrinsic signals reshape T cell function to mitigate abnormal immune T cell responses.

MicroRNAs targeting TGF-ß signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-ß) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-ß signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-ß signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-ß-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-ß-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-ß signaling may restore immune homeostasis in MS patients.

CD8+ Tregs kill pathogenic cells to avert autoimmunity.

Mouse Ly49+CD8+ regulatory T cells (Tregs) can subdue autoreactive CD4+ T cells to suppress autoimmunity. Recently, Li et al. demonstrated that killer-cell immunoglobulin-like receptor (KIR)+CD8+ T cells are the human equivalent of Ly49+CD8+ regulatory T cells and kill pathogenic CD4+ T cells, which can be increased in certain human autoimmune diseases and viral infections.

Tolerogenic CD11c+dendritic cells regulate CD4+Tregs in replacing delayed ischemic preconditioning to alleviate ischemia-reperfusion acute kidney injury.

Ischemia-reperfusion injury (IRI) is one of the primary clinical causes of acute kidney injury (AKI). The key to IRI lies in immune-inflammatory damage, where dendritic cells (DCs) play a central role in eliciting immune responses within the context of inflammation induced by ischemia-reperfusion. Our previous study has confirmed that delayed ischemic preconditioning (DIPC) can reduce the kidney injury by mediating DCs to regulate T-cells. However, the clinical feasibility of DIPC is limited, as pre-clamping of the renal artery is not applicable for the prevention and treatment of ischemia-reperfusion acute kidney injury (I/R-AKI) in clinical patients. Therefore, the infusion of DCs as a substitute for DIPC presents a more viable strategy for preventing renal IRI. In this study, we further evaluated the impact and mechanism of infused tolerogenic CD11c+DCs on the kidneys following IRI by isolating bone marrow-derived dendritic cells and establishing an I/R-AKI model after pre-infusion of DCs. Renal function was significantly improved in the I/R-AKI mouse model after pre-infused with CD11c+DCs. The pro-inflammatory response and oxidative damage were reduced, and the levels of T helper 2 (Th2) cells and related anti-inflammatory cytokines were increased, which was associated with the reduction of autologous DCs maturation mediated by CD11c+DCs and the increase of regulatory T-cells (Tregs). Next, knocking out CD11c+DCs, we found that the reduced immune protection of tolerogenic CD11c+DCs reinfusion was related to the absence of own DCs. Together, pre-infusion of tolerogenic CD11c+DCs can replace the regulatory of DIPC on DCs and T-cells to alleviate I/R-AKI. DC vaccine is expected to be a novel avenue to prevent and treat I/R-AKI.

Characterization of novel CD8+ regulatory T cells and their modulatory effects in murine model of inflammatory bowel disease.

Dysregulation of mucosal immune system has been proposed to be critical in the pathogenesis of inflammatory bowel diseases (IBDs). Regulatory T cells (Tregs) play an important role in regulating immune responses. Tregs are involved in maintaining intestinal homeostasis and exerting suppressive function in colitis. Our previous studies showed that a novel forkhead box protein P3 (Foxp3) negative Tregs (Treg-of-B cells), induced by culturing naïve CD4+ T cells with B cells, could protect against colitis and downregulate T helper (Th) 1 and Th17 cell cytokines in T cell-mediated colitis. In the present study, we aimed to induce Treg-of-B cells in the CD8+ T-cell population and investigate their characteristics and immunomodulatory functions. Our results showed that CD8+ Treg-of-B cells expressed Treg-associated markers, including lymphocyte-activation gene-3 (LAG3), inducible co-stimulator (ICOS), programmed death-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), tumor necrosis factor receptor superfamily member-4 (TNFRSF4, OX40), and tumor necrosis factor receptor superfamily member-18 (TNFRSF18, GITR), but did not express Foxp3. CD8+ Treg-of-B cells produced higher concentration of inhibitory cytokine interleukin (IL)-10, and expressed higher levels of cytotoxic factor granzyme B and perforin after stimulation, compared to those of CD8+CD25- T cells. Moreover, CD8+ Treg-of-B cells suppressed T cell proliferation in vitro and alleviated colonic inflammation in chronic dextran sulfate sodium (DSS)-induced colitis. In conclusion, our study identified a novel subpopulation of CD8+ Tregs with suppressive effects through cell contact. These CD8+ Treg-of-B cells might have therapeutic potential for IBDs.

Loss of chromosome Y in regulatory T cells.

BACKGROUND: Mosaic loss of chromosome Y (LOY) in leukocytes is the most prevalent somatic aneuploidy in aging humans. Men with LOY have increased risks of all-cause mortality and the major causes of death, including many forms of cancer. It has been suggested that the association between LOY and disease risk depends on what type of leukocyte is affected with Y loss, with prostate cancer patients showing higher levels of LOY in CD4 + T lymphocytes. In previous studies, Y loss has however been observed at relatively low levels in this cell type. This motivated us to investigate whether specific subsets of CD4 + T lymphocytes are particularly affected by LOY. Publicly available, T lymphocyte enriched, single-cell RNA sequencing datasets from patients with liver, lung or colorectal cancer were used to study how LOY affects different subtypes of T lymphocyte. To validate the observations from the public data, we also generated a single-cell RNA sequencing dataset comprised of 23 PBMC samples and 32 CD4 + T lymphocytes enriched samples. RESULTS: Regulatory T cells had significantly more LOY than any other studied T lymphocytes subtype. Furthermore, LOY in regulatory T cells increased the ratio of regulatory T cells compared with other T lymphocyte subtypes, indicating an effect of Y loss on lymphocyte differentiation. This was supported by developmental trajectory analysis of CD4 + T lymphocytes culminating in the regulatory T cells cluster most heavily affected by LOY. Finally, we identify dysregulation of 465 genes in regulatory T cells with Y loss, many involved in the immunosuppressive functions and development of regulatory T cells. CONCLUSIONS: Here, we show that regulatory T cells are particularly affected by Y loss, resulting in an increased fraction of regulatory T cells and dysregulated immune functions. Considering that regulatory T cells plays a critical role in the process of immunosuppression; this enrichment for regulatory T cells with LOY might contribute to the increased risk for cancer observed among men with Y loss in leukocytes.

Tregs protect against invariant NKT cell-mediated autoimmune colitis and hepatitis.

Immunomodulatory T cells play a pivotal role in protection against (auto)immune-mediated diseases that open perspectives for therapeutic modulation. However, how immune regulatory networks operate in vivo is less understood. To this end, we focused on FOXP3+CD4+CD25+ regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells, two lymphocyte populations that independently regulate adaptive and innate immune responses. In vitro, a functional interplay between Tregs and iNKT cells has been described, but whether Tregs modulate the function and phenotype of iNKT cell subsets in vivo and whether this controls iNKT-mediated autoimmunity is unclear. Taking advantage of the conditional depletion of Tregs, we examined the in vivo interplay between iNKT and Treg cells in steady state and in preclinical models of liver and gut autoimmunity. Under non-inflamed conditions, Treg depletion enhanced glycolipid-mediated iNKT cell responses, with a general impact on Type 1, 2 and 17 iNKT subsets. Moreover, in vivo iNKT activation in the absence of Tregs suppressed the induction of iNKT anergy, consistent with a reduction in programmed cell death receptor 1 (PD-1) expression. Importantly, we unveiled a clear role for an in vivo Treg-iNKT crosstalk both in concanavalin A-induced acute hepatitis and oxazolone-induced colitis. Here, the absence of Tregs led to a markedly enhanced liver and gut pathology, which was not observed in iNKT-deficient mice. Taken together, these results provide evidence for a functional interplay between regulatory T cell subsets critical in controlling the onset of autoimmune disease.

CCR5-mediated homing of regulatory T cells and monocytic-myeloid derieved suppressor cells to dysfunctional endothelium contributes to early atherosclerosis.

A disbalance between immune regulatory cells and inflammatory cells is known to drive atherosclerosis. However, the exact mechanism is not clear. Here, we investigated the homing of immune regulatory cells, mainly, regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) subsets in asymptomatic coronary artery disease (CAD) risk factor-exposed young individuals (dyslipidemia [DLP] group) and stable CAD patients (CAD group). Compared with healthy controls (HCs), Tregs frequency was reduced in both DLP and CAD groups but expressed high levels of CCR5 in both groups. The frequency of monocytic-myeloid-derived suppressor cells (M-MDSCs) was increased while polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were decreased in CAD patients only. Interestingly, although unchanged in frequency, M-MDSCs of the DLP group expressed high levels of CCR5. Serum levels of chemokines (CCL5, CX3CL1, CCL26) and inflammatory cytokines (IL-6, IL-1ß, IFN-γ, TNF-α) were higher in the DLP group. Stimulation with inflammatory cytokines augmented CCR5 expression in Tregs and M-MDSCs isolated from HCs. Activated endothelial cells showed elevated levels of CX3CL1 and CCL5 in vitro. Blocking CCR5 with D-Ala-peptide T-amide (DAPTA) increased Treg and M-MDSC frequency in C57Bl6 mice fed a high-fat diet. In accelerated atherosclerosis model, DAPTA treatment led to the formation of smooth muscle-rich plaque with less macrophages. Thus, we show that CCR5-CCL5 axis is instrumental in recruiting Tregs and M-MDSCs to dysfunctional endothelium in the asymptomatic phase of atherosclerosis contributing to atherosclerosis progression. Drugs targeting CCR5 in asymptomatic and CAD risk-factor/s-exposed individuals might be a novel therapeutic regime to diminish atherogenesis.

Single-cell transcriptomic and T cell antigen receptor analysis of human cytomegalovirus (hCMV)-specific memory T cells reveals effectors and pre-effectors of CD8+- and CD4+-cytotoxic T cells.

Latent human cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals. Although T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in hCMV immunity is lacking. Here, in an unbiased manner, we characterized over 8000 hCMV-reactive peripheral memory T cells isolated from seropositive human donors, at a single-cell resolution by analysing their single-cell transcriptomes paired with the T cell antigen receptor (TCR) repertoires. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental and functional memory T cell subsets such as, long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs) of both CD4 and CD8 origin. The hCMV-specific TREGs, in addition to being enriched for molecules known for their suppressive functions, showed enrichment for the interferon response signature gene sets. The hCMV-specific CTLs were of two types, the pre-effector- and effector-like. The co-clustering of hCMV-specific CD4-CTLs and CD8-CTLs in both pre-effector as well as effector clusters suggest shared transcriptomic signatures between them. The huge TCR clonal expansion of cytotoxic clusters suggests a dominant role in the protective immune response to CMV. The study uncovers the heterogeneity in the hCMV-specific memory T cells revealing many functional subsets with potential implications in better understanding of hCMV-specific T cell immunity. The data presented can serve as a knowledge base for designing vaccines and therapeutics.