Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

A Macrophage Colony-Stimulating-Factor-Producing γδ T Cell Subset Prevents Malarial Parasitemic Recurrence.

Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αß T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αß T cells have declined.

Aging unconventionally: γδ T cells, iNKT cells, and MAIT cells in aging.

Unconventional T cells include γδ T cells, invariant Natural Killer T cells (iNKT) cells and Mucosal Associated Invariant T (MAIT) cells, which are distinguished from conventional T cells by their recognition of non-peptide ligands presented by non-polymorphic antigen presenting molecules and rapid effector functions that are pre-programmed during their development. Here we review current knowledge of the effect of age on unconventional T cells, from early life to old age, in both mice and humans. We then discuss the role of unconventional T cells in age-associated diseases and infections, highlighting the similarities between members of the unconventional T cell family in the context of aging.

Speed and navigation control of thymocyte development by the fetal T-cell gene regulatory network.

T-cell differentiation is a tightly regulated developmental program governed by interactions between transcription factors (TFs) and chromatin landscapes and affected by signals received from the thymic stroma. This process is marked by a series of checkpoints: T-lineage commitment, T-cell receptor (TCR)ß selection, and positive and negative selection. Dynamically changing combinations of TFs drive differentiation along the T-lineage trajectory, through mechanisms that have been most extensively dissected in adult mouse T-lineage cells. However, fetal T-cell development differs from adult in ways that suggest that these TF mechanisms are not fully deterministic. The first wave of fetal T-cell differentiation occurs during a unique developmental window during thymic morphogenesis, shows more rapid kinetics of differentiation with fewer rounds of cell division, and gives rise to unique populations of innate lymphoid cells (ILCs) and invariant γδT cells that are not generated in the adult thymus. As the characteristic kinetics and progeny biases are cell-intrinsic properties of thymic progenitors, the differences could be based on distinct TF network circuitry within the progenitors themselves. Here, we review recent single-cell transcriptome data that illuminate the TF networks involved in T-cell differentiation in the fetal and adult mouse thymus.

The multisensory regulation of unconventional T cell homeostasis.

Unconventional T cells typically group γδ T cells, invariant Natural Killer T cells (NKT) and Mucosal Associated Invariant T (MAIT) cells. With their pre-activated status and biased tropism for non-lymphoid organs, they provide a rapid (innate-like) and efficient first line of defense against pathogens at strategical barrier sites, while they can also trigger chronic inflammation, and unexpectedly contribute to steady state physiology. Thus, a tight control of their homeostasis is critical to maintain tissue integrity. In this review, we discuss the recent advances of our understanding of the factors, from neuroimmune to inflammatory regulators, shaping the size and functional properties of unconventional T cell subsets in non-lymphoid organs. We present a general overview of the mechanisms common to these populations, while also acknowledging specific aspects of their diversity. We mainly focus on their maintenance at steady state and upon inflammation, highlighting some key unresolved issues and raising upcoming technical, fundamental and translational challenges.

γδ TCR Recognition of MR1: Adapting to Life on the Flip Side.

Nonclassical class I MHC-like molecules are ligands for several unconventional T cell populations. Recently, Le Nours et al. identified human γδ T cells recognising MHC-related protein-1 (MR1) via their T cell receptor (TCR). Also recognised by the αß-TCR of mucosal associated invariant T cells, MR1 interacts with specific γδ-TCRs using strikingly diverse binding modes, suggesting fundamental differences in γδ T cell recognition.

Hyperactivation and enhanced cytotoxicity of reduced CD8+ gamma delta T cells in the intestine of patients with Crohn's disease correlates with disease activity.

BACKGROUND AND AIMS: We aimed to investigate the immune characteristics of intestinal CD8+ gamma delta T (CD8+ γδ T) cells in Crohn's disease (CD) and their correlation with disease activity. METHODS: The study cohorts included 21 CD patients and 21 healthy individuals. CD8+ γδ T cells were isolated from human ileal mucosa for detection by flow cytometry. The activation or inhibition status of cells was detected by detecting the expression of activation marker HLA-DR and the immunosuppressive molecule PD-1 on cells. The cytotoxicity of cells was assessed by detecting the expression of cytotoxic molecules (Perforin, Granzyme B, and TRAIL) in cells. Ratios of investigated cells were calculated as prediction factors by receiver operating characteristic curve (ROC) analysis. RESULTS: The study revealed a reduction in intestinal CD8+ γδT cells among active CD patients, with a more pronounced reduction observed in moderately active patients compared to mildly active patients. Moreover, active CD patients exhibited heightened activation levels in their intestinal CD8+ γδT cells, whereas the activation was comparatively weakened in moderately active patients compared with mildly active patients. Additionally, the cytotoxicity of intestinal CD8+ γδT cells was enhanced solely in mildly active patients, while it was impaired in moderately active patients compared with mildly active patients. Furthermore, HLA-DR+ CD8+ γδT cell ratio, CD8+ γδT ratio, and CD8+ γδT count were identified as indicators in the diagnosis of active CD. Meanwhile, the ratios of Granzyme B+ CD8+ γδT cell and Perforin+ CD8+ γδT cell were identified as indicators that distinguish mildly moderately active CD cases. CONCLUSIONS: Intestinal CD8+ γδT was reduced in active CD patients, but their activation and cytotoxicity were enhanced. However, with increased disease activity, intestinal CD8+ γδ T cells became dysfunctional. CD-specific perturbations observed in various phenotypic markers in CD8+ γδ T cells can be used as indicators to assist in diagnosing CD patients.

Deciphering the regulatory landscape of fetal and adult γδ T-cell development at single-cell resolution.

γδ T cells with distinct properties develop in the embryonic and adult thymus and have been identified as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases, and tissue homeostasis. Despite their potential value for immunotherapy, differentiation of γδ T cells in the thymus is incompletely understood. Here, we establish a high-resolution map of γδ T-cell differentiation from the fetal and adult thymus using single-cell RNA sequencing. We reveal novel sub-types of immature and mature γδ T cells and identify an unpolarized thymic population which is expanded in the blood and lymph nodes. Our detailed comparative analysis reveals remarkable similarities between the gene networks active during fetal and adult γδ T-cell differentiation. By performing a combined single-cell analysis of Sox13, Maf, and Rorc knockout mice, we demonstrate sequential activation of these factors during IL-17-producing γδ T-cell (γδT17) differentiation. These findings substantially expand our understanding of γδ T-cell ontogeny in fetal and adult life. Our experimental and computational strategy provides a blueprint for comparing immune cell differentiation across developmental stages.

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

T cells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, T cells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted T cells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted T cells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

Surface protein and functional analyses identify CD4+CD39+ TCR αß+ and activated TCR Vδ1+ cells with distinct pro-inflammatory functions in Crohn's disease lesions.

Crohn's disease (CD) is a chronic immune-mediated disorder of the gastrointestinal tract. Extensive screening studies have revealed the accumulation of immune cell subsets with unique plasticity and immunoregulatory properties in patients with CD. We performed phenotypic and functional studies on inflamed and non-inflamed bioptic tissue to investigate the presence of distinct T cells in the intestinal mucosa of CD patients. We analysed hundreds of surface molecules expressed on cells isolated from the intestinal tissue of CD patients using anti-CD45 mAbs-based barcoding. A gene ontology enrichment analysis showed that proteins that regulate the activation of T cells were the most enriched group. We, therefore, designed T-cell focused multicolour flow-cytometry panels and performed clustering analysis which revealed an accumulation of activated TEM CD4+CD39+ T cells producing IL-17 and IL-21 and increased frequency of terminally differentiated TCR Vδ1+ cells producing TNF-α and IFN-γ in inflamed tissue of CD patients. The different functional capacities of CD4+ and TCR Vδ1+ cells in CD lesions indicate their non-overlapping contribution to inflammation. The abnormally high number of terminally differentiated TCR Vδ1+ cells suggests that they are continuously activated in inflamed tissue, making them a potential target for novel therapies.

OMIP-099: 31-color spectral flow cytometry panel to investigate the steady-state phenotype of human T cells.

We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using cryopreserved peripheral blood mononuclear cells (PBMC). The markers included in this panel were chosen in order to characterize the steady-state phenotype of T cells and includes markers (CD45RA, CD45RO, CCR7, CD95) to distinguish the main subsets (e.g., naïve, TEM , TCM , TEMRA , TSCM etc.) of CD4, CD8, and γδ T cells. This panel also includes markers for the identification of differentiation status (CD27, CD28), activation/antigen experience status (CD11a, CD49d, CD38, HLA-DR, CD56, and CD39), co-inhibitory marker expression (PD-1, TIM-3), and CD4 T helper subsets (CXCR3, CXCR5, CCR4, CCR6, Foxp3, CD25, and CD127). This optimized panel provides a broad assessment of the steady-state phenotype of human T cells.

A new algorithm for coeliac disease based on the 'long forgotten' TCRγδ+ intra-epithelial lymphocytes detected with an antibody working on FFPE sections.

AIMS: Diagnosis of coeliac disease (CD) with mild mucosal changes is difficult for all parties involved. We aimed to determine the power of T cell receptor (TCR)γδ+ intra-epithelial lymphocytes (IELs) in discriminating CD from other causes of intra-epithelial lymphocytosis using a new monoclonal antibody. METHODS: A total of 167 cases categorised as coeliac (117 untreated CD, classified according to Marsh, updated by Ensari, including 29 type 1, 29 type 2, 39 type 3 and 20 treated CD), and non-coeliac groups (24 controls and 26 non-coeliac IELosis) based on clinical, serological and histological data were studied for IEL counts enumerated per 100 enterocytes using haematoxylin and eosin, CD3, TCR δ-stains. RESULTS: TCRγδ+ IELs were significantly higher in CD (24.83 ± 16.13) compared to non-CD (6.72 ± 6.32) and were correlated with the degree of mucosal damage. Both γδ+ IEL count and ratio showed higher performance in differentiating untreated coeliacs from controls, with a sensitivity of 83.76; 85.57 and specificity of 95.83; 79.17, respectively. TCRγδ+ IEL counts distinguished type 1 CD (20.41 ± 13.57) from non-coeliac IELosis (9.42 ± 7.28) (p = 0.025). Discriminant analysis revealed that villus/crypt ratio, γδ+ and CD3+ IEL counts, γδ+/CD3+IEL ratio, IEL distribution pattern were potent discriminants and correctly classified 82.3% of cases while the algorithm accurately diagnosed 93.4% of cases. CONCLUSIONS: The new antibody detecting γδ+ IELs in FFPE sections revealed thresholds of 10.5 for γδ+ IELs and 14% for γδ+/CD3+IEL ratio which distinguished coeliacs from non-coeliacs with high sensitivity and specificity, particularly in cases with normal villus/crypt axis including type 1 CD, non-CD IELosis and controls. A 'coeliac algorithm' based on γδ+ IELs is proposed with the hope that it will be used in the histopathological diagnostic approach by the pathology community.

Prominent tissue hemophagocytic lymphohistiocytosis obscuring primary cutaneous gamma/delta (γδ) T-cell lymphoma.

Primary cutaneous gamma/delta (γδ) T-cell lymphoma (PCGDTCL) is a rare, aggressive malignant neoplasm of γδ T lymphocytes arising within the skin and subcutis. We present a challenging case of PCGDTCL diagnosed in a 35-year-old male soldier who presented with constitutional symptoms, pancytopenia, hemophagocytic lymphohistiocytosis (HLH), disseminated lymphadenopathy, and cutaneous lesions on his extremities and back following a deployment to Iraq and Syria. Histopathologic evaluation of an excisional biopsy showed that PCGDTCL can be focal, localized to the subcutaneous adipose tissue, and obscured by predominant HLH in the surrounding tissues. Pathologists should recognize that the diagnosis of PCGDTCL may be confounded by florid HLH and require multiple biopsies and a comprehensive immunohistochemical panel.

The Role of Gamma Delta T Lymphocytes in Physiological and Pathological Condition-Focus on Psoriasis, Atopic Dermatitis, Autoimmune Disorders, Cancer and Lymphomas.

Gamma delta (γδ) T cells are a heterogeneous population of cells that play roles in inflammation, host tissue repair, clearance of viral and bacterial pathogens, regulation of immune processes, and tumor surveillance. Recent research suggests that these are the main skin cells that produce interleukin-17 (I-17). Furthermore, γδ T cells exhibit memory-cell-like characteristics that mediate repeated episodes of psoriatic inflammation. γδ T cells are found in epithelial tissues, where many cancers develop. There, they participate in antitumor immunity as cytotoxic cells or as immune coordinators. γδ T cells also participate in host defense, immune surveillance, and immune homeostasis. The aim of this review is to present the importance of γδ T cells in physiological and pathological diseases, such as psoriasis, atopic dermatitis, autoimmune diseases, cancer, and lymphoma.

From thymus to periphery: Molecular basis of effector γδ-T cell differentiation.

The contributions of γδ T cells to immune (patho)physiology in many pre-clinical mouse models have been associated with their rapid and abundant provision of two critical cytokines, interferon-γ (IFN-γ) and interleukin-17A (IL-17). These are typically produced by distinct effector γδ T cell subsets that can be segregated on the basis of surface expression levels of receptors such as CD27, CD44 or CD45RB, among others. Unlike conventional T cells that egress the thymus as naïve lymphocytes awaiting further differentiation upon activation, a large fraction of murine γδ T cells commits to either IFN-γ or IL-17 expression during thymic development. However, extrathymic signals can both regulate pre-programmed γδ T cells; and induce peripheral differentiation of naïve γδ T cells into effectors. Here we review the key cellular events of "developmental pre-programming" in the mouse thymus; and the molecular basis for effector function maintenance vs plasticity in the periphery. We highlight some of our contributions towards elucidating the role of T cell receptor, co-receptors (like CD27 and CD28) and cytokine signals (such as IL-1ß and IL-23) in these processes, and the various levels of gene regulation involved, from the chromatin landscape to microRNA-based post-transcriptional control of γδ T cell functional plasticity.

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling.

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his âºß T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.

Peripheral T Cell Populations are Differentially Affected in Familial Mediterranean Fever, Chronic Granulomatous Disease, and Gout.

Both innate errors of immunity, such as familial Mediterranean fever (FMF) and chronic granulomatous disease (CGD), and the common inflammatory disease gout are characterized by episodes of sterile inflammatory attacks in the absence of an infection. While these disorders encompass distinct pathologies due to differentially affected metabolic pathways and inflammasome activation mechanisms, their common features are the excessive production of interleukin (IL)-1ß and innate immune cell hyperreactivity. On the other hand, the role of T cells and innate-like lymphocytes such as gamma delta (γδ) T cells in these pathologies is ill-defined. In order to widen our understanding of T cell involvement in CGD, FMF and gout pathology, we developed multicolour immunophenotyping panels for flow cytometry to characterize γδ T cells as well as CD4 and CD8 T cell populations in terms of their cytokine production, activation status, memory or naive phenotypes, exhaustion status, homing receptor expression, and cytotoxic activity. Our study is the first deep immunophenotyping analysis of T cell populations in CGD, FMF, and gout patients. We found that CGD affects the frequencies and activation status of T cells, while gout impairs the cytokine production capacity of Vδ2 T cells. FMF was characterized by decreased percentages of regulatory T cells in circulation and attenuated IFN-γ production capacity by Vδ2 T cells. Autoinflammatory syndromes and congenital defects of phagocyte differentially affect T cell compartments. Future studies are warranted to assess whether these phenotypical changes are relevant for disease pathology.