Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4- CD8- unconventional αß T cells (UTCαß). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαß associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαß polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors.

The paradox of aging: Aging-related shifts in T cell function and metabolism.

T cell survival, differentiation after stimulation, and function are intrinsically linked to distinct cellular metabolic states. The ability of T cells to readily transition between metabolic states enables flexibility to meet the changing energy demands defined by distinct effector states or T cell lineages. Immune aging is characterized, in part, by the loss of naïve T cells, accumulation of senescent T cells, severe dysfunction in memory phenotype T cells in particular, and elevated levels of inflammatory cytokines, or 'inflammaging'. Here, we review our current understanding of the phenotypic and functional changes that occur with aging in T cells, and how they relate to metabolic changes in the steady state and after T cell activation. We discuss the apparent contradictions in the aging T cell phenotype - where enhanced differentiation states and metabolic profiles in the steady state can correspond to a diminished capacity to adapt metabolically and functionally after T cell activation. Finally, we discuss key recent studies that indicate the enormous potential for aged T cell metabolism to induce systemic inflammaging and organism-wide multimorbidity, resulting in premature death.

Highlight of 2023: Unconventional T cells: recent insights on development, trafficking and target cell recognition.

In this article for the Highlights of 2023 Series, we discuss recent research on unconventional T cells with a focus on gamma delta T cell development and cancer cell targeting, as well as the contributions of MAIT cells to wound repair.

Conventional and unconventional T-cell responses contribute to the prediction of clinical outcome and causative bacterial pathogen in sepsis patients.

Sepsis is characterized by a dysfunctional host response to infection culminating in life-threatening organ failure that requires complex patient management and rapid intervention. Timely diagnosis of the underlying cause of sepsis is crucial, and identifying those at risk of complications and death is imperative for triaging treatment and resource allocation. Here, we explored the potential of explainable machine learning models to predict mortality and causative pathogen in sepsis patients. By using a modelling pipeline employing multiple feature selection algorithms, we demonstrate the feasibility of identifying integrative patterns from clinical parameters, plasma biomarkers, and extensive phenotyping of blood immune cells. While no single variable had sufficient predictive power, models that combined five and more features showed a macro area under the curve (AUC) of 0.85 to predict 90-day mortality after sepsis diagnosis, and a macro AUC of 0.86 to discriminate between Gram-positive and Gram-negative bacterial infections. Parameters associated with the cellular immune response contributed the most to models predictive of 90-day mortality, most notably, the proportion of T cells among PBMCs, together with expression of CXCR3 by CD4+ T cells and CD25 by mucosal-associated invariant T (MAIT) cells. Frequencies of Vδ2+ γδ T cells had the most profound impact on the prediction of Gram-negative infections, alongside other T-cell-related variables and total neutrophil count. Overall, our findings highlight the added value of measuring the proportion and activation patterns of conventional and unconventional T cells in the blood of sepsis patients in combination with other immunological, biochemical, and clinical parameters.

Function of unconventional T cells in oral lichen planus revealed by single-cell RNA sequencing.

OBJECTIVE: We intended to map the single-cell profile of OLP, explore the molecular characteristics of unconventional T cells in OLP tissues. METHODS: Buccal mucosa samples from OLP patients and healthy individuals were used to prepare single-cell suspension. Single-cell RNA sequencing was used to analyze the proportion of all the cells, and the molecular characteristics of unconventional T cells. Immunohistochemical staining was used to detect the expression of unconventional T cells marker genes. RESULTS: The cell clusters from buccal mucosa were categorized into immune cells, fibroblasts, endothelial cells, and epithelial cells. Unconventional T cells with phenotype of CD247+TRDC+NCAM1+ were identified. Immunohistochemical staining revealed higher expression of unconventional T cell marker genes in OLP tissue, predominantly in the lamina propria. In OLP, unconventional T cells are in a unique stress response state, exhibited enhanced NF-κB signaling and apoptosis inhibition, enhanced heat shock protein genes expression, weakened cytotoxic function. A large number of ligand-receptor pairs were found between unconventional T cells and other cells, particularly with fibroblasts and endothelial cells. CONCLUSIONS: This study mapped the single-cell profile of OLP, delineated the molecular characteristics of unconventional T cells in OLP, and uncovered that these unconventional T cells are in a stress response state.

Landscape of unconventional γδ T cell subsets in cancer.

T cells are broadly categorized into two groups, namely conventional and unconventional T cells. Conventional T cells are the most prevalent and well-studied subset of T cells. On the other hand, unconventional T cells exhibit diverse functions shared between innate and adaptive immune cells. During recent decades, γδ T cells have received attention for their roles in cancer immunity. These cells can detect various molecules, such as lipids and metabolites. Also, they are known for their distinctive ability to recognize and target cancer cells in the tumor microenvironment (TME). This feature of γδ T cells could provide a unique therapeutic tool to fight against cancer. Understanding the role of γδ T cells in TME is essential to prepare the groundwork to use γδ T cells for clinical purposes. Here, we provide recent knowledge regarding the role γδ T cell subsets in different cancer types.

Immune recognition of phosphoantigen-butyrophilin molecular complexes by γδ T cells.

Gamma-delta (γδ) T cells are an important component of the immune system. They are often enriched in non-lymphoid tissues and exhibit diverse functional attributes including rapid activation, cytokine production, proliferation, and acquisition of cytotoxicity following both TCR-dependent and TCR-independent stimulation, but poor capacity for immunological memory. They can detect a broad range of antigens, although typically not peptide-MHC complexes in contrast to alpha-beta (αß) T cells. In humans, a prominent population of γδ T cells, defined as Vγ9Vδ2+ cells, reacts to small phosphorylated non-peptide "phosphoantigens" (pAgs). The molecular mechanism underpinning this recognition is poorly defined, but is known to involve butyrophilin family members and appears to involve indirect pAg recognition via alterations to butyrophilin molecular complexes. In this review, we discuss recent advances in our understanding of pAg recognition by γδ T cells including the role of butyrophilins and in particular, a newly described role for butyrophilin 2A1.

T-cell receptor αß+ double-negative T cells in the kidney are predominantly extravascular and increase in abundance in response to ischemia-reperfusion injury.

T-cell receptor+ CD4- CD8- double-negative (DN) T cells are a population of T cells present in low abundance in blood and lymphoid organs, but enriched in various organs including the kidney. Despite burgeoning interest in these cells, studies examining their abundance in the kidney have reported conflicting results. Here we developed a flow cytometry strategy to clearly segregate DN T cells from other immune cells in the mouse kidney and used it to characterize their phenotype and response in renal ischemia-reperfusion injury (IRI). These experiments revealed that in the healthy kidney, most DN T cells are located within the renal parenchyma and exhibit an effector memory phenotype. In response to IRI, the number of renal DN T cells is unaltered after 24 h, but significantly increased by 72 h. This increase is not related to alterations in proliferation or apoptosis. By contrast, adoptive transfer studies indicate that circulating DN T cells undergo preferential recruitment to the postischemic kidney. Furthermore, DN T cells show the capacity to upregulate CD8, both in vivo following adoptive transfer and in response to ex vivo activation. Together, these findings provide novel insights regarding the phenotype of DN T cells in the kidney, including their predominant extravascular location, and show that increases in their abundance in the kidney following IRI occur in part as a result of increased recruitment from the circulation. Furthermore, the observation that DN T cells can upregulate CD8 in vivo has important implications for detection and characterization of DN T cells in future studies.

Immunobiology of the biliary tract system.

The biliary tract is a complex tubular organ system spanning from the liver to the duodenum. It is the site of numerous acute and chronic disorders, many of unknown origin, that are often associated with cancer development and for which there are limited treatment options. Cholangiocytes with proinflammatory capacities line the lumen and specialised types of immune cells reside in close proximity. Recent technological breakthroughs now permit spatiotemporal assessments of immune cells within distinct niches and have increased our understanding of immune cell tissue residency. In this review, a comprehensive overview of emerging knowledge on the immunobiology of the biliary tract system is provided, with a particular emphasis on the role of distinct immune cells in biliary disorders.

Transcriptome landscape of double negative T cells by single-cell RNA sequencing.

CD4 and CD8 coreceptor double negative TCRαß+ T (DNT) cells are increasingly being recognized for their critical and diverse roles in the immune system. However, their molecular and functional signatures remain poorly understood and controversial. Moreover, the majority of studies are descriptive because of the relative low frequency of cells and non-standardized definition of this lineage. In this study, we performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of male C57BL/6 mice using strict fluorescence-activated cell sorting. The data was replicated in a subsequent study. Our analysis revealed five transcriptionally distinct naïve DNT cell clusters, which expressed unique sets of genes and primarily performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, in comparison with CD4+, CD8+ T cells and NK cells, Ikzf2 was highly expressed by both naïve and activated cytotoxic DNT cells. In conclusion, we provide a map of the heterogeneity in naïve and active DNT cells, addresses the controversy about DNT cells, and provides potential transcription signatures of DNT cells. The landscape approach herein will eventually become more feasible through newer high throughput methods and will enable clustering data to be fed into a systems analysis approach. Thus the approach should become the "backdrop" of similar studies in the myriad murine models of autoimmunity, potentially highlighting the importance of DNT cells and other minor lineage of cells in immune homeostasis. The clear characterization of functional DNT subsets into helper DNT, cytotoxic DNT and innate DNT will help to better understand the intrinsic roles of different functional DNT subsets in the development and progression of autoimmune diseases and transplant rejection, and thereby may facilitate diagnosis and therapy.

Distinct MHC class I-like interacting invariant T cell lineage at the forefront of mycobacterial immunity uncovered in Xenopus.

The amphibian Xenopus laevis is to date the only species outside of mammals where a MHC class I-like (MHC-like) restricted innate-like (i) T cell subset (iVα6 T cells) reminiscent of CD1d-restricted iNKT cells has been identified and functionally characterized. This provides an attractive in vivo model to study the biological analogies and differences between mammalian iT cells and the evolutionarily antecedent Xenopus iT cell defense system. Here, we report the identification of a unique iT cell subset (Vα45-Jα1.14) requiring a distinct MHC-like molecule (mhc1b4.L or XNC4) for its development and function. We used two complementary reverse genetic approaches: RNA interference by transgenesis to impair expression of either XNC4 or the Vα45-Jα1.14 rearrangement, and CRISPR/Cas9-mediated disruption of the Jα1.14 gene segment. Both XNC4 deficiency that ablates iVα45T cell development and the direct disruption of the iVα45-Jα1.14 T cell receptor dramatically impairs tadpole resistance to Mycobacterium marinum (Mm) infection. The higher mortality of Mm-infected tadpoles deficient for iVα45T cells correlates with dysregulated expression responses of several immune genes. In contrast, iVα45-Jα1.14-deficient tadpoles remain fully competent against infection by the ranavirus FV3, which indicates a specialization of this unique iT cell subset toward mycobacterial rather than viral pathogens that involve iVα6 T cells. These data suggest that amphibians, which are evolutionarily separated from mammals by more than 350 My, have independently diversified a prominent and convergent immune surveillance system based on MHC-like interacting innate-like T cells.

γδ T cell responses: How many ligands will it take till we know?

γδ T cells constitute a sizeable and non-redundant fraction of the total T cell pool in all jawed vertebrates, but in contrast to conventional αß T cells they are not restricted by classical MHC molecules. Progress in our understanding of the role of γδ T cells in the immune system has been hampered, and is being hampered, by the considerable lack of knowledge regarding the antigens γδ T cells respond to. The past few years have seen a wealth of data regarding the TCR repertoires of distinct γδ T cell populations and a growing list of confirmed and proposed molecules that are recognised by γδ T cells in different species. Yet, the physiological contexts underlying the often restricted TCR usage and the chemical diversity of γδ T cell ligands remain largely unclear, and only few structural studies have confirmed direct ligand recognition by the TCR. We here review the latest progress in the identification and validation of putative γδ T cell ligands and discuss the implications of such findings for γδ T cell responses in health and disease.

Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.

Although many flow cytometers can analyze 30-50 parameters, it is still challenging to develop a 40+ color panel for the phenotyping of immune cells using fluorochrome conjugated antibodies due to limitations in the availability of spectrally unique fluorochromes that can be excited by the commonly used laser lines (UV, Violet, Blue, Green/Yellow-green, and Red). Spectral flowcytometry is capable of differentiating fluorochromes with significant overlap in the emission spectra, enabling the use of spectrally similar fluorochrome pairs such as Brilliant Blue 515 and FITC in a single panel. We have developed a 43 color panel to characterize most of the immune subsets within the peripheral immune system, including conventional T cells, unconventional T cells such as invariant natural killer T cells (iNKT), Gamma delta (γδ) T-cell subsets (TCR Vδ2, TCR Vγ9) and mucosal-associated invariant T cells (MAIT), B-cell subsets, natural killer (NK) cells, plasmacytoid dendritic cells, dendritic cell subsets, hematopoietic progenitor cells, basophils, and innate lymphoid cell (ILC) subsets (CD117, CRTH2). The panel includes surface markers to analyze activation (CD38, HLA-DR, ICOS/CD278), differentiation (CD45RA, CD27, CD28, CD57), expression of cytokine and chemokine receptors (CD25, CD127, CCR10, CCR6, CCR4, CXCR3, CXCR5, CRTH2/CD294), and co-inhibitory molecules and exhaustion (PD-1, CD223/LAG-3, TIGIT), which enables a deep characterization of PBMCs from peripheral blood. Cells were analyzed on a 5-laser Cytek Aurora and data analysis was done using FlowJo. This panel can help to make a thorough interpretation of immune system, specifically when specimen quantity is low. The panel has not been completely optimized but would rather act as a guide toward the development of a workflow for optimized multicolor immunophenotyping panel.

Evolutionary Underpinnings of Innate-Like T Cell Interactions with Cancer.

Cancers impose a significant health and economic burden. By harnessing the immune system, current immunotherapies have revolutionized the treatment against human cancers and potentially offer a long-term cure. Among others, innate-like T (iT) cells, including natural killer T cells, are promising candidates for immunotherapies. Unlike conventional T cells, iT cells regulate multiple immune processes and express an invariant T cell receptor that is shared among different individuals. However, the conditions that activate the pro- and antitumor functions of iT cells are partially understood. These gaps in knowledge hamper the use of iT cell in clinics. It might be beneficial to examine the roles of iT cells in an alternative animal model - the amphibian Xenopus whose immune system shares many similarities to that of mammals. Here, we review the iT cell biology in the context of mammalian cancers and discuss the challenges currently found in the field. Next, we introduce the advantages of Xenopus as a model to investigate the role of iT cells and interacting major histocompatibility complex (MHC) class I-like molecules in tumor immunity. In Xenopus, 2 specific iT cell subsets, Vα6 and Vα22 iT cells, recognize and fight tumor cells. Furthermore, our recent data reveal the complex functions of the Xenopus MHC class I-like (XNC) gene XNC10 in tumor immune responses. By utilizing reverse genetics, transgenesis, and MHC tetramers, we have a unique opportunity to uncover the relevance of XNC genes and iT cell in Xenopus tumor immunity.

MAIT cells and microbial immunity.

Mucosal-associated invariant T (MAIT) cells, the most abundant T-cell subset in humans, are increasingly being recognized for their importance in microbial immunity. MAIT cells accumulate in almost every mucosal tissue examined, including the lung, liver and intestinal tract, where they can be activated through T-cell receptor (TCR) triggering as well as cytokine stimulation in response to a host of microbial products. In this review, we specifically discuss MAIT cell responses to bacterial and fungal infections, with a focus on responses that are both MR1-dependent and -independent, the evidence for diversity in MAIT TCR usage in response to discrete microbial products, protective immunity induced by MAIT cells, and MAIT cell antimicrobial functions in the context of these infections.

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RATIONALE: The development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis. OBJECTIVES: To identify T-cell-dependent mechanisms that are protective in tuberculosis. METHODS: Multicolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood. MEASUREMENTS AND MAIN RESULTS: At two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%). CONCLUSIONS: Our data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis.

Evolution of innate-like T cells and their selection by MHC class I-like molecules.

Until recently, major histocompatibility complex (MHC) class I-like-restricted innate-like αßT (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire were thought to be a recent evolutionary acquisition restricted to mammals. However, molecular and functional studies in Xenopus laevis have demonstrated that iT cells, defined as MHC class I-like-restricted innate-like αßT cells with a semi-invariant TCR, are evolutionarily conserved and prominent from early development in amphibians. As these iT cells lack the specificity conferred by conventional αß TCRs, it is generally considered that they are specialized to recognize conserved antigens equivalent to pathogen-associated molecular patterns. Thus, one advantage offered by the MHC class I-like iT cell-based recognition system is that it can be adapted to a common pathogen and function on the basis of a relatively small number of T cells. Although iT cells have only been functionally described in mammals and amphibians, the identification of non-classical MHC/MHC class I-like genes in other groups of endothermic and ectothermic vertebrates suggests that iT cells have a broader phylogenetic distribution than previously envisioned. In this review, we discuss the possible role of iT cells during the emergence of the jawed vertebrate adaptive immune system.

The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αß T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αß TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR ß-chain contacts are mostly through the variable CDR3ß loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αß T-cell recognition of MHC and MHC-like ligands.

Dysfunctional states of unconventional T-cell subsets in cancer.

Unconventional T cells represent a promising therapeutic agent to overcome the current limitations of immunotherapies due to their universal T-cell receptors, ability to respond directly to cytokine stimulation, and capacity to recruit and modulate conventional immune cells in the tumor microenvironment. Like conventional T cells, unconventional T cells can enter a dysfunctional state, and the functional differences associated with this state may provide insight into the discrepancies observed in their role in antitumor immunity in various cancers. The exhaustive signature of unconventional T cells differs from conventional αß T cells, and understanding the differences in the mechanisms underlying exhaustive differentiation in these cell types may aid in the discovery of new treatments to improve sustained antitumor responses. Ongoing clinical trials investigating therapies that leverage unconventional T-cell populations have shown success in treating hematologic malignancies and reducing the immunosuppressive tumor environment. However, several hurdles remain to extend these promising results into solid tumors. Here we discuss the current knowledge on unconventional T-cell function/dysfunction and consider how the incorporation of therapies that modulate unconventional T-cell exhaustion may aid in overcoming the current limitations of immunotherapy. Additionally, we discuss how components of the tumor microenvironment alter the functions of unconventional T cells and how these changes can affect tumor infiltration by lymphocytes and alter conventional T-cell responses.