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Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population., Competing Interests: Competing interests statement:R.L.P., V.A.D.S., A.W., D.G.-H., R.K., J.R.F., N.C.M., M.M., W.B., M.D., M.M.-U., T.G., C.B., D.L., C.B.-R., R.P., N. Chepisiuk, P.K.S., A.P.W., A.S.P., M.L.M., M.S., L.D., A.K., R.R., M.L., S.L., and L.F.G. are or were employees of Immunocore Ltd. A patent has been filed on the sequence and utility of the ImmTAB-inhA molecules described in this study. The authors have no additional financial interests.

T cell exhaustion develops in human immunodeficiency virus (HIV) infection due to chronic viral antigenic stimulation. This adaptive response primarily affects virus-specific CD8 + T cells, which may remain dysfunctional despite viral load-reducing antiretroviral therapy; however, abnormalities may also be evident in non-HIV-specific populations. Both could limit the efficacy of cell therapies against viral reservoirs. Here, we show that bulk (polyclonal) CD8 + T cells from people living with HIV (PLWH) express proposed markers of dysfunctional HIV-specific T cells at high levels yet form lytic immunological synapses (IS) and eliminate primary resting infected (HIV Gag lo ) CD4 + T cells, when redirected by potent bispecific T cell-retargeting molecules, Immune mobilising monoclonal T cell receptors (TCR) Against Virus (ImmTAV). While PLWH CD8 + T cells are functionally impaired when compared to CD8 + T cells from HIV-naïve donors, ImmTAV redirection enables them to eliminate Gag lo CD4 + T cells that are insensitive to autologous HIV-specific cytolytic T cells. ImmTAV molecules may therefore be able to target HIV reservoirs, which represent a major barrier to a cure., (© 2022. The Author(s).)

Immunotherapy approaches that target peptide-human leukocyte antigen (pHLA) complexes are becoming highly attractive because of their potential to access virtually all foreign and cellular proteins. For this reason, there has been considerable interest in the development of the natural ligand for pHLA, the T-cell receptor (TCR), as a soluble drug to target disease-associated pHLA presented at the cell surface. However, native TCR stability is suboptimal for soluble drug development, and natural TCRs generally have weak affinities for pHLAs, limiting their potential to reach efficacious receptor occupancy levels as soluble drugs. To overcome these limitations and make full use of the TCR as a soluble drug platform, several protein engineering solutions have been applied to TCRs to enhance both their stability and affinity, with a focus on retaining target specificity and selectivity. Here, we review these advances and look to the future for the next generation of soluble TCR-based therapies that can target monomorphic HLA-like proteins presenting both peptide and nonpeptide antigens., (© 2021 Immunocare. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the TCR complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell-T cell interface, inhibition of proximal TCR signaling events, and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8+ T cell-mediated cytotoxicity in vitro. Crucially, in soluble form, the PD-1 ImmTAAI molecules were inactive and, hence, could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

Background and Aims: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA)., Approach and Results: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA., Conclusions: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials., (© 2020 Immunocore Ltd. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR-peptide-HLA crystal structures and, using a single-chain peptide-HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1 157-165 -HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual's limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Cellular immunity is governed by a complex network of migratory cues that enable appropriate immune cell responses in a timely and spatially controlled fashion. This review focuses on the chemokines and their receptors regulating the steady-state localisation of immune cells within healthy peripheral tissues. Steady-state immune cell traffic is not well understood but is thought to involve constitutive (homeostatic) chemokines. The recent discovery of tissue-resident memory T cells (T RM cells) illustrates our need for understanding how chemokines control immune cell mobilisation and/or retention. These studies will be critical to unravel novel pathways for preserving tissue function (aging) and preventing tissue disease (vaccination)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human skin harbors two major T cell compartments of equal size that are distinguished by expression of the chemokine receptor CCR8. In vitro studies have demonstrated that CCR8 expression is regulated by TCR engagement and the skin tissue microenvironment. To extend these observations, we examined the relationship between CCR8 + and CCR8 - skin T cells in vivo. Phenotypic, functional, and transcriptomic analyses revealed that CCR8 + skin T cells bear all the hallmarks of resident memory T cells, including homeostatic proliferation in response to IL-7 and IL-15, surface expression of tissue localization (CD103) and retention (CD69) markers, low levels of inhibitory receptors (programmed cell death protein 1, Tim-3, LAG-3), and a lack of senescence markers (CD57, killer cell lectin-like receptor subfamily G member 1). In contrast, CCR8 - skin T cells are heterogeneous and comprise variable numbers of exhausted (programmed cell death protein 1 + ), senescent (CD57 + , killer cell lectin-like receptor subfamily G member 1 + ), and effector (T-bet hi , Eomes hi ) T cells. Importantly, conventional and high-throughput sequencing of expressed TCR β-chain ( TRB ) gene rearrangements showed that these CCR8-defined populations are clonotypically distinct, suggesting unique ontogenies in response to separate antigenic challenges and/or stimulatory conditions. Moreover, CCR8 + and CCR8 - skin T cells were phenotypically stable in vitro and displayed similar levels of telomere erosion, further supporting the likelihood of a nonlinear differentiation pathway. On the basis of these results, we propose that long-lived memory T cells in human skin can be defined by the expression of CCR8., (Copyright © 2018 The Authors.)

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100-300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells ( i.e., failed to trigger chemotaxis and Ca 2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.-Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4., (© The Author(s).)

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study, we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak, whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin, and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction, exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin., (Copyright © 2015 The Authors.)

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. Although the mechanisms controlling memory T-cell migration to peripheral tissues are poorly understood, the current paradigm includes the localized secretion of "imprinting" signals from tissue-resident dendritic cells in the draining lymph nodes. Here we show that CCR8 expression by newly activated naive T cells is regulated by skin-specific factor(s) derived primarily from epidermal keratinocytes, thereby providing a mechanism for the preferential expression of CCR8 by skin-resident memory T cells. Importantly, no such effects were observed after coculture with primary cells from skin-unrelated epithelia, including mesothelium and small intestine. The keratinocyte-derived CCR8-inducing factor(s) were soluble, and independent of vitamins A and D. Furthermore, the induction of CCR8 under these conditions correlated with an increase in cutaneous lymphocyte-associated antigen expression. Our findings challenge current tissue homing paradigms, especially those involving CCR10, and emphasize the importance of steady-state epidermis rather than tissue-resident dendritic cells in controlling the localization of memory T cells within human skin.

Irrespective of the immune status, the vast majority of all lymphocytes reside in peripheral tissues whereas those present in blood only amount to a small fraction of the total. It has been estimated that T cells in healthy human skin outnumber those present in blood by at least a factor of two. How lymphocytes within these two compartments relate to each other is not well understood. However, mounting evidence suggest that the study of T cell subsets present in peripheral blood does not reflect the function of their counterparts at peripheral sites. This is especially true under steady-state conditions whereby long-lived memory T cells in healthy tissues, notably those in epithelial tissues at body surfaces, are thought to fulfill a critical immune surveillance function by contributing to the first line of defense against a series of local threats, including microbes, tumors, and toxins, and by participating in wound healing. The relative scarcity of information regarding peripheral T cells and the factors regulating their localization is primarily due to inherent difficulties in obtaining healthy tissue for the extraction and study of immune cells on a routine basis. This is most certainly true for humans. Here, we review our current understanding of T cell homing to human skin and compare it when possible with gut-selective homing. We also discuss candidate chemokines that may account for the tissue selectivity in this process and present a model whereby CCR8, and its ligand CCL1, selectively regulate the homeostatic migration of memory lymphocytes to skin tissue.

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome.

Two previous reports that receptor-interacting protein (RIP)-2 knockout (RIP2-/-) mice had defective nuclear factor-kappa B (NF-kappaB) signaling and T helper (Th)1 immune responses had led us to believe that this putative serine-threonine kinase might be a possible target for transplant immunosuppression. Thus, we tested whether RIP2-/- mice were able to reject vascularized allografts. Surprisingly, we found that T cells from RIP2-/- mice proliferated and produced interferon (IFN)-gamma after allostimulation in vitro. Moreover, naïve RIP2-/- CD4+ T cells differentiated normally into Th1 or Th2 cells under appropriate cytokine microenvironments. Consistent with these findings, no difference in allograft survival was observed between wild-type and RIP2-/- recipient mice, and rejection had similar pathology and cytokine profiles in both types of recipients. RIP2 deficiency was associated with defective NOD signaling, but this did not affect T-cell receptor (TCR)-dependent activation of the canonical NF-kappaB signaling or expression of NF-kappaB genes in rejecting allografts. Our data demonstrate that RIP2-deficient mice have intact canonical NF-kappaB signaling and can mount Th1-mediated alloresponses and reject vascularized allografts as efficiently as wild-type mice, thus arguing against RIP2 as a primary target for immunosuppression.

Biological markers of disease have become increasingly important for the clinician to diagnose, predict and monitor progression, and assess the therapeutic effect of interventions on underlying pathogenic mechanisms. Robust and specific biomarkers would be very useful in inflammation, where they may facilitate early identification of tissue injury, predict disease progression and help to modify disease outcomes. However, at present, there are no robust biomarkers to predict the course of inflammation. Here, we discuss emerging data indicating that RIP2, a putative serine/threonine protein kinase, may serve as a biomarker for the resolution of peritoneal dialysis-associated peritonitis and, more generally, of the acute inflammatory response to infection.

Background: Early upregulation of receptor-interacting protein-2 (RIP2) expression during peritoneal dialysis (PD)-associated peritonitis correlates with a favorable clinical outcome, while failure to upregulate RIP2 correlates with a protracted course. We noticed that patients who do not upregulate RIP2 during PD-associated peritonitis have more peritoneal macrophages during the early phase of infection., Methods: To study the mechanism behind this observation, we examined the role of RIP2 in the immune response to bacterial challenge in a mouse model of acute peritonitis. We injected RIP2(+/+) and RIP2(-/-) mice intraperitoneally with a Staphylococcus epidermidis cell free-preparation, and peritoneal cells were isolated 3, 6 and 24 h after challenge., Results: Surprisingly, RIP2(-/-) mice had a comparable influx of inflammatory leukocytes, but had a significantly higher number of peritoneal macrophages at 3 h, indicating delayed emigration of these cells. No significant differences were seen at later times suggesting that migration was delayed but not inhibited. In addition, RIP2(-/-) macrophages were more permissive to intracellular infection by Staphylococcus aureus, indicating that, in the absence of RIP2, resident peritoneal macrophages could become reservoirs of bacteria., Conclusion: These findings provide a mechanism for the observation that upregulation of RIP2 expression is required for rapid resolution of peritonitis, by decreasing intracellular infection and by regulating the migration of antigen-presenting cells in the early stages of an inflammatory response., (Copyright 2008 S. Karger AG, Basel.)

There are no predictive factors for peritoneal dialysis-associated peritonitis; however, its resolution correlates with a cell-mediated Th1 immune response. We tested the hypothesis that induction of receptor-interacting protein 2 (RIP2), an assumed kinase linked with Th1 responses, is a useful marker in this clinical setting. Basal RIP2 expression was measured in human immune cells and during dialysis-associated peritonitis. RIP2 increased with bacterial toxin cell activation and the temporal profile for this differed depending on immune cell involvement in the innate or adaptive phases of the response. Importantly, RIP2 expression increased in peritoneal immune cells during dialysis-associated peritonitis and this upregulation correlated with clinical outcome. An early induction in peritoneal CD14(+) cells correlated with rapid resolution, whereas minimal induction correlated with protracted infection and with catheter loss in 36% of patients. These latter patients had higher levels of MCP-1 consistent with a delayed transition from innate to adaptive immunity. Our study shows that upregulation of RIP2 is a useful marker to monitor dialysis-associated peritonitis and in predicting the clinical outcome of these infections.

During the past few years, there has been a substantial increase in the understanding of innate immunity. Dendritic cells are emerging as key players in the orchestration of this early phase of immune responses, with a role that will translate into the subsequent type of adaptive immune response against infection. Here we provide an overview of dendritic cell differentiation and function, with particular emphasis on those features unique to the immune defense of the peritoneal cavity and in the context of peritoneal dialysis-associated immune responses. The reader is referred to the primary references included in the accompanying list for specific details in this fascinating field.

Scattered evidence suggests that the human peritoneal cavity contains cells of the dendritic cell (DC) lineage but their characterization is missing. Here, we report that the peritoneal cavity of normal subjects and of stable patients on peritoneal dialysis (PD) contains a population of CD14(+) cells that can differentiate into DCs or macrophages. Within this pool, we characterized a CD14(+)CD4(+) cell subset (2.2% of the peritoneal cells) fulfilling the definition of myeloid DC precursors or pre-DC1 cells. These cells expressed high levels of HLA-DR, CD13, CD33, and CD86, and low levels of CD40, CD80, CD83, CD123, CD209, TLR-2 and TLR-4. These cells retained CD14 expression until late stages of differentiation, despite concomitant up-regulation of DC-SIGN (CD209), CD1a, CD80 and CD40. Peritoneal pre-DC1 cells had endocytic capacity that was down-regulated upon LPS/IFN-gamma stimulation, were more potent allo-stimulators than peritoneal CD14(+)CD4(-/lo) cells and monocyte-derived macrophages, and induced Th1 cytokine responses. More importantly, the number of peritoneal pre-DC1 cells increased during PD-associated peritonitis, with a different profile for Gram positive and Gram negative peritonitis, suggesting that these cells participate in the induction of peritoneal adaptive immune responses, and may be responsible for the bias towards Th1 responses during peritonitis.