T‐cell regulation through a basic suppressive mechanism targeting low‐density lipoprotein receptor‐related protein 1

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T‐cell adhesion, and notably also T‐cell receptor (TCR) ‐induced activation, are subject to constant suppression through shedding of low‐density lipoprotein receptor‐related protein 1 (LRP1). The broad‐spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR‐induced activation and adhesion to β₁ and β₂ integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full‐length thrombospondin‐1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non‐adhering cells and expression of TSP170 correlated with T‐cell motility. The pro‐adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP‐1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP‐1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP‐1 in integrin‐dependent adhesion and TCR‐induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP‐1‐dependent pathway and ligation of CD28 antagonizes shedding independent of TSP‐1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T‐cell level.     

The who's who of T‐cell differentiation: Human memory T‐cell subsets

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single‐cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T‐cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T‐cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.     

Deciphering new mechanisms on T‐cell costimulation by human mast cells

It is well established that full activation of T cells to recognize a specific antigen requires additional signals. These secondary signals are generated by the interaction of costimulatory molecules expressed on APCs. Classical APCs include DCs, macrophages, Langerhans cells, and B cells. However, in recent years, several haematopoietic and nonhaematopoietic cells have been described to express MHC class II antigens and, in appropriate conditions, costimulatory molecules. In this issue, Suurmond et al. [Eur. J. Immunol. 2016. 46: 1132–1141] show, for the first time, that human mast cells not only express costimulatory molecules of the TNF‐receptor and CD28 families, but can also costimulate T cells through a yet‐to‐be‐defined CD28‐independent interaction.     

LKB1 is essential for the proliferation of T‐cell progenitors and mature peripheral T cells

The serine/threonine kinase LKB1 has a conserved role in Drosophila and nematodes to co‐ordinate cell metabolism. During T lymphocyte development in the thymus, progenitors need to synchronize increased metabolism with the onset of proliferation and differentiation to ensure that they can meet the energy requirements for development. The present study explores the role of LKB1 in this process and shows that loss of LKB1 prevents thymocyte differentiation and the production of peripheral T lymphocytes. We find that LKB1 is required for several key metabolic processes in T‐cell progenitors. For example, LKB1 controls expression of CD98, a key subunit of the L ‐system aa transporter and is also required for the pre‐TCR to induce and sustain the regulated phosphorylation of the ribosomal S6 subunit, a key regulator of protein synthesis. In the absence of LKB1 TCR‐β‐selected thymocytes failed to proliferate and did not survive. LBK1 was also required for survival and proliferation of peripheral T cells. These data thus reveal a conserved and essential role for LKB1 in the proliferative responses of both thymocytes and mature T cells.     

L‐type amino acid transporter‐1 and CD98 expression in bone and soft tissue tumors

L‐type amino acid transporter‐1 (LAT1) is expressed in many cancers. We examined LAT1 and CD98 expression immunohistochemically in surgically resected specimens of various bone and soft tissue tumors. Out of 226 cases, 79 (35%) were LAT1⁺ and 95 (42%) were CD98⁺. In bone tumors, LAT1 was highly expressed in osteoblastoma (89%), chondrosarcoma (50%), and osteosarcoma (60%); in soft tissue tumors, LAT1 was highly expressed in rhabdomyosarcoma (80%), synovial sarcoma (63%), Ewing's sarcoma (60%), epithelioid sarcoma (100%) and angiosarcoma (100%). In malignant soft tissue tumors, LAT1 expression was associated with higher histological grade. High CD98 expression was seen in many bone tumors of intermediate and high malignancy. Among soft tissue tumors, CD98 was expressed in tendon sheath giant cell tumor and malignant peripheral nerve sheath tumor (57%), Ewing's sarcoma (50%) and undifferentiated sarcoma (64%). Some of the malignant soft tissue tumors expressed both LAT1 and CD98. This study showed that LAT1 and CD98 was expressed in many malignant and intermediate bone tumors, and some malignant soft tissue tumors.     

Viral inoculum dose impacts memory T‐cell inflation

Memory T‐cell inflation develops during certain persistent viral infections and is characterized by the accumulation and maintenance of large numbers of effector‐memory T cells, albeit with varying degrees in size and phenotype among infected hosts. The underlying mechanisms that control memory T‐cell inflation are not yet fully understood. Here, we dissected CMV‐specific memory T‐cell formation and its connection to the initial infectious dose by varying the inoculum size. After low dose inoculum with mouse CMV, the accumulation of inflationary memory T cells was severely hampered and correlated with reduced reservoirs of latent virus in nonhematopoietic cells and diminished antigen‐driven T‐cell proliferation. Moreover, lowering of the initial viral dose turned the characteristic effector memory‐like inflationary T cells into more central memory‐like cells as evidenced by the cell‐surface phenotype of CD27^high, CD62L⁺, CD127⁺, and KLRG1^?, and by improved secondary expansion potential. These data show the impact of the viral inoculum on the degree of memory T‐cell inflation and provide a rationale for the observed variation of human CMV‐specific T‐cell responses in terms of magnitude and phenotype.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Retinoic acid promotes the development of Arg1‐expressing dendritic cells for the regulation of T‐cell differentiation

Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in DCs requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid‐responsive elements in the 5′ noncoding region of the Arg1 gene. Arg1, produced by DCs in response to retinoic acid, promotes the generation of FoxP3⁺ regulatory T (Treg) cells. Importantly, blocking the retinoic acid receptor makes DCs hypo‐responsive to known inducers of Arg1 such as IL‐4 and GM‐CSF in Arg1 expression. We found that intestinal CD103⁺ DCs that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in DCs.     

Lack of transglutaminase 2 diminished T‐cell responses in mice

Transglutaminase 2 (TG2) has been reported to play a role in dendritic cell activation and B‐cell differentiation after immunization. Its presence and role in T cells, however, has not been explored. In the present study, we determined the expression of TG2 on mouse T cells, and evaluated its role by comparing the behaviours of wild‐type and TG2^?/? T cells after activation. In our results, naive T cells minimally expressed TG2, expression of which was increased after activation. T‐cell proliferation, expression of activation markers such as CD69 and CD25, and secretions of interleukin‐2 and interferon‐γ were suppressed in the absence of TG2, presumably due, in part, to diminished nuclear factor‐κB activation. These effects on T cells seemed to be reflected in the in vivo immune response, the contact hypersensitivity reaction elicited by 2,4‐dinitro‐1‐fluorobenzene, with lowered peak responses in the TG2^?/? mice. When splenic T cells from mice immunized with tumour lysate‐loaded wild‐type dendritic cells were re‐challenged ex vivo with the same antigen, the profile of surface markers including CD44, CD62L, and CD127 strongly indicated lesser generation of memory CD8⁺ T cells in TG2^?/? mice. In the TG2^?/? CD8⁺ T cells, moreover, Eomes expression was markedly decreased. These results indicate possible roles of TG2 in CD8⁺ T‐cell activation and CD8⁺ memory T‐cell generation.     

Chitin induces upregulation of B7‐H1 on macrophages and inhibits T‐cell proliferation

Chitin is a highly abundant glycopolymer, which serves as structural component in fungi, arthropods and crustaceans but is not synthesized by vertebrates. However, vertebrates express chitinases and chitinase‐like proteins, some of which are induced by infection with helminths suggesting that chitinous structures may be targets of the immune system. The chitin‐induced modulations of the innate and adaptive immune responses are not well understood. Here, we demonstrate that intranasal administration of OVA and chitin resulted in diminished T‐cell expansion and Th2 polarization as compared with OVA administration alone. Chitin did not promote nor attenuate Th2 polarization in vitro. Chitin‐exposed macrophages inhibited proliferation of CD4⁺ T cells in a cell–cell contact‐dependent manner. Chitin induced upregulation of the inhibitory ligand B7‐H1 (PD‐L1) on macrophages independently of MyD88, TRIF, TLR2, TLR3, TLR4 and Stat6. Inhibition of T‐cell proliferation was largely dependent on B7‐H1, as the effect was not observed in cocultures with cells from B7‐H1‐deficient mice.     

Proinflammatory stimuli induce galectin‐9 in human mesenchymal stromal cells to suppress T‐cell proliferation

Human multipotent mesenchymal stromal cells (MSCs) are clinically applied to treat autoimmune diseases and graft‐versus‐host disease due to their immunomodulatory properties. Several molecules have been identified to mediate these effects, including constitutively expressed galectin‐1. However, there are indications in the literature that MSCs exert enhanced immunosuppressive functions after interaction with an inflammatory environment. Therefore, we analyzed how inflammatory stimuli influence the expression of the galectin network in MSCs and functionally tested the relevance for the immunomodulatory effects of MSCs. We found that galectin‐9 was strongly induced in MSCs upon interaction with activated PBMCs. Proinflammatory cytokines, such as interferon‐gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α), and also ligands of the Toll‐like receptors (TLRs) TLR2, TLR3, and TLR4 elicited similar induction of galectin‐9 in activated PBMCs. Galectin‐9 was not only upregulated intracellularly, but also released by MSCs in significant amounts into the supernatant after exposure to proinflammatory stimuli. In proliferation assays, MSCs with a galectin‐9 knockdown lost a significant portion of their antiproliferative effects on T cells. In conclusion, we found that unlike constitutively expressed galectin‐1, galectin‐9 is induced by several proinflammatory stimuli and released by MSCs. Thus, galectin‐9 contributes to the inducible immunomodulatory functions of MSCs.     

The DC‐HIL ligand syndecan‐4 is a negative regulator of T‐cell allo‐reactivity responsible for graft‐versus‐host disease

Acute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo-reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen-presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan-4 (SD-4) on effector T cells mediates the inhibitory function of DC-HIL on APC, we proposed that SD-4 negatively regulates the T-cell response to allo-stimulation in acute GVHD, using SD-4 knockout mice. Although not different from their wild-type counterparts in responsiveness to anti-CD3 stimulation, SD-4^−/− T cells lost the capacity to mediate the inhibitory function of DC-HIL and were hyper-reactive to allogeneic APC. Moreover, infusion of SD-4^−/− T cells into sub-lethally γ-irradiated allogeneic mice worsened mortality, with hyper-proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD-4 deletion had no deleterious effect on T-cell-suppressive activity compared with SD-4^+/+ regulatory T cells. We conclude that SD-4, as the T-cell ligand of DC-HIL, is a potent inhibitor of allo-reactive T cells responsible for GVHD and a potentially useful target for treating this disease.     

Comparative analysis of murine T‐cell receptor repertoires

For understanding the rules and laws of adaptive immunity, high‐throughput profiling of T‐cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T‐cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V‐gene and J‐gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

Differential T‐cell receptor signals for T helper cell programming

Upon encounter with their cognate antigen, naive CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen‐presenting cells, cytokines and co‐stimulatory molecules. The strength of the T‐cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength.