Ramesh C. Halder

Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide

Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Vα14+ NK T cells, and a panel of Vα3/Vα8+ CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d+ antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-γ and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases.

Type II NKT cell–mediated anergy induction in type I NKT cells prevents inflammatory liver disease

Because of the paucity of known self lipid-reactive ligands for NKT cells, interactions among distinct NKT cell subsets as well as immune consequences following recognition of self glycolipids have not previously been investigated. Here we examined cellular interactions and subsequent immune regulatory mechanism following recognition of sulfatide, a self-glycolipid ligand for a subset of CD1d-restricted type II NKT cells. Using glycolipid/CD1d tetramers and cytokine responses, we showed that activation of sulfatide-reactive type II NKT cells and plasmacytoid DCs caused IL-12- and MIP-2-dependent recruitment of type I, or invariant, NKT (iNKT) cells into mouse livers. These recruited iNKT cells were anergic and prevented concanavalin A-induced (ConA-induced) hepatitis by specifically blocking effector pathways, including the cytokine burst and neutrophil recruitment that follow ConA injection. Hepatic DCs from IL-12(+/+) mice, but not IL-12(-/-) mice, adoptively transferred anergy in recipients; thus, IL-12 secretion by DCs enables them to induce anergy in iNKT cells. Our data reveal what we believe to be a novel mechanism in which interactions among type II NKT cells and hepatic DCs result in regulation of iNKT cell activity that can be exploited for intervention in inflammatory diseases, including autoimmunity and asthma.

PD-1/PD-L Blockade Prevents Anergy Induction and Enhances the Anti-Tumor Activities of Glycolipid-Activated Invariant NKT Cells

Invariant NKT (iNKT) cells recognize glycolipid Ags, such as the marine sponge-derived glycosphingolipid α-galactosylceramide (αGalCer) presented by the CD1d protein. In vivo activation of iNKT cells with αGalCer results in robust cytokine production, followed by the acquisition of an anergic phenotype. Here we have investigated mechanisms responsible for the establishment of αGalCer-induced iNKT cell anergy. We found that αGalCer-activated iNKT cells rapidly up-regulated expression of the inhibitory costimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of αGalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of αGalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1/PD-L pathway failed to prevent bacterial- or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1/PD-L interactions enhanced the antimetastatic activities of αGalCer. Collectively, our findings reveal a critical role for the PD-1/PD-L costimulatory pathway in the αGalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.

Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a β-linked self-glycolipid

TCR-mediated recognition of β-linked self-glycolipids bound to CD1d is poorly understood. Here, we have characterized the TCR repertoire of a CD1d-restricted type II NKT cell subset reactive to sulfatide involved in the regulation of autoimmunity and antitumor immunity. The sulfatide/CD1d-tetramer+ cells isolated from naïve mice show an oligoclonal TCR repertoire with predominant usage of the Vα3/Vα1-Jα7/Jα9 and Vβ8.1/Vβ3.1-Jβ2.7 gene segments. The CDR3 regions of both the α- and β-chains are encoded by either germline or nongermline gene segments of limited lengths containing several conserved residues. Presence of dominant clonotypes with limited TCR gene usage for both TCR α- and β-chains in type II NKT cells reflects specific antigen recognition not found in the type I NKT cells but similar to the MHC-restricted T cells. Although potential CD1d-binding tyrosine residues in the CDR2β region are conserved between most type I and type II NKT TCRs, CDR 1α and 3α regions differ significantly between the two subsets. Collectively, the TCR repertoire of sulfatide-reactive type II NKT cells exhibits features of both antigen-specific conventional T cells and innate-like cells, and these findings provide important clues to the recognition of β-linked glycolipids by CD1d-restricted T cells in general.

Tissue-specific expansion of NKT and CD5+B cells at the onset of autoimmune disease in (NZB×NZW)F1 mice

Natural killer T (NKT) cells and CD5+B cells were searched for in various immune organs of autoimmune prone (NZB×NZW)F1 (NZB/W F1) mice. The number of lymphocytes increased in the liver, spleen, and peritoneal cavity after the onset of disease (at the age of 30 weeks) while the number of thymocytes decreased at that time. Prominent changes of lymphocyte subsets were seen in the liver and peritoneal cavity, namely, expansion of IL‐2Rβ+TCRα βint cells in the liver and of CD5+B220+ cells in the peritoneal cavity. The majority of TCRα βint cells in the liver were NK1.1+, and CD5+B cells in the peritoneal cavity were CD1d+. Proteinuria became prominent in NZB/W F1 mice with the progression of disease. In parallel with this progression, the proportion of NKT cells decreased slightly in the liver, but their absolute number remained at a high level in this organ. These NKT cells were CD4+ and used an invariant chain of Vα14Jα281 for TCRα. Reflecting the elevation of CD5+B cells, autoantibodies against hepatocyte cytoplasmand denatured DNA were detected in sera. Although NKT cells are known to be immunoregulatory cells in some autoimmune mice, the present results raise the possibility that NKT cells as well as CD5+B cells might be associated with the onset of autoimmune diseases in NZB/W F1 mice. Indeed, NKT cells in F1 mice had a high potential to induce autoimmune‐like inflammationwhen α–galactosylceramide was administered or when active NKT cells were transferred into young F1 mice.

Essential Role of Extrathymic T Cells in Protection Against Malaria

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1−TCRint cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 × 103 per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1−TCRint cells (IL-2Rβ+TCRαβ+). Unconventional CD8+ NKT cells (Vα14−) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.

Expansion of unconventional T cells with natural killer markers in malaria patients

Immunological states during human malarial infection were examined. In parallel with parasitemia and anemia, granulocytosis was induced in the blood of patients, especially those infected with Plasmodium (P.) falciparum. At that time, the level of lymphocytes remained unchanged or slightly increased in the blood. However, the distribution of lymphocyte subsets was modulated, showing that the proportion of CD56(+)T cells, CD57(+)T cells, and gammadeltaT cells (i.e. all unconventional T cells) had increased in patients infected with P. falciparum or P. vivax. This phenomenon occurred at the early phase of infection and disappeared in the course of recovery. The data from patients with multiple attacks of P. vivax infection showed that there was no augmentation of these responses. In adult cases, the increase in the proportion of unconventional T cells seemed to closely parallel disease severity. However, all these responses were weak in children, even those infected with P. falciparum. In conjunction with accumulating evidence from mouse malaria experiments, the present results suggest that the immunological state induced by malarial infection might mainly be an event of unconventional T cells and that the immunological memory might not be long-lasting, possibly due to the properties of unconventional T cells.

Intensive generation of NK1.1– extrathymic T cells in the liver by injection of bone marrow cells isolated from mice with a mutation of polymorphic major histocompatibility complex antigens

Whether intermediate TCR (TCRint) cells and natural killer T (NKT or NK1.1+TCRint) cells are extrathymically generated remains controversial. This arises from the fact that there are few of these T cells in athymic nude mice and neonatally thymectomized mice. However, when athymic mice were provided with appropriate microenvironments or stimulation, many TCRint cells (mainly NK1.1−) were found to arise in the liver. NKT cells are known to be positively selected by monomorphic major histocompatibility complex (MHC) ‐like antigens (e.g. CD1d). This is true even if they are CD4+. In other words, a MHC class I‐like antigen is restricted to CD4 antigen. This rule is somewhat different from that seen in conventional T cells (i.e. the restriction of class II with CD4 and that of class I and CD8). In the case of NK1.1−TCRint cells, they were selected by polymorphic MHC antigens, but their MHC restriction to CD4 or CD8 antigen was incomplete. This was revealed by experiments of bone marrow transfer with class I (bm 1) or II (bm 12) disparity. Depending on the disparity, a unique cytokine profile in sera was detected. These results suggest that the development of T lineage lymphocytes and MHC restriction to CD4 and CD8 might have occurred in parallell as a phylogenic event, and that NK1.1− extrathymic T cells (i.e. NK1.1−TCRint) are at an intermediate position between NKT cells and conventional T cells in phylogeny.

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c‐kit+ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3− IL‐2Rβ+) and extrathymic T cells (CD3int IL‐2Rβ+) before and after irradiation. To determine their function, post‐irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8+ CD3int IL‐2Rβ+ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1+ and NK1.1− subsets of CD3int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1+ subset of CD3int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1− subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.

Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients

We have investigated transcriptional and epigenetic differences in peripheral blood mononuclear cells (PBMCs) of monozygotic female twins discordant in the diagnosis of amyotrophic lateral sclerosis (ALS). Exploring DNA methylation differences by reduced representation bisulfite sequencing (RRBS), we determined that, over time, the ALS twin developed higher abundances of the CD14 macrophages and lower abundances of T cells compared to the non‐ALS twin. Higher macrophage signature in the ALStwinwas also shownby RNA sequencing (RNA‐seq). Moreover, the twins differed in the methylome at loci near several genes, including EGFR and TNFRSF11A, and in the pathways related to the tretinoin and H3K27me3 markers. We also tested cytokine production by PBMCs. The ALS twins PBMCs spontaneously produced IL‐6 and TNF‐α, whereas PBMCs of the healthy twin produced these cytokines only when stimulated by superoxide dismutase (SOD)‐1. These results and flow cytometric detection of CD45 and CD127 suggest the presence of memory T cells in both twins, but effector T cells only in the ALS twin. The ALS twins PBMC supernatants, but not the healthy twins, were toxic to rat cortical neurons, and this toxicity was strongly inhibited by an IL‐6 receptor antibody (tocilizumab) and less well by TNF‐α and IL‐1β antibodies. The putative neurotoxicity of IL‐6 and TNF‐α is in agreement with a high expression of these cytokines on infiltrating macrophages in the ALS spinal cord. We hypothesize that higher macrophage abundance and increased neurotoxic cytokines have a fundamental role in the phenotype and treatment of certain individuals with ALS.—Lam, L., Chin, L., Halder, R.C., Sagong, B., Famenini, S., Sayre, J., Montoya, D., Rubbi L., Pellegrini, M., Fiala, M. Epigenetic changes in T‐cell and monocyte signatures and production of neurotoxic cytokines in ALS patients. FASEB J. 30, 3461–3473 (2016). www.fasebj.org