γδ T Cells Express Activation Markers in the Central Nervous System of Mice with Chronic-relapsing Experimental Autoimmune Encephalomyelitis

In this study, we assessed the expression of activation markers on γδ T cells in central nervous system (CNS) lesions of SJL mice adoptively sensitized to develop experimental autoimmune encephalomyelitis (EAE) using myelin basic protein-reactive T cells. Although disease expression is known to be dependent upon T cells that express the αβ T cell receptor (TCR), a role for γδ T cells has been implicated in some studies but not in others. Using three-color flow cytometric analysis of both total and γδ T cells in spleen and CNS, the data showed that expression of CD69 (early activation marker), CD62L (lymphocyte homing receptor), CD25 (IL-2Rα), CD122 (IL-2Rβ) and CD95/CD95L (Fas/FasL), fluctuated on γδ T cells in EAE lesions in a disease-related fashion. Furthermore, the pattern of expression for these markers on γδ T cells was distinct from that found on the total lymphocyte population. Cytokine analysis of γδ T cells in the CNS demonstrated a bias towards a Th1-like cytokine profile. From these data, we conclude that γδ T cells in EAE lesions display an activated phenotype and form a dynamic component of the total lymphocyte population in the CNS, supporting a contributory role for these cells.     

Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

γ/δ T Cells in Multiple Sclerosis: Chemokine and Chemokine Receptor Expression

γ/δ T cells are enriched in multiple sclerosis (MS) brain lesions and have been postulated to contribute to the pathogenesis of the disease. Increased expression of the chemokine receptors CCR5 and CXCR3 on T cells and raised amounts of the chemokines RANTES and IP-10 have been noted in the CSF and brain tissue of MS patients, but the contribution of γδ T cells to these increases is unknown. We therefore compared intracellular RANTES and IP-10 production as well as CCR5, CXCR3, and CXCR1 expression by γδ T cells derived from the blood and CSF of patients with MS and healthy controls (HC). We observed higher RANTES production by MS γδ than by αβ T cell lines. Most of the MS as well as the HC γδ and αβ T cell lines expressed CXCR3, while expression of CXCR1 was low. Interestingly, MS γδ T cell lines, compared to lines from HC, expressed lower levels of CCR5. Furthermore, CSF-derived γδ T cells had even lower CCR5 expression than blood-derived ones. The higher RANTES production by MS γδ T cell lines, together with a lower expression of CCR5, may reflect an autoregulatory loop, caused by an increased production of its ligands (RANTES, MIP-1α, and MIP-1β) or due to other pro-inflammatory cytokines. Alternatively, we show that lower CCR5 expression could also reflect the result of repeated in vivo stimulation of γδ T cells by autoantigens.     

Human γδ T Cells Induce Dendritic Cell Maturation

γδ T cells are known to be involved in the innate immune defenses against infectious microorganisms. Herein, we considered that γδ T cells could also influence adaptative immunity by interacting with dendritic cells (DC) in the early phase of the immune response. To investigate this hypothesis, γδ T cells isolated from the peripheral blood of healthy volunteers were cocultured with autologous monocyte-derived dendritic cells, which were subsequently analyzed for their expression of key surface molecules and for their production of IL-12. First, we found that γδ T cells induced the upregulation of HLA-DR, CD86, and CD83 on DC. This effect did not require cell to cell contact and could be blocked by a neutralizing anti-TNF antibody. We then observed that γδT cells activated by the synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) induced the production of IL-12 (p40) and IL-12 (p70) by DC, an effect that involved IFN-γ production. The relevance of this finding to DC function was demonstrated by the increased production of IFN-γ by alloreactive T cells when stimulated in a mixed leucocyte reaction with DC preincubated with activated γδ T cells. We conclude that γδ T cell activation might result in DC maturation and thereby in enhanced αβ T cell responses.     

Induction of Thymus-Derived γδ T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both γδ and αβ T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of γδ T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing γδ T cells were detected. γδ T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of γδ T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like αβ T cells. γδ T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal γδ and αβ T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. γδ T cells induced by the B subunit are Th2-type cells derived from the thymus. These γδ T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of αβ T cells.     

Recovery from Mouse Hepatitis Virus Infection Depends on Recruitment of CD8 Cells Rather Than Activation of Intrahepatic CD4αβTCR or NK-T Cells

Mouse hepatitis virus (MHV) provides an excellent animal model for the study of the immunopathological mechanisms involved in hepatic viral diseases. We previously generated an attenuated viral variant, YAC-MHV3, which induces a subclinical disease and recovery within 15 days. In contrast, the L2-MHV3 strain induces the development of a fulminant hepatitis, leading to death within 3 days. In this paper, we document intrahepatic and splenic T cell subpopulations involved in the hepatitis process and viral elimination identified in attenuated or pathogenic MHV3-infected C57BL/6 mice. Percentages of intrahepatic CD4⁺ cells decreased in attenuated YAC-MHV3-infected mice, while they increased in mice infected with pathogenic L2-MHV3, compared with uninfected animals. Moreover, in YAC-MHV3-infected mice, the percentages of intrahepatic CD8⁺ cells slightly decreased at 24 h pi, then increased until 15 days pi. In contrast, the CD4/CD8 ratios of splenic lymphoid subpopulations increased in the first days of infection and returned to normal values at 15 days pi. Intrahepatic NK1.1⁺αβ − TCR^inter cells decreased in both virally infected groups of mice, while CD4⁺αβ − TCR^inter LFA-1^high cells increased in L2-MHV3-infected mice, in contrast with what was seen in YAC-MHV3-infected mice. However, these cells became anergic following Con A or PHA stimulation. Ex vivo studies showed that only the intrahepatic CD8⁺ cells that were increased in YAC-MHV3-infected mice could be stimulated by lectins. In addition, in vitro viral infections revealed that L2-MHV3 viral infection led to an increase of intrahepatic CD4⁺αβ − TCR^inter cells in the absence of CD8⁺ cells only. These results indicate that the attenuated phenotype of the YAC-MHV3 virus is related to two different mechanisms: the first involves no increase of intrahepatic CD4⁺αβ − TCR^inter or NK-T cells, while the second favors the recruitment and activation of CD8⁺ cells in liver. The results are discussed in relation to the integrity of intrahepatic immune tolerance mechanisms and immune-mediated viral elimination.     

γδ T-Cell Function in Pathogenesis of Cerebral Malaria in Mice Infected with Plasmodium berghei ANKA

Mice depleted of γδ T cells by monoclonal antibody treatment and infected with Plasmodium berghei ANKA did not develop cerebral malaria (CM). In striking contrast, δ^0/0 mice infected with P. berghei developed CM despite their γδ T-cell deficiency. γδ T cells appear to be essential for the pathogenesis of CM in mice having experienced normal ontogeny but not in mice genetically deprived of γδ T cells from the beginning of life.     

Role of γδ T Cells in Immunopathology of Pulmonary Mycobacterium avium Infection in Mice

Several studies have shown that γδ T cells influence granuloma development after infection with intracellular pathogens. The role of γδ T cells in controlling the influx of inflammatory cells into the lung after Mycobacterium avium infection was therefore examined with gene-disrupted mice (K/O). The mice were infected with either M. avium 724, a progressively replicating highly virulent strain of M. avium, or with M. avium 2-151 SmT, a virulent strain that induces a chronic infection. γδ-K/O mice infected with M. avium 2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. γδ-K/O mice infected with M. avium 724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates of M. avium that induce protective T-cell-specific immunity are largely unaffected by the absence of γδ T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of γδ T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.     

Streptozotocin-induced diabetes in mice lacking αβ T cells

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) α-chain were therefore used to assess whether TCR αβ⁺ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR αβ cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR αβ cells tipping the balance between tolerable and clinically damaging action on islet cells.     

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity

CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca²⁺ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca²⁺ influx and membrane potential (V_m)-induced Ca²⁺ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.     

γδ T lymphocyte responses to HIV

Natural immunity may be involved in controlling viral spread in hosts infected with HIV. A panel of γδ T cell receptor-positive lymphocyte clones was isolated from the peripheral blood of healthy HIV⁻ donors and tested for anti-HIV cytotoxic responses. Twelve of 30 (40%) Vγ9⁺Vδ2⁺ T cell clones, but none of seven Vδ1⁺ T cell clones, displayed lytic activity against HIV-infected cells. The Vγ9⁺/Vδ2⁺ clones cytotoxic for HIV-infected cells also lysed Daudi cells. However, not all Vγ9⁺/Vδ2⁺ clones which lysed Daudi targets had the capacity to lyse HIV-infected cells. Some of the γδ T cell clones were also investigated for potential proliferative responses to HIV-infected cells. One Vγ9⁺/Vδ2⁺ T cell clone (ME8-7) and one Vδ1⁺ T cell clone (ME18-2) demonstrated proliferative responses toward HIV-infected cells. Another Vγ9⁺/Vδ2⁺ clone (VM39) proliferated in response to cell-free HIV. Taken together, these results provide direct evidence of anti-HIV γδ T cell responses in healthy, HIV⁻ persons.     

T cells recognize a peptide derived from α-gliadin presented by the celiac disease-associated HLA-DQ (α10501, β10201) heterodimer

CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis- or trans-encoded HLA-DQ heterodimer; i.e., DQ(α1^*0501, β1^*0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(α1^*, β1^*0201) heterdimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic α-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(α1^*0501, β1^*0201) heterdimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31–47 of α-gliadin, which is included in the part of α-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other α-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(α1^*0501, β1^*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.     

Role of γδ T lymphocytes in the development of Behçet's disease

Phenotypic and functional properties of γδ T cells, which play an important role in mucocutaneous immunity, were examined to elucidate whether immunological abnormality in Behc¸et's disease may be related to a specific T cell population. We found that CD45RA⁺Vγ9⁺Vδ2⁺γδ T cells, which constitute a minor population of γδ T cells in healthy individuals, were increased in number in Behc¸et's disease irrespective of disease activity. This CD45RA⁺ subset of γδ T cells in the active, but not inactive, phase of this disease expressed IL-2Rβ and HLA-DR, suggesting that they are activated in vivo in active Behc¸et's disease. In addition, the CD45RA⁺γδ T cells produced extreme amounts of tumour necrosis factor and contained perforin granules. These data indicate that a phenotypically distinct subset of γδ T cells, CD45RA⁺CD45RO⁻Vγ9⁺Vδ2⁺, may contribute to immunological abnormalities which may lead to complexity of pathophysiology in Behc¸et's disease.     

Protective Roles of γδ T Cells and Interleukin-15 in Escherichia coli Infection in Mice

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.     

β-galactosylceramide alters invariant natural killer T cell function and is effective treatment for lupus

NZB/W female mice spontaneously develop systemic lupus, an autoantibody mediated disease associated with immune complex glomerulonephritis. Natural killer (NK) T cells augment anti-dsDNA antibody secretion by NZB/W B cells in vitro, and blocking NKT cell activation in vivo with anti-CD1 mAb ameliorates lupus disease activity. In the current study, we show that β-galactosylceramide reduces the in vivo induction of serum IFN-γ and/or IL-4 by the potent NKT cell agonist α-galactosylceramide and reduces NKT cell helper activity for IgG secretion. Treatment of NZB/W mice with the β-galactosylceramide ameliorated lupus disease activity as judged by improvement in proteinuria, renal histopathology, IgG anti-dsDNA antibody formation, and survival. In conclusion, β-galactosylceramide, a glycolipid that reduces the cytokine secretion induced by a potent NKT cell agonist ameliorates lupus in NZB/W mice.     

Single-cell analysis reveals the origins and intrahepatic development of liver-resident IFN-γ-producing γδ T cells

γδ T cells are heterogeneous lymphocytes located in various tissues. However, a systematic and comprehensive understanding of the origins of γδ T cell heterogeneity and the extrathymic developmental pathway associated with liver γδ T cells remain largely unsolved. In this study, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively catalog the heterogeneity of γδ T cells derived from murine liver and thymus samples. We revealed the developmental trajectory of γδ T cells and found that the liver contains γδ T cell precursors (pre-γδ T cells). The developmental potential of hepatic γδ T precursor cells was confirmed through in vitro coculture experiments and in vivo adoptive transfer experiments. The adoptive transfer of hematopoietic progenitor Lin⁻Sca-1⁺Mac-1⁺ (LSM) cells from fetal or adult liver samples to sublethally irradiated recipients resulted in the differentiation of liver LSM cells into pre-γδ T cells and interferon-gamma⁺ (IFN-γ⁺) but not interleukin-17a⁺ (IL-17a⁺) γδ T cells in the liver. Importantly, thymectomized mouse models showed that IFN-γ-producing γδ T cells could originate from liver LSM cells in a thymus-independent manner. These results suggested that liver hematopoietic progenitor LSM cells were able to differentiate into pre-γδ T cells and functionally mature γδ T cells, which implied that these cells are involved in a distinct developmental pathway independent of thymus-derived γδ T cells.     

TcR-α/β CD4CD8 T Cells in Humans with the Autoimmune Lymphoproliferative Syndrome Express a Novel CD45 Isoform That Is Analogous to Murine B220 and Represents a Marker of Altered O-Glycan Biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor α/β⁺ CD4⁻CD8⁻ T cells (α/β⁺ double-negative T cells [α/β⁺-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The α/β⁺-DNT cells are immunophenotypically and functionally similar to α/β⁺-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, α/β⁺-DNT cells express the B-cell-specific CD45R isoform B220. We show that α/β⁺-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4⁺ T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell–cell interactions, and access to alternative apoptosis pathways.     

Origin and development of the γδ T-cell system in sheep: a critical role for the thymus in the generation of TcR diversity and tissue tropism

Ruminant γδ T cells are concentrated at epithelial surfaces and share many features in common with species such as mice and humans which contain relatively fewer γδ T cells than ruminants. To date no γδ T cells with invariant TcR have been found in sheep and the generation of γδ TcR diversity which is thymic dependent follows a developmentally regulated sequence. Analysis of thymic export of γδ T cells shows that emigration of γδ T-cell subsets increases markedly during fetal life and after birth suggesting intrathymic processes leading to mature γδ T cells may change during development. Skin homing γδ T cells acquire their tissue tropism inside the thymus and pathways of recirculation of γδ T cells to skin are laid down during fetal development independent of antigen and remain stable through into adult life.