The regulatory role of interferon‐γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin‐induced pulmonary fibrosis

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild‐type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)‐γ and interleukin (IL)‐17A. Histological and biochemical analyses showed that bleomycin‐induced IP was more severe in T cell receptor (TCR‐δ‐deficient (TCRδ^–/–) mice than WT mice. In TCRδ^–/– mice, pulmonary IL‐17A⁺CD4⁺ Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ^–/– mice infused with γδT cells from WT mice, the number of pulmonary IL‐17A⁺ CD4⁺ T cells was lower than in TCRδ^–/– mice. The examination of IL‐17A^–/– TCRδ^–/– mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL‐17A⁺CD4⁺ T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4⁺ cells isolated from TCRδ^–/– mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN‐γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN‐γ‐producing γδT cells through the suppression of pulmonary IL‐17A⁺CD4⁺ T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin‐induced IP mouse model via the suppression of IL‐17A production.     

Peroxisome proliferator‐activated receptor‐β/δ modulates mast cell phenotype

The peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) is known to have multiple anti‐inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARβ/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARβ/δ modulates mast cell phenotype. Bone‐marrow‐derived mast cells (BMMCs) and peritoneal mast cells from Pparβ/δ^+/+ mice expressed higher levels of high‐affinity IgE receptor (FcεRI) compared with Pparβ/δ^−/− mice. BMMCs from Pparβ/δ^+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparβ/δ^−/− mice. Resting BMMCs from Pparβ/δ^+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal‐regulated kinases compared with Pparβ/δ^−/− mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARβ/δ expression. This study demonstrates that PPARβ/δ is an important regulator of mast cell phenotype.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)‐CD40 signaling by γδ T cells induces protective immunity against the blood‐stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T‐cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1⁺ cells, we saw that Vγ1⁺ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1⁺ γδ T cells preferentially expand and express CD40L, and both Vγ1⁺ and Vγ4⁺ γδ T cells produce IFN‐γ during infection. Although expression of CD40L on Vγ1⁺ γδ T cells is maintained during infection, the IFN‐γ positivity of Vγ1⁺ γδ T cells is reduced in late‐phase infection due to γδ T‐cell dysfunction. In Plasmodium‐infected IFN‐γ signaling‐deficient mice, DC activation is reduced, resulting in the suppression of γδ T‐cell dysfunction and the dampening of γδ T‐cell expansion in the late phase of infection. Our data suggest that Vγ1⁺ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1⁺ γδ T‐cell response is dependent on IFN‐γ‐activated DCs.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

γδ T cells come to stay: Innate skin memory in the Aldara model

The term immunological memory has long been a trademark restricted to adaptive lymphocytes such as memory B cells and plasma cells as well as memory CD8⁺ αβ T cells. In recent years, innate lymphocytes such as NK cells have also been shown to adapt to their environment by antigen‐specific expansion and selective survival. However, whether γδ T cells mount comparable memory responses to pathogenic stimuli is less well understood. In this issue of European Journal of Immunology, Hartwig et al. [Eur. J. Immunol. 2015. 45: 3022–3033] identify a subset of IL‐17‐producing γδ T cells that are capable of establishing long‐lived memory in the skin of mice exposed to imiquimod in the Aldara psoriasis model. These γδ T cells uniformly express a Vγ4⁺Vδ4⁺ TCR. They produce IL‐17A/F and persist in the dermis for long periods of time, also at untreated distal sites. Upon secondary challenge, experienced Vγ4⁺Vδ4⁺ cells show enhanced effector functions and mediate exacerbated secondary inflammation. These findings showcase innate γδ T‐cell memory that uses a single conserved public TCR combination. Furthermore, they provide mechanistic insight to the observed psoriatic relapses in patients in response to topical treatment with imiquimod.     

Th17‐related cytokines contribute to recall‐like expansion/effector function of HMBPP‐specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination

Whether cytokines can influence the adaptive immune response by antigen‐specific γδ T cells during infections or vaccinations remains unknown. We previously demonstrated that, during BCG/Mycobacterium tuberculosis (Mtb) infections, Th17‐related cytokines markedly upregulated when phosphoantigen‐specific Vγ2Vδ2 T cells expanded. In this study, we examined the involvement of Th17‐related cytokines in the recall‐like responses of Vγ2Vδ2 T cells following Mtb infection or vaccination against TB. Treatment with IL‐17A/IL‐17F or IL‐22 expanded phosphoantigen 4‐hydroxy‐3‐methyl‐but‐enyl pyrophosphate (HMBPP)‐stimulated Vγ2Vδ2 T cells from BCG‐vaccinated macaques but not from naïve animals, and IL‐23 induced greater expansion than the other Th17‐related cytokines. Consistently, Mtb infection of macaques also enhanced the ability of IL‐17/IL‐22 or IL‐23 to expand HMBPP‐stimulated Vγ2Vδ2 T cells. When evaluating IL‐23 signaling as a prototype, we found that HMBPP/IL‐23‐expanded Vγ2Vδ2 T cells from macaques infected with Mtb or vaccinated with BCG or Listeria ΔactA prfA*‐ESAT6/Ag85B produced IL‐17, IL‐22, IL‐2, and IFN‐γ. Interestingly, HMBPP/IL‐23‐induced production of IFN‐γ in turn facilitated IL‐23‐induced expansion of HMBPP‐activated Vγ2Vδ2 T cells. Furthermore, HMBPP/IL‐23‐induced proliferation of Vγ2Vδ2 T cells appeared to require APC contact and involve the conventional and novel protein kinase C signaling pathways. These findings suggest that Th17‐related cytokines can contribute to recall‐like expansion and effector function of Ag‐specific γδ T cells after infection or vaccination.     

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCR^low subset had a higher CD3 to TCR ratio and was enriched in Vδ2⁺ cells, whereas most Vδ1⁺ cells belonged to the γδTCR^high subset. In PB from TB, most γδTCR^high were CD45RA⁺CCR7^‐ and γδTCR^low were CD45RA^+/?CCR7⁺CXCR3⁺. In the pleural space the proportion of CD45RA^‐CCR7⁺CXCR3⁺ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCR^low cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.     

The proportion of different interleukin‐17‐producing T‐cell subsets is associated with liver fibrosis in chronic hepatitis C

Studies have suggested the pivotal role of T helper type 1 (Th1) ‐related cytokines on the outcome of hepatitis C virus (HCV) infection. Nevertheless, the role of different interleukin‐17 (IL‐17) ‐secreting T cells on chronic hepatitis C (CHC) is less clear. Here, the in vivo IL‐1β, IL‐6, and IL‐17 levels were positively correlated with both alanine transaminase (ALT) levels and hepatic lesions. When compared with the control group, CHC patients showed a lower proportion of IL‐17‐secreting (CD4⁺ and CD8⁺) T cells capable of simultaneously producing IL‐21. Moreover, the percentage of IL‐10‐secreting Th17 cells was also lower in CHC patients. Notably, advanced liver lesions were observed among those patients with lower percentage levels of IL‐17‐producing T cells positive for IL‐21, interferon‐γ (IFN‐γ) and IL‐10. In contrast, the severity of hepatic damage was associated with peripheral single IL‐17⁺ T cells. The percentage of IL‐17⁺ IL‐21^– IFN‐γ⁺ (CD4⁺ and CD8⁺) T‐cell phenotypes was positively associated with plasma CD14 levels. Finally, elevated levels of circulating CD14 were detected among CHC patients with extensive liver damage. In summary, although preliminary, our results suggest that a balance between different IL‐17‐producing T cells, associated with peripheral levels of CD14, may be a progress marker for liver disease in chronically HCV‐infected patients.     

T helper type 1 polarizing γδ T cells and Scavenger receptors contribute to the pathogenesis of Pemphigus vulgaris

γδ T cells and Scavenger receptors are key parts of the innate immune machinery, playing significant roles in regulating immune homeostasis at the epithelial surface. The roles of these immune components are not yet characterized for the autoimmune skin disorder Pemphigus vulgaris (PV). Phenotyping and frequency of γδ T cells estimated by flow cytometry have shown increased frequency of γδ T cells (6·7% versus 4·4%) producing interferon‐ γ (IFN‐γ; 35·2% versus 26·68%) in the circulation of patients compared with controls. Dual cytokine‐secreting (IFN‐γ and interleukin‐4) γδ T cells indicate the plasticity of these cells. The γδ T cells of patients with PV have shown higher cytotoxic potential and the higher frequency of γδ T cells producing IFN‐γ shows T helper type 1 polarization. The increased expression of Scavenger receptors expression (CD36 and CD163) could be contributing to the elevated inflammatory environment and immune imbalance in this disease. Targeting the inflammatory γδ T cells and Scavenger receptors may pave the way for novel therapeutics.     

Dermal IL‐17‐producing γδ T cells establish long‐lived memory in the skin

Conventional αβ T cells have the ability to form a long‐lasting resident memory T‐cell (T_RM) population in nonlymphoid tissues after encountering foreign antigen. Conversely, the concept of ‘innate memory’, where the ability of nonadaptive branches of the immune system to deliver a rapid, strengthened immune response upon reinfection or rechallenge, is just emerging. Using the αβ T‐cell‐independent Aldara psoriasis mouse model in combination with genetic fate‐mapping and reporter systems, we identified a subset of γδ T cells in mice that is capable of establishing a long‐lived memory population in the skin. IL‐17A/F‐producing Vγ4⁺Vδ4⁺ T cells populate and persist in the dermis for long periods of time after initial stimulation with Aldara. Experienced Vγ4⁺Vδ4⁺ cells show enhanced effector functions and mediate an exacerbated secondary inflammatory response. In addition to identifying a unique feature of γδ T cells during inflammation, our results have direct relevance to the human disease as this quasi‐innate memory provides a mechanistic insight into relapses and chronification of psoriasis.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

4–1BB signal stimulates the activation,expansion, and effector functions of γδ T cells in mice and humans

We show here that the expression of 4–1BB is rapidly induced in γδ T cells following antigenic stimulation in both mice and humans, and ligation of the newly acquired 4–1BB with an agonistic anti‐4–1BB augments cell division and cytokine production. We further demonstrate that γδ rather than αβ T cells protect mice from Listeria monocytogenes (LM) infection and 4–1BB stimulation enhances the γδ T‐cell activities in the acute phase of LM infection. IFN‐γ produced from γδ T cells was the major soluble factor regulating LM infection. Vγ1⁺ T cells were expanded in LM‐infected mice and 4–1BB signal triggered an exclusive expansion of Vγ1⁺ T cells and induced IFN‐γ in these Vγ1⁺ T cells. Similarly, 4–1BB was induced on human γδ T cells and shown to be fully functional. Combination treatment with human γδ T cells and anti‐hu4–1BB effectively protected against LM infection in human γδ T cell‐transferred NOD‐SCID mice. Taken together, these data provide evidence that the 4–1BB signal is an important regulator of γδ T cells and induces robust host defense against LM infection.     

Modulation of γδ T‐cell activation by neutrophil elastase

γδ T cells are non‐conventional, innate‐like T cells, characterized by a restricted T‐cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up‐regulation of CD69 expression, and the production of interferon‐γ and tumour necrosis factor‐α induced by anti‐CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase‐1 and neutralization of interleukin‐18 did not affect neutrophil‐mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T‐cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T‐cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T‐cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease‐activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T‐cell activation.     

Interleukin‐18 activates Vγ9Vδ2+ T cells from HIV‐positive individuals: recovering the response to phosphoantigen

The study aimed to identify an immunoregulatory factor that restores the phosphoantigen response of Vγ9Vδ2⁺ T cells from HIV‐positive individuals on antiretroviral therapy. It was designed to characterize the effects of interleukin‐18 (IL‐18) on proliferation and effector function in Vγ9Vδ2 T cells from HIV‐negative individuals and test whether exogenous IL‐18 reconstitutes the Vγ9Vδ2 T‐cell response to phosphoantigen from HIV‐positive donors. Vγ9Vδ2 T cells from HIV‐negative individuals responded strongly to phosphoantigen or aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMC), whereas cells with similar T‐cell receptor profiles from HIV‐positive individuals only responded to aminobisphosphonate. Interleukin‐18 was higher after aminobisphosphonate stimulation due to activation of the inflammasome pathway. Both IL‐18 and IL‐18 receptor levels were measured and the activity of exogenous IL‐18 on HIV‐negative and HIV‐positive PBMC was evaluated in terms of Vγ9Vδ2 T‐cell proliferation, memory subsets, cytokine expression and CD107a expression. Interleukin‐18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV‐negative controls. When Vγ9Vδ2 T cells from HIV‐positive individuals were stimulated with isopentenyl pyrophosphate in the presence of IL‐18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D and CD107a (markers of cytotoxic effector phenotype). Interleukin‐18 stimulation specifically expanded the Vγ9‐JγP⁺ subset of Vγ9Vδ2 T cells, as was expected for normal responses to phosphoantigen. Interleukin‐18 is a potent stimulator of Vγ9Vδ2 T‐cell proliferation and effector function. Therapies directed at reconstituting Vγ9Vδ2 T‐cell activity in HIV‐positive individuals should include stimulators of IL‐18 or direct cytokine supplementation.