Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

Liver distribution of γδ‐T‐cells in patients with chronic hepatitis of different etiology

The γδ T cells represent a minor unique T‐cell subpopulation long been considered as innate‐like immune cells. They are found in increased numbers in tissues from various inflammatory conditions. Their role in chronic hepatitis, however, is still discussed controversially. Fresh frozen tissues from 50 patients (18 cases hepatitis B infection, 25 hepatitis C, three cases with co‐infection of hepatitis B and C and four patients with autoimmune hepatitis) were investigated. Immunohistochemistry with primary antibodies detecting αβ and γδ TCR was used to evaluate their incidence and distribution in the different histological structures of the liver. The inflammatory infiltrate in all cases of chronic hepatitis was dominated by αβ T cells and was mainly localized in the portal tracts with formation of an interface hepatitis (95.3%αβ T cells; 4.7%γδ T cells). There were neither significant differences between inflammatory infiltrate nor the amount or percentage of γδ T cells between hepatitis B, C or autoimmune hepatitis. No accumulation of γδ T cells could be observed in cases of chronic hepatitis of different etiologies. The immune‐mediated phenomena in chronic hepatitis are dominated by αβ T cells. Thus, the adapted immune system is responsible for the inflammatory processes in chronic hepatitis.     

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.     

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.     

Neutrophils suppress γδ T‐cell function

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN‐γ, and the proliferation of γδ T cells induced by (E)‐1‐hydroxy‐2‐methylbut‐2‐enyl 4‐diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil‐derived ROS. We also show that the ROS‐generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi‐directional cross‐talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.     

NKG2D regulates production of soluble TRAIL by ex vivo expanded human γδ T cells

Soluble TRAIL (sTRAIL) can be produced by myeloid‐derived cells to kill cancer cells. Whether this mechanism is used by T cells, and if so, how sTRAIL production is regulated, remains unclear. Our previous studies showed that ex vivo expanded human γδ T cells express TRAIL and NK receptor group 2 (R2), member D (NKG2D), and possess potent anticancer activities both in vitro and in vivo. Here, we investigated in greater detail the mechanisms by which γδ T cells utilize TRAIL and NKG2D to kill lung cancer cells. We demonstrate that human lung cancer cells express TRAIL R2 and NKG2D ligands. Blocking TRAIL or NKG2D during γδ T‐cell‐lung cancer cell co‐cultures significantly reduced γδ T‐cell‐mediated cytotoxicity. Cross‐linking NKG2D with anti‐NKG2D antibody to mimic ligand binding promoted γδ T cells to produce sTRAIL, which induced apoptosis in lung cancer cells through TRAIL R2. Either neutralizing sTRAIL or blocking lung cancer cell TRAIL R2 significantly reduced γδ T‐cell‐mediated cytotoxicity to lung cancer cells. This study demonstrates that γδ T cells can mediate anticancer immunity via NKG2D‐regulated production of sTRAIL.     

Respiratory syncytial virus protects against the subsequent development of ovalbumin‐induced allergic responses by inhibiting Th2‐type γδ T cells

Respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans still remain inconclusive. The association between RSV infection and allergic diseases may be dependent on an atopic background and previous history of RSV infection. It has been reported that RSV infection before sensitization to an allergen decreased the production of Th2‐like cytokines in the lung and the levels of allergen‐specific Th2‐type antibodies in the serum. However, the underlying mechanisms are largely unknown. In the present study, the role of pulmonary γδ T cells in RSV‐affected, allergen‐induced airway inflammation was investigated. BALB/c mice were sensitized to or challenged with ovalbumin (OVA) and infected with RSV either before or after the sensitization period. It became clear that sensitization and challenge of mice with OVA induced a large influx of γδ T cells to the lungs. However, prior RSV infection inhibited the infiltration of γδ T cells as well as activated γδ T cells, characterized by expression of CD40L or CD69 molecular in the cell surface. Moreover, prior RSV infection elevated the type 1 cytokine gene expression but suppressed type 2 cytokine expression in the lung γδ T cells. Adoptive transfer of γδ T cells from OVA‐sensitized and challenged mice increased airway inflammation, suggesting that γδ T cells may play a proinflammatory role in allergic responses. These results described here support the idea of an unknown γδ T cell‐dependent mechanism in the regulation of RSV‐affected, allergen‐induced allergic airway responses. J. Med. Virol. 85:149–156, 2012. © 2012 Wiley Periodicals, Inc.     

Complement C5a regulates IL‐17 by affecting the crosstalk between DC and γδ T cells in CLP‐induced sepsis

Complement 5a (C5a) and Interleukin‐17 (IL‐17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL‐17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)‐induced sepsis and decreased IL‐17 expression in vivo. IL‐17 was secreted mainly by γδ T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL‐17 secretion by γδ T cells. Dendritic cells (DC) were found to act as a “bridge” between C5a and γδ T cells in a mechanism involving IL‐6 and transforming growth factor β (TGF‐β). These results imply that C5a affects the crosstalk between DC and γδ T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL‐17.     

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.     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.     

Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti‐tumour necrosis factor‐α therapy

Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn's disease (CD) who received anti‐tumour necrosis factor (TNF)‐α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti‐TNF‐α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty‐five men and 10 women who were treated with anti‐TNF‐α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR‐γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti‐TNF‐α‐treated patients than naive patients, suggesting that anti‐TNF‐α therapy may promote the clonal selection of γδ T cells in psoriasis patients.     

Mechanisms of chorioamnionitis‐associated preterm birth: interleukin‐1β inhibits progesterone receptor expression in decidual cells

In chorioamnionitis (CAM), a major cause of preterm birth (PTB), maternal–fetal inflammation of the decidua and amniochorion cause the release of cytokines that elicit cervical ripening, fetal membrane rupture and myometrial activation. We posit that this inflammatory milieu triggers PTB by inhibiting progesterone receptor (PR) expression and increasing decidual prostaglandin (PG) production. Immunohistochemical staining of decidua detected significantly lower PR levels in decidual cells (DCs) from CAM‐complicated PTB. Incubation of DCs with IL‐1β decreased PR expression and significantly increased PGE₂ and PGF_2α production and COX‐2 expression. The addition of PGF_2α to DC cultures also suppressed PR expression. However, the COX inhibitor, indomethacin, did not reverse IL‐1β suppression of PR expression in DC cultures. Although IL‐1β treatment activated the NF‐K B, ERK1/2 and p38 MAPK signalling cascades in DCs, inhibition of ERK1/2 MAPK signalling alone was sufficient to completely reverse the suppression of PR levels by IL‐1β. These findings suggest that CAM‐associated PTB is induced at least in part by IL‐1β‐mediated functional progesterone withdrawal. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

In vivo application of mAb directed against the γδ TCR does not deplete but generates “invisible” γδ T cells

mAb targeting the γδ TCR have been used for γδ T‐cell depletion with varying success. Although the depletion‐capacity of the anti‐γδ TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use γδ T‐cell depletion protocols involving the mAb clone UC7‐13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7‐13D5 antibodies does not deplete γδ T cells in vivo, but rather leads to TCR internalization and thereby generates “invisible” γδ T cells. We addressed this issue using anti‐γδ TCR mAb injections into WT mice as well as into reporter TCR delta locus‐histone 2B enhanced GFP knock‐in mice, in which γδ T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus‐histone 2B enhanced GFP mice provided here for the first time direct evidence that the “depleted” γδ T cells were actually still present. Our results show further that GL3 and UC7‐13D5 mAb are in part cross‐competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of γδ T cells through mAb. We conclude that γδ T‐cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of γδ T cells.     

Differential chemokine and cytokine production by neonatal bovine γδ T‐cell subsets in response to viral toll‐like receptor agonists and in vivo respiratory syncytial virus infection

γδ T cells respond to stimulation via toll‐like receptors (TLR). Bovine γδ T cells express TLR3 and TLR7, receptors that are key for the recognition of viruses such as bovine respiratory syncytial virus (BRSV); however, responses of γδ T cells to stimulation via these receptors, and their role during viral infections, remains unclear. Here, we demonstrate that neonatal bovine γδ T cells exhibit robust chemokine and cytokine production in response to the TLR3 agonist, Poly(I:C), and the TLR7 agonist, Imiquimod. Importantly, we observe a similar phenotype in γδ T‐cell subsets purified from calves infected with BRSV. Bovine γδ T cells are divided into subsets based upon their expression of WC1, and the response to TLR stimulation and viral infection differs between these subsets, with WC1.1⁺ and WC1^neg γδ T cells producing macrophage inflammatory protein‐1α and granulocyte–macrophage colony‐stimulating factor, and WC1.2⁺ γδ T cells preferentially producing the regulatory cytokines interleukin‐10 and transforming growth factor‐β. We further report that the active vitamin D metabolite 1,25‐dihydroxyvitamin D3 does not alter γδ T‐cell responses to TLR agonists or BRSV. To our knowledge, this is the first characterization of the γδ T‐cell response during in vivo BRSV infection and the first suggestion that WC1.1⁺ and WC1^neg γδ T cells contribute to the recruitment of inflammatory populations during viral infection. Based on our results, we propose that circulating γδ T cells are poised to rapidly respond to viral infection and suggest an important role for γδ T cells in the innate immune response of the bovine neonate.     

CCR7‐mediated migration in the thymus controls γδ T‐cell development

αβ T‐cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C‐C chemokine receptor (CCR)7‐deficient (Ccr7^?/?) and CCR9‐deficient mice (Ccr9^?/?) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T‐cell development. γδ T‐cell frequencies and numbers were decreased in CCR7‐deficient and increased in CCR9‐deficient mice. Transfer of CCR7‐ or CCR9‐deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell‐intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T‐cell output in CCR7‐deficient mice. In vitro, CCR7‐deficient precursors showed normal γδ T‐cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T‐cell localization within thymic medulla or cortex, respectively. However, γδ T‐cell motility was unaltered in CCR7‐ or CCR9‐deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T‐cell development.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.