IL-18R signaling is required for γδ T cell response and confers resistance to Trypanosoma cruzi infection.

IFN-γ-producing γδ T cells have been suggested to play an important role in protection against infection with Trypanosoma cruzi. However, little is known about the mechanisms leading to functional differentiation of this T cell subset in this model. In the current work, we investigated the possibility that the IL-18/MyD88 pathway is central for the generation of effector γδ T cells, playing a role for resistance against infection. We found that splenic γδ+ CD3+ cells were rapidly expanded (10-14 days post infection), which was accompanied by an early γδ T cell infiltration into the heart. In the following days, intracardiac parasitism was reduced, the protective immunity being accompanied by decreased γδ T cells tissue infiltration. As predicted, there was a drastic reduction of γδ T cells in Myd88- and Il18r1-deficient mice, both transgenic strains displaying a susceptible phenotype with increased intracardiac parasitism. In vivo and in vitro assays confirmed that IL-18R deficiency hampered γδ T cell proliferation. Further characterization revealed that T. cruzi infection up-regulates IL-18R expression in WT γδ+ T cell population whereas Il18r1-/- mice showed impaired generation of cytotoxic GzB+ and IFN-γ-producing γδ T cells. Consistently, in vitro cytotoxicity assay confirmed that cytolytic function was impaired in Il18r1-deficient γδ T cells. As a proof of concept, adoptive transfer of WT γδ T cells rescues Il18r1-deficient mice from susceptibility, reducing parasitemia and abrogating the mortality. Collectively, our findings implicate the IL-18R-MyD88 signaling in the mechanisms underlying generation of immunoprotective γδ T cells response in experimental Trypanosoma cruzi infection.

Maternal IgG impairs the maturation of offspring intrathymic IL-17-producing γδT cells: Implications for murine and human allergies.

BACKGROUND: The precise mechanism involved in the acquisition of the IL-17+ profile of γδT cells, the ligands responsible for this change, and whether this default is acquired during intrathymic maturation need to be elucidated. OBJECTIVE: This study aimed to evaluate whether IL-17-producing γδT cells are present in the airways of tolerant offspring from allergen-sensitized mothers and the possible implication of maternal IgG in the generation of these cells. METHODS: Female mice were immunized or not, and the allergic response, frequency of γδT cell subsets and cytokine production of the offspring were analysed by flow cytometry. The effects of passive in vivo transfer of purified IgG were investigated in offspring. A translational approach was employed to analyse γδT cells in the thymus and PBMCs from humans. RESULTS: Maternal immunization reduced the frequency of spontaneous IL-17-producing γδT cells in the thymus, spleen and lung of offspring. This effect was mimicked by the in vivo treatment of females with purified IgG. IgG directly interacted with γδT cell membranes. The modulatory effect of human IgG on human infant intrathymic and adult peripheral γδT cells showed similarities to murine γδT cells, which is rarely reported in the literature. CONCLUSIONS & CLINICAL RELEVANCE: Together, our results reveal that IgG from potentially tolerant atopic mothers can influence offspring thymic IL-17-producing γδT cell maturation. Furthermore, we suggest that IgG is an unprecedented modulatory factor of murine and human γδT cells. These observations may support the future development of IgG-based immunoregulatory therapeutic strategies.

Dendritic Cells Exposed to Triiodothyronine Deliver Pro-Inflammatory Signals and Amplify IL-17-Driven Immune Responses.

BACKGROUND/AIMS: Although a cross-talk between immune and endocrine systems has been well established, the precise pathways by which these signals co-regulate pro- and antiinflammatory responses on antigen-presenting cells remain poorly understood. In this work we investigated the mechanisms by which triiodothyronine (T3) controls T cell activity via dendritic cell (DC) modulation. METHODS: DCs from wild-type (WT) and IL-6-deficient mice were pulsed with T3. Cytokine production and programmed death protein ligands (PD-L) 1 and 2 expression were assayed by flow cytometry and ELISA. Interferon-regulatory factor-4 (IRF4) expression was evaluated by RT-qPCR and flow cytometry. The ability of DCs to stimulate allogenic splenocytes was assessed in a mixed lymphocyte reaction and the different profile markers were analyzed by flow cytometry and ELISA. For in vivo experiments, DCs treated with ovalbumin and T3 were injected into OTII mice. Proliferation, cytokine production, frequency of FoxP3+ regulatory T (Treg) cells and PD-1+ cells were determined by MTT assay, ELISA and flow cytometry, respectively. RESULTS: T3 endows DCs with pro-inflammatory potential capable of generating IL-17-dominant responses and down-modulating expression of PD-L1 and 2. T3-stimulated WT-DCs increased the proportion of IL-17-producing splenocytes, an effect which was eliminated when splenocytes were incubated with T3-treated DCs derived from IL-6-deficient mice. Enhanced IL-17 expression was recorded in both, CD4- and CD4+ populations and involved the IRF-4 pathway. Particularly, γδ-T cells but not natural killer (NK), NKT, B lymphocytes nor CD8+ T cells were the major source of IL-17-production from CD4- cells. Moreover, T3-conditioned DCs promoted a decrease of the FoxP3+ Treg population. Furthermore, T3 down-modulated PD-1 expression on CD4- cells thereby limiting inhibitory signals driven by this co-inhibitory pathway. Thus, T3 acts at the DC level to drive proinflammatory responses in vitro. Accordingly, we found that T3 induces IL-17 and IFNγ-dominant antigen-specific responses in vivo. CONCLUSION: These results emphasize the relevance of T3 as an additional immune-endocrine checkpoint and a novel therapeutic target to modulate IL-17-mediated pro-inflammatory responses.

The atypical chemokine receptor ACKR2 drives pulmonary fibrosis by tuning influx of CCR2+ and CCR5+ IFNγ-producing γδT cells in mice.

Chemokines coordinate lung inflammation and fibrosis by acting on chemokine receptors expressed on leukocytes and other cell types. Atypical chemokine receptors (ACKRs) bind, internalize, and degrade chemokines, tuning homeostasis and immune responses. ACKR2 recognizes and decreases the levels of inflammatory CC chemokines. The role of ACKR2 in fibrogenesis is unknown. The purpose of the study was to investigate the role of ACKR2 in the context of pulmonary fibrosis. The effects of ACKR2 expression and deficiency during inflammation and fibrosis were analyzed using a bleomycin-model of fibrosis, ACKR2-deficient mice, bone marrow chimeras, and antibody-mediated leukocyte depletion. ACKR2 was upregulated acutely in response to bleomycin and normalized over time. ACKR2-/- mice showed reduced lethality and lung fibrosis. Bone marrow chimeras showed that lethality and fibrosis depended on ACKR2 expression in pulmonary resident (nonhematopoietic) cells but not on leukocytes. ACKR2-/- mice exhibited decreased expression of tissue-remodeling genes, reduced leukocyte influx, pulmonary injury, and dysfunction. ACKR2-/- mice had early increased levels of CCL5, CCL12, CCL17, and IFNγ and an increased number of CCR2+ and CCR5+ IFNγ-producing γδT cells in the airways counterbalanced by low Th17-lymphocyte influx. There was reduced accumulation of IFNγ-producing γδT cells in CCR2-/- and CCR5-/- mice. Moreover, depletion of γδT cells worsened the clinical symptoms induced by bleomycin and reversed the phenotype of ACKR2-/- mice exposed to bleomycin. ACKR2 controls the CC chemokine expression that drives the influx of CCR2+ and CCR5+ IFNγ-producing γδT cells, tuning the Th17 response that mediated pulmonary fibrosis triggered by bleomycin instillation.

Immunophenotypic Shifts in Primary Cutaneous γδ T-Cell Lymphoma Suggest Antigenic Modulation: A Study of Sequential Biopsy Specimens.

Primary cutaneous γδ T-cell lymphoma (PCGD TCL), an aggressive type of lymphoma, accounts for approximately 1% of all primary cutaneous lymphomas. We have occasionally observed changes in T-cell antigen expression (immunophenotypic [IP] shift) over time, a phenomenon that is considered rare in T-cell lymphoma including cutaneous T-cell lymphoma. Therefore, we assessed sequential biopsies of PCGD TCL for possible IP shifts of the lymphoma cells. We searched for cases of PCGD TCL with consecutive biopsies to perform a comprehensive immunohistochemical analysis of paired specimens. A median of 12 markers per case was tested. We evaluated the percentage of neoplastic lymphocytes and determined the differential expression of antigens (gain, loss, increase or decrease). We identified 9 patients with PCGD TCL with consecutive biopsies. All (100%) cases had IP shifts of at least 1 antigen, whereas overall 22 pairs of markers were shifted: gain of reactivity occurred in 7 (31.8%) and loss in 3 (13.6%); increased reactivity in 4 (18.2%) and decreased in 8 (36.4%). Molecular analysis of TCRγ showed identically sized monoclonal rearrangements between biopsy pairs in 4/4 (100%) patients. There was no correlation between IP shifts and the clinical appearance of lesions, histopathologic or cytologic features, or molecular rearrangements. IP shifts are common in PCGD TCL, occurring in all patients in this study and involving a variety of antigens. IP shifts do not seem to be linked to changes in the T-cell clone and are without obvious clinical or morphologic correlates. The occurrence of IP shifts in PCGD TCL suggests that antigen modulation may be involved in pathogenesis. IP shifts are somewhat frequent in T-cell lymphoma; however, it does not suggest a second neoplasm, and molecular studies can be used to determine clonal identity.

Elucidating the function and tolerance mechanism of gamma delta (γ δ) T cells in a Helicobacter pylori infection model.

In this study, the functions and mechanisms of γ δ T cells were analyzed in patients infected with Helicobacter pylori. Peripheral blood was collected from gastritis patients in the Gastroenterology Department of Ningbo No. 2 Hospital. Preliminary analyses revealed 24 H. pylori-positive and 17 H. pylori-negative patients. The wild-type and γ δ T knockout mice were infected with cultured H. pylori cells (obtained from the H. pylori-positive patients). H. pylori in mice was quantified by polymerase chain reaction; gastritis was confirmed by hematoxylin and eosin staining. The TCR-δ(-/-) mice were treated with vein adoptive immunotherapy 24 h prior to H. pylori inoculation; the same method was used to detect the extent of gastritis and bacterial colonization. The γ δ T knockout mice showed high levels of H. pylori infection than the wild-type mice; in addition, the knockout mice showed severe disease pathology. γ δ T knockout mice also displayed increased matrix metalloproteinase-9 (MMP-9) and decreased MMP-7 expression in the gastric mucosa. γ δ T cells play a protective role in patients infected with H. pylori. γ δ T cell [responsible for the production of interleukin-17 (IL-17) and IL-22] expression was increased in H. pylori-positive patients, indicating statistical significance. However, there was no significant difference in interferon-gamma + γ δ T expression between the positive and negative patients. This study demonstrated the probable involvement of γ δ T cells in the immune response of an organism, via the secretion of IL-17 and IL-22.

Molecular and biochemical characterization of the Mexican axolotl CD3 (CD3ε and CD3γ/δ).

In mammals, the T-cell receptor (TCR) complex expressed on mature T-cells consists of α/ß or γ/δ clonotypic heterodimers non-covalently associated with four invariant chains forming the CD3 complex (CD3γ, CD3δ, CD3ε and CD3ζ). The TCR is the unit implicated in the antigenic peptide recognition whereas the CD3 subunits present as three different dimers (δ-ε, γ-ε and ζ-ζ) in the receptor complex participate to the signal transduction and are indispensable for the expression of the TCR at the cell surface. We report the cloning, characterization and expression analysis of CD3γ/δ and CD3ε genes in an amphibian urodele, the Mexican axolotl. Amino acid comparisons show that important motifs and residues were preserved between the axolotl CD3 chains and various vertebrate CD3ε, CD3γ, CD3δ and CD3γ/δ chains. During ontogeny, CD3ε transcripts are first detected in the dorsal region of tail-bud embryos before thymus organogenesis. CD3γ/δ transcripts are first detected in the head of 4-week-old larvae. A cross-reactive polyclonal anti-CD3ε antibody was used for the co-immunoprecipitation of the two CD3 proteins of 25 and 29 kDa, respectively, associated with the 90-kDa αß TCR heterodimer.

Leukotriene B4 mediates gammadelta T lymphocyte migration in response to diverse stimuli.

Herein, we investigated the involvement of the 5-LO-derived lipid mediator LTB(4) in gammadelta T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB(4) triggered gammadelta T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB(4) in pleural cavities. The in vivo inhibition of LTB(4) biosynthesis by the 5-LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS-induced gammadelta T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit gammadelta T cells into the inflammatory site after i.pl. LPS. Antagonists of the high-affinity LTB(4) receptor BLT1, CP105,696, and LY292476 also attenuated LPS-induced gammadelta T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS-simulated mice. LTB(4)/BLT1 also accounted for gammadelta T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS-recruited gammadelta T cells. Isolated gammadelta T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB(4) in vitro, confirming that gammadelta T lymphocytes can respond directly to LTB(4). In addition to its direct effect on gammadelta T cells, LTB(4) triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that gammadelta T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB(4)/BLT1.

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.

Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.

Hepatosplenic gammadelta T-cell lymphoma following seven malaria infections.

Hepatosplenic gammadelta T-cell lymphoma (HSTL) is a clinicopathological entity associated with an immunocompromised status in approximately 25% of patients. Herein is described a case of HSTL in a 53-year-old Brazilian man with seven previous malaria infections, initially misdiagnosed as a hyperreactive splenomegaly due to chronic malaria. A characteristic lymphoid infiltrate was observed in spleen, liver and bone marrow sinusoids/sinuses. Neoplastic cells had a CD45RO+, CD2+, CD7+, CD3+, CD5-, CD8+, CD56+, perforin+, FasL-negative, T-cell receptor (TCR)alphabeta-negative, TCRgammadelta+ profile. Analyses of gamma and delta TCR rearrangements confirmed diagnosis of gammadelta T-cell lymphoma by detecting VgammaI/Vdelta1-Jdelta1 clonal rearrangements. Sensitive polymerase chain reaction (PCR) for Plasmodium falciparum, Epstein-Barr virus and herpesvirus-8 failed to demonstrate infection. The disease progressed to a fatal outcome following cutaneous infiltration and leukemic proliferation. The authors also comment on the association of lymphoma and infection, focusing on PCR diagnosis of TCRgamma and delta clonal rearrangements and the presumed pathogenic events leading to HSTL in the context of chronic malaria infection. Initial lymphomagenic stages might not be direct consequences of antigenic stimulation of Vdelta1 T-cells, but might depend on interactions between gammadelta T and B cells during cooperative or regulatory responses to Plasmodium sp.

Blastic CD4 NK cell leukemia/lymphoma: a distinct clinical entity.

We report the findings of three new cases of a distinct clinicopathologic natural killer (NK) cell malignancy characterized by cutaneous, nodal and bone marrow infiltration by CD3-CD4+CD56+ NK blastic cells. Tumor cells were detected in bone marrow and in peripheral blood smears and showed finely distributed nuclear chromatin with nucleoli and a moderate amount of cytoplasm. Epstein-Barr virus (EBV) DNA was negative in the two tested cases. The immunophenotypes determined by flow cytometry were identical concerning mCD3-cytCD3-CD4+weakCD56+ HLA-DR+. The TCR was in germline configuration in the two cases tested. NK cell activity was demonstrated only in one out of the two cases tested. The negative reactions with alpha-naphthyl-acetate-esterase (ANAE), CD11b and CD14 strongly suggested that the tumor cells were not of the monocytic lineage.

Structure, diversity and expression of the TCRdelta chains in the Mexican axolotl.

Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). alpha beta T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and gamma delta T cells recognize native peptide or non-peptide antigens independently of MHC. Very little is known about gamma delta T cells in ectothermic vertebrates. We have cloned and characterized the TCRdelta chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cdelta domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Valpha regions that can associate with Calpha (Valpha2, Valpha3, Valpha5 and Valpha6) can also associate with Cdelta, but no specific Vdelta regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has been present in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jdelta regions were identified, but no Ddelta segments were clearly recognized at the Vdelta-Jdelta junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vdelta-Jdelta junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Valpha/delta6-Cdelta and Vbeta7-Cbeta junctions showed that both the TCRdelta and the TCRbeta chains were limited in diversity in thymectomized axolotls.

CD4+ TCRalphabeta T cells are critically involved in the control of experimental murine cysticercosis in C57BL/6J mice.

Taenia crassiceps cysticerci develop in the peritoneal cavity of BALB/cAnN mice and, to a lesser extent, in C57BL/6J mice. The mechanisms involved in the immunity to this murine cysticercosis seem to be mainly mediated by T cells. To gain further insight into the mechanisms of cysticercal immunity, the susceptibility of mice deficient in different immunologically relevant genes was compared with that of the respective wild type. Mice were classified according to the parasite load and survival after infection: highly susceptible (HS), with an increased parasite load and mortality rate (CD4-/-, TCRalpha-/-, TCRbeta-/-, RAG1-/-), susceptible, with only increased parasite load (TCRdelta-/-, BALB/cAnN), and relatively resistant, with a lower number of parasites (CD8-/-, WT). Neither specific proliferative response nor Th2 cytokine or antibody responses were observed in HS mice. These data strongly suggest that CD4+TCRalphabeta+ T cells have a critical role in the control of T. crassiceps murine cysticercosis.

Suppression of asthma-like responses in different mouse strains by oral tolerance.

In this study we examined the effect of oral antigen (Ag) administration on the development of experimental asthma in different mouse strains. We selected BALB/c, BP2, CBA/Ca interleukin (IL)-5 transgenic, and BALB/c T-cell receptor-delta-deficient mouse strains because they exhibit different aspects of the asthma syndrome. Mice exposed to 1% ovalbumin (OVA), dissolved in the drinking water for 5 consecutive days, became unresponsive to subsequent immunogenic OVA challenges. This regimen of OVA administration induced Ag-specific unresponsiveness in all mouse strains tested, including gammadelta-deficient mice that are said to be resistant to tolerance induction. The Ag-specific unresponsiveness was characterized by reduced (almost absent) airway eosinophilic inflammation, airway hyperreactivity, and mucus production; also by low levels of T helper (Th) 2-type cytokines in bronchoalveolar lavage fluid, and decreased immunoglobulin (Ig) G1 and IgE OVA-specific antibody production. The unresponsive state was not associated with increased levels of the suppressive cytokines IL-10 and transforming growth factor (TGF)-beta or with immune deviation toward the Th1 pathway due to increased levels of interferon-gamma and IL-12. Moreover, treatment with anti- TGF-beta antibodies did not abrogate oral tolerance. Oral Ag administration was quite effective in suppressing the development of key features of asthma when initiated after primary immunization (Day 0) or after booster (Day 7), but not after challenge (Day 14) when it increased allergic responses. Collectively, our findings show for the first time the beneficial and detrimental effects of oral Ag administration on the development of experimental asthma.

Childhood B lineage acute lymphoblastic leukemia clonality study by the polymerase chain reaction.

PURPOSE: B cell precursors acute lymphoblastic leukemia (ALL) present rearrangements in the heavy chain immunoglobulin and T cell receptor genes, especially in the complementarity determining region 3 (CDR-3) and T cell receptor delta (TCR delta) (V delta 2 D delta 3) regions. These rearrangements may be amplified by the polymerase chain reaction (PCR) and used as clonal markers of B lineage ALL. Our purpose was to study clonality at the DNA level by PCR in B lineage ALL. PATIENTS AND METHODS: Fifty-three pediatric patients (36 with B lineage ALL, 7 with ALL-T, and 10 with nonlymphocytic disease) were investigated using consensus primers for the CDR-3 regions of IgH and TCR delta. RESULTS: Clonality was detected in 86.1% of the patients with B lineage ALL when the primers for the CDR-3 regions were used, in 41.6% when the primers for TCR delta were used, and in 91.6% when the two primers were used together. Biclonality was found in 22.5% and 6.6% of patients that have shown clonality for CDR-3 and TCR delta, respectively. Clonality was not detected in any other samples using these primers. CONCLUSIONS: PCR using CDR-3 and TCR delta primers can be used as an aid for B lineage ALL diagnosis and clonal evolution of theses disease.

A maternally-inherited alteration in the T cell repertoire of balb/c mice

Se ha demostrado que los virus exógenos del tumor mamario murino (MMTV) transmitidos por leche, inducen la expresión de diferentes superantígenos en los huéspedes infectados. Cada uno de estos superantígenos es capaz de inducir la deleción clonal progresiva de las células T portadoras de determinados elementos Vß de su receptor (TCR). En este trabajo se describe la existencia de una alteración en el repertorio T de los ratones BALB/c de una colonia. Dicha alteración, transmitida por vía materna, involucra la deleción de las células T CD4+ que expresan las cadenas Vß2 y Vß14 del TCR y correlaciona con una alta incidencia de tumores de mama. Estos resultados indican la transmisión materna de un superantígeno(s), probablemente asociado a la presencia de virus MMTV en la leche

A maternally-inherited alteration in the T cell repertoire of balb/c mice

Se ha demostrado que los virus exógenos del tumor mamario murino (MMTV) transmitidos por leche, inducen la expresión de diferentes superantígenos en los huéspedes infectados. Cada uno de estos superantígenos es capaz de inducir la deleción clonal progresiva de las células T portadoras de determinados elementos Vß de su receptor (TCR). En este trabajo se describe la existencia de una alteración en el repertorio T de los ratones BALB/c de una colonia. Dicha alteración, transmitida por vía materna, involucra la deleción de las células T CD4+ que expresan las cadenas Vß2 y Vß14 del TCR y correlaciona con una alta incidencia de tumores de mama. Estos resultados indican la transmisión materna de un superantígeno(s), probablemente asociado a la presencia de virus MMTV en la leche (AU)