αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

Expansion of CD56+ NK T and γδ T cells from cord blood of human neonates

A particular T cell population expressing NK cell markers, CD56 and CD57, exists in humans. Many CD56⁺ T and CD57⁺ T cells (i.e. NK T cells) exist in the liver and increase in number in the blood with ageing. They may be a human counterpart of extrathymic T cells, similar to NK1.1⁺ CD3^int cells seen in mice. We investigate here the existence of such NK T cells in human cord blood and the in vitro expansion of these cells by the stimulation of human recombinant IL-2 (rIL-2). There were very small populations (< 1.0%) of CD56⁺ T cells, CD57⁺ T cells, and γδ T cells in cord blood. However, all of these populations increased in number after birth and with ageing. When lymphocytes in cord blood were cultured with rIL-2 (100 U/ml) for 14 days, CD56⁺ T cells expanded up to 25% of T cells. CD57⁺ T cells were never expanded by these in vitro cultures. The expansion of γδ T cells (mainly Vγ9^? non-adult type) also occurred in the in vitro culture. A considerable proportion of CD56⁺ T cells was found to use Vα24 (i.e. equivalent to invariant Vα14 chain used by murine NK T cells) for TCR αβ. These results suggest that neonatal blood contains only a few NK T cells but CD56⁺ NK T cells and γδ T cells are able to expand in vitro.     

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+ and CD4+ αβ+ human T cell subset responses upon stimulation with various Mycobacterium tuberculosis soluble extracts

By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for human γδ⁺ T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their γδ⁺ T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85°C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of γδ⁺ T cells after 6–7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121°C, 25 min) and then washed by centrifugation, a predominant proportion of CD4⁺ αβ⁺ T cells was achieved in the responding population. The stimulatory activity for γδ⁺ T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving of M. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human γδ⁺ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on γδ⁺ T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of γδ⁺ T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4⁺ αβ⁺ T cells only. The γδ⁺ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of γδ⁺ T cells on CD4⁺ αβ⁺ T cells.     

NK1.1+ CD4+ CD8- thymocytes with specific lymphokine secretion

CD4+8^? or CD4^?8⁺ thymocytes have been regarded as direct progenitors of peripheral T cells. However, recently, we have found a novel NK1.1⁺ subpopulation with skewed T cell antigen receptor (TcR) Vβ family among heat-stable antigen negative (HSA^?) CD4⁺8^? thymocytes. In the present study, we show that these NK1.1⁺ CD4+8^? thymocytes, which represent a different lineage from the major NK1.1^? CD4⁺8^? thymocytes or CD4⁺ lymph node T cells, vigorously secrete interleukin (IL)-4 and interfron (IFN)-γ upon stimulation with immobilized anti-TcR-αβ antibody. On the other hand, neither NK1.1^? CD4+8^?thymocytes nor CD4⁺ lymph node T cells produced substantial amounts of these lymphokines. A similar pattern of lymphokine secretion was observed with the NK1.1⁺ CD4⁺ T cells obtained from bone marrow. The present findings elucidate the recent observations that HSA^? CD4⁺8^? thymocytes secrete a variety of lymphokines including IFN-γ, IL-4, IL-5 and IL-10 before the CD4⁺8^? thymocytes are exported from thymus. Our evidence indicates that NK1.1⁺ CD4⁺8^? thymocytes are totally responsible for the specific lymphokine secretions observed in the HSA- CD4⁺8^? thymocytes.     

The major diversification of Vγ1.1+ and Vγ2+ thymocytes in mice occurs after commitment to the γδ T‐cell lineage

γδ T cells are a heterogeneous cell population with different subsets playing specialized and often opposing roles during immune responses. A key question is whether γδ thymocytes are determined for their effector function already at an early stage, before their commitment to the γδ T‐cell lineage, or are instructed during their later development. Here, we show that the adult Vγ1.1⁺ and Vγ2⁺ γδ T‐cell subsets both go through a CD73⁺CD24⁺ development stage, and that the gene regulation involved in lineage commitment is shared by both subsets. We demonstrate that the major subset diversification first occurs after the cells have committed to the γδ T‐cell lineage, strongly supporting an instructive model for functional programming of γδ T cells. In conclusion, we show that the two major adult γδ T‐cell subsets in mice develop through a shared pathway utilizing similar cellular machinery and that they diverge after the CD24⁺CD73⁺ maturity stage.     

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.     

Evidence for programmed cell death of self-reactive γδ T cell receptor-positive thymocytes

The negative selection of T cells expressing the γδ T cell antigen receptor (γδ T cells) was studied using transgenic mice expressing a γδ receptor with specificity for an H-2T-linked class I major histocompatibility complex molecule from H-2^b mice. The potentially self-reactive γδ thymocytes in H-2^b/d transgenic mice are larger and have lower levels of γδ T cell receptor expression than γδ thymocytes from H-2^d mice. H-2^b/d γδ thymocytes do not respond to H-2^b antigen-presenting cells, and thus are inactive compared to H-2^d γδ thymocytes. However, the H-2^b/d γδ thymocyte population, but not the H-2^d γδ thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self-reactive γδ thymocytes undergo programmed cell death. This in vitro programmed cell death of self-reactive γδ thymocytes may reflect the clonal deletion process that results in a depletion of γδ T cells in the peripheral lymphoid organs of adult H-2^b/d mice. We also present evidence that self-reactive γδ T cells, similarly to αβ T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.     

The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γ δ T cell population is due to cellular selection

Thy-1^dull γ δ T cells are an unusual subset of mature TCRγ δ T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vγ1 gene together with that of a member of the Vδ6 subfamily (the Vδ6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRγ δ repertoire, we have cloned all Vδ6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1^dull and Thy-1^bright γ δ thymocyte populations. Furthermore, we have also cloned non-functional Vδ6DδJδ1 rearrangements present in the Thy-1^dull γ δ T cell population and compared their Vδ6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRδ repertoire expressed by the Thy-1^dull γ δ thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRγ δ repertoire analysis of Thy-1^dull γ δ T cells in β2 -microglobulin (β₂m)-deficient mice indicated that these putative ligands are not β₂m-dependent major histocompatibility complex class I or class I-like molecules.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Selective expansion of activated Vδ4+ cells during experimental infection of mice with Leishmania major

Previous work from this laboratory has revealed that infection of mice with Leishmania major leads to an expansion of γδ⁺ T cells in the spleen. Further examination of the γδ⁺ T cells expanding in infected mice has shown that the majority of these cells in the spleen, lymph nodes, blood and liver expressed the Vβ4 gene segment. Cell cycle analysis, using propidium iodide incorporation, demonstrated that while only 1% of αβ⁺ T cells in the spleen were in either S + G2/M phase, up to 10% of the γδ⁺ T cells were in cycling phase 8 weeks after infection. Comparison of the state of activation of the two populations in different organs after infection, confirmed that γδ⁺ T cells are actively dividing in lymph nodes, liver and blood, but not in the thymus or among intraepithelial lymphocytes. Examination of the expression of different activation markers on the surface of γδ⁺ T cells in the spleen of both normal and chronically infected BALB/c mice by FACS analysis, revealed increased expression of LFA-1, CD25, CD44, 4F2, CD28 and the heat-stable antigen, whereas Thy-1 and CD5 decreased. Collectively, these results suggest an oligoclonal expansion and activation of γδ⁺ T cells in response to L. major infection.     

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.     

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.     

A population of CD62Llow NK1.1− CD4+ T cells that resembles NK1.1+ CD4+ T cells

In MHC class II−/− C57BL/6 (II−/−) mouse spleen, a small population of CD4⁺ T cells is present of which NK1.1⁺ CD4⁺ (NK) T cells comprise 40 to 45 %. We report here that many of the NK1.1⁻ CD4⁺ T cells derived from II−/− mice are also NK T cells. They produce large amounts of IL-4 in response to anti-CD3 ligation and do so without any requirement for the presence of IL-4 in the priming culture, a property characteristic of NK T cells. Their IFN-γ production is large and is enhanced by IL-12. In addition, II−/− NK1.1⁻ CD4⁺ T cells produce IL-4 as a result of culture with L cells expressing murine CD1 (L-CD1). We report that CD49b, a component of integrin VLA-2, is expressed on the majority of both NK1.1⁺ and NK1.1⁻ NK T cells. NK1.1⁻ NK T cells also exist in wild-type C57BL/6 mice. Evidence supporting this is that Vβ8 usage by CD62L^low NK1.1⁻ CD4⁺ T cells was ∼ 5 % higher than that by CD62L^high CD4⁺ T cells in wild-type mice in keeping with the estimated proportion of NK1.1⁻ NK T cells in the CD62L^low population. CD62L^low CD4⁺ T cells from β2-m−/− mice, which lack NK T cells, showed no increase in Vβ8 usage. When activated by anti-CD3 or L-CD1, CD62L^low NK1.1⁻ CD4⁺ T cells from conventional but not β2-m−/− and CD1−/− mice produce IL-4 in a manner indistinguishable from II−/− NK1.1⁻ CD4⁺ T cells. NK1.1⁻ NK T cells in normal mouse spleens are approximately as numerous as NK1.1⁺ NK T cells.     

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.