Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Increased Th2-like Invariant Natural Killer T cells in Peripheral Blood From Patients With Asthma

Purpose

Invariant natural killer T (iNKT) cells might play an important role in asthma pathogenesis in humans. Our previous study found no difference in the number of blood iNKT cells between asthma patients and controls. However, few studies have examined the function of blood iNKT cells in human asthma.

Methods

Twenty asthma patients and eight controls were included in this study. Blood iNKT cells were identified using double staining with anti-Vα24 and anti-Vβ11 monoclonal antibodies (mAbs) or with 6B11 and anti-Vβ11 mAbs. Intracellular IL-4, IL-10, and IFN-γ cytokines were stained in blood iNKT cells using their respective mAbs and isotypes. In addition, their relationships with clinical parameters were analyzed.

Results

The number of Vα24+Vβ11+ iNKT cells or 6B11+Vβ11+ iNKT cells did not differ between asthma patients and controls. However, among Vα24+Vβ11+iNKT cells, the proportion of IL-4+iNKT cells was increased in asthma patients compared to controls (7.0±3.0% vs 0.5%±0.4%, P<0.05). There were no differences in the proportions of IL-10+or IFN-γ+iNKT cells between the groups. The proportion of IL-4+ cells among 6B11+Vβ11+iNKT cells inversely correlated with FEV1, expressed as a percentage predicted value in asthma patients (Rs=-0.64, P<0.05, n=19).

Conclusions

Blood iNKT cells are thought to be Th2-like, and IL-4-producing iNKT cells may be associated with lung function in human asthma.     

T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR Vα-Vβ genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption

Human epidermis contains a phenotypically heterogeneous population of T cells. No information, however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed αβ TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-αβ complex. Comparative analyses of TCR Vα and Vβ expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of Vα and Vβ genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare.     

Characterization of T cells specific for an epitope of human 60-kD heat shock protein (hsp) in patients with Behçet's disease (BD) in Japan

BD is prevalent in the area of the Silk Route. It has been shown that hsp are involved in the T cell activation in patients with BD in the UK, where this disease has developed sporadically. We have thus examined whether the T cell response to the hsp-derived peptides may be induced in patients with BD in Japan, an east pole of the Silk Route. As with patients in the UK, the human 60-kD hsp peptide 336–351 also yielded vigorous proliferation of T cells in Japanese patients with BD, but neither in normal subjects nor in patients with rheumatoid arthritis (RA); there was significant association between proliferation by this peptide and the presence of ocular lesion, but not any other symptoms of BD. To clarify whether the peptide stimulates T cells as a polyclonal activator, a specific antigen or a superantigen-like substance, we analysed T cell receptor (TCR) usage of responding T cells by means of MoAbs specific for TCR Vβ subfamily and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP)-based technique. We found that T cells with certain TCR Vβ subfamilies (including Vβ5.2–3, 8, 13.6, 18, 21.3) were increased in circulation and responded to the hsp peptide in an antigen-specific fashion. In addition, TCR Vβ gene-amplified products of freshly isolated T cells of patients with BD formed several bands in the PCR-SSCP analysis; some of them became prominent after stimulation with the peptide. This suggests that T cells in patients with this disease have already been expanded oligoclonally in vivo, which may be a result of stimulation by triggering antigens, including the hsp peptide. In addition, hsp peptide stimulation induced proinflammatory cytokine mRNA expression in peripheral blood mononuclear cells, including IL-8, tumour necrosis factor-alpha (TNF-α) and TNF-β in eight out of eight patients studied. Taken together, the results suggest that hsp antigen may play a role in the pathogenesis of BD, not only in the area of the Silk Route, but also outside the Silk Route area.     

CD30 Is Required for Activation of a Unique Subset of Interleukin-17A-Producing γδ T Cells in Innate Immunity against Mycobacterium bovis Bacillus Calmette-Guérin Infection

Interleukin-17A (IL-17A)-producing γδ T cells are known to be activated following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Here, we show that CD30, a member of the tumor necrosis factor (TNF) receptor superfamily, is important for activation of IL-17A-producing γδ T cells after BCG infection. Vγ1⁻ Vγ4⁻ γδ T cells preferentially expressing Vγ6/Vδ1 genes were identified as the major source of IL-17A in the peritoneal cavity during the early stage of BCG infection. The number of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 increased in peritoneal exudate cells (PEC) of wild-type (WT) mice but not in those of CD30 knockout (KO) mice in response to BCG infection. Consistently, CD30 ligand (CD30L) or CD30 expression, predominantly by Vγ1⁻ Vγ4⁻ γδ T cells, was rapidly upregulated after BCG infection. Inhibition of CD30L/CD30 signaling by in vivo administration of a soluble CD30 and immunoglobulin fusion protein (CD30-Ig) severely impaired activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells in WT mice, while stimulating CD30L/CD30 signaling by in vivo administration of agonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A production by Vγ1⁻ Vγ4⁻ γδ T cells in CD30L KO mice after BCG infection. These results suggest that CD30 signaling plays an important role in the activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 at an early stage of BCG infection.     

Stimulation of peripheral blood lymphocytes with Campylobacter jejuni generates a γδ T cell response in patients with Guillain–Barré syndrome

In three patients whose Guillain–Barré syndrome (GBS) was preceded by gastrointestinal infection due to Campylobacter jejuni, γδ T cells were generated from peripheral blood in response to in vitro stimulation with C. jejuni. In one of the patients, where a diagnostic sural nerve biopsy was performed, γδ T cells were also isolated following culture of the nerve tissue. Studies with healthy volunteers and C. jejuni gastroenteritis patients also showed preferential enrichment for γδ T cells in peripheral blood cells stimulated with C. jejuni, although the response was significantly lower than that seen in GBS patients. In two out of three GBS patients and all of the controls, γδ T cell receptor (TCR) gene usage was shown to be Vγ9/Vδ2⁺. In the GBS patient where nerve-infiltrating γδ T cells were isolated, these and C. jejuni-specific peripheral blood cells had similar TCR gene usage, predominantly consisting of Vγ5/Vδ1⁺ cells. Sequencing the Vδ1 products from nerve and peripheral blood showed similarities in CDR3 length, but the single Vδ1 sequence obtained from nerve was not identified in peripheral blood. These results suggest that the generation of γδ T cells is part of a normal immune response to C. jejuni, which, in patients with GBS, may contribute to the pathogenesis of their inflammatory neuropathy.     

Characterization of Heat Shock Protein-Specific T Cells in Atherosclerosis

A role for infection and inflammation in atherogenesis is widely accepted. Arterial endothelium has been shown to express heat shock protein 60 (HSP60) and, since human (hHSP60) and bacterial (GroEL) HSP60s are highly conserved, the immune response to bacteria may result in cross-reactivity, leading to endothelial damage and thus contribute to the pathogenesis of atherosclerosis. In this study, GroEL-specific T-cell lines from peripheral blood and GroEL-, hHSP60-, and Porphyromonas gingivalis-specific T-cell lines from atherosclerotic plaques were established and characterized in terms of their cross-reactive proliferative responses, cytokine and chemokine profiles, and T-cell receptor (TCR) Vβ expression by flow cytometry. The cross-reactivity of several lines was demonstrated. The cytokine profiles of the artery T-cell lines specific for GroEL, hHSP60, and P. gingivalis demonstrated Th2 phenotype predominance in the CD4 subset and Tc0 phenotype predominance in the CD8 subset. A higher proportion of CD4 cells were positive for interferon-inducible protein 10 and RANTES, with low percentages of cells positive for monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR Vβ5.2 family in all lines. These cytokine, chemokine, and Vβ profiles are similar to those demonstrated previously for P. gingivalis-specific lines established from periodontal disease patients. These results support the hypothesis that in some patients cross-reactivity of the immune response to bacterial HSPs, including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may explain the apparent association between atherosclerosis and periodontal infection.     

CD4+ CD25high Foxp3+ regulatory T cells downregulate human Vδ2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

Vδ2⁺ T cells, the major circulating T-cell receptor-γδ-positive (TCR-γδ⁺) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vδ2⁺ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4⁺ T cells. CD4⁺ CD25^high Foxp3⁺ T cells (Tregs) comprise 5–10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-αβ⁺ lymphocytes, Tregs suppress Vδ2⁺ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vδ2⁺ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vδ2⁺ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-γ secretion by Vδ2⁺ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vδ2⁺ T-cell interferon-γ responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vδ2⁺ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vδ2⁺ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vδ2⁺ T-cell functional deficiencies observed in TB.     

A new effect of IL-4 on human γδ T cells: promoting regulatory Vδ1 T cells via IL-10 production and inhibiting function of Vδ2 T cells

Interleukin 4 (IL-4) has a variety of immune functions, including helper T-cell (Th-cell) differentiation and innate immune-response processes. However, the impact of IL-4 on gamma delta (γδ) T cells remains unclear. In this study, we investigate the effects of IL-4 on the activation and proliferation of γδ T cells and the balance between variable delta 1 (Vδ1) and Vδ2 T cells in humans. The results show that IL-4 inhibits the activation of γδ T cells in the presence of γδ T-cell receptor (TCR) stimulation in a STAT6-dependent manner. IL-4 promoted the growth of activated γδ T cells and increased the levels of Vδ1 T cells, which in turn inhibited Vδ2 T-cell growth via significant IL-10 secretion. Vδ1 T cells secreted significantly less interferon gamma (IFNγ) and more IL-10 relative to Vδ2. Furthermore, Vδ1 T cells showed relatively low levels of Natural Killer Group 2D (NKG2D) expression in the presence of IL-4, suggesting that Vδ1 T cells weaken the γδ T cell-mediated anti-tumor immune response. For the first time, our findings demonstrate a negative regulatory role of IL-4 in γδ T cell-mediated anti-tumor immunity.     

The glycolipid exoantigen derived from Chlamydia muridarum activates invariant natural killer T cells

The chlamydial glycolipid exoantigen (GLXA), a glycolipid antigen derived from Chlamydia muridarum, has been implicated in chlamydial–host cell interaction. Although glycolipid antigens from Sphingomonas and related bacteria have been shown to activate invariant natural killer T (iNKT) cells, it is not yet known whether GLXA can activate these cells. In this study, we have for the first time investigated the role of GLXA in iNKT cell activation using in vitro as well as in vivo settings. First, we examined the effect of GLXA on iNKT cell activation in a cell-free antigen-presentation assay, and found that GLXA specifically stimulated iNKT1.4 hybridoma cell produce enhanced amounts of IL-2. Next, we analyzed the effect of pharmacological activation of iNKT cells by GLXA using iNKT cell-deficient (iNKT knockout (KO)) mice and bone marrow-derived dendritic cell (BMDC)–liver mononuclear cell (LMC) coculture system. On stimulation with GLXA, iNKT cells produced higher quantities of cytokines in a CD1d-dependent fashion. More importantly, iNKT cells from GLXA-treated, but not from cell mock-treated, mice showed higher expression of activation marker, CD69, and enhanced production of interferon (IFN)-γ and IL-4 in vivo. Cumulatively, these data provide evidence on the pharmacological ability of GLXA in specifically activating iNKT cells.     

Multifunctional immune responses of HMBPP-specific Vγ2Vδ2 T cells in M. tuberculosis and other infections

Vγ2Vδ2 T (also known as Vγ9Vδ2 T) cells exist only in primates, and in humans represent a major γδ T-cell sub-population in the total population of circulating γδ T cells. Results from recent studies suggest that while (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen from Mycobacterium tuberculosis (Mtb) and other microbes activates and expands primate Vγ2Vδ2 T cells, the Vγ2Vδ2 T-cell receptor (TCR) recognizes and binds to HMBPP on antigen-presenting cells (APC). In response to HMBPP stimulus, Vγ2Vδ2 TCRs array to form signaling-related nanoclusters or nanodomains during the activation of Vγ2Vδ2 T cells. Primary infections with HMBPP-producing pathogens drive the evolution of multieffector functional responses in Vγ2Vδ2 T cells, although Vγ2Vδ2 T cells display different patterns of responses during the acute and chronic phases of Mtb infection and in other infections. Expanded Vγ2Vδ2 T cells in primary Mtb infection can exhibit a broader TCR repertoire and a greater clonal response than previously assumed, with different distribution patterns of Vγ2Vδ2 T-cell clones in lymphoid and non-lymphoid compartments. Emerging in vivo data suggest that HMBPP activation of Vγ2Vδ2 T cells appears to impact other immune cells during infection.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Biased T-Cell Antigen Receptor Repertoire in Lyme Arthritis

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) β-chain variable (Vβ) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vβ2 and Vβ6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells with B. burgdorferi provoked active proliferation but not a statistically significant increase in expression of any TCR Vβ, including Vβ2 and Vβ6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specific Borrelia reaction.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.     

Self-activation of Vγ9Vδ2 T cells by exogenous phosphoantigens involves TCR and butyrophilins

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.     

Brief Treatment with iNKT Cell Ligand α-Galactosylceramide Confers a Long-term Protection Against Lupus

CD1d presents glycolipid antigens such as α-galactosylceramide (αGalCer) to invariant natural killer T cells (iNKT). We have reported that activated iNKTs inhibit IL-10-producing autoreactive B cells, while promoting or leaving intact the normal B cell responses, making iNKT modulation an attractive therapeutic modality. Here, we report that a brief treatment of young lupus-prone (NZB/NZW)F1 (BWF1) mice with two injections of αGalCer conferred a long-term protection against lupus. Long-term repeated administrations of αGalCer, however, afforded no clinical benefit. These disparate clinical effects correlated with iNKT responsiveness. While a brief treatment with αGalCer enhanced iNKT responses upon in vitro recall, the long-term αGalCer treatment resulted in reduced iNKT responses in BWF1 mice. The improvement in disease with αGalCer treatment was associated with the reduced IL-10 production. Furthermore, iNKTs directly inhibited IL-10-secreting cells in vivo in reconstituted SCID mice and inhibited IL-10-secreting B cells in vitro in co-cultures. Thus, a brief treatment with a CD1d-binding glycolipid enhances iNKT responses, reduces IL-10 production, and delays the onset of lupus, whereas long-term repeated treatments induce marked iNKT hyporesponsiveness and do not affect disease outcome in BWF1 mice. Identifying glycolipid regimens that can modulate iNKT responsiveness will have important implications for developing iNKT-based therapies for autoimmune diseases.     

Evolution of a CD3+/CD+ α/ β T-Cell Receptor+ Mature T-Cell Clone from CD3-CD7+ Sorted Human Bone Marrow Cells

In order to study extrathymic differentiation in vitro, CD7⁺CD3^- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCRδ1 (TCRγ/δ-specific) or WT31 (TCR2, α/β-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3⁺, CD4⁺, CD8^-, TCR2⁺, TCR1^-, and was further characterized as CD2⁺, CD45RO⁺, CD16^-, and CD56^-. The presence of mRNA for TCR α and γ but not ,and γ chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted “natural killer (NK)-like” lysis of K562 target cells, with no autocytotoxicity detected. Tle NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-δ, and GM-CSF in these cells after stimulation with PHA and B-LCL.These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.     

Inverted Vδ1/Vδ2 ratio within the T cell receptor (TCR)-γδ T cell population in peripheral blood of heart transplant recipients

We investigated the levels of TCR-γδ T cells and their subpopulations Vδ1 and Vδ2 in the peripheral blood lymphocytes (PBL) of 28 heart transplant (HTx) patients. Patients (n = 10) receiving cyclosporin A (CsA) for treatment of a nephrotic syndrome (NS) and 10 healthy individuals served as controls. There was no difference in levels of TCR-γδ T cells between the different groups. However, an elevated proportion of Vδ1⁺γδ T cells was found in the PBL of HTx patients, especially when these cells were present in their graft-infiltrating lymphocyte (GIL) cultures. Vδ1⁺γδ T cells of HTx patients showed normal expression of CD45RO and lacked the activation markers CD25 and HLA-DR. After expanding in IL-2-containing medium, PBL cultures of HTx patients more often were dominated by Vδ1 cells than PBL cultures of controls, in which Vδ2 cells were predominantly grown. The aberrant composition of the TCR-γδ population in HTx patients was not a result of immunosuppressive medication, since the proportion Vδ1⁺γδ T cells was normal in the PBL of the NS patients receiving a similar dose of CsA. It is postulated that long-term antigenic stimulation by the graft, at low level, might be responsible for the altered composition of the γδ pool in the HTx patients. Since no donor HLA-specific γδ T cells have been detected, other ligands, such as heat shock proteins, may be involved.