Copy number alterations and copy-neutral loss of heterozygosity in Ukrainian patients with primary myelofibrosis.

AIM: To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis (PMF). MATERIALS AND METHODS: We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScan HD microarray platform. Statistical significance was evaluated by the Fisher exact test. RESULTS: We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p = 0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. CONCLUSION: We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).

Imaging, procedural and clinical variables associated with tumor yield on bone biopsy in metastatic castration-resistant prostate cancer.

BACKGROUND: Understanding the mechanisms driving disease progression is fundamental to identifying new therapeutic targets for the treatment of men with metastatic castration-resistant prostate cancer (mCRPC). Owing to the prevalence of bone metastases in mCRPC, obtaining sufficient tumor tissue for analysis has historically been a challenge. In this exploratory analysis, we evaluated imaging, procedural and clinical variables associated with tumor yield on image-guided bone biopsy in men with mCRPC. METHODS: Clinical data were collected prospectively from men with mCRPC enrolled on a phase II trial with serial metastasis biopsies performed according to standard clinical protocol. Imaging was retrospectively reviewed. We evaluated the percent positive biopsy cores (PPC), calculated as the number of positive cores divided by the total number of cores collected per biopsy. RESULTS: Twenty-nine men had 39 bone biopsies. Seventy-seven percent of bone biopsies had at least one positive biopsy core. We determined that lesion size and distance from the skin to the lesion edge correlated with tumor yield on biopsy (median PPC 75% versus 42% for lesions >8.8 cm(3) versus ⩽ 8.8 cm(3), respectively, P=0.05; median PPC 33% versus 71% for distance ⩾ 6.1 versus <6.1 cm, respectively, P = 0.02). There was a trend towards increased tumor yield in patients with increased uptake on radionuclide bone scan, higher calcium levels and shorter duration of osteoclast-targeting therapy, although this was not statistically significant. Ten men had 14 soft tissue biopsies. All soft tissue biopsies had at least one positive biopsy core. CONCLUSIONS: This exploratory analysis suggests that there are imaging, procedural and clinical variables that have an impact on image-guided bone biopsy yield. In order to maximize harvest of prostate cancer tissue, we have incorporated a prospective analysis of the metrics described here as part of a multi-institutional project aiming to use the molecular characterization of mCRPC tumors to direct individual therapy.

Androgen receptor functions in castration-resistant prostate cancer and mechanisms of resistance to new agents targeting the androgen axis.

The metabolic functions of androgen receptor (AR) in normal prostate are circumvented in prostate cancer (PCa) to drive tumor growth, and the AR also can acquire new growth-promoting functions during PCa development and progression through genetic and epigenetic mechanisms. Androgen deprivation therapy (ADT, surgical or medical castration) is the standard treatment for metastatic PCa, but patients invariably relapse despite castrate androgen levels (castration-resistant PCa, CRPC). Early studies from many groups had shown that AR was highly expressed and transcriptionally active in CRPC, and indicated that steroids from the adrenal glands were contributing to this AR activity. More recent studies showed that CRPC cells had increased expression of enzymes mediating androgen synthesis from adrenal steroids, and could synthesize androgens de novo from cholesterol. Phase III clinical trials showing a survival advantage in CRPC for treatment with abiraterone (inhibitor of the enzyme CYP17A1 required for androgen synthesis that markedly reduces androgens and precursor steroids) and for enzalutamide (new AR antagonist) have now confirmed that AR activity driven by residual androgens makes a major contribution to CRPC, and led to the recent Food and Drug Administration approval of both agents. Unfortunately, patients treated with these agents for advanced CRPC generally relapse within a year and AR appears to be active in the relapsed tumors, but the molecular mechanisms mediating intrinsic or acquired resistance to these AR-targeted therapies remain to be defined. This review outlines AR functions that contribute to PCa development and progression, the roles of intratumoral androgen synthesis and AR structural alterations in driving AR activity in CRPC, mechanisms of action for abiraterone and enzalutamide, and possible mechanisms of resistance to these agents.

Adiponectin signals in prostate cancer cells through Akt to activate the mammalian target of rapamycin pathway.

Adiponectin has received much attention due to its beneficial effects on insulin sensitivity, and epidemiologic studies have further shown an inverse association between adiponectin levels and risk for multiple tumors, which is independent of the IGF system or other risk factors. Previous studies have shown that adiponectin can activate AMP-activated protein kinase (AMPK) in myocytes, hepatocytes, and adipocytes, suggesting that adiponectin may suppress tumor development through AMPK activation and subsequent inhibition of mammalian target of rapamycin (mTOR). However, the mechanisms through which adiponectin affects cancer cells are not understood, and it remains to be determined whether adiponectin is linked to the same downstream targets in all cells types, and in particular in cancer cells. In the present study, we demonstrate that while adiponectin stimulates AMPK in phosphatase and tensin homolog deleted on chromosome ten (PTEN) deficient LNCaP prostate cancer cells, it also increases mTOR activity as assessed by phosphorylation of two downstream targets, p70 S6 kinase and ribosomal protein S6. This adiponectin stimulation of mTOR was mediated through phosphatidylinositol 3-kinase (PI3 kinase) and Akt activation. These results show that adiponectin can activate both AMPK and PI3 kinase/Akt pathways, and that cell type-specific factors such as PTEN status may determine which of these pathways will have the dominant effect on mTOR. Therefore, while it is possible that high endogenous adiponectin levels could be protective against cancer by direct mechanisms or indirect systemic mechanisms, our results indicate that adiponectin may also directly stimulate signaling pathways that enhance the growth of some tumors.

Androgen regulation of soluble guanylyl cyclasealpha1 mediates prostate cancer cell proliferation.

The growth and progression of prostate cancer are dependent on androgens and androgen receptor (AR), which act by modulating gene expression. Utilizing a gene microarray approach, we have identified the alpha1-subunit gene of soluble guanylyl cyclase (sGC) as a novel androgen-regulated gene. A heterodimeric cytoplasmic protein composed of one alpha and one beta subunit, sGC mediates the widespread cellular effects of nitric oxide (NO). We report here that, in prostate cancer cells, androgens stimulate the expression of sGCalpha1. A cloned human sGCalpha1 promoter is activated by androgen in an AR-dependent manner, suggesting that sGCalpha1 may be a direct AR target gene. Disruption of sGCalpha1 expression severely compromises the growth of both androgen-dependent and androgen-independent AR-positive prostate cancer cells. Overexpression of sGCalpha1 alone is sufficient for stimulating prostate cancer cell proliferation. Interestingly, the major growth effect of sGCalpha1 is independent of NO and cyclic guanosine monophosphate, a major mediator of the sGC enzyme. These data strongly suggest that sGCalpha1 acts in prostate cancer via a novel pathway that does not depend on sGCbeta1. Tissue studies show that sGCalpha1 expression is significantly elevated in advanced prostate cancer. Thus, sGCalpha1 may be an important mediator of the procarcinogenic effects of androgens.

Targeting steroid hormone receptor pathways in the treatment of hormone dependent cancers.

Sex steroid hormones play a central role in the development and progression of prostate and breast cancers. The biological functions of these and other steroid hormones are mediated by a family of closely related steroid hormone receptors (SHRs), with the androgen receptor (AR) mediating the effects of testosterone and related androgens, and the classical estrogen receptor (ERalpha) mediating the effects of estradiol. Recent studies have begun to elucidate the complex pathways through which SHRs regulate gene expression, and their interaction with other cellular pathways. These studies have also begun to reveal molecular mechanisms underlying the diverse spectrum of effects mediated by steroid hormone analogues in different tissues. A major advance has been the finding that certain drugs induce unique conformational changes in SHRs that alter their interactions with transcriptional coactivator and corepressor proteins, resulting in cell type specific responses. These unique conformational changes appear responsible for the tissue specific effects of the selective estrogen receptor modulators (SERMs) in breast cancer. SHRs are clearly well established therapeutic targets in cancer, and drug development has continued to focus on agents that either block steroid hormone production or bind to and modulate their receptors. The identification of multiple proteins and pathways that mediate the downstream functions of SHRs may eventually provide additional therapeutic targets. This review outlines the basic biology of SHR structure and function, with a focus on AR and ERalpha. Hormonal therapies in prostate and breast cancer that directly target AR and ERalpha, respectively, are then presented and possible novel drug targets in the SHR pathway are discussed.

A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, approximately 50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-gamma and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d(+) lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.

SRY interacts with and negatively regulates androgen receptor transcriptional activity.

This study investigated interactions between SRY, the Y chromosome encoded male sex determining factor, and the androgen receptor (AR). Coexpression of AR and SRY caused marked repression of AR transcriptional activity on a series of androgen-responsive reporter genes. Mammalian one- and two-hybrid experiments demonstrated an AR-SRY interaction mediated by the AR DNA binding domain. Precipitations with glutathione S-transferase fusion proteins indicated that AR-SRY interactions were direct and mediated by the AR DNA binding domain and the SRY high mobility group box DNA binding domain. Transient expression of SRY in LNCaP prostate cancer cells repressed expression of an androgen-dependent prostate-specific antigen (PSA) reporter gene and stable SRY expression repressed the endogenous PSA gene. SRY protein expression was increased by proteosome inhibitors and by the androgen-liganded AR in transient and stable transfectants. AR transcriptional activity was also repressed by DAX1, and the effects of SRY and DAX1 on the AR were additive. These findings indicate that interactions between the AR, SRY, and DAX1 contribute to normal male development and function and suggest a general role for protein-protein interactions between high mobility group box proteins and steroid hormone receptors in regulating tissue-specific gene expression.

Prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium.

Prostate cancer (PCa) is an androgen dependent disease that can be treated by androgen ablation therapy, and clinical trials are under way to prevent PCa through the reduction of androgen receptor (AR) activity. However, there are no animal models of AR-mediated prostatic neoplasia, and it remains unclear whether the AR is a positive or negative regulator of cell growth in normal prostate secretory epithelium. To assess the direct effects of the AR in prostate epithelium, a murine AR transgene regulated by the rat probasin promoter (Pb) was used to generate transgenic mice expressing increased levels of AR protein in prostate secretory epithelium. The prostates in younger (<1 year) Pb-mAR transgenic mice were histologically normal, but Ki-67 immunostaining revealed marked increases in epithelial proliferation in ventral prostate and dorsolateral prostate. Older (>1 year) transgenic mice developed focal areas of intraepithelial neoplasia strongly resembling human high-grade prostatic intraepithelial neoplasia (PIN), a precursor to PCa. These results demonstrate that the AR is a positive regulator of cell growth in normal prostate epithelium and provide a model system of AR-stimulated PIN that can be used for assessing preventative hormonal therapies and for identifying secondary transforming events relevant to human PCa.

Loss of IFN-gamma production by invariant NK T cells in advanced cancer.

Invariant NK T cells express certain NK cell receptors and an invariant TCRalpha chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-gamma, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (alpha-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-gamma and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-gamma production and a striking decrease in their IFN-gamma:IL-4 ratio. The IFN-gamma deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-gamma and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Prevalence of an ulcerative colitis-associated CD8+ T cell receptor beta-chain CDR3-region motif and its association with disease activity.

The normal human intestinal mucosa contains clonal T cell expansions. Clonal populations of T cells can be determined through evaluation of the idiotypic, hypervariable region of their T cell receptor (TCR). We have previously reported that there exists a highly conserved TCR pattern among intestinal CD8+ T cells in the majority of ulcerative colitis (UC) patients undergoing colectomy that was not present in normal control individuals. This TCR pattern, or motif, was characterized by particular beta-chain usage (TCRBV3 and TCRBJ1S6) and a defined length in the hypervariable third complementarity determining region (CDR3). The aim of this study was to assess the motif's relationship to disease activity. Subjects were 66 with UC, 19 with Crohn's disease, 14 inflammatory controls, and 6 normal controls. cDNA and gDNA were prepared from colonic biopsies and paraffin blocks, respectively, obtained from study subjects and used to assess TCRBV CDR3 region length and usage, as well as for cloning and sequencing of TCRs. The TCRBV CDR3 region was present in 25 of a series of 48 UC subjects but only 3 of 19 Crohn's disease patients and 3 of 14 inflammatory controls. The motif was more common in UC than either Crohn' s disease or inflammatory controls (chi2 = 7.5, P = 0.006, and chi2 = 4.1, P = 0.04, respectively). The motifs presence was not dependent upon histologic disease activity (either active or inactive UC). Clinical UC disease activity was also not significantly associated with an increased presence of the motif in 14 paired biopsies, which were taken during times of clinical activity or inactivity. There was a trend toward persistence of the motif, as it was present in 6 of 14 subjects over a 3- to 6-month time period. The previously described UC-associated TCRBV CDR3 region motif located in the intestinal CD8+ T-cell subset is found in a significant proportion of UC subjects. The TCR motif does not significantly discriminate active from inactive disease states. The persistent and diffuse nature of this TCR-associated motif in UC suggests that an ongoing T-cell response to a particular antigen(s) is occuring in this disorder.

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

A multi-institutional phase ii study of SU101, a platelet-derived growth factor receptor inhibitor, for patients with hormone-refractory prostate cancer.

In a multi-institutional Phase II trial, we evaluated the efficacy of a platelet-derived growth factor receptor (PDGF-r) inhibitor, SU101, in patients with hormonerefractory prostate cancer. The patients received a 4-day i.v. loading dose of SU101 at 400 mg/m(2) for 4 consecutive days, followed by 10 weekly infusions at 400 mg/m(2). The primary study end points were a decline in prostate-specific antigen (PSA) and a decrease in measurable tumor. Secondary end points were time to progression and an effect on pain as measured by the Brief Pain Survey. Expression of PDGF-r was examined in both metastatic and archival primary prostate tumor samples. Forty-four patients were enrolled at four centers. The median age was 72 years, the median PSA was 223 ng/ml, and 21 patients had at least one prior chemotherapy. Thirty-nine patients are evaluable for PSA, and three patients demonstrated a PSA decline >50% from baseline (55-99.9% decrease). The median time to progression was 90 days. Of 19 patients evaluable for measurable disease, 1 patient had a partial response. Nine of 35 evaluable patients had significant improvement in pain. The most frequent adverse events were asthenia (75%), nausea (55%), anorexia (50%), and anemia (41%). PDGF-r expression was detected in 80% of the metastases and 88% of primary prostate cancers. The results of this trial may warrant further clinical studies with other PDGF-r inhibitors.

Human CD1d functions as a transplantation antigen and a restriction element in mice.

To study the potential functions of human CD1d (hCD1d), we developed transgenic (Tg) mice that ectopically express hCD1d under the control of H-2K(b) promoter. High levels of hCD1d expression were detected in all Tg tissues tested. Skin grafts from the K(b)/hCD1d Tg mice were rapidly rejected by MHC-matched non-Tg recipient mice, suggesting that hCD1d can act as transplantation Ags. Furthermore, we were able to elicit hCD1d-restricted CD8(+) CTLs from mice immunized with K(b)/hCD1d Tg splenocytes. These CTLs express TCR rearrangements that are distinct from invariant TCR of NK T cells, and secrete significant amounts of IFN-gamma upon Ag stimulation. Analysis with various hCD1d-expressing targets and use of Ag presentation inhibitors indicated the recognition of hCD1d by CTLs did not involve species or tissue-specific ligands nor require the processing pathways of endosomes or proteasomes. Additionally, the reactivity of hCD1d-specific CTLs was not affected by acid stripping followed by brefeldin A treatment, suggesting that CTLs may recognize a ligand/hCD1d complex that is resistant to acid denaturation, or empty hCD1d molecules. Our results show that hCD1d can function as an alloantigen for CD8(+) CTLs. The hCD1d Tg mice provide a versatile model for the study of hCD1d-restricted cytolytic responses to microbial Ags.

Involvement of CD1 in peripheral deletion of T lymphocytes is independent of NK T cells.

During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8(+) T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either beta(2)-microglobulin or the TCR element J alpha 281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.

Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice.

BACKGROUND & AIMS: CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of natural killer (NK)1.1(+) T cells. These NK T cells exhibit important immunoregulatory functions in several autoimmune disease models. METHODS: To investigate whether CD1d and NK T cells have a similar role in intestinal inflammation, the effects of the glycolipid, alpha-galactosylceramide (alpha-GalCer), on dextran sodium sulfate (DSS)-induced colitis were examined. Wild-type (WT), CD1d(-/-), and RAG(-/-) mice were examined for their response to either alpha-GalCer or the control analogue, alpha-mannosylceramide (alpha-ManCer). RESULTS: WT mice, but not CD1d(-/-) and RAG(-/-) mice, receiving alpha-GalCer had a significant improvement in DSS-induced colitis based on body weight, bleeding, diarrhea, and survival when compared with those receiving alpha-ManCer. Elimination of NK T cells through antibody-mediated depletion resulted in a reduction of the effect of alpha-GalCer. Furthermore, adoptive transfer of NK T cells preactivated by alpha-GalCer, but not alpha-ManCer, resulted in diminished colitis. Using a fluorescent-labeled analogue of alpha-GalCer, confocal microscopy localized alpha-GalCer to the colonic surface epithelium of WT but not CD1d(-/-) mice, indicating alpha-GalCer binds CD1d in the intestinal epithelium and may be functionally active at this site. CONCLUSIONS: These results show an important functional role for NK T cells, activated by alpha-GalCer in a CD1d-restricted manner, in regulating intestinal inflammation.

CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes.

Human T cells expressing CD161 and an invariant T-cell receptor (TCR) alpha-chain (Valpha24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T-cell population, and whether alterations in CD1d expression contribute to the regulation of Valpha24invt T-cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Valpha24invt T-cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d-, but low-level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d-. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation. These results demonstrate constitutive CD1d expression by human professional antigen-presenting cells and that post-translational processing of CD1d may contribute to regulation of the activity of CD1d-specific T cells.

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.

CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site.

Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber-associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1(+) antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.