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By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific Th cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other Treg-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-β induced Treg cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12α/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12α, A-498 cells were found to express CXCR4. More importantly, real-time RT–PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12α and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12α was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12α. Comparing gene expression profiles in rhCXCL12α stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention. British Journal of Cancer (2002) 86 , 1250–1256. DOI: 10.1038/sj/bjc/6600221 www.bjcancer.com © 2002 Cancer Research UK

IntroductionThe steroid hormone 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ) is known for its important role in regulating calcium homeostasis and bone mineralization. 1 1␣,25(OH) 2 D 3 acts through a nuclear receptor, the vitamin D receptor (Vdr), which is a member of the steroid and thyroid hormone receptor superfamily. More recently, evidence has accumulated that the hormone can have important functions in the immune system. Expression of Vdr was found in different immune effector cells of the myeloid and lymphoid lineage under resting and activating conditions. 2,3 These findings contributed to the hypothesis that locally produced 1␣,25(OH) 2 D 3 may perform regulatory functions on those cells. Indeed, over the past few years it has been demonstrated that 1␣,25(OH) 2 D 3 can act as an important immunosuppressive modulator. 1␣,25(OH) 2 D 3 has been shown to suppress T-cell proliferation 4 and to decrease the production of the T helper type 1 (Th1) cytokines interleukin 2, interferon ␥ (IFN-␥), and tumor necrosis factor ␣ (TNF-␣), leading to the inhibition of Th1 cell development. 5 Besides its direct effects on T cells, 1␣,25(OH) 2 D 3 and its analogs are potent inhibitors of dendritic cell (DC) differentiation and maturation and can impair the capacity of DCs to induce alloreactive T-cell activation. 6,7 In line with this, Vdr-deficient mice have been shown to have an increased frequency of mature DCs in lymph nodes. 8 Additional support for the immunomodulatory role of 1␣,25(OH) 2 D 3 in vivo came from studies of autoimmune diseases in several different animal models. It has been demonstrated that 1␣,25(OH) 2 D 3 can prevent or suppress experimental autoimmune encephalomyelitis, 9 rheumatoid arthritis, 10 systemic lupus erythematosus, 11 type 1 diabetes, 12 and inflammatory bowel disease, 13,14 further supporting its potent suppressive effects on the immune system.In contrast to its well-characterized effects on adaptive immune responses, much less is known about the effects of 1␣,25(OH) 2 D 3 on effectors of innate immunity, especially on macrophages. It has been shown that 1␣,25(OH) 2 D 3 can induce the differentiation of myeloid progenitors into macrophages. 15,16 However, the effects of 1␣,25(OH) 2 D 3 on mature and activated macrophages that are involved in inflammatory reactions have not been characterized yet. Such possible effects might be of especial importance since it was demonstrated that macrophages can release biologically active 1␣,25(OH) 2 D 3 on activation with IFN-␥. 17,18 The production of 1␣,25(OH) 2 D 3 by activated macrophages is regulated by the IFN-␥-mediated induction of 1␣-hydroxylase expression, the enzyme controlling the last step of 1␣,25(OH) 2 D 3 synthesis. 17,18 In Supported by the National German Genome Network (NGFN; 01GR0439), EU FP5 project EUMORPHIIA (QLG2- CT-2002-00930), and Volkswagenstiftung.L.H. performed research and wrote the paper; J.B., J.E., and S.S. performed research; T.F. contributed reagents and analytical tools; R.G. and M.P.K analyzed data; R.B. initiate...

The transmembrane protein CD83 has been initially described as a maturation marker for dendritic cells. Moreover, there is increasing evidence that CD83 also regulates B cell function, thymic T cell maturation, and peripheral T cell activation. Herein, we show that CD83 expression confers immunosuppressive function to CD4+ T cells. CD83 mRNA is differentially expressed in naturally occurring CD4+CD25+ regulatory T cells, and upon activation these cells rapidly express large amounts of surface CD83. Transduction of naive CD4+CD25− T cells with CD83 encoding retroviruses induces a regulatory phenotype in vitro, which is accompanied by the induction of Foxp3. Functional analysis of CD83-transduced T cells in vivo demonstrates that these CD83+Foxp3+ T cells are able to interfere with the effector phase of severe contact hypersensitivity reaction of the skin. Moreover, adoptive transfer of these cells prevents the paralysis associated with experimental autoimmune encephalomyelitis, suppresses proinflammatory cytokines IFN-γ and IL-17, and increases antiinflammatory IL-10 in recipient mice. Taken together, our data provide the first evidence that CD83 expression can contribute to the immunosuppressive function of CD4+ T cells in vivo.

By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human activated regulatory CD4 þ CD25 hi T cells (T reg ). Retroviral transduction of UBD in human allo-reactive effector CD4 þ T helper (T h ) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T h cell proliferation without upregulation of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in T h cells leads to the induction of IL1R2 that resemble FOXP3-transduced T h cells and naturally derived T reg cells. A comparison of the transcriptome of FOXP3-and UBD-transduced T h cells with T reg cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4 þ CD25 hi derived T reg cells and FOXP3-transduced T h cells, whereas little was induced in UBD-transduced T h cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4 þ CD25 hi derived regulatory T cells.