Functional characterisation of decoy receptor 3 in Crohn's disease.

AIMS: Both epithelial barrier dysfunction and apoptosis resistance of immune cells contribute to the pathogenesis of Crohn's disease. The soluble decoy receptor 3 (DcR3) acts in an anti-apoptotic manner by neutralising the death ligand CD95L. Here, we investigated the possible involvement of DcR3 in Crohn's disease. METHODS: The epithelial fraction of human small intestinal mucosa samples was obtained by laser microdissection. Expression of DcR3 was examined by global gene expression profiling, quantitative reverse transcription polymerase chain reaction, immunoblot analysis, and immunohistochemistry. DcR3 concentrations in the serum of patients with Crohn's disease were measured by enzyme-linked immunosorbent assay. Apoptosis assays were performed to study the effects of DcR3 in intestinal epithelial cells and lamina propria T cells. RESULTS: DcR3 is over-expressed in the epithelial layer of ileum specimens in patients with Crohn's disease, both at actively inflamed and non-active sites. DcR3 serum levels are significantly elevated in patients with active and non-active Crohn's disease as compared to healthy controls. The expression of DcR3 in intestinal epithelial cells is induced by tumour necrosis factor alpha. Increased DcR3 expression is associated with activation of nuclear factor kappa B (NF-kappaB) and results in protection of intestinal epithelial cells and lamina propria T cells from CD95L-induced apoptosis. CONCLUSIONS: DcR3 may promote inflammation in Crohn's disease by inhibiting CD95L-induced apoptosis of epithelial and immune cells as well as by inducing NF-kappaB activation.

Up-regulation of the phosphoinositide 3-kinase pathway in human lamina propria T lymphocytes.

Human intestinal lamina propria T lymphocytes (LPT), when investigated ex vivo, exhibit functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). One prominent feature represents their enhanced sensitivity to CD2 stimulation when compared to PBT. Given that LPT are hyporesponsive to T cell receptor (TCR)/CD3 stimulation, an alternative activation mode, as mimicked by CD2 triggering in vitro, may be functional in mucosal inflammation in vivo. This study provides insight into signalling events associated with the high CD2 responsiveness of LPT. When compared to PBT, LPT show an increased activation of the phosphoinositide 3/protein kinase B/glycogen synthase kinase 3beta (PI3-kinase/AKT/GSK-3beta) pathway in response to CD2 stimulation. Evidence is provided that up-regulation of this pathway contributes to the enhanced CD2-induced cytokine production in LPT. Given the importance of TCR-independent stimulation for the initiation of intestinal immune responses analysis of signalling pathways induced by 'co-stimulatory' receptors may provide valuable information for therapeutic drug design.

Interleukin-18 is increased only in a minority of patients with active Crohn's disease.

BACKGROUND AND AIMS: It has been suggested that Crohn's Disease (CD) is associated with an elevated T helper 1 response as manifested by increased production of interleukin-18 (IL-18). Local concentrations of neutralizing IL-18 binding proteins (IL-18 bp) may counteract biological functions of mature IL-18 in mucosal inflammation. Therefore, we investigated the IL-18/IL-18 bp system in a large group of patients with active inflammatory bowel disease (IBD) to identify patients that could respond theoretically to IL-18 neutralizing treatment strategies. PATIENT/METHODS: IL-18 and IL-18 bp messenger RNA (mRNA) expression in colonic mucosa from patients with active CD (n = 72), active ulcerative colitis (UC; n = 32), and non-IBD controls (infectious colitis or diverticulitis; n = 19) and normal, non-diseased controls (n = 20) were measured by reverse-transcribed real-time polymerase chain reaction. Mature IL-18 protein and IL-18 bp expression in inflamed mucosa were assessed by Western blotting. RESULTS/FINDINGS: Although IL-18 mRNA was increased in some patients with CD, the increase was not statistically significant. Densitometric evaluation of IL-18/alpha-actin ratio in patients with active CD (n = 20) and patients with UC (n = 10) demonstrated an increased ratio of IL-18 protein in CD when compared to UC (1.04 vs 0.72 [median]). On closer inspections, only 7/20 CD patients had an increased IL-18 protein expression in inflamed areas compared to noninflamed mucosa. INTERPRETATION/CONCLUSION: IL-18 expression in active CD is heterogeneous, only a minority of patients expresses elevated levels. Further treatment strategies targeting IL-18 expression in active CD should be concentrated on this subgroup of patients.

Increased cytokine transcripts in pouchitis reflect the degree of inflammation but not the underlying entity.

BACKGROUND AND AIMS: After ileopouch anal anastomosis (IPAA), 10-40% of patients with ulcerative colitis (UC) but only 5% of patients with familial adenomatous polyposis (FAP) develop pouchitis. Immunoregulatory abnormalities might be of importance in the pathogenesis of the disease. Therefore, we characterized cytokine and chemokine transcripts in inflamed and non-inflamed pouches in patients with UC compared to those with FAP and Crohn's disease (CD). PATIENTS AND METHODS: Mucosal biopsies were taken from 87 patients with IPAA [UC (n=70), CD (n=8) or FAP (n=9)]. Patients with active ileal CD (n=14), active UC (n=17) and non-inflammatory conditions (n=12) served as controls. The expression of 20 gene transcripts was quantified using real-time polymerase chain reaction. RESULTS AND FINDINGS: Pro-inflammatory cytokines and chemokines are significantly increased in IPAA patients with acute pouchitis. This increase is independent of the underlying disease (UC or CD) and reflects the degree of inflammation. A good correlation between pouchitis activity (using the Pouchitis Disease Activity Index) and the MRP-14, interleukin-8, macrophage inflammatory protein-2alpha and matrix metalloproteinase-1 transcripts was observed. INTERPRETATIONS AND CONCLUSIONS: Our data support the view that pouchitis reflects an inflammatory process that is different from that of underlying inflammatory bowel diseases, as the cytokine and chemokine patterns in pouchitis are neither typical of CD nor of UC, but maybe due to bacterial intestinal microflora overgrowth in the pouch lumen. Quantification of transcript levels allows an estimation of the extent of mucosal inflammation and may become helpful in the evaluation of the disease, especially in clinical trials.

Detection of cardiac allograft rejection by real-time PCR analysis of circulating mononuclear cells.

BACKGROUND: Detection of cardiac allograft rejection is based on the histological examination of endomyocardial biopsies (EMB). We have explored the possibility of whether graft rejection could be detected by characteristic gene expression patterns in peripheral blood mononuclear cells (PBMC) of heart-transplant recipients. METHODS: The study included 58 blood samples of 44 patients. On the day of EMB, mononuclear cells were isolated from peripheral blood, and gene expression was measured by quantitative real-time PCR. Thirty-nine parameters, including cytokine and chemokine genes were analyzed. Gene expression results were correlated with histological assessment of concomitant evaluated EMB according to International Society for Heart and Lung Transplantation (ISHLT) nomenclature. RESULTS: Gene expression of perforin, CD95 ligand, granzyme B, RANTES, CXCR3, COX2, ENA 78 and TGF-beta1 was significantly different in PBMC of patients with mild to moderate degrees of allograft rejection (> or =grade 2) compared with patients exhibiting no or minor forms of rejection ( or =grade 2 vs.

[CD80-modified tumor cell lines: implications for cell-based vaccinations in breast cancer patients]. / Eine CD80-modifizierte Tumorzelllinie als therapeutische Vakzine beim Mammakarzinom--Vakzinedesign und Studienkonzeption.

A number of genetic alterations are required for malignant transformation. However, these mutations provide the source for tumor-associated antigens which can be recognized by cellular effectors of the immune system. Recent advances in tumor immunology - such as the improved understanding of antigen presentation as well as T cell activation - have opened new perspectives for cancer immunotherapy. The advantage of using tumor cell based vaccines is that these comprise the complete antigen pool of an individual tumor for activating polyclonal immune responses. However, the induction of antigen-specific immune responses is impaired by the fact that T cell activation is depending on additional nonspecific costimulatory signals provided by the antigen-presenting cell. The majority of solid human tumors does not express costimulatory molecules and is unable to deliver all signals required for T cell activation. In contrast, tumors often induce immunologic tolerance. Therefore, the introduction of genes encoding costimulatory molecules such as CD80 or cytokines is aimed at conferring the immunostimulatory potential of tumor cells. We have undertaken efforts at endowing a breast carcinoma cell line expressing at least seven known tumor associated antigens with immunostimulatory competence by CD80 gene transfer. In preclinical studies this cell line was demonstrated to induce specific immune responses. We designed a phase I/II trial to administer the CD80-modified cell line to patients with metastatic breast cancer to determine the toxicities of the vaccination protocol and nature of the vaccine-induced immune response.

Activation of beta(1) integrins mediates proliferation and inhibits apoptosis of intestinal CD4-positive lymphocytes.

A characteristic of lamina propria lymphocytes (LPL) is their low proliferative response to stimuli of the CD3 pathway. beta(1) integrins were expressed on LPL; however, their function is unknown. Therefore, we determined whether beta(1) integrins contribute to T cell responses by providing costimulatory signals. Integrins on CD4(+) LPL of controls and patients with inflammatory bowel disease were characterized by flow cytometry. Cells were stimulated by anti-CD3 or anti-CD2 antibodies either alone or in combination with a stimulatory beta(1) integrin antibody (12G10). Proliferation and apoptosis were measured by [(3)H]thymidine pulsing or flow cytometry. Cytokine mRNA and apoptosis-related transcripts were quantified by reverse transcriptase-PCR. We demonstrated that beta(1) integrin costimulation restored CD3-induced proliferation of CD4(+) LPL and reduced activation-induced apoptosis. Activation of beta(1) integrins by addition of 12G10 antibody to CD3-stimulated cells restored their capacity to express proinflammatory cytokine transcripts. Further, expression of the activated form of beta(1) integrins was significantly elevated on LPL from inflamed mucosa. These studies demonstrate that beta(1) integrin costimulation modulates the response of LPL after TCR stimulation. An increased expression of activated beta(1) integrins on LPL in intestinal inflammation may abolish their unresponsiveness to antigens and perpetuate the inflammatory process.

Simultaneous ligation of CD5 and CD28 with monoclonal antibodies restores impaired immunostimulatory function in human renal cell carcinoma.

Tumor cells, including renal cell carcinoma (RCC) cells, do not effectively stimulate T lymphocyte responses against specific antigens presented on their surface. Reasons for this low immunogenicity may include low or absent expression of MHC class I and/or class II molecules, as well as accessory and costimulatory molecules. We used tumor cell pretreatment with cytokines, together with monoclonal antibodies (mAbs) directed at receptors for costimulatory molecules, to render RCC cells immunostimulatory. Interferon-gamma or tumor necrosis factor-alpha pretreatment enhanced expression of MHC class I and class II molecules, as well as CD54, but had only minimal effects on T cell activation. A CD28 mAb, or an even more effective combination of CD28 and CD5 mAb, induced strong primary proliferative responses of allogeneic resting T lymphocytes. Cytokine pretreatment further augmented this T cell response in vitro and allowed T cell expansion and establishment of T cell lines. Stimulation of T cells with autologous RCC cells resulted in a similar T cell activation but with the expansion of cytolytic T cells directed at autologous MHC class II molecules. These experiments demonstrate that cytokines combined with costimulatory mAbs are useful for increasing the immunogenicity of tumor cells. They also indicate. however, that autologous MHC class II expression on tumor cells, together with strong costimulation, may lead to the activation of autoreactive T cells.

The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes.

Cofilin, an actin-depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co-stimulation through accessory receptors (e.g. CD2 or CD28) - however, not following TCR/CD3 stimulation alone - cofilin undergoes dephosphorylation. The subcellular localization as well as the actin-binding activity of cofilin are regulated by the phosphorylation state of serine-3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM-kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM-kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19-kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506-resistant co-stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co-stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).

Thiol-mediated redox regulation of intestinal lamina propria T lymphocytes.

Intestinal lamina propria T lymphocytes (LP-Ts) have a markedly low proliferative potential both in vivo and in vitro. Here, we have identified that the capacity of antigen-presenting cells to release cysteine upon receptor-ligand interactions represents a critical parameter for proliferation of LP-Ts. The availability of cysteine is limiting for the intracellular production of glutathione, which in turn is essential for cell cycle progression. When cysteine is provided either directly or by addition of the reducing agent 2-mercaptoethanol to cystine-containing culture medium, proliferation of LP-T is fully restored. Importantly, coculture with peripheral blood monocytes that easily take up cystine, reduce cystine, and secrete cysteine also restores reactivity of LP-Ts to T cell receptor/CD3 stimulation. In marked contrast, lamina propria macrophages lack this capacity to elaborate cysteine, and thereby secure physiological unresponsiveness to antigen exposure in the intestinal microenvironment. The well-documented local recruitment of blood monocytes in inflammatory bowel disease (IBD) may thus represent an important parameter underlying hyperresponsiveness of T cells, an essential component of the pathogenesis of IBD.

Cofilin: a missing link between T cell co-stimulation and rearrangement of the actin cytoskeleton.

The actin cytoskeletal network plays a regulatory role in receptor-mediated signal-transducing events. Recently, we have shown that the small actin-depolymerizing protein cofilin represents a component of a co-stimulatory signaling pathway in human T cells. Cofilin is dephosphorylated on phosphoserine residues following co-stimulation via accessory receptors such as CD2, CD4, CD8 or CD28, but not in response to TCR engagement alone. Here we demonstrate that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton. Only the dephosphorylated form of cofilin binds to cytoskeletal actin in vivo. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 block dephosphorylation of cofilin and its association with the actin cytoskeleton. These results suggest that cofilin provides an as yet missing link between functionally crucial T cell surface receptors and rearrangements of the actin cytoskeleton.

Protection of trinitrobenzene sulfonic acid-induced colitis by an interleukin 2-IgG2b fusion protein in mice.

BACKGROUND & AIMS: We have shown in previous studies that an interleukin 2 (IL-2)-IgG2b fusion protein suppresses both humoral and cellular immune reactions in a murine model of DTH reaction. We now analyze the effects of IL-2-IgG2b in a model of intestinal inflammation in mice induced by the hapten reagent 2,4, 6-trinitrobenzene sulfonic acid (TNBS) that mimics immunologic characteristics of human Crohn's disease. METHODS: In TNBS-induced colitis, colonic and splenic T-cell subsets were characterized by immunohistochemistry and flow cytometry. Cytokine synthesis was studied by semiquantitative reverse-transcription polymerase chain reaction and intracellular cytokine staining in CD4(+) T cells. RESULTS: When mice were treated with IL-2-IgG2b, improvement in both wasting disease and histopathologic signs of colonic inflammation was observed. An increase in the number of colonic CD4(+)/CD25(+) T cells and increased synthesis of the immunosuppressive cytokine IL-10 also occurred. The protective role of IL-10 was demonstrated by the finding that neutralization of IL-10 in vivo using IL-10-specific antibodies inhibited the IL-2-IgG2b effects in TNBS-induced colitis. CONCLUSIONS: These studies show for the first time that the IL-2-IgG2b fusion protein can abrogate experimental colitis by local induction of IL-10-secreting T cells.

Tumor therapy with bispecific antibody: the targeting and triggering steps can be separated employing a CD2-based strategy.

For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 x tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab')2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.

Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma.

One possible reason for the poor immunogenicity of tumors is the induction of peripheral tolerance by tumor cells that fail to deliver costimulatory signals. Furthermore, T cells stimulated with wild-type tumor cells often fail to secrete cytokines. The present study has been undertaken to identify cytokines that cooperate with CD80 in T-cell activation in vitro toward human breast and ovarian carcinoma cell lines. Tumor cell-mediated T-lymphocyte activation was analyzed directly in allogeneic mixed lymphocyte/tumor cell cultures as proliferation and effector functions were assessed in cytotoxic T-cell assays. Interleukin-7 (IL-7) amplified the proliferative response toward CD80-transfected breast and ovarian carcinomas and stimulated predominantly CD4+ T lymphocytes. IL-12 represses the proliferative response of naive T cells but cooperates with CD80-mediated activation during secondary stimulations. In long-term T-cell cultures, IL-12 synergizes with CD80 expression to stimulate cytolytic CD8+ T-cell lines, which recognize a breast carcinoma line in a human histocompatibility leukocyte antigen-restricted manner. These studies illustrate that costimulation is necessary for tumor cells to function as alloantigen-presenting cells. Furthermore, when added after the priming of T cells with CD80-transfected tumor cells, IL-12 could be helpful in propagating sufficient T-cell numbers to be used in adoptive transfers during cellular immunotherapy.

Potential of CD80-transfected human breast carcinoma cells to induce peptide-specific T lymphocytes in an allogeneic human histocompatibility leukocyte antigens (HLA)-A2.1+-matched situation.

Allogeneic human histocompatibility leukocyte antigen (HLA)-matched tumor cell lines that have been made immunogenic by the transfer of genes encoding for costimulatory molecules such as CD80 are considered to be potential vaccines for the induction of systemic immune reactions in cancer patients. We used a human HLA-A2.1+ CD80-transfected breast carcinoma cell line (KS-CD80) and investigated in vitro the efficiency at which antigen (Ag)-specific responses were induced following the stimulation of allogeneic HLA-A2.1-matched T lymphocytes. The influenza matrix protein M1 was used as a model Ag. It was either endogenously expressed or exogenously loaded as a peptide (matrix protein), and the frequency of the generated specific T cells was determined. The expression of CD80 in KS cells was required for an effective activation and expansion of Ag-specific T cells. This response was augmented following the pretreatment of KS-CD80 cells with interferon-gamma and tumor necrosis factor-alpha. Interleukin-4 (IL-4), IL-7, and IL-12 further increased T-cell expansion. IL-7 was best at supporting the generation of T cells with Ag specificity. This investigation demonstrates that allogeneic CD80+ tumor cells can induce Ag-specific, HLA-restricted T lymphocytes at a high frequency. Our study supports the use of allogeneic cell lines for the induction of specific T-cell responses in tumor patients.

Prolonged allograft survival but no tolerance induction by modulating CD28 antibody JJ319 after high-responder rat heart transplantation.

BACKGROUND: Allograft rejection depends on T cell immune responses requiring antigen recognition and costimulatory signals through accessory T cell receptors, including CD28. Inhibition of CD28 signaling with a CTLA-4-immunoglobulin (Ig) fusion protein has resulted in immunosuppression and occasional T cell anergy in mouse transplant models, but not in rats. Because this approach also inhibits a potentially tolerizing signal through CTLA-4, selective blockade of CD28 ligation might induce more profound immunosuppression and transplant tolerance. METHODS: The effects of escalating doses of the rat CD28 monoclonal antibody JJ319 on allograft survival were studied after vascularized heterotopic heart transplantation in a high responder strain combination (DA to Lewis). CD28 antigen modulation and circulating antibody levels were monitored by flow cytometry. RESULTS: CD28 antibody JJ319 markedly prolonged cardiac graft survival compared with untreated controls (7 days, range: 6-8). A strictly dose-dependent increase in median graft survival time was demonstrated with a maximum of 36 days (range: 30-40; p <0.001) after the administration of 8 x 1 mg JJ319 i.p. (days -1 to +6 before/after transplantation). However, indefinite graft survival and tolerance could not be induced by JJ319 treatment. At the maximal dose, flow cytometry showed complete down modulation of the CD28 receptor for 10-14 days without T cell depletion in close temporal relation to antibody presence in serum. In vitro, CD28-modulated T cells showed significantly reduced responses to activation. CONCLUSIONS: CD28 antibody JJ319 induces profound immunosuppression after rat heart transplantation, however without development of transplant tolerance. The underlying mechanism seems to be receptor modulation during primary alloantigen recognition. While still potentially applicable clinically, there are no qualitative or quantitative differences to the treatment with CTLA-4/lg or the blockade of CD2 or LFA-1, as reported elsewhere. Thus, a CD28-modulating approach seems not to allow therapeutic exploitation of a tolerizing signal delivered by CTLA-4 but may still be clinically applicable, especially in combined immune interventions.

Cyclosporin A-resistant transactivation of the IL-2 promoter requires activity of okadaic acid-sensitive serine/threonine phosphatases.

Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-kappa B, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced.

In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease.

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohn's disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1beta mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1beta in the lamina propria compartment.

Gene transfer of costimulatory molecules into a human colorectal cancer cell line: requirement of CD54, CD80 and class II MHC expression for enhanced immunogenicity.

Colorectal cancer is considered a non-immunogenic malignany. One strategy to augment the immunogenicity of such tumours is represented by the expression of costimulatory molecules by gene transfer. Using transfected variants of the human colorectal cancer cell line SW480 we tested various costimulatory molecules (CD80, CD86, CD54) and a class II major histocompatibility complex (MHC) allele (HLA-DR3) alone or in combination on their ability to support primary T-lymphocyte activation in vitro. Expression of CD80 or CD86 similarly as the combination of both was not sufficient to induce proliferation of human allogeneic T cells. Expression of CD54 together with CD80 strongly augmented the costimulatory function of CD80, as observed in the presence of a CD3 monoclonal antibody (mAb), but did not lead directly to a T-cell response against modified tumour cells. Importantly, SW480 cells coexpressing CD54, CD80 and the HLA-DR3 allele effectively promoted T-lymphocyte proliferation. Moreover, the use of such CD54+/CD80+/HLA-DR3+ SW480 variants for repetitive stimulations resulted in the generation of T-cell lines predominantly composed of CD8+ T cells exhibiting class I MHC restricted cytolytic activity towards untransfected SW480 tumour cells. This demonstrates that the generation of immunogenic tumour cell variants, i.e. for the use as cellular vaccines, requires multiple genetic alterations in the case of non-immunogenic human tumours cells, such as colorectal cancer cells.

Differential immunosuppressive activity of monoclonal CD2 antibodies on allograft rejection versus specific antibody production.

CD2 is a co-stimulatory receptor involved in T cell activation. Here we report on immunosuppressive effects of three mouse CD2 monoclonal antibodies (OX34, OX54, OX55) directed against non-overlapping epitopes of the rat CD2 receptor on various modes of T cell activation in vitro and in vivo. Although non-ligand-blocking OX54 and OX55, in concert, activated T cells through CD2 in vitro, they individually suppressed the mixed lymphocyte reaction (MLR) and significantly prolonged allograft survival after rat heart transplantation in vivo. Phenotype analysis revealed that OX55 significantly down-modulated CD2 in vivo, whereas OX54 depleted T cells. Graft rejection coincided with re-expression of CD2 and clearance of OX55 from serum, whereas T cell depletion by OX54 outlasted the period of graft survival. The most suppressive antibody, OX34, down-modulated CD2 and inhibited T cell activation through the TCR or CD2 and the MLR and prolonged median allograft survival time from 7 days in controls to 45 days in the absence of any additional treatment. Graft survival was clearly dose dependent and correlated with the duration of CD2 down-modulation and the presence of circulating CD2 antibody in serum. Importantly, the specific antibody production to a T cell-dependent antigen as demonstrated by immunization with keyhole limpet hemocyanin in vivo remained unaffected after treatment with OX34. These results demonstrate the pivotal role of CD2 signaling in mediating allogeneic immune reactions after vascularized organ transplantation while allowing specific humoral immune responses in vivo.