Cetuximab (IMC-C225, Erbitux ImClone Systems Inc, New York, NY) is a recombinant, human/mouse chimeric monoclonal antibody (MAb) that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR) on both normal and tumor cells, and competitively inhibits the binding of epidermal growth factor (EGF) as well as other ligands. Cetuximab binding to the EGFR blocks phosphorylation and activation of receptor-associated kinases and their associated downstream signalling (MAPK, PI3K/Akt, Jak/Stat pathways) resulting in inhibition of many cellular processes such as induction of apoptosis, cell growth, decreased Matrix Metallo-Proteinase (MMPs) and vascular endothelial growth factor (VEGF) production. Cetuximab is also able to display cytotoxic effect through antibody-dependent cellular cytotoxicity (ADCC). In vitro and in vivo experiments elucidated a wide range of biological properties attributed to cetuximab, these include: direct inhibition of EGFR tyrosine kinase activity, inhibition of cell cycle progression, inhibition of angiogenesis, invasion and metastatization processes, activation of pro-apoptotic molecules and synergic cytotoxicity effect with chemotherapy and radiotherapy. Several studies have shown cetuximab is able to inhibit growth of EGFR-expressing tumor cells in vitro as well as in nude mice bearing xenografts of human cancer cell lines. Moreover, numerous clinical trials demonstrated cetuximab efficacy in different tumor types and it is approved by Food and Drugs Administration (FDA) for use in the treatment of metastatic colorectal cancer (mCRC) as single agent or in combination with chemotherapy, for locally/regionally advanced head and neck squamous cell carcinoma (HNSCC) in combination with radiotherapy, and as monotherapy for recurrent/metastatic HNSCC after failing platinum-based chemotherapy. This review will illustrate pre-clinical and clinical data on biological properties of cetuximab focusing on the predictive markers of clinical response to this drug.

1053 Background: Recently, it has been demonstrated that RANK is overexpressed in about 65% of primary breast tumors. Here we explored the clinical significance of RANK expression in early breast cancer. For this purpose we included in the study a series of early breast cancer primary tissues and correlated IHC RANK expression with skeletal clinical outcome. Methods: This study included 93 patients with breast cancer with complete clinicopathological information and up to 2 years of follow-up. Samples contained 15 lobular carcinomas and 77 ductal carcinomas; N0-1 versus N2-3: 63/29; HER2/neu ±/unknown: 25/55/13; sites of metastases: 16 patients only skeletal, 16 only visceral, 29 skeletal plus visceral, 32 without metastases. No patient was treated with neoadjuvant therapy. RANK protein expression was determined using immunohistochemistry. We considered RANK-positive patients when more than 50% of tumoral tissues was scored as grade 2 (intensity higher than internal control -macrophages-) or 3 (intensity much higher than internal control). Results: RANK was expressed in 38 (41%) of the primary tumor samples. RANK expression was independent from histotype (p:0.23), HER2/neu expression (p:0.47), and grading (p:0.39) but was dependent on nodes status (p:0.05). RANK-positive patients showed a higher risk to develop skeletal metastases (p:0.023). Moreover, RANK expression was associated with accelerated bone metastasis formation (p > 0.034). Multivariate analysis confirmed that RANK is an independent prognostic indicator for early bone metastasis development (p > 0.037). Conclusions: RANK is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis and with increased probability to respond to anti-RANKL monoclonal therapy (denosumab). This is the first time that RANK has been linked to the bone metastasis process in breast cancer. No significant financial relationships to disclose.

BACKGROUND KRAS wild-type mutational status is necessary but not sufficient to get benefit from epidermal growth factor receptor inhibition. Predictive markers are currently being evaluated. In this study, we investigated early hypomagnesemia as a predictor of efficacy and outcome in terms of time to progression (TtP) and overall survival (OS) in a cohort of patients affected by advanced colorectal adenocarcinoma KRAS wild-type cetuximab-treated. PATIENTS AND METHODS One hundred and forty-three patients affected by stage IV colorectal adenocarcinoma KRAS wild type receiving cetuximab + irinotecan (CTX+IRI) as third-line anticancer treatment and resistant to oxaliplatin- and irinotecan-based chemotherapy were retrospectively included. Magnesium plasma levels were measured before the first day and 7, 14, 21 and 28 days after CTX+IRI infusion. RESULTS The median magnesium basal value showed a statistically significant decrease after the start of CTX+IRI treatment (at 28 days, P < 0.0001). Patients with an early decrease of magnesium levels >50% compared with the basal level had a higher tumor response rate (55.8% versus 16.7%, P < 0.0001), a longer TtP (6.3 versus 3.6, P < 0.0001) and a longer median OS (11.0 versus 8.1, P = 0.002). CONCLUSIONS We have shown that early hypomagnesemia could be a predictor of efficacy and outcome in those patients. Magnesium circulating level is an easy and inexpensive biomarker to routinely be detected in patients treated with cetuximab.

Bone metastases have a major impact on morbidity and on mortality in cancer patients. Despite its clinical relevance, metastasis remains the most poorly elucidated aspect of carcinogenesis. The biological mechanisms leading to bone metastasis establishment have been referred as "vicious circle," a complex network between cancer cells and the bone microenvironment. This review is aimed to underline the new molecular targets in bone metastases management other than bisphosphonates. Different pathways or molecules such as RANK/RANKL/OPG, cathepsin K, endothelin-1, Wnt/DKK1, Src have recently emerged as potential targets and nowadays preclinical and clinical trials are underway. The results from those in the advanced clinical phases are encouraging and underlined the need to design large randomised clinical trials to validate these results in the next future. Targeting the bone by preventing skeletal related events (SREs) and bone metastases has major clinical impact in improving survival in bone metastatic patients and in preventing disease relapse in adjuvant setting.

3564 Background: KRAS wild-type mutational status is necessary but not sufficient to get benefit from EGFR inhibition. Predictive markers are currently being evaluated including other EGFR downstream pathways and EGFR ligands. In the present study we reported hypomagnesemia as a predictor of efficacy and outcome in terms of TTP and OS in a selected cohort of patients affected by advanced colorectal adenocarcinoma KRAS wild-type cetuximab-treated. Methods: 143 patients affected by stage IV and histologically confirmed colorectal adenocarcinoma KRAS wild-type receiving cetuximab plus irinotecan as third-line anticancer treatment and resistant to oxaliplatin- and irinotecan-based chemotherapy were included. Magnesium plasma levels were measured before and 1, 7, 14 21, 28 days after cetuximab plus irinotecan infusion. Results: The median magnesium basal value showed a statistically significant decrease after the start of cetuximab plus irinotecan anticancer treatment (at 28 days, p < 0.0001). Patients with a decrease of magnesium levels > 50% compared to the basal level had a higher tumor response rate (55.8% vs. 16.7%, p < 0.0001), a longer TTP (6.3 vs. 3.6, p < 0.0001) and a longer median OS (11.0 vs. 8.1, p = 0.002) compared with those patients without magnesium modifications. Conclusions: We have shown that hypomagnesemia could be a predictor of efficacy and outcome in those patients harboring a wild type KRAS status treated with cetuximab plus irinotecan. There is growing evidence magnesium plays a key role in tumor biology. Criteria in selecting patients who are likely to benefit from cetuximab-based therapies are growing. Magnesium circulating level is an easy and economically inexpensive biomarker to routinely and serially be detected in patients cetuximab-treated. No significant financial relationships to disclose.

More than 30%–50% of women at breast cancer diagnosis are vitamin D deficient. The importance of preserving bone health in survivors of breast cancer has gained predominant priority because these women have higher than average rates of bone loss and fracture as they age and they have a 15% higher fracture risk than women without a history of breast cancer. Recently, prospective cohort studies reported an inverse association between serum 25-hydroxyvitamin D (25-OHD) levels and breast cancer prognosis [1.Freedman D.M. Looker A.C. Chang S.C. et al.Prospective study of serum vitamin D and cancer mortality in the United States.J Natl Cancer Inst. 2007; 99: 1594-1602Crossref PubMed Scopus (306) Google Scholar]. We evaluate the fluctuations of vitamin D circulating levels during sequential anthracycline- and docetaxel-based adjuvant chemotherapy in early-stage breast cancer patients. The levels of 25-OHD at diagnosis (before chemotherapy) and during adjuvant chemotherapy (after four anthracycline-based cycles and, at the end of adjuvant chemotherapy, after four docetaxel-based administrations) in seven pre- and 13 postmenopausal women (median age was 53 years) with early-stage breast cancer following radical surgery were assessed. Our results highlighted that all patients were deficient in vitamin D plasma levels at baseline. The median serum 25-OHD level at baseline was 9.03 ng/ml [95% confidence interval (CI) 8.20–11.82 ng/ml] and at 63 days it was 6.75 ng/ml [95% CI 5.67–10.96 ng/ml; median percent reduction (MPR) = 25.3%]; its reduction was not statistically significant when compared with basal levels (P = 0.114). The median serum 25-OHD at 147 days was 6.14 ng/ml (95% CI 5.48–9.29 ng/ml; MPR = 32.0%) and it was statistically significant (P = 0.047) when compared with the basal value. We may hypothesize that the significant reduction of vitamin D plasma levels could be related to an increase of body mass index in terms of adipose tissue observed in our patients, and generally in early-stage breast cancer patients undergoing adjuvant chemotherapy. An increased storage of 25-OHD in adipose tissue results in lower circulating levels of it. This is a plausible explanation for increased rates of vitamin D deficiency in obese individuals [2.Wortsman J. Matsuoka L.Y. Chen T.C. et al.Decreased bioavailability of vitamin D in obesity.Am J Clin Nutr. 2000; 72: 690-693Crossref PubMed Scopus (2325) Google Scholar]. Alternatively, increases in total body fat may also be the consequence of low vitamin D levels, as it has been hypothesized that low vitamin D status, by causing parathyroid hormone excess and calcium influx into adipocytes, may promote weight gain. An additional plausible hypothesis may be a direct effect of anthracycline- and docetaxel-based regimens on 25-OHD circulating levels. A notable change in 25-OHD concentration after the same chemotherapy regimens was not observed in two studies [3.Kailajarvi M.E. Salminen E.K. Paija O.M. et al.Serum bone markers in breast cancer patients during 5-fluorouracil, epirubicin and cyclophosphamide (FEC) therapy.Anticancer Res. 2004; 24: 1271-1274PubMed Google Scholar, 4.Crew K.D. Shane E. Cremers S. et al.High prevalence of vitamin D deficiency despite supplementation in premenopausal women with breast cancer undergoing adjuvant chemotherapy.J Clin Oncol. 2009; 27: 2151-2156Crossref PubMed Scopus (126) Google Scholar] but in both studies patients were supplemented with vitamin D. In our population, vitamin D supplement was forbidden. This consideration is particularly important because all tested patients were deficient in vitamin D at baseline. In addition to its crucial effects on bone and mineral metabolism, vitamin D has pleiotropic anticancer effects. In a recent large cohort study of early-stage breast cancer patients, low levels of vitamin D were significantly associated with an increased risk of distant recurrence and death in univariate analyses [5.Goodwin P.J. Ennis M. Prithard K.I. et al.Prognostic effects of 25-hydroxyvitamin D levels in early breast cancer.J Clin Oncol. 2009; 27: 3757-3763Crossref PubMed Scopus (293) Google Scholar]. We strongly suggest oncologists to assess circulating levels of vitamin D 25-OHD in all early breast cancer patients before starting adjuvant chemotherapy and to supplement them with adequate doses of vitamin D before and during chemotherapy. The authors confirm that they do not have any conflicts of interest to declare.

This system constituted of the Receptor Activator of nuclear Factor-kB Ligand (RANKL), the Receptor Activator of Nuclear Factor-kB (RANK) and by the decoy Receptor Osteoprotegerin (OPG) plays a central role in bone resorption. Denosumab (AMG 162) is an investigational fully human monoclonal antibody with a high affinity and specificity for RANKL. This review will critically describe and discuss the recent results of clinical trial investigating denosumab in different settings of medical oncology. In particular, we will report the recently published data of clinical trials investigating denosumab in prevention of cancer treatment induced bone loss (CTIBL), in prevention of skeletal related events (SREs) in bone metastatic patients and the ongoing studies in prevention of disease recurrence in the adjuvant setting of solid tumours. The clinical data that will be reported in this review represent the first step in a path that will conduct us to explore new horizons in the field of bone health care in cancer patients.

In the present study we investigated the potential role of Toll-like receptor 4 (TLR-4) Asp299Gly and Thr399Ile polymorphisms as risk factors in the development of gastric cancer. TLR-4 Asp299Gly and Thr399Ile polymorphisms were investigated in 171 Italian patients with sporadic gastric cancer and in 151 controls. Unconditional regression (odds ratio and 95% confidence intervals) were used to investigate the association of the studied polymorphisms with gastric cancer. TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer (P = 0.023 and hazard ratio = 3.62). No significant association for TLR-4 Asp299Gly polymorphism was found. In the subgroup of patients with intestinal-type gastric cancer, a significant risk of gastric cancer was associated with TLR-4 Thr399Ile genotype (P = 0.006). Our results demonstrated that TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer. An increased risk for intestinal gastric cancer in carriers of the TLR4 Thr399Ile allele was observed. Future epidemiological studies should consider the possible interactions between proinflammatory genotypes (such as TLR and interleukin-1R polymorphisms) and other risk factors for cancer such as dietary habits and/or exposure to environmental carcinogens.

Introduction: Magnesium plays a role in a large number of cellular metabolic reactions. Cetuximab is able to induce hypomagnesemia by interfering with magnesium (Mg2+) transport in the kidney. We designed this trial to investigate if Mg2+ serum level modifications may be related with clinical response and outcome in advanced colorectal cancer patients during treatment with cetuximab plus irinotecan. Experimental Design: Sixty-eight heavily pretreated metastatic colorectal cancer patients were evaluated for Mg2+ serum levels at the following time points: before; 6 hours; and 1, 7, 14, 21, 50, and 92 days after the start of treatment. Results: Basal Mg2+ median levels were significantly decreased just 7 days after the first anticancer infusion and progressively decreased from the 7th day onward, reaching the highest significance at the last time point (P < 0.0001). Twenty-five patients showed a reduction in median Mg2+ circulating levels of at least 20% within the 3rd week after the first infusion. Patients with this reduction showed a response rate of 64.0% versus 25.6% in the nonreduced Mg2+ group. The median time to progression was 6.0 versus 3.6 months in the reduced Mg2+ group and in that without reduction, respectively (P < 0.0001). Overall survival was longer in patients with Mg2+ reduction than in those without (10.7 versus 8.9 months). Conclusions: Our results confirm that cetuximab treatment may induce a reduction of Mg2+ circulating levels and offer the first evidence that Mg2+ reduction may represent a new predictive factor of efficacy in advanced colorectal cancer patients treated with cetuximab plus irinotecan.

V gamma 9V delta 2 T cells have the ability to produce inflammatory cytokines involved in protective immunity against intracellular pathogens and tumours and to display strong cytolytic as well as bactericidal activities. This suggests a direct involvement of V gamma 9V delta 2 T lymphocytes in immune control of cancer and infections. These observations have recently aided development of novel immunotherapeutic approaches aimed at V gamma 9V delta 2 T cell activation. Nitrogen-containing bisphosphonates (N-BPs) play a crucial role in V gamma 9V delta 2 T lymphocyte activation and in the acquisition of effector functions. The preliminary results of these innovative strategies are encouraging. Moreover, compelling evidence in the literature supports the hypothesis that the antitumour effect of bisphosphonates is exerted through direct as well as indirect mechanisms. An additional and not yet well explored mechanism by which N-BPs may display antineoplastic effect is related to their immunomodulatory properties. It is fascinating that N-BPs influence the immune system in various but interrelated ways, being able to enhance the innate and to promote the adaptive immune responses. For all these reasons, V gamma 9V delta 2 T lymphocytes represent a particularly interesting target for immunotherapeutic protocols based on N-BP administration. All these unexpected effects of N-BPs on the immune system have opened new and intriguing possibilities of therapeutic use for these drugs.

3558 Purpose: Recent data have demonstrated in preclinical tumor models an antiangiogenic and antitumor activity of low weekly doses of ZA. As a consequence, the purpose of this study was to confirm these data, evaluating in cancer patients the modifications in angiogenic cytokines levels following repeated weekly low doses of ZA. Experimental Design: 26 consecutive cancer patients with bone metastases treated, for the first time, with four weekly doses of 1 mg of ZA followed by standard doses (4 mg every 28 days) were prospectively evaluated for circulating levels of vascular endothelial growth factor (VEGF) at different time points: just before and after 1, 7, 14, 21, 28, 56 and 84 days following the first disphosphonate infusion. Results: Basal serum VEGF median levels were significantly decreased just after 7 days (-29.7%) (with only one weekly infusion) (P=0.038), This significant decrease of circulating VEGF levels persisted 14(-33.2%), 21 (-39.4%), 28(-31.8%), 56(-33.6%) and 84(-27.9%) days after the first infusion (respectively, P=0.002, P=0.001, P=0.008, P=0.002, P=0.014). Conclusions: This study confirms, for the first time in humans, that weekly low doses of zoledronic acid could have antiangiogenic properties through a significant and long lasting decrease of VEGF serum levels. No significant financial relationships to disclose.

BACKGROUND:Bisphosphonate (BP) therapy has become a standard of therapy for patients with malignant bone disease. In vivo preclinical and preliminary clinical data indicate that BPs may prevent cancer treatment-induced bone loss and the onset of malignant bone disease in patients with early-stage cancer.DESIGN:This review will describe the preclinical evidences of action of BPs on osteoclasts and tumor cells. In addition, the effects of principal BPs on skeletal disease progression in patients with breast cancer, prostate cancer, non-small-cell lung cancer and other cancers will be reported. The preliminary clinical data from retrospective trials on the effect of zoledronic acid (ZA) on survival will be described and the ongoing adjuvant phase III trial will be analyzed.CONCLUSIONS:This review will describe the preliminary clinical evidences from prospective studies on the effect of ZA treatment on the prevention of bone metastasis.

Attempts to enhance patients’ immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8+ T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8+ T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68+ tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

Objective To evaluate the prevalence of cardiac alterations by trans thoracic echocardiography (TTE) and the possible role of aPLs in determining heart damage in SLE patients.Patients and methods We investigated 34 consecutive Caucasian SLE patients and 34 age and sex- matched controls. All patients underwent TTE. Lupus anticoagulant (LA) was assayed. IgG and IgM antiphospholipid antibodies against cardiolipin (aCL), phosphatidylinositol (aPI), phosphatidylserine (aPS), phosphatidic acid (aft, and anti-beta 2-glycoprotein I antibodies (alpha beta 2GPI) were determined by ELISA.Results Nineteen (56%) SLE patients showed at least one cardiac abnormality (P < 0.0001 - RR 19; OR 41.8; 95% CI 5.1 - 342). The predominant valve dysfunctions were represented by mitral (21%) and tricuspidal (18%) regurgitation. Aortic regurgitation was observed in 12% of patients, pericardial effusion and left atrial enlargement were identified in 15% and 12% of cases, respectively. Mitral valvular strands were detected in one patient. The prevalence of cardiac abnormalities correlated with disease duration. Echocardiographic alterations were more common in aPLs positive than in aPLs negative patients (P = 0.02 - RR 2.5; OR 6.1; 95% CI 1.2-30.1). Patients with IgG-aPA,-aPI and -aPS had a higher prevalence of left atrial enlargement (P < 0.05); IgG-aPA and -aPI were significantly associated with increased interventricular septum thickness (P < 0.05).Conclusions Our findings confirm that the heart is one of the main target in SLE patients. The association between aPLs and cardiac impairment suggests an adjunctive role of these autoantibodies in determining heart damage. SLE vasculopathy is a multifactorial process leading to accelerated atherosclerosis. Heart involvement over the course of disease requires a comprehensive screening and management of traditional and new cardiovascular risk factors to prevent cardiac damage, which represents the primary cause of morbidity and mortality in SLE patients.

Alloantigen specific T suppressor/regulatory cells have been shown to play an important role in the maintenance of peripheral and central tolerance, thereby preventing allograft rejection. We have previously demonstrated the existence of a distinct population of alloantigen specific CD8+CD28-FOXP3+ T suppressor (TS) cells that tolerize dendritic and endothelial cells (EC) by upregulating the cell surface expression of inhibitory receptors, Immunoglobulin Like Transcripts (ILT)3 and ILT4. In this study, we followed 14 heart transplant recipients (8 patients with no episodes and 6 with one or more episodes of grade 3A rejection) at fixed time intervals (340 serial determinations) from the day of transplantation to 8 months later. In rejection-free patients, we observed an increase in the frequency of the CD8+CD28− T cell population within 2 months after transplantation. This increase was associated with FOXP3 expression in these cells which acquired the ability to induce the upregulation of ILT3 and ILT4 on the donor matched EC in an alloantigen specific manner. In 3 of the 6 patients, who had at least one episode of rejection, an increased frequency of CD8+CD28− T cells was seen 5±1 months after successful treatment of the last episode of grade 3A rejection. This expansion coincided with the expression of FOXP3 and the ability of these CD8+CD28− T cells to induce the expression of ILT3 and ILT4 in EC sharing HLA class I antigens with the donor. These patients remained rejection free after having developed allospecific CD8+CD28− TS. This finding in conjunction with the observation that patients who developed TS within 2 months post transplantation had an uneventful course, emphasizing the dynamic characteristic relationship between regulatory and effector T cells. Our data indicate that the development of TS is not necessarily compromised after an early rejection episode. Monitoring the generation of TS in organ allograft recipients may provide useful information for their clinical management.

OBJECTIVE:To investigate the thrombotic tendency in patients with systemic lupus erythematosus (SLE) by evaluating congenital or acquired abnormalities associated with an increased risk of venous and/or arterial thrombosis. METHODS:A total of 57 patients with SLE were included in the study. Twenty-one patients (37%) had a history of arterial and/or venous thrombosis and 36 patients (63%) did not have such a history. Sera from 50 healthy controls were examined. Protein C, protein S, antithrombin, D-dimer, fibrinogen, homocysteine, anticardiolipin antibodies (aCL), lupus anticoagulant (LAC), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T gene mutation were evaluated. RESULTS:Protein C, antithrombin, fibrinogen, D-dimer, and homocysteine levels were significantly higher in patients with SLE than in controls. A prothrombin mutation was observed in 2 (4%) of 50 controls and in 6 (11%) of 57 patients. A significantly higher prevalence (P = 0.036) of MTHFR homozygous mutation was observed in patients with SLE (14 [25%] of 57) in comparison with controls (4 [8%] of 50). IgG-aCL and IgM-aCL levels were significantly higher in patients with SLE than in controls (P < 0.0001). The presence of medium-high (> or = 20 IgG phospholipid units/ml) IgG-aCL antibody titers was significantly higher (P = 0.005) in patients with thrombosis (11 [52%] of 21) than in patients without (5 [14%] of 36) thrombosis. LAC was present in 22 (38.5%) of 57 patients and in none of 50 controls. CONCLUSION:In this study, we confirm the association between thrombosis and IgG-aCL at medium-high titers and suggest that the coexistence of other risk factors can affect the expression of thrombosis in patients with SLE.

T suppressor and regulatory cells have been shown to play an important role in the maintenance of central and peripheral tolerance thereby preventing allograft rejection, autoimmunity and allergy. We have previously described a distinct population of antigen-specific CD8+CD28− T suppressor cells (TS). These CD8+CD28− TS cells can be generated in vitro after multiple rounds of stimulation of human peripheral blood mononuclear cells (PBMCs) with either allogenic- or xenogeneic-donor APCs. CD8+CD28− TS cells are FOXP3+, MHC class I-restricted and tolerize both professional antigen presenting cells, such as dendritic cells (DC) and nonprofessional APC such as endothelial cells (EC) by up-regulating the cell surface expression of inhibitory receptors immunoglobulin-like transcript (ILT)-3 and ILT4 and down-regulating the expression of costimulatory molecules such as CD58 and CD86. Tolerized ILT3high, ILT4high APC anergize CD4+ TH cells and can induce the generation of antigen-specific CD4+CD25+ T regulatory cells (TR) cells and CD8+CD28− TS cells. In this review, we present our recent studies on the molecular characterization of these antigen specific T suppressor cells and tolerogenic APC.

P928 Aims: Allograft rejection is mediated by T cells with direct and indirect allorecognition capacity. The direct pathway involves T cells which interact directly with professional (dendritic cells, macrophages) or nonprofessional (endothelial cells) Antigen Presenting Cells (APC) of the donor. The indirect pathway involves T cells which recognize dominant allopeptides derived from the processing of necrotic or apoptotic cells of the graft by host APC. Both in the periphery and within the graft, the frequency of T cells with direct allorecognition capacity is 10,000 times higher than that of allopeptide reactive T cells at all times. We have explored the hypothesis that tolerogenic endothelial inhibit the direct recognition pathway. Methods: Human endothelial cells (EC) were conditioned by use of IFN-γ and used for T cell priming. Conditioned EC were incubated with allospecific CD4+CD25+CD45RO+ FOXP3+ T regulatory cells (TR) or CD8+CD28- T suppressor cells. The expression on EC of CD31, CD40, CD54, CD62E CD106 CD58, HLA class I, HLA class II, ILT3, and ILT4, prior and following exposure to TS or TR was measured using flow cytometry and RT-PCR. T suppressor cells were tested in proliferation assays for their capacity to inhibit TH reactivity. Results: The ILT3high, ILT4high phenotype is characteristic of tolerogenic DC which induce regulatory T cells inhibiting the direct allorecognition pathway. However, after the departure of donor APC from the graft, rejection can be triggered by activated endothelial cells (EC). We have explored the hypothesis that EC may be involved in the maintenance of quiescence. Our studies have shown that alloantigen specific CD8+CD28- T cells with suppressor activity are FOXP3 positive and interact with human dendritic cells as well as EC. The interaction of TS with EC results in the induction of the inhibitory receptor ILT4, down-regulation of costimulatory and adhesion molecules and tolerization of EC. In turn, tolerized ILT4high EC elicit the differentiation of CD8+CD28- FOXP3+ T suppressor cells. Similarly alloantigen specific TR elicited the transcriptional activation of ILT3 and ILT4 in EC. Alloantigen specific CD8+CD28- FOXP3+ T cells, which trigger the transcriptional activation of ILT4 in EC, were found in the circulation of 21 heart allograft recipients in quiescence. Patients with a history of early acute rejection episodes (N=7) and/or chronic rejection (N=3) showed no TS or TR. The assay which we developed facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppression, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Conclusions: Endothelial cells are an important target of antigen specific T effector cells not only in transplantation but also in numerous autoimmune diseases. Agents which inhibit EC activation and/or render them tolerogenic are likely to have a beneficial effect in treatment of rejection, autoimmunity and possibly atherosclerosis.