Induction of circulating tumor-reactive CD8+ T cells after vaccination of melanoma patients with the gp100 209-2M peptide.

Patients with stage I-III melanoma were vaccinated with the modified HLA-A2-binding gp100(209-2M)-peptide after complete surgical resection of their primary lesion and sentinel node biopsy. Cytoplasmic interferon-gamma production by freshly thawed peripheral blood mononuclear cells (direct ex vivo analysis) or by peripheral blood mononuclear cells subjected to 1 cycle of in vitro sensitization with peptide, interleukin-2, and interleukin-15 was measured following restimulation with the modified and native gp100 peptides, and also A2gp100 melanoma cell lines. Peptide-reactive and tumor-reactive T cells were detected in 79% and 66% of selected patients, respectively. Patients could be classified into 3 groups according to their vaccine-elicited T-cell responses. One group of patients responded only to the modified peptide used for immunization, whereas another group of patients reacted to both the modified and native gp100 peptides, but not to naturally processed gp100 antigen on melanoma cells. In the third group of patients, circulating CD8 T cells recognized A2gp100 melanoma cell lines and also both the modified and native peptides. T cells with a low functional avidity, which were capable of lysing tumor cells only if tumor cells were first pulsed by the exogenous administration of native gp100(209-217) peptide were identified in most patients. These results indicate that vaccination with a modified gp100 peptide induced a heterogeneous group of gp100-specific T cells with a spectrum of functional avidities; however, high avidity, tumor-reactive T cells were detected in the majority of patients.

Der Radioimmunoassay zur Ermittlung des carcinoembryonalen Antigens (CEA), seine Bedeutung und Limitierung während der postoperativen Kontrollperiode von Patienten mit colorectalem Carcino

Carcinoembryonic antigen (CEA) is a tumor marker defined by specific heterologous antisera. Elevated levels of circulating CEA can be detected by radioimmunoassay in most cases of colorectal carcinoma, depending on the degree of tumor spread. The fact that elevation of CEA level can also be observed in other types of carcinomas and in several nonmalignant conditions greatly limits the value of the CEA test for the early diagnosis of colorectal carcinomas. Repeated CEA measurements and their critical interpretation, however, appear to be of importance after tumor resection for the detection of tumor recurrence during the postoperative follow-up period.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab.

Metastatic melanoma has a poor prognosis with high resistance to chemotherapy and radiation. Recently, the anti-CTLA-4 antibody ipilimumab has demonstrated clinical efficacy, being the first agent to significantly prolong the overall survival of inoperable stage III/IV melanoma patients. A major aim of patient immune monitoring is the identification of biomarkers that predict clinical outcome. We studied circulating myeloid-derived suppressor cells (MDSC) in ipilimumab-treated patients to detect alterations in the myeloid cell compartment and possible correlations with clinical outcome. Lin(-) CD14(+) HLA-DR(-) monocytic MDSC were enriched in peripheral blood of melanoma patients compared to healthy donors (HD). Tumor resection did not significantly alter MDSC frequencies. During ipilimumab treatment, MDSC frequencies did not change significantly compared to baseline levels. We observed high inter-patient differences. MDSC frequencies in ipilimumab-treated patients were independent of baseline serum lactate dehydrogenase levels but tended to increase in patients with severe metastatic disease (M1c) compared to patients with metastases in skin or lymph nodes only (M1a), who had frequencies comparable to HD. Interestingly, clinical responders to ipilimumab therapy showed significantly less lin(-) CD14(+) HLA-DR(-) cells as compared to non-responders. The data suggest that the frequency of monocytic MDSC may be used as predictive marker of response, as low frequencies identify patients more likely benefitting from ipilimumab treatment. Prospective clinical trials assessing MDSC frequencies as potential biomarkers are warranted to validate these observations.

NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Circulating Tumor-reactive CD8(+) T cells in melanoma patients contain a CD45RA(+)CCR7(-) effector subset exerting ex vivo tumor-specific cytolytic activity.

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer(+) CD8(+) T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells.

Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia.

Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

CD14 expression on monocytes and TNF alpha production in patients with septic shock, cardiogenic shock or bacterial pneumonia.

OBJECTIVES: In patients with septic shock, circulating monocytes become refractory to stimulation with microbial products. Whether this hyporesponsive state is induced by infection or is related to shock is unknown. To address this question, we measured TNF alpha production by monocytes or by whole blood obtained from healthy volunteers (controls), from patients with septic shock, from patients with severe infection (bacterial pneumonia) without shock, and from patients with cardiogenic shock without infection. MEASUREMENTS: The numbers of circulating monocytes, of CD14+ monocytes, and the expression of monocyte CD14 and the LPS receptor, were assessed by flow cytometry. Monocytes or whole blood were stimulated with lipopolysaccharide endotoxin (LPS), heat-killed Escherichia coli or Staphylococcus aureus, and TNF alpha production was measured by bioassay. RESULTS: The number of circulating monocytes, of CD14+ monocytes, and the monocyte CD14 expression were significantly lower in patients with septic shock than in controls, in patients with bacterial pneumonia or in those with cardiogenic shock (p < 0.001). Monocytes or whole blood of patients with septic shock exhibited a profound deficiency of TNF alpha production in response to all stimuli (p < 0.05 compared to controls). Whole blood of patients with cardiogenic shock also exhibited this defect (p < 0.05 compared to controls), although to a lesser extent, despite normal monocyte counts and normal CD14 expression. CONCLUSIONS: Unlike patients with bacterial pneumonia, patients with septic or cardiogenic shock display profoundly defective TNF alpha production in response to a broad range of infectious stimuli. Thus, down-regulation of cytokine production appears to occur in patients with systemic, but not localised, albeit severe, infections and also in patients with non-infectious circulatory failure. Whilst depletion of monocytes and reduced monocyte CD14 expression are likely to be critical components of the hyporesponsiveness observed in patients with septic shock, other as yet unidentified factors are at work in this group and in patients with cardiogenic shock.

Correlation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with clinical and pathological relationships of patients with oral squamous cell carcinoma

Despite advances in the diagnosisand treatment of head and neck cancer,survival rates have not improvedover recent years. New therapeuticstrategies, including immunotherapy,are the subject of extensive research.In several types of tumors, the presenceof tumor infiltrating lymphocytes(TILs), notably CD8+ T cellsand dendritic cells, has been correlatedwith improved prognosis. Moreover,some T cells among TILs havebeen shown to kill tumor cells in vitroupon recognition of tumor-associatedantigens. Tumor associated antigensare expressed in a significant proportionof squamous cell carcinoma ofthe head and neck and apparently mayplay a role in the regulation of cancercell growth notably by inhibition ofp53 protein function in some cancers.The MAGE family CT antigens couldtherefore potentially be used as definedtargets for immunotherapy andtheir study bring new insight in tumorgrowth regulation mechanisms. Between1995 - 2005 54 patients weretreated surgically in our institution forsquamous cell carcinoma of the oralcavity. Patient and clinical data wasobtained from patient files and collectedinto a computerized database.For each patient, paraffin embeddedtumor specimens were retrieved andexpression of MAGE CT antigens,p53, NY-OESO-1 were analyzed byimmunohistochemistry. Results werethen correlated with histopathologicalparameter such as tumor depth,front invasion according to Bryne andboth, local control and disease freesurvival. MAGE-A was expressed in52% of patients. NY-ESO-1 and p53expression was found in 7% and 52%cases respectively. A higher tumordepth was significantly correlatedwith expression of MAGE-Aproteins(p = 0.03). No significant correlationcould be made between the expressionof both p53 andNY-OESO-1 andhistopathological parameters. Expressionof tumor-associated antigendid not seem to impact significantlyon patient prognosis. As does thedemonstration of p53 function inhibitionby CT antigens of MAGE family,our results suggest, that tumor associatedantigens may be implicated in tumorprogression mechanisms. Thishypothesis need further investigationto clarify the relationship betweenhost immune response and local tumorbiology.

Involvement of circulating CEA in liver metastases from colorectal cancers re-examined in a new experimental model.

Both experimental and clinical data show evidence of a correlation between elevated blood levels of carcinoembryonic antigen (CEA) and the development of liver metastases from colorectal carcinomas. However, a cause-effect relationship between these two observations has not been demonstrated. For this reason, we developed a new experimental model to evaluate the possible role of circulating CEA in the facilitation of liver metastases. A CEA-negative subclone from the human colon carcinoma cell line CO115 was transfected either with CEA-cDNA truncated at its 3' end by the deletion of 78 base pairs leading to the synthesis of a secreted form of CEA or with a full-length CEA-cDNA leading to the synthesis of the entire CEA molecule linked to the cell surface by a GPI anchor. Transfectants were selected either for their high CEA secretion (clone CO115-2C2 secreting up to 13 microg CEA per 10(6) cells within 72 h) or for their high CEA membrane expression (clone CO115-5F12 expressing up to 1 x 10(6) CEA molecules per cell). When grafted subcutaneously, CO115-2C2 cells gave rise to circulating CEA levels that were directly related to the tumour volume (from 100 to 1000 ng ml(-1) for tumours ranging from 100 to 1000 mm3), whereas no circulating CEA was detectable in CO115 and CO115-5F12 tumour-bearing mice. Three series of nude mice bearing a subcutaneous xenograft from either clone CO115-2C2 or the CO115-5F12 transfectant, or an untransfected CO115 xenograft, were further challenged for induction of experimental liver metastases by intrasplenic injection of three different CEA-expressing human colorectal carcinoma cell lines (LoVo, LS174T or CO112). The number and size of the liver metastases were shown to be independent of the circulating CEA levels induced by the subcutaneous CEA secreting clone (CO115-2C2), but they were directly related to the metastatic properties of the intrasplenically injected tumour cells.

AA-amyloidosis caused by visceral leishmaniasis in a human immunodeficiency virus-infected patient.

AA-amyloidosis in the setting of chronic visceral leishmaniasis (VL) has been reported in animal models but documentation in humans is unavailable. Here, we report on a Portuguese man who in 1996 was diagnosed with both human immunodeficiency virus (HIV)-infection and VL. Antiretroviral treatment led to sustained suppression of HIV viremia but CD4+ lymphocytes rose from 8 to only 160 cells/mL. Several courses of antimony treatment did not prevent VL relapses. Renal failure developed in 2006 and renal biopsy revealed AA-amyloidosis. The patient had cryoglobulinemia and serum immune complexes containing antibodies directed against seven leishmanial antigens. Antimony plus amphotericin B, followed by oral miltefosine resulted in a sustained VL treatment response with elimination of circulating Leishmania infantum DNA and CD4+ recovery. The concomitant reduction of serum AA levels and disappearance of circulating leishmanial immune complexes suggests that prolonged VL may lead to AA-amyloidosis in immunocompromised humans.

Selected polymorphisms in sex hormone-related genes, circulating sex hormones and risk of endometrial cancer.

BACKGROUND: The role of estrogen and progesterone in the development of endometrial cancer is well documented. Few studies have examined the association of genetic variants in sex hormone-related genes with endometrial cancer risk. METHODS: We conducted a case-control study nested within three cohorts to examine the association of endometrial cancer risk with polymorphisms in hormone-related genes among 391 cases (92% postmenopausal at diagnosis) and 712 individually-matched controls. We also examined the association of these polymorphisms with circulating levels of sex hormones and SHBG in a cross-sectional analysis including 596 healthy postmenopausal women at blood donation (controls from this nested case-control study and from a nested case-control study of breast cancer in one of the three cohorts). RESULTS: Adjusting for endometrial cancer risk factors, the A allele of rs4775936 in CYP19 was significantly associated (OR(per allele)=1.22, 95% CI=1.01-1.47, p(trend)=0.04), while the T allele of rs10046 was marginally associated with increased risk of endometrial cancer (OR(per allele)=1.20, 95% CI=0.99-1.45, p(trend)=0.06). PGR rs1042838 was also marginally associated with risk (OR(per allele)=1.25, 95% CI=0.96-1.61, p(trend)=0.09). No significant association was found for the other polymorphisms, i.e. CYP1B1 rs1800440 and rs1056836, UGT1A1 rs8175347, SHBG rs6259 and ESR1 rs2234693. Rs8175347 was significantly associated with postmenopausal levels of estradiol, free estradiol and estrone and rs6259 with SHBG and estradiol. CONCLUSION: Our findings support an association between genetic variants in CYP19, and possibly PGR, and risk of endometrial cancer.

Complement facilitates early prion pathogenesis.

New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.