Characterization of human malignant melanoma cell lines. III. Membrane immunofluorescence reactivity with sera from patients with melanoma

Summary Seven well-characterized human malignant melanoma cell lines have been evaluated in terms of their reactivity in membrane immunofluorescence tests with sera from 48 patients with melanoma, 23 patients with other forms of cancer and 28 normal controls. There was a significantly greater degree of reactivity of melanoma sera (33.7%) than of sera of normal controls (22.2%) or of sera from patients with other forms of cancer (24.2%). The incidence of strong reactors among the melanoma patients was found to be inversely proportional to the extent of disease in the melanoma patients: Stage I, 54.5%, Stage III, 36.8% and Stage IV, 29.4%. Reactivity against non-melanoma cell lines was similar in the three subject groups and was unaffected by stage of disease in the melanoma patients. No single cell line showed preferential reactivity with melanoma sera. There was an increased overall incidence of reactivity of all three subject groups against non-pigmented cell lines.A-B-0 antigens and heterophile antigens were excluded as a cause of seropositivity. The antigen(s) was trypsin-sensitive and neuraminidase-resistant.These data suggest that long term cultures of human melanoma may contain melanoma-associated antigens which may be useful in the further study and search for melanoma-specific antigens.     

Immunity to the melanoma inhibitor of apoptosis protein (ML-IAP; livin) in patients with malignant melanoma

Therapeutic targeting of melanoma antigens frequently focuses on the melanocyte differentiation or cancer-testis families. Antigen-loss variants can often result, as these antigens are not critical for tumor cell survival. Exploration of functionally relevant targets has been limited. The melanoma inhibitor of apoptosis protein (ML-IAP; livin) is overexpressed in melanoma, contributing to disease progression and treatment resistance. Improved understanding of the significance of ML-IAP immune responses in patients has possible therapeutic applications. We found ML-IAP frequently expressed in melanoma metastases by immunohistochemistry. To assess spontaneous immunity to ML-IAP, an overlapping peptide library representing full-length protein was utilized to screen cellular responses in stage I–IV patients and healthy controls by ELISPOT. A broad array of CD4⁺ and CD8⁺ cellular responses against ML-IAP was observed with novel class I and class II epitopes identified. Specific HLA-A*0201 epitopes were analyzed further for frequency of reactivity. The generation of specific CD4⁺ and cytotoxic T cells revealed potent functional capability including cytokine responsiveness to melanoma cell lines and tumor cell killing. In addition, recombinant ML-IAP protein used in an ELISA demonstrated high titer antibody responses in a subset of patients. Several melanoma patients who received CTLA-4 blockade with ipilimumab developed augmented humoral immune responses to ML-IAP as a function of treatment which was associated with beneficial clinical outcomes. High frequency immune responses in melanoma patients, associations with favorable treatment outcomes, and its essential role in melanoma pathogenesis support the development of ML-IAP as a disease marker and therapeutic target.     

Adherent indomethacin-sensitive suppressor cells in malignant melanoma

Summary Twenty-five patients with malignant melanoma were studied to determine the mechanisms underlying decreased cellular immunity in this disease. Sixteen patients were examined following surgical resections of tumor, and nine patients had evidence of metastases. Patients with metastatic disease (group II) had decreased lymphocyte transformation to concanavalin A (48,255±30,074) compared with controls (83,550±41,277) and patients free of disease (group I) 110,231±59,990) (P>0.01). Rigorous depletion of monocytes by adherent techniques resulted in an improvement in blastogenesis in seven of nine group II patients, whereas controls and group I patients had marked decreases in reactivity. Indomethacin also improved lymphocyte reactivity; a mean of 166.4% was recorded in five metastatic disease patients studied, as against a mean of 2% in group I and 9.65% in controls. In seven patients followed serially the presence of adherent suppressor cells tended to correlate with progressive disease, shorter survival, and decreased skin test reactivity.     

Inhibition of dark repair of ultraviolet damage in DNA by caffeine and 8-chlorocaffeine. Kinetics of inhibition

Summary The inhibition of dark repair by caffeine and 8-chlorocaffeine was investigated by a cellular enzyme kinetics system. The results suggest that the caffeine and 8-chlorocaffeine act by inhibiting the excision enzyme of the dark repair system.This work was supported by National Insitutes of Health Research Grant AI-05340, the University of Kansas Research Fund, and N.I.H. Training Grant 5 T1 GM-73.     

Serologic analysis of human tumor antigens

Summary The immune adherence (IA) assay was used to measure serum reactivity of patients with melanoma and osteosarcoma against paired cell lines (tumor cells and normal skin fibroblasts obtained from the same individuals) grown in tissue culture. Sera from 224 patients with various stages of melanoma were compared with sera from 100 normal age- and sex-matched donors. None of the 18 stage I sera (0%), 23 of 166 (14%) stage II sera, 3 of 40 (7%) stage III sera, and 3 of 100 (3%) normal sera were highly reactive to a standard allogeneic melanoma-fibroblast pair. None of the sera exhibited unique activity against melanoma. There was no correlation between stage of melanoma and high serum reactivity, nor was this reactivity predictive of recurrence. Sera from 39 tumor-bearing osteosarcoma patients prior to amputation were compared with sera from 50 normal age-and sex-matched donors. Eight of 39 (21%) patient sera and 1 of 50 (2%) normal sera were highly reactive to an osteosarcoma-fibroblast pair. No sera had reactivity uniquely directed against osteosarcoma. Eight osteosarcoma and two melanoma patients were tested simultaneously against their autologous cultured tumor and skin cells. Only one of these patients exhibited high reactivity towards autologous cells, and this reactivity was equal against both osteosarcoma and normal cells. None of seven highly reactive osteosarcoma or six highly reactive melanoma sera had residual tumor-specific reactivity against allogeneic osteosarcoma or melanoma after absorption with cultured fibroblasts, cultured fetal fibroblasts, or fetal calf serum.     

The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but short-lived, immune responses in mice and humans

Understanding the basic immunology of an infectious disease requires insight into the pattern of T cell reactivity and specificity. Although lymphatic filariasis is a major tropical disease, the predominant T cell Ags of filarial species such as Brugia malayi are still undefined. We have now identified a prominent T cell Ag from B. malayi microfilariae (Mf) as Bm-SPN-2, a serpin secreted exclusively by this stage. Mf-infected mice mounted strong, but short-lived, Bm-SPN-2-specific Th1 responses, measured by in vitro production of IFN-gamma, but not IL-4 or IL-5, 14 days postinfection. By day 35, responsiveness to Bm-SPN-2 was lost despite enhanced reactivity to whole Mf extract. Single immunization with Mf extract also stimulated typical Th1 reactions to Bm-SPN-2, but IgG1 Ab responses dominated after repeated immunizations. Human patients displayed potent humoral responses to Bm-SPN-2 in both IgG1 and IgG4 subclasses. Thus, 100% (20 of 20) of the microfilaremic (MF(+)) patients bore IgG4 responses to Bm-SPN-2, while only 30% of endemic normal subjects were similarly positive. Following chemotherapy, Bm-SPN-2-specific Abs disappeared in 12 of 13 MF(+) patients, although the majority remained seropositive for whole parasite extract. PBMC from most, but not all, endemic subjects were induced to secrete IFN-gamma when stimulated with Bm-SPN-2. These findings demonstrate that Bm-SPN-2 is recognized by both murine and human T and B cells and indicate that their responses are under relatively stringent temporal control. This study also provides the first example of a stage-specific secreted molecule that acts as a major T cell Ag from filarial parasites and is a prime candidate for a serodiagnostic probe.     

Virus-augmented delayed hypersensitivity skin tests in gynecological malignancies

Summary Cultured human tumor cells of various histologic origins were infected with PR8/A/34 influenza virus. Nonviable crude membrane extracts were derived from the infected and uninfected cells. The extracts were coded and tested for their ability to produce delayed hypersensitivity skin reactions (DHSR) in allogeneic patients with squamous uterine cervical carcinoma, epithelial ovarian carcinoma, and malignant melanoma. Augmented antigen sensitivity to the virus-modified extracts compared with virus alone or to the unmodified extracts was observed in all patient groups. There was insufficient specificity to delineate a response by individual tumor type and related tumor extract, but some of the observed responses suggested tumor or organ site associations. Cervical carcinoma patients reacted more frequently to the virus-modified cervix extract, which also produced a high frequency of response in patients with ovarian carcinoma and melanoma. Ovarian carcinoma patients demonstrated increased sensitivity to both virus-modified ovarian carcinoma extracts, although 14 of 21 patients also showed responsiveness to one of the unmodified ovarian extracts. Malignant melanoma patients showed increased sensitivity to all virus-modified extracts except one of two derived from the ovarian carcinoma, and demonstrated a significantly augmented response to the virus-modified melanoma extract when the response to this extract was compared with that in ovarian carcinoma patients.The augmented reactions appear to be due to an association of the PR8 virus and as yet undetermined cellular components rather than to the virus alone. The possible involvement of tumor-associated determinants and the clinical significance of this phenomenon require further investigation.     

Tumor-reactive CD4+ T cell responses to the melanoma-associated chondroitin sulphate proteoglycan in melanoma patients and healthy individuals in the absence of autoimmunity

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.     

Autologous and homologous immunofluorescent antibody to established breast cancer cell lines

Summary Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients. This work was supported in part by Grant CA 16789 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

Interaction of TaqI polymorphism at exon 9 of the vitamin D receptor gene with the negative lepromin response may favor the occurrence of leprosy

Controversies over the vitamin D receptor (VDR) acting as a susceptibility factor in Mycobacterium sp. infections may be the result of incorrect population stratification. The risk of leprosy occurrence conditioned by VDR polymorphism was investigated by stratifying the population of a highly endemic Brazilian region into negative and positive Mitsuda responses. Leprosy patients (102) and a group of healthy nonconsanguineous household contacts (68) were genotyped for the VDR TaqI polymorphism (T/t). TT and Tt genotypes were not considered to be risk factors as their odds ratios (OR) were not different from those presented by the negative Mitsuda response individuals. The combination of the tt genotype and the negative Mitsuda test provided an occurrence rate 13 times higher in leprosy patients than in controls with positive Mitsuda responses. This suggests that there is a higher risk of leprosy development when individuals carry this unfavorable combination, and demonstrates a possible synergistic role of these two variables in leprosy susceptibility via effects on cellular immunity.     

A multifaceted analysis of immune-endocrine-metabolic alterations in patients with pulmonary tuberculosis

Our study investigated the circulating levels of factors involved in immune-inflammatory-endocrine-metabolic responses in patients with tuberculosis with the aim of uncovering a relation between certain immune and hormonal patterns, their clinical status and in vitro immune response. The concentration of leptin, adiponectin, IL-6, IL-1β, ghrelin, C-reactive protein (CRP), cortisol and dehydroepiandrosterone (DHEA), and the in vitro immune response (lymphoproliferation and IFN-γ production) was evaluated in 53 patients with active untreated tuberculosis, 27 household contacts and 25 healthy controls, without significant age- or sex-related differences. Patients had a lower body mass index (BMI), reduced levels of leptin and DHEA, and increased concentrations of CRP, IL-6, cortisol, IL-1β and nearly significant adiponectin values than household contacts and controls. Within tuberculosis patients the BMI and leptin levels were positively correlated and decreased with increasing disease severity, whereas higher concentrations of IL-6, CRP, IL-1β, cortisol, and ghrelin were seen in cases with moderate to severe tuberculosis. Household contacts had lower DHEA and higher IL-6 levels than controls. Group classification by means of discriminant analysis and the k-nearest neighbor method showed that tuberculosis patients were clearly different from the other groups, having higher levels of CRP and lower DHEA concentration and BMI. Furthermore, plasma leptin levels were positively associated with the basal in vitro IFN-γ production and the ConA-driven proliferation of cells from tuberculosis patients. Present alterations in the communication between the neuro-endocrine and immune systems in tuberculosis may contribute to disease worsening.     

Vitiligo- and melanoma-associated hypopigmentation: a similar appearance but a different mechanism

The significance of the association between the appearance of hypopigmentation in patients with melanoma and the prognosis is still not clear. It was postulated that, in melanoma, an immune response is responsible for the destruction of the malignant as well as the normal pigmented cells, and that the eventual development of vitiligo-like patches in melanoma patients improves their prognosis. We studied the level of anti-melanoma antibodies in the sera of patients with melanoma with hypopigmentation and compared it to the titer in patients with melanoma only, to the titer of patients with vitiligo, and to that of healthy subjects. Only IgG-type antibodies were found in the sera of patients with vitiligo, with melanoma, or with melanoma with hypopigmentation. No significant differences in the titer of anti-melanoma antibodies could be found between the patients with melanoma when subgrouped according to the initial stage and the status of the disease at the time when the test was carried out. Statistically significantly (P<0.001) higher titers of antibodies were detected in the sera of patients with vitiligo in comparison to the lower titers in the other groups. Our results point to a similar immunobiological status, which probably does not give any advantage to patients with melanoma with hypopigmentation compared to patients without it. The appearance of hypopigmentary plaques in melanoma patients should be regarded, in our opinion, as a concomitant immunological phenomenon of the disease.This work was supported by a grant given by the Israeli Chief Scientist, Ministry of Health, Israel     

Identification of human populations with a high incidence of cellular immunity against breast carcinoma

Summary Cell mediated immunity specifically directed against breast carcinoma, but not against osteogenic sarcoma or other carcinomas, was found to occur in the household contacts of patients with active breast carcinoma (primary and metastatic). Such immunity was not found in the normal population, nor in household contacts of patients with osteogenic sarcoma; it was also not found in household contacts of breast carcinoma patients who had been disease free for two or more years. Patients with breast carcinoma had cytotoxicity indexes (CI) comparable to the normal population, regardless of their clinical condition.     

Cell-mediated immunity in Cryptococcosis

Cell-mediated immune responses in patients who had recovered from cryptococcosis were compared to those of healthy subjects. Cryptococcal patients were mildly lymphopenic but showed no defect in percentage of thymus-derived lymphocytes. One-third had positive delayed skin test reactions to cryptococcal antigen. Their skin test reactivity to two commonly used noncryptococcal antigens was less intense than healthy control subjects. Strongly positive and specific lymphocyte transformation occurred in the presence of an extract of Cryptococcus neoformans (cryptococcin) in half of the patients. In contrast, few healthy subjects had positive transformation responses to cryptococcin. One patient who was followed sequentially through treatment of cryptococcal meningitis acquired strong cryptococcin reactivity during the course of treatment. Cellular immunologic response to cryptococcin identifies many subjects who have had C. neoformans exposure, and may be of value for assessing immunologic status of patients undergoing therapy. These studies also indicate that most patients with cryptococcosis have a degree of deficiency in cell-mediated response to fungal antigens even when a specific underlying disease process cannot be identified.     

Immunoglobulin G response against 10-kDa and 65-kDa heat-shock proteins in leprosy patients and their household contacts

Abstract We measured antibody responses to recombinant Mycobacterium leprae 65-kDa (rML65) and 10-kDa (rML10) by indirect ELISA in sera from leprosy patients, household contacts and healthy controls in a leprosy-endemic area in the north east of Argentina. Serum antibody levels to those antigens were correlated with IgM anti-phenolic glycolipid I (PGL-I) levels, with bacterial index and the period of time under chemotherapy. Bacterial index positive (BI⁺) patients showed higher mean values when compared with BI negatives (BI⁻). Among lepromatous patients a positive correlation was observed between IgG antibody responses to both recombinant antigens and IgM antibody response to PGL-I. Anti-rML10 test detected a higher percentage of positive/total than anti-rML65 in all leprosy groups and healthy contacts. Bacterial load, leprosy clinical form and the time under chemotherapy were factors which could influence levels of the antibody response. The contribution of these antibody studies for a precise and early diagnosis in leprosy is discussed.     

Household Contact Screening Adherence among Tuberculosis Patients in Northern Ethiopia

BackgroundHousehold contacts of active tuberculosis cases are at high risk of getting tuberculosis disease. Tuberculosis detection rate among contacts of household members is high. Hence, this study investigated household contact screening adherence and associated factors among tuberculosis patients in Amhara region, Ethiopia.MethodsA cross-sectional study was conducted from April 10 - June 30, 2013 in five urban districts of Amhara region, where 418 patients receiving treatment at tuberculosis clinic were interviewed. All patients were interviewed using structured and pre-tested questionnaire. Bringing at least one household contact to TB clinic was regarded as adherent to household contacts screening. Bivariate and multiple logistic regressions were used to investigate association.ResultsThe overall adherence to household contact screening in Amhara region was 33.7%. Adherence was higher among Muslims than Christians. Adherence was high if patient took health education from Health Care Worker [AOR: 3.22, 95% CI: 1.88 to 5.51] and 2.17 times higher if patient had sufficient knowledge on tuberculosis [AOR: 2.17, 95% CI: 1.29 to 3.67] during interview. Relationship with contact was a significant [AOR: 0.4, 95% CI: 0.2 to 0.9] social related factor.ConclusionOne third of tuberculosis patients adhered to household contact screening in health facilities during their treatment course. Promoting knowledge of tuberculosis in the community and continuous health education to tuberculosis patients are recommended.     

Intermolecular antigen spreading occurs during the preclinical period of human type 1 diabetes

Intra- and intermolecular spreading of T cell responses to autoantigens has been implicated in the pathogenesis of autoimmune diseases. Therefore, we questioned whether T cell responses from subjects identified as at-risk (positive for autoantibody reactivity to islet proteins) for the development of type 1 diabetes, a cell-mediated autoimmune disease, would demonstrate intermolecular Ag spreading of T cell responses to islet cell proteins. Previously, we have demonstrated that by the time subjects develop type 1 diabetes, they have T cell responses to numerous islet proteins, whereas T cells from normal controls respond to a limited number of islet proteins. Initial testing of PBMC responses from 25 nondiabetic at-risk subjects demonstrated that 16 of the 25 subjects have PBMC responses to islet proteins similar to controls. Fourteen of these 16 subjects were available for follow-up. Eleven of the 14 developed T cell responses to increasing numbers of islet proteins, and 6 of these subjects developed type 1 diabetes. In the nine subjects who already demonstrated T cell Ag spreading at the initial visit, four were available for follow-up. Of these four, two had increases in T cell reactivity to islet proteins, while two maintained their initial levels of T cell reactivity. We also observed Ag spreading in autoantibody reactivity to islet proteins in nine of the 18 at-risk subjects available for follow-up. Our data strongly support the conclusion that intermolecular spreading of T cell and Ab responses to islet proteins occurs during the preclinical period of type 1 diabetes.     

Normal tissue depresses while tumor tissue enhances human T cell responses in vivo to a novel self/tumor melanoma antigen,OA1

Antitumor T cells often recognize targets that are nonmutated "self" tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a "knockout." In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-"knockout") individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.