Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

A monoclonal antibody recognizing HLA-DR2 on malignant and activated cells

A monoclonal antibody, designated NDS15.38, which recognizes a polymorphic determinant of HLA-DR, was produced from a fusion in which mice were immunized with the human B lymphoblastoid cell line GIR₂ (HLA type A1, B8, 27, Cw2, DR2, 7). NDS15.38 functions efficiently as an affinity column and purifies a two-chain complex of molecular weight 33 000 and 30 000 under reducing conditions. The monoclonal antibody reacts with HLA-DR2-positive B lymphoblastoid cell lines and B lymphocytes from patients with chronic lymphatic leukemia in an indirect radioactive binding assay. However, NDS15.38 does not appear to react with peripheral blood B lymphocytes from normal individuals. Using a peroxidase staining technique, NDS15.38 was shown to react with phytohemagglutin (PHA)-stimulated lymphocytes and with apparently activated B cells in the germinal centers of lymph nodes from individuals who were tissue typed as HLA-DR2. Thus it appears that NDS15.38 recognizes a polymorphic determinant of HLA-DR on malignant and stimulated cells, but not on resting cells.     

Requirement of Ia-positive accessory cells in the MLR response against class II antigen on human B cell tumor line

In this report we have made a comparative study of the capacity of normal human stimulator cells and Epstein-Barr virus-transformed human B cell line Wa (EBV-Wa) cells to stimulate alloreactive T cells. Class II antigen (presumably HLA-DR4 determinant) on EBV-Wa cells was shown to act as a stimulating molecule in the mixed lymphocyte reaction (MLR) through a blocking study by using anti-Ia antibodies. Furthermore, it was found that HLA-DR-positive accessory cells in the responder population were required to elicit MLR responses against HLA-DR antigen on EBV-Wa cells. In contrast, HLA-DR-positive accessory cells in the responding cell population were not essential for elicitation of MLR responses against HLA-DR antigen on normal allogeneic peripheral blood mononuclear cells, as reported. The cell-cell interaction between responder HLA-DR-positive accessory cells and responding T cells in a major histocompatibility complex (MHC)-restricted manner was required for eliciting MLR responses against class II antigen on EBV-Wa cells such as antigen-presenting cell-T cell interaction in soluble antigen-specific T cell proliferative responses. The function of HLA-DR-positive accessory cells in the responder population could not be substituted for by the presence of interleukin 1. Furthermore, there was no obvious correlation between the degree of surface HLA-DR antigen expression on EBV-Wa cells and its stimulating ability. Thus, two distinct types of allo-class II, antigen-specific T cell activation between normal human stimulator cells and EBV-Wa cells were shown to exist.     

Selective extraction by 1-butanol of surface glycoprotein antigens from human melanoma cells

Summary The butanol extraction method has previously been used to achieve selective release of tumor-specific transplantation antigens from mouse sarcoma cells. In this study we investigated the feasibility of this method for extracting four surface glycoprotein antigens (87K, 95–150K, HLA-DR, and HLA-A,B,C) from cultured human melanoma cells. Of the four antigens examined, only 95–150K and HLA-DR antigens could readily be detected in material extracted by 2%, 3%, or 5% butanol. The 3% butanol was found to be most effective in releasing these two antigens. Treatment of melanoma cells with 3% butanol did not decrease the viability of extracted cells as judged by either Trypan Blue dye exclusion or plating efficiency. Thus the noncytolytic butanol extraction method offers a promising approach to the isolation of certain glycoproteins such as 95–150K and HLA-DR from viable human melanoma cells for further purification and structural analysis.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.     

Enzyme histochemistry of human melanomas and pigmented naevi with special reference to alpha-D-mannosidase activity

Summary A histochemical study of-d-mannosidase revealed that normal human melanocytes (resting state, activated, lentigo simplex) exhibit either no or just detectable activity, as do melanocytes in the initial phase of lentigo maligna. Junctional, or occasionally zone A naevocytes displayed a very low enzyme activity. On the other hand, melanocytes in the initial stage of neoplastic transformation (dysplastic naevi, advanced stage of lentigo maligna) and also melanoma cells in disorders of low malignant potential (initial naevogenic melanoma, superficial spreading melanoma) displayed a high activity uniformly throughout the cell population. In the malignant forms (nodular melanoma, recurrences, metastases), the enzyme activity was remarkably heterogeneous, suggesting a breakdown of uniformity during malignant transformation. The significance of -mannosidase activity induction in the course of melanocyte neoplastic transformation is not clear at present. The results of biochemical assays suggest that the lysosomal isoenzyme is mainly responsible. Other lysosomal enzymes, and dehydrogenases studied concomitantly, did not display any comparable phenomena of induction or similar behaviour. However, the results of a comparison of-mannosidase with the melanocyte reference enzyme tyrosinase suggested activity patterns in the enzyme pair which may provide a better insight into the biochemical differentiation of human melanocytes in neoplastic disorders. The possible relationship of-mannosidase to melanogenesis is also discussed.     

Gamma-interferon-induced monocyte major histocompatibility complex class II antigen expression individuals with acquired immune deficiency syndrome

Twelve patients with the acquired immune deficiency syndrome (AIDS) and Kaposi's sarcoma were treated with recombinant human gamma-interferon (rIFN-gamma). A rapid, substantial increase in the fraction of HLA-DQ-positive monocytes was noted after treatment with rIFN-gamma. The rIFN-gamma-induced increase in monocyte HLA-DQ was seen throughout the course of treatment, with the percentage of HLA-DQ-positive monocytes dropping slightly following each week's treatment with rIFN-gamma and then rapidly increasing following the next course of treatment. Although the percentage of HLA-DR-positive monocytes was unchanged (HLA-DR was expressed on greater than 80% of monocytes prior to treatment), the density of HLA-DR on monocytes also increased following rIFN-gamma treatment. Following rIFN-gamma treatment, no changes were seen in CD3, CD4, CD8 T cell numbers, in T cell subset ratio (CD4/CD8), in Leu 7 or CD16 (Leu 11) cell number, in spontaneous Ig secretion, in PHA-induced in vitro proliferation, or in NK activity. These results indicate that exposure to rIFN-gamma in vivo led to the increased expression of class II antigens on monocytes in patients with AIDS.     

Human tumor and normal tissue reactivity of the anti-(breast cancer) monoclonal antibody BA-Br-3 and its similarity to the anti-(epithelial membrane antigen) monoclonal antibody E29

Summary A mouse monoclonal antibody (BA-Br-3) raised against the breast carcinoma cell line CAMA-1 was previously shown to react with a 300-kDa globule-like glycoprotein from human milk fat also expressed in the cytoplasm and on the surface of human carcinoma cells of different histological types. In this report the reactivity of this mAb with a large number of normal and malignant human tissues was analyzed using immunoperoxidase techniques. When tested on sections of both fresh-frozen tissues and formalin-fixed, paraffin-embedded tissues, BA-Br-3 reacted with a formalin-resistant antigenic determinant expressed by normal and malignant epithelial cells. Preferential reactivity was observed at the apical portion of ductal epithelial cells in normal breast and in glandular epithelia distributed in several other organs. Reactivity with mucin-like secretions in the lumina of ducts was also found. BA-Br-3 reacted mostly in heterogenous staining patterns with 88% of 49 breast carcinoma specimens tested, regardless of their histological type or whether they were primary or secondary neoplasms. Testing of epithelial malignant tumors other than breast carcinomas with this antibody showed that 127 of 151 (84%) were also reactive. mAb BA-Br-3 and E29 (a commercially available anti-(epithelial membrane antigen) shared very similar staining patterns and distributions of reactivity with breast and other epithelial tumors. However, BA-Br-3 showed a significantly higher percentage of reactivity with melanoma (33% versus 6%,P = 0.003) and a trend toward a higher percentage of reactivity with sarcoma (55% versus 27%,P >0.05). This antibody, therefore, defines a molecule that is a member of the mucin-like epithelial membrane antigen family. Further studies are warranted to determine its usefulness in antibody-directed cancer diagnosis, prognosis, and immunotherapy.     

DropArray™, a Wall‐Less 96‐Well Plate for Uptake and Immunofluorescence Microscopy,Confirms CD22 Recycles

CD22 is a cell surface glycoprotein restricted to normal and malignant B‐cells and is the target of several anti‐CD22 antibody‐based cancer therapies. For therapeutic antibody‐payload conjugates, it is important to understand the subcellular trafficking of anti‐CD22 antibodies to optimize antibody and/or linker–drug properties to maximize antitumor efficacy. It is agreed that anti‐CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell‐type dependent. No studies examined anti‐CD22 trafficking to either pathway in B‐cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray?, a novel wall‐less 96‐well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10–20 μL) volumes. We examined the time‐course of trafficking of five different anti‐CD22 antibodies in eight B‐cell lines representing four B‐cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti‐CD22 antibodies conjugated to different therapeutic payloads.      

An anatomical basis for prognosis of malignant melanoma.

Clinical observations during surgery have led us to speculate that an anatomic basis may in fact exist for the poor prognosis associated with malignant melanoma arising in certain recognized "high-risk" areas. In our study we have treated and followed 45 patients with primary malignant melanoma for over 5 years. During the course of treatment, we identified variations in anatomic characteristics at the tumor sites. Criteria were established for high- and low-risk locations by the neurovascular structure encountered. We speculate that these sites that have neurovascular windows provide a readily accessible vascular pathway for the dissemination of malignant cells to deeper visceral structures and may account for the poor prognosis associated with primary lesions in these locations. Thirty patients were classified as being at high risk for developing metastasis, whereas 13 were classified as being at low risk; 2 patients were in a special-risk category. To date, 12 of the 30 patients with "high-risk" melanoma have gone on to develop metastatic disease, which represents 40 percent of that group, whereas none of the patients classified as "low risk" have developed metastases during the same period.     

Down-regulation of HLA class II and costimulatory CD86/B7-2 on circulating monocytes from melanoma patients

Antigen-presenting cells are crucial for the induction of an antigen-specific antitumoral immune response. Deteriorations in the expression pattern of cell surface molecules important for the presentation of antigens might therefore be indicative of an impaired immune response status in cancer patients. In the present study we investigated the expression of MHC class I and class II molecules, of the costimulatory molecules CD80/B7-1 and CD86/B7-2, of the adhesion molecule CD11c, and of the marker of activation CD71 on CD14⁺ peripheral blood monocytes (PBMs) from 144 melanoma patients in different stages of disease and 43 healthy controls, by flow cytometric analysis. We found a decreased expression of HLA-DR (p<0.0005), HLA-DQ (p=0.006), HLA-DP (p<0.0005), and CD86/B7-2 (p=0.001) on PBMs from melanoma patients compared with healthy controls, whereas no significant difference could be detected in the expression of HLA class I antigens and CD80/B7-1. This down-regulated expression was associated with disease progression. In contrast, CD71 expression was stage-dependently increased on PBMs from melanoma patients compared with healthy controls (p=0.024). No correlation was found between the PBM surface expression pattern and age, gender, tumor load, and current mode of therapy of the patients. The observed down-regulation of HLA class II and CD86/B7-2 on melanoma patients PBMs might reflect an ineffective antigen-presenting function contributing to an impaired antigen-specific immune response in these patients.Both authors S. Ugurel and D. Uhlig contributed equally to this work     

Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.     

T lymphocyte subsets in patients with malignant melanoma

Summary T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.     

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability

 From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2⁺ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5×10⁷ and 15×10⁷ irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5×10⁷ cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed; in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients. Received: 20 December 1996 / Accepted: 27 February 1997     

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

Background and Objective Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. Methods and Results CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. Conclusion These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.     

HLA class I expression in metastatic melanoma correlates with tumor development during autologous vaccination

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and beta2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively.     

Human IgG and IgM monoclonal antibodies against autologous melanoma produced by Epstein-Barr-virus-transformed B lymphocytes

Summary The serum antibody response to human melanoma has prognostic and potential physiological consequences. The specificity of the host B cell antibody response may be an important determinant of disease outcome. We have utilized Epstein-Barr virus (EBV) transformation to analyze the repertory of the host B cell response to melanoma. Production of antibody that binds selectively to autologous (eight cases) or allogeneic (four cases) short-term-cultured melanoma cells was assessed from EBV-transformed B lymphoblastoid cells. Forty-two cultures of EBV-transformed B cells that secreted IgM and 23 that secreted IgG antibodies gave patterns of differential reactivity with autologous or allogeneic melanoma. Antibodyforming B cells persisted in producing melanoma-reactive IgG and IgM for 8–21 weeks. Preselection of B cells by adsorption to tumor cell antigens before transformation enhanced the frequency of antibody secretion. The specificity of the antibody produced by the longest-producing culture appears to be restricted to a subset of melanomas. The patient from whom this tumor-restricted IgG-producing B cell was retrieved was unusual, having had a transient serum IgG of similar specificity, and having manifest a syndrome of vitiligo at the time of her development of serum antimelanoma antibody, followed by disease-free survival of resected recurrent metastatic melanoma to the present (more than 6 years). This study has given support to findings of conventional serology, revealing the production of melanoma-reactive antibody from B cells of patients who have demonstrable serological response to tumor.Supported by the American Cancer Society grant IM 433