Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide AE37 vaccine in breast cancer patients to prevent recurrence.

BACKGROUND: AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccine's efficacy. METHODS: Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed. RESULTS: The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12). CONCLUSION: The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted.

Responder T cells depleted of alloreactive cells react to antigen presented on allogeneic macrophages from nonresponder strains.

T cells from strains responder to the antigen poly(Glu40 Ala60) (GA) were depleted of alloreactive cells by bromo-deoxyuridine and light treatment, and were subsequently primed in vitro in GA presented by allogeneic macrophages from nonresponder strains. Antigen-specific secondary proliferative responses restricted by allogeneic Ia molecules of the macrophages were obtained in all strain combinations tested. These data indicate that Ir gene-controlled nonresponsiveness cannot be the result of a failure of antigen presentation.

H-2-controlled suppression of T cell response to lactate dehydrogenase B. Characterization of the lactate dehydrogenase B suppressor pathway.

We characterized the cell types involved in the H-2-controlled suppression of T cell response to lactate dehydrogenase B (LDHB). The suppressor effector (Tse) was found to be an Lyt-1+2+, J+ cell that recognizes antigen together with Ek molecules of antigen-presenting cells (APC). To become functional, the Tse cell requires a second signal from a nonspecific, Lyt-1+2-, J+ suppressor-inducer (Tsi) cell. The Tsi-Tse interaction is not subject to any genetic restriction. The target cell of suppression is an Lyt-1+2-, J- (most likely T helper [Th]) cell that recognizes LDHB in the context of A molecules on APC. The suppression is manifested in inhibition of the antigen-specific, A-restricted proliferation of Th cells. The interaction between Tse and Th is restricted by the A region of the H-2 complex. Because this restriction is determined by the receptor of Th cells, the mechanism of Th-Tse interaction most likely involves a concomitant recognition of LDHB and A region-controlled molecules by Th cells on the surface of Tse cells.

Monoclonal suppressor factor specific for lactate dehydrogenase B. I. Mechanism of interaction between the factor and its target cells.

Hybridomas secreting a monoclonal T suppressor-effector factor (TseF) were produced by fusion of a lactate dehydrogenase B (LDHB)-specific long-term T suppressor-effector (Tse) cell line with the BW5147 thymoma. A short exposure (4 h) to TseF completely suppresses the antigen-specific and A-restricted proliferation of LDHB-primed Lyt-1+2- [possibly helper (Th)] cells. The action of TseF on Th cells, as that of the Tse cells themselves, is antigen-specific and A-restricted. The interaction of TseF with Th cells involves two binding events, of which one occurs via antigen bridge, and the other represents the recognition of a factor-derived Ak-like moiety by the anti-Ak receptor of Th cells. The Ak-like moiety of the TseF carries the determinants that serve as restriction elements for antigen recognition by Th cells, and additional determinants demonstrable by T cell-specific monoclonal "anti-Ak" antibodies, however, it lacks serologically detectable determinants of the B cell-derived A alpha A beta class II Mhc molecules.

Tumor-infiltrating and circulating granulocytic myeloid-derived suppressor cells correlate with disease activity and adverse clinical outcomes in mycosis fungoides.

PURPOSE: Cutaneous T cell lymphomas (CTCL) are rare and histologically diverse lymphoproliferative neoplasms, with mycosis fungoides (MF) representing the most common disease subset. Given the emerging role of myeloid-derived suppressor cells (MDSC) as a clinically applicable biomarker in solid tumors, we sought to investigate the presence of tumor-infiltrating and circulating MDSC in early- and advanced-stage MF patients and evaluate their prognostic significance in patient overall survival. METHODS: Tumor-infiltrating MDSC were assessed immunohistochemically with Arginase-1 in 31 MF and 14 non-MF skin punch biopsies. Circulating MDSC were assessed with flow cytometry in freshly isolated PBMC from 29 MF patients. Granulocytic MDSC (G-MDSC) were defined as CD11b+CD14-CD15+ and monocytic MDSC (M-MDSC) were defined as CD11b+CD14+HLA-DRlow/-. RESULTS: MDSC infiltration occurred in approximately one-third (35.5%) of CTCL lesions, with a predilection for non-MF lesions (p < 0.05). The predominant morphology of MDSC was granulocytic. Although in MF lesions the presence of MDSC infiltrates did not correlate with clinical stage, it conferred significantly worse overall survival outcomes (p < 0.05). Circulating G-MDSC were significantly higher in MF patients compared to healthy donor controls (p < 0.0001), while M-MDSC did not show any statistically significant difference. G-MDSC were significantly higher in patients with active disease compared to patients who were in partial remission (p < 0.01). As with tumor-infiltrating MDSC, clinical stage did not correlate with circulating G-MDSC levels, while prospective overall survival analysis showed that patients with high levels of circulating G-MDSC have significantly inferior outcomes (p < 0.01). CONCLUSIONS: This study shows that G-MDSC could represent a novel and easily assessable biomarker in MF, which mirrors disease activity and can predict patient subgroups with aggressive clinical features.

The prognostic significance of immune changes in patients with renal cell carcinoma treated with interferon alfa-2b.

PURPOSE: To evaluate the response rate and the immunorestorative properties of subcutaneously administered interferon alfa-2b (IFN-A2b) in patients with advanced renal cell carcinoma (RCC) and to correlate the immune status with the clinical responses. PATIENTS AND METHODS: Twenty-six patients with advanced RCC were treated with recombinant IFN-A2b. The dose was increased progressively from 5 x 10(6) IU the first week to 10 x 10(6) IU the second week, and thereafter to 15 x 10(6) IU subcutaneously. RESULTS: Four patients (15%) achieved partial responses (PRs), and five patients (19%) had stable disease (S), whereas 17 patients (65%) progressed. In all patients, blood was withdrawn before IFN treatment and monthly thereafter. T lymphocytes after isolation from peripheral blood were tested for proliferation in the autologous mixed lymphocyte reaction (autoMLR) and allogeneic mixed lymphocyte reaction (alloMLR), interleukin-2 (IL-2) production, expression of IL-2 receptors during the alloMLR, and the production of interleukin-1 (IL-1) by peripheral-blood monocytes. Twelve patients were assessable, four patients had a PR, one patient had S, and seven patients had progressive disease. Striking increases were demonstrated in all parameters 1 month after treatment with IFN-A2b in the four patients who responded and the patient with S. Namely, the autoMLR responses showed a mean increase of 250%, the IL-2 production 247%, the expression of IL-2-specific receptors 446%, the alloMLR responses 160%, and the production of IL-1 262%. On the contrary, the nonresponders did not show any change in their overall immune status, and in some, deterioration of the already depressed immunologic functions was observed. CONCLUSIONS: Administration of IFN-A2b results in a marked potentiation of deficient cellular immune response in vitro in those patients with RCC who respond to the treatment. This may have prognostic significance, and certainly more patients are required to be studied for definite conclusions.

Granulocyte-macrophage colony-stimulating factor improves immunological parameters in patients with refractory solid tumours receiving second-line chemotherapy: correlation with clinical responses.

In this report, we studied the immunorestorative properties of subcutaneously administered granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with refractory solid tumours receiving second-line chemotherapy. Such patients exhibit abnormal immune responses in vivo and in vitro and, therefore, it was of interest to examine the effect of GM-CSF-induced immunomodulation on clinical response. We examined patients with primary malignant carcinomas (head and neck, n = 10; urogenital tract, n = 17; penis n = 6; colorectal, n = 8) who were treated with carboplatin (JM8), 300 ng/m2 on days 1 and 22, leucovorin (LV), 200 mg/m2 plus 5-fluoracil (5-FU), 500 mg/m2 on days 8, 15 and 29 and four cycles of daily injections with placebo or GM-CSF, 300 micrograms/day on days 3-6, 10-13, 17-20 and 24-27. Peripheral blood was collected from the patients one day after the end of each of the four-cycle injections with placebo or GM-CSF, namely on days 7, 14, 21 and 28. Peripheral blood mononuclear cells (PBMC) were tested in the autologous mixed lymphocyte reaction (AMLR) and for natural killer (NK) or lymphokine-activated killer (LAK) cell activity. Cytokine levels in serum were measured by immunoenzymatic (ELISA) assay. A total of 21 patients received a four-cycle regimen with GM-CSF (Group 1) and 20 were similarly treated with placebo (Group 2). All received standard chemotherapy as outlined above. Before GM-CSF treatment, all patients exhibited increased serum levels of interleukin-1 (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and prostaglandin E2 (PGE2) and decreased serum levels of IL-2. Cellular immune responses (AMLR, NK- and LAK-cytotoxicity) were also low in all patients. Five patients from Group 1 had a PR (partial response), 2 patients had CR (complete response), and 14 patients had stable disease. Seven patients from Group 2 showed progressive disease, 3 had a PR and 10 had stable disease. All immune parameters were significantly improved during treatment in Group 1 but remained unchanged or even deteriorated in Group 2. Administration of GM-CSF during treatment of cancer patients with conventional chemotherapeutic drugs results in a marked potentiation of deficient cellular immune responses in vitro and a change towards normalisation of cytokine serum levels. The results reported herein support the use of GM-CSF as immunopotentiator during chemotherapy, but more patients must be studied before definite conclusions can be drawn.

An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.

The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initially used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodimer-1, a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

Prothymosin-alpha enhances HLA-DR antigen expression on monocytes from patients with multiple sclerosis.

Monocytes from patients with multiple sclerosis (MS) express decreased numbers of class II major histocompatibility complex (MHC) antigens in peripheral blood and are poor stimulators in the autologous mixed lymphocyte reaction (autoMLR). We assessed the effect of prothymosin-alpha (ProT alpha) on the expression of MHC class II antigens by monocytes. Immediately after isolation, monocytes were analyzed for MHC class II antigen expression using a radiolabelled monoclonal antibody specific for a monomorphic determinant on HLA-DR antigens. After incubation with ProT alpha we observed significant increases in HLA-DR antigens on MS monocytes (1.5- to 4-fold increase compared to freshly isolated monocytes). Kinetic analysis revealed that enhancement peaked after 2 days of incubation with ProT alpha. The increase in HLA-DR antigen on MS monocytes resulted in the restoration of the deficient autoMLR in MS patients. This is the first demonstration suggesting a link between HLA-DR antigen expression and cellular immune defects in MS. The significance of low autoMLR responses for T suppressor levels in MS patients is discussed.

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis.

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Decreased expression of HLA-DR antigens on monocytes in patients with multiple sclerosis.

Immunofluorescence, cell binding assays and enzyme immunoassays were used to investigate the expression of class II major histocompatibility antigens on peripheral blood monocytes in 67 patients with multiple sclerosis. Monocytes from patients with active disease expressed fewer HLA-DR molecules on their surface than normal monocytes; furthermore the percentage of cells which exhibited detectable amounts of surface HLA-DR antigens was decreased in patients with active multiple sclerosis. During the inactive stage of the disease both deficiencies were milder, probably representing secondary pathogenetic phenomena. Quantitation of monocyte surface HLA-DR antigen expression could be valuable in assessing the clinical disease activity. The demonstration of a molecular defect in patients with multiple sclerosis will improve our understanding of the pathogenesis of the disease.

Cytokine serum levels, autologous mixed lymphocyte reaction and surface marker analysis in never medicated and chronically medicated schizophrenic patients.

A number of immunological parameters were studied in 82 DSM-III-R diagnosed schizophrenic patients (53 first drug-naive and 29 medicated chronic patients) as well as 62 healthy blood donors. The serum levels of interleukin-2 (IL-2), interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were measured and correlated with cellular immunity, as assessed by the autologous mixed lymphocyte reaction (AMLR). T lymphocyte subsets were also examined. The above immune parameters were reassessed in a subgroup of 11 first-episode, drug-naive patients 1month after neuroleptic medication. IL-2 serum levels were significantly lower, and IL-1beta and TNF-alpha were significantly higher in schizophrenic patients compared with healthy donors (P<0.001); no significant difference was observed between the two patient groups (medicated and not medicated). Abnormal cytokine serum levels were associated with decreased AMLR responses in vitro. Increased percentages of activated CD4+ and CD16+ natural killer cells, as well as cells expressing ICAM-1 adhesion molecules and IL-2 specific receptors, were detected in the patients. Immunophenotype studies revealed a higher percentage of cells expressing IL-2 receptors in medicated chronic schizophrenic patients compared with drug-naive patients. The abnormal cytokine production in vivo, along with the low AMLR responses in vitro, and the high percentage of activated CD4+ lymphocytes presented in this study suggest alterations in the immune system of schizophrenic patients (medicated or not medicated) consistent with immune activation.

Monocyte disorders associated with T cell defects in patients with solid tumors.

T cells proliferate in response to autologous monocytes in the autologous mixed lymphocyte reaction (AMLR). AMLR was found to be impaired in patients with advanced cancer (stages III and IV), whereas normal values were found in the early stages of the disease (stages I and II). Peripheral T lymphocytes from patients with advanced stages also exhibited a decreased ability to produce Interleukin-2 (IL-2) during an AMLR response, whereas production of IL-2 by T cells in stages I and II was comparable to that of normal donors. The impaired IL-2 production by T lymphocytes in the AMLR was associated with high concentrations of soluble interleukin-2 receptor (sIL-2R) in culture supernatants and reduced expression of membrane-bound interleukin-2 receptors (IL-2R) on the same AMLR-activated T lymphocytes. These abnormalities in T cells from cancer patients were demonstrated to be associated with dysfunctions of autologous monocytes. Thus monocytes from patients with advanced cancer exhibited diminished expression of HLA-DR antigens and produced low levels of Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor a (TNFa). No changes were detected in the expression of HLA-A, -B, -C antigens. The results presented here demonstrate that decreased in vitro T cell responses may be attributed to monocyte dysfunctions in these patients and provide new information for a better understanding of the impaired T cell function in cancer patients.

Local immunotherapy with interferon-alpha in metastatic pleural and peritoneal effusions: correlation with immunologic parameters.

Interferon-alpha (IFN-alpha) has been found to exert multiple enhancing effects in the immune response in vitro, IFN-alpha has been also used in clinical trials with variable response rates. The aim of the present study was to assess the effectiveness of IFN-alpha in the treatment of 25 patients with malignant pleural or peritoneal effusions caused by lung, and metastatic breast and ovarian cancer. Clinical responses were correlated with a) the ratio of malignant effusion (ME)-associated tumor cells to ME-associated mononuclear cells (MEMNC), b) MEMNC-derived cytotoxic responses against autologous or allogeneic tumor targets, and c) major histocompatibility complex (MHC) antigen expression on tumor cells. After partial drainage of pleural or peritoneal fluid, the patients were allocated to receive 10 million units of IFN-alpha by intrapleural or intraperitoneal injection at weekly intervals. The treatment was terminated if the malignant effusion disappeared or the patients had received four to six consecutive procedures. None of the patients received concomitant systemic chemotherapy or radiation therapy. MEMNC and tumor cells were isolated by centrifugation on discontinous percoll density gradients. Cytotoxic and phenotypic profiles of MEMNG were analyzed before and after treatment with IFN-alpha. An improvement was observed in patients with increased ratios of tumor cells to malignant effusion-associated mononuclear cells (MEMNC) in the effusions. In the same patients MEMNC were overpopulated by CD8+ T lymphocytes. In this group of patients the administration of IFN-alpha was associated with 25% complete response and 75% partial response rates. In contrast only 17% partial responses were achieved in patients whose effusions had decreased tumor cell to MEMNC ratios. The immunomodulation induced by IFN-alpha in vivo was also tested. Thus in a group of 6 patients, treatment with IFN-alpha resulted in the induction of CD8+ cell-mediated lysis against autologous tumor cells which was associated with PR (two patients). Natural killer (NK)-cell activity, and MHC class I antigen expression on effusion-associated tumor cells were also enhanced during treatment, but were not correlated with the outcome of the therapy since similar findings were also observed in the 4 non-responders. Local infusions of IFN-alpha provide an effective alternative treatment for malignant effusion in patients with lung, breast, and ovarian cancer. Increased ratios of tumor cells to MEMNC and the presence of CD8+ T lymphocytes within the malignant effusions may play an important role in the outcome of such a treatment with IFN-alpha but more patients need to be studied for definite conclusions.

A novel culture environment for generating mature human dentritic cells from peripheral blood CD14+ cells.

BACKGROUND: We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (PBMC) activated with anti-CD3-specific antibody (ACD3S) can induce, upon brief exposure, tumor-reactive lymphocytes in cancer patients. Here, we report that ACD3S can also induce rapid and stable maturation of dendritic cells (DC) which can be used as antigen presenting cells in in vitro protocols and for cancer immunotherapy in vivo. MATERIALS AND METHODS: A short (4-day) priming of CD14+ monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by only a 24 hour-incubation in ACD3S, is sufficient to generate fully mature and stable DC. RESULTS: These DC (i) stimulated strong T cell proliferative responses in the mixed lymphocyte reaction, (ii) when pulsed with unfractionated peptides from autologous tumor membrane extracts activated CD4+ T cells which proliferated in response to the autologous tumor and CD8+ cytotoxic T cells (CTL) which specifically lyse autologous tumor targets and (iii) produced high levels of IL-12. CONCLUSION: ACD3S-treated DC are functionally superior to monocyte-conditioned medium (MCM)-treated DC generated under the same short-term protocol and as efficient as DC induced by the standard 10-day protocol. Our data present an efficient and effective method for generating in a very short period of time, highly mature and functionally competent DC.

The prognostic value of alpha-thymosins in breast cancer.

The prognosis of breast cancer is of major clinical importance and several histopathological, biochemical and immunological variables have been reported to be useful prognostic factors. In the present study, we investigated the clinical significance of the levels of alpha-thymosins in relation to established prognostic factors, both in breast cancer and non-malignant breast lesions, alpha-thymosin levels were measured in breast tissue extracts by specific radioimmunoassays (RIAs) developed for human prothymosin alpha (ProT alpha) and parathymosin alpha (ParaT alpha) and were found to be significantly higher (up to 17.2-fold) in malignant but not in benign breast lesions, as compared to the values of the neighbouring tissues. When alpha-thymosin levels of the tumor samples were correlated with various known prognostic parameters a statistically significant correlation (p < 0.05) was observed between the levels of ProT alpha in malignant tissues to the grade of cancer and the lymph node status of the patient. An association between ProT alpha levels with increase in risk of death from breast cancer was also noticed. These results suggest that the expression of alpha-thymosins in human breast cancer a) depends on the proliferation status of the tumor, b) associates with established prognostic factors describing the metastatic potential of the tumor and c) is related to the overall survival of the patient. The fact that such relationships hold only for cancer tissues encourages the future use of alpha-thymosins as potent prognostic factors in breast cancer.

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD4+ and CD8+ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD4+ lymphocytes showed significantly higher SCE frequencies than autologous CD8+ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD4+ and CD8+ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD4+ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD8+ lymphocytes. Abnormalities in CD4+ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.

Gamma-interferon enhances the cytotoxic activity of interleukin-2-induced peripheral blood lymphocyte (LAK) cells, tumor infiltrating lymphocytes (TIL), and effusion associated lymphocytes.

The effect of gamma-interferon (IFN-gamma) on the induction of interleukin-2 (IL-2) activated killer cell activity was studied: (I) in peripheral blood lymphocytes (LAK cells) from cancer patients and healthy donors, (II) in lymphocytes infiltrating solid tumors (TIL) from melanoma and breast cancer patients, and (III) in pleural effusion associated lymphocytes (EAL) from patients with lung adenocarcinoma. The coculture of LAK, TIL and pleural effusion mononuclear cells (MNC) with several doses of IFN-gamma (10, 50, 250, and 1250 U/ml) and a low dose of IL-2 (10 U/ml) for 5 days resulted in a synergistic effect on the cytotoxicity of these cells against several tumor cell lines. Furthermore there was a potentiation in the proliferation of MNC after a 5-day culture. The induction of lymphocyte cytotoxicity by a combination of IFN-gamma with low doses of IL-2 may be helpful in designing more effective cancer immunotherapeutic protocols with LAK, TIL or EAL.

Monocyte defect causes decreased autoMLR in multiple sclerosis patients.

We have recently demonstrated that peripheral blood monocytes from patients with multiple sclerosis (MS) have a defect in stimulating autologous and allogeneic T lymphocytes. This defect was found to correlate with disease activity. We report here that T4+ cells from MS patients proliferate weakly in the autologous mixed lymphocyte reaction (autoMLR). Furthermore, we provide direct proof that the depressed T4+ cell proliferation is due to the monocyte functional (stimulatory) defect.

Production and characterization of a monoclonal antibody against epirubicin.

Epirubicin is an anthracyclinic antibiotic that has been increasingly used in the treatment of a variety of malignancies. A hybridoma producing monoclonal antibody (MAb) against the drug was obtained by cell fusion. The MAb is of the IgM isotype and has an affinity constant of 1.4 x 10(-7) M. Inhibition analysis showed that the antibody recognizes an epitope related to the C 4'-hydroxyl group in the amino sugar moiety, distinguishing epirubicin from the closely related doxorubicin. Since the precise mechanism of anthracycline action as well as its immunomodulating effects are still under scrutiny, powerful tools for their study are clearly needed. Moreover, this MAb can be useful in monitoring the levels of epirubicin in treated patients, as well as for the construction of bispecific antibodies in tumor-targeting immunotherapy.