Immunological effects of tumor vaccines: II. T cell responses directed against cellular antigens in the viral oncolysates

Peripheral blood mononuclear cells (PBMC) from patients with epithelial adenocarcinoma of the ovary treated in vivo with tumor vaccines administered as viral oncolysates (VO) exhibited significant proliferative responses in vitro to VO as well as to cellular oncolysates (CO). These responses were dependent on the concentration of VO or CO. VO consisted of lysates from the same ovarian tumor cell lines 2774 and CaOV3 infected in vitro with the avirulent strain of influenza virus A/PR8/34. CO were lysates from the same ovarian tumor cell lines without virus. Depletion experiments with the OKT3 monoclonal antibody plus complement demonstrated that these proliferative responses are T cell specific and under the control of the HLA-D region. Furthermore, these T cell responses are directed against both tumor tumor cellular components and tumor HLA class I molecules. These responses can be detected as early as two weeks after the first intraperitoneal injection of VO and reach a maximum 12-16 weeks after the first application of VO for treatment. PBMC from ovarian patients that received in vivo VO exhibited insignificant proliferative responses to CO prepared from human fibroblasts or tumor cell lines of hematopoietic origin. In contrast, they exhibited significant proliferative responses to CO prepared from a human cervix tumor cell line. These results demonstrate systemic T cell activation by antigens in the tumor vaccines in patients with epithelial ovarian carcinoma after in vivo intraperitoneal administration of VO.     

Functional and phenotypic heterogeneity of electrophoretically separated leukaemic B cells from patients with chronic lymphocytic leukaemia.

E-rosette negative largely leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL) were separated by density gradient electrophoresis, on the basis of their surface charge. The separated cells were pooled in seven fractions, according to their relative position in the electrophoretic distribution, and analysed by functional tests and cell-surface phenotypes. The ability of electrophoretically separated B cells from patients with CLL to differentiate into plasma cells in the presence of T cells from normal donors and in the pokeweed mitogen induced differentiation system was investigated. Lymphocytes able to differentiate into plasma cells under these conditions were highly enriched in the low-mobility fractions V, VI and VII. These plasma cells were of leukaemic origin, because they expressed only the light chain present on the cell surface of the leukaemic B cells before stimulation. Lymphocytes with Fc (IgG) receptors were relatively enriched in the high and intermediate mobility fractions I-IV, but they were present in the remaining of the fractions in smaller proportions. Lymphocytes with Fc (IgM) receptors were present in all fractions, but only in very small proportions in the very high mobility fraction I. Cells with complement receptors I and II were present in all fractions. Analysis of the density of cell surface immunoglobulin expression using the fluorescence-activated cell sorter, revealed that fractions of high and intermediate electrophoretic mobility (I-V) contained cells with both low and intermediate density of surface immunoglobulin, whereas the low mobility fractions VI and VII contained predominantly cells with low density of surface immunoglobulin. These results revealed significant phenotypic and functional heterogeneity of B cells from patients with CLL, suggesting the presence of subpopulations of leukaemic B cells in different differentiation or maturation stages.     

Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.

We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.     

HLA-DQB1*0301 association with increased cutaneous melanoma risk

Susceptibility to a variety of malignancies has been linked to human leukocyte antigen (HLA) genes, including the HLA class II allele DQBI*0301. To determine whether melanoma risk is associated with HLA class II alleles, molecular oligotyping of HLA class II-DRBI, -DQAI and -DQAI genes was performed for 45 patients with melanoma. The DQBI *0301 allele was present in 56% of melanoma patients vs. 27% of 200 local Caucasian controls. This difference was highly significant (Bonferroni'scorrected chi-square p = 0.003, OR = 3.4). No other class II allele tested was present at significantly increased or decreased frequency in melanoma patients. Furthermore, presence of DQBI*0301 in melanoma patients was associated with advanced disease. Melanoma patients carrying the DQBI*0301 allele presented on average with thicker primary tumors (mean 3.7 mm vs. 1.8 mm, 2-tailed p = 0.02) and were more likely to present with regional or distant metastatic disease (stages III-IV, 44% vs. 5%, chi-square p = 0.003), compared to melanoma patients without DQBI*0301. Risk of melanoma incidence or progression may be influenced by DQBI*0301 or a closely linked gene. © 1994 Wiley-Liss, Inc.