T-cell receptor biases and clonal proliferations in blood and pleural effusions of patients with lung cancer.

We sought evidence that pulmonary carcinomas mediate a cellular immunologic response by analyzing T-cell antigen receptor beta-chain variable gene (TCRBV) repertoires of lymphocytes from peripheral blood (PBL) and malignant pleural effusions (PEL) of five lung cancer patients. Expression levels of 27 TCRBV were quantitated by multiprobe RNase protection assay (RPA), and clonal expansions were identified by sequence enrichment nuclease assay (SENA) and junctional region sequencing. Abnormal TCRBV expansions were identified in all subjects by RPA (mean 6.9 +/- 1.7/patient), and their number closely correlated with elapsed time since initial diagnosis (r = 0.97). SENA, performed in specimens from three patients, confirmed the presence of mono or oligoclonality in 48% of abnormal RPA expansions, and further identified T-cell clones among TCRBV with normal expression levels. The majority of clonal expansions were among PEL, and were nearly equally divided between CD4 and CD8. These data show that T-cell repertoires of lung cancer patients are characterized by marked abnormalities and frequent clonal expansions, most likely representing responses to unique, tumor-specific antigens (TSA). Moreover, this process appears exaggerated among PEL, further suggesting that malignant effusions include local proliferations of tumor reactive T cells. These findings imply the presence of lung cancer TSA capable of eliciting cellular immune responses and raise the possibility that selective immunotherapies can ultimately be developed.

T cell receptor biases and clonal proliferations among lung transplant recipients with obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the most serious late complication of lung transplantation, but the pathogenesis of this disorder has not been elucidated. We sought evidence that OB is mediated by a cellular immunologic response by characterizing T cell antigen receptor beta-chain variable gene (TCRBV) repertoires in lung allograft recipients. Expression levels of 27 TCRBV among recipients were determined by multiprobe RNase protection assay after PCR amplification. In comparison to recipients with no evidence of rejection (n = 9), the PBL TCRBV repertoires of OB subjects (n = 16) exhibited more frequent expansions (16 vs. 9% of all measured TCRBV, P < 0.02), and the magnitudes of these abnormalities were greater (8.2 +/- 0.8 vs. 4.5 +/- 0.3 SD from mean normal values, P < 0.01). TCRBV sequencing showed these expansions were composed of clonal or oligoclonal populations. Thus, T cell responses in the recipients are marked by highly selective clonal expansions, presumably driven by indirect recognition of a limited number of immunodominant alloantigens. These processes are exaggerated among allograft recipients with OB, implying that cognate immune mechanisms are important in the pathogenesis of the disorder. Furthermore, the prominence of finite, distinct TCR phenotypes raise possibilities for development of novel diagnostic modalities and targeted immunotherapies for OB and other manifestations of chronic allograft rejection.

Intrathymic injection of polynucleosomes delays autoantibody production in BXSB mice.

T-cell dependent autoimmunization with nucleosomes appears to be an early event in the induction of lupus anti-chromatin antibodies. We investigated this phenomenon by injecting H1-stripped chromatin polynucleosomes into the thymuses of BXSB male lupus-prone mice. In comparison to uninjected controls, the production of IgG antichromatin, anti-native DNA, and anti-denatured DNA were significantly reduced among the injected animals for a period of 8 to 10 weeks. Peripheral T-cells from intrathymic (i.t.)-treated animals showed decreased proliferative responses to polynucleosomes compared to those from uninjected controls. Treatment did not affect T-cell antigen receptor V beta profiles, excluding the possibility that results were due to superantigen-imposed deletions. In situ staining using the TUNEL method demonstrated that generation and phagocytosis of apoptotic material in thymuses of unmanipulated BXSB mice were similar to normal controls. These findings show that polynucleosomes likely comprise the antigens for helper T-cell engagement and induction of lupus-associated anti-chromatin antibodies. Bypassing the underlying defect of T-cell tolerance for polynucleosomal antigens among BXSB mice, by i.t. administration of exogenous polynucleosomes, results in abrogation of autoantibody production.

Monoclonal antibody KS1/4-methotrexate immunoconjugate studies in non-small cell lung carcinoma.

The antigen reactive with murine monoclonal antibody (MAb) KS1/4 is expressed on epithelial malignancies and some normal epithelial tissues. Studies were undertaken to evaluate KS1/4-methotrexate (KS1/4-MTX) immunoconjugate in patients with advanced non-small cell carcinoma of the lung. Eleven patients in two different groups received KS1/4-MTX in two different escalating dose infusion schedules with a maximal tolerated dose of 1,750 mg/M2 and a cumulative dose of MTX of 40 mg/M2. Toxicities were similar in both groups and included fever, anorexia, nausea, vomiting, diarrhea, abdominal pain, guaiac positive stool, and hypoalbuminemia. Two patients had an associated aseptic meningitis. One patient had a 50% decrease in two lung nodules without a change in lymphangitic infiltrates. This patient received a second course of treatment and developed an immune complex-mediated arthritis and serum sickness. Four patients mounted a human antimouse antibody response. Post-treatment tumor biopsies documented binding of MAb KS1/4. These studies document the feasibility and potential usefulness of a MAb directed against tumor-associated antigens with the targeting of chemotherapeutic drugs in patients with non-small cell lung carcinoma.

Biosynthesis and glycosylation of the carcinoma-associated antigen recognized by monoclonal antibody KS1/4.

Monoclonal antibody KS1/4 recognizes an epitope expressed on the cell surface of human adenocarcinoma cells and certain epithelia. Western blotting analyses of tumor cell extracts utilizing KS1/4 reveal staining of a major Mr 40,000 band and a minor Mr 42,000 band. Both components are also detectable in KS1/4 immunoprecipitates of L-[35S]methionine- and D-[3H]glucosamine-labeled human lung tumor cell extracts. When synthesis occurs in the presence of tunicamycin or when the immunoprecipitates are treated with peptide:N-glycosidase F, a single polypeptide component (Mr 37,000) is precipitated. Immediately following translation, digestion of Mr 40,000 and Mr 42,000 glycoproteins with endo-beta-N- acetylglucosaminidase H also yields a single polypeptide component at Mr 37,000. However, over a 3-h period beginning at 10 min posttranslation, a Mr 39,000 major component and a Mr 41,000 minor component gradually appear in the endo-beta-N-acetylglucosaminidase H digests as the Mr 37,000 component gradually disappears. Analysis of tryptic glycopeptides derived from the Mr 40,000 and 42,000 components suggests that the two components differ by the addition of one extra oligosaccharide to the Mr 42,000 component. Nonequilibrium pH gradient electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of KS1/4 immunoprecipitates resolves each of the two components into multiple spots. Digestion of the KS1/4 immunoprecipitates with neuraminidase prior to two-dimensional analysis or immunoprecipitation of short pulse-labeled extracts reduces the number of spots to three each at the Mr 40,000 and Mr 42,000 positions. Digestion of the KS1/4 immunoprecipitates with peptide:N-glycosidase F, immunoprecipitation of extracts labeled in the presence of tunicamycin, or endo-beta-N-acetylglucosaminidase H digestion of immunoprecipitates of short pulse-labeled extracts prior to two-dimensional analysis results in a single series of Mr 37,000 spots, suggesting that the polypeptide portions of the Mr 40,000 and Mr 42,000 components may be identical. Endo-beta-N-acetylglucosaminidase H digestion of KS1/4 immunoprecipitates of short pulse-labeled extracts, followed by nonequilibrium pH gradient electrophoresis, V8 protease digestion, and polyacrylamide gel electrophoresis revealed an apparently identical set of polypeptides derived from each of the three Mr 37,000 spots, suggesting that the three spots derive from highly similar polypeptides.

Antigenic mimicry of a human cellular polypeptide by Mycoplasma hyorhinis.

A 46-kilodalton (kDa) polypeptide was immunoprecipitated from radiolabeled extracts of human cell lines infected with Mycoplasma hyorhinis by murine monoclonal antibodies PF/2A and ML77. Both of these antibodies also reacted in an enzyme-linked immunosorbent assay (ELISA) with M. hyorhinis cells and with human and nonhuman cell lines infected with M. hyorhinis but failed to react with A7573 cells infected with any of 10 other species of the order Mycoplasmatales. PF/2A also reacted in the ELISA with certain human cell lines that were demonstrated to be free of mycoplasma infection. From extracts of these lines, a polypeptide antigen that appeared as a 24-kDa doublet on polyacrylamide gels was immunoprecipitated by PF/2A. When the PF/2A-reactive human cell lines were infected by M. hyorhinis, both the 46- and 24-kDa antigens were immunoprecipitated by PF/2A. ML77 did not react in the ELISA with any noninfected human cells tested and failed to immunoprecipitate a 24-kDa component from any human cells. In Western blotting analyses of extracts of M. hyorhinis cells, both PF/2A and ML77 stained a 46-kDa band. PF/2A also stained 24-kDa bands in Western blotting analyses of reactive human cells and M. hyorhinis cells, although a 24-kDa component was not precipitated from extracts of M. hyorhinis cells by PF/2A.

Antigens associated with human squamous cell lung carcinoma defined by murine monoclonal antibodies.

A panel of 12 monoclonal antibodies that preferentially react with human squamous lung carcinoma cells has been produced. All are reactive with fresh frozen sections of squamous cell lung carcinoma tissues in immunoperoxidase assays and are unreactive with lymphoblastoid cells, red blood cells, and fibroblasts in enzyme-linked immunosorbent assay. At least eight of these antibodies interact with cell surface components. These reagents can be subdivided into four groups based upon their reactivities. Groups 1 to 3 are unreactive with normal liver, lung, kidney, colon, spleen, and pancreas in immunoperoxidase assays. Group 1 antibodies (PF1/A, PF1/B, PF1/C, PF1/D, and PF1/E) are all of IgG3 subclass and immunoprecipitate nonsulfated glycoprotein components with molecular weights of 80,000 and 180,000 and a nonglycosylated polypeptide with a molecular weight of 38,000. Group 1 antibodies are also reactive with some lung adenocarcinomas and, with the exception of PF1/E, stain certain differentiated strata within normal adult plantar and fetal epidermis. Group 2 antibodies (PF2/A and PF2/B) react also with breast, gastric, and colonic adenocarcinomas and some tumors of neuroectodermal origin. Group 2 antibodies, which are both of IgG3 subclass immunoprecipitate a nonglycosylated Mr 24,000 polypeptide. Group 3 antibodies (PF3/A, an IgG1; PF3/B, an IgGM; and PF3/C, an IgG2a) react additionally with certain other tumors, as well as with normal adult and fetal epidermis. Group 4 antibodies (PF4/A, an IgG2a; and PF4/B, an IgG1) are less specific than those of the preceding groups, as they react with some normal tissues, including pancreatic islets and pneumocytes, as well as with a variety of adenocarcinomas and tumors of neuroectodermal origin. PF4/A and PF4/B immunoprecipitate Mr 100,000 and 95,000 glycoproteins, respectively.