Roberto S. Accolla

Subsets of human Ia-like molecules defined by monoclonal antibodies

Abstract Two human Ia molecule subsets were denned by their activity with hybridoma antibodies raised against Daudi cell membranes. The first subset (NG-1) was recognized by two distinct hybridoma products D1-11 and D1-12. The second subset (NG-2) was recognized by the other hybridoma, D4-22. The two subsets are present in all Ia preparations tested irrespective of their HLA-DR phenotype. In addition, each particular HLA-DR phenotype can be expressed on both subsets. The relative contribution of these subsets shows considerable variations. Since both D1-12 and D4-22 recognize small Ia subunit, the two subsets appear to carry alternative forms of this subunit

Targeted Delivery of Tumor Necrosis Factor-α to Tumor Vessels Induces a Therapeutic T Cell–Mediated Immune Response that Protects the Host Against Syngeneic Tumors of Different Histologic Origin

Purpose: We sought to demonstrate that a single systemic administration of L19mTNFα (a fusion protein constituted by the scFv L19 specific for the oncofetal ED-B domain of fibronectin and tumor necrosis factor α, TNFα) in combination with melphalan induced complete and long-lasting tumor eradication in tumor-bearing mice and triggered the generation of a specific T cell–based immune response that protects the animals from a second tumor challenge, as well as from challenges with syngeneic tumor cells of different histologic origin. Experimental Design and Results: Treatment with L19mTNFα, in combination with melphalan, induced complete tumor regression in 83% of BALB/c mice with WEHI-164 fibrosarcoma and 33% of animals with C51 colon carcinoma. All cured mice rejected challenges with the same tumor cells and, in a very high percentage of animals, also rejected challenges with syngeneic tumor cells of different histologic origin. In adoptive immunity transfer experiments, the splenocytes from tumor-cured mice protected naive mice both from C51 colon carcinoma and from WEHI-164 fibrosarcoma. Similar results were also obtained in adoptive immunity transfer experiments using severely immunodepressed mice. Experiments using depleted splenocytes showed that T cells play a major role in tumor rejection. Conclusions: The results show that the selective targeting of mTNFα to the tumor enhances its immunostimulatory properties to the point of generating a therapeutic immune response against different histologically unrelated syngeneic tumors. These findings predicate treatment approaches for cancer patients based on the targeted delivery of TNFα to the tumor vasculature.

CIITA-Induced MHC Class II Expression in Mammary Adenocarcinoma Leads to a Th1 Polarization of the Tumor Microenvironment, Tumor Rejection, and Specific Antitumor Memory

Purpose: We have shown previously that the MHC class II–negative murine TS/A adenocarcinoma is rejected in vivo if induced to express MHC class II molecules by transfection of the MHC class II transactivator CIITA. In this study, we explored the immunologic basis of tumor rejection and the correlation between histopathology of tumor tissue and immune rejection. Experimental Design: Stable TS/A-CIITA transfectants were generated and injected into mice. In vivo cell depletion, immunohistochemistry of tumor tissues, and immune functional assays were done to assess the cellular and immunologic basis of rejection. Results: Ninety-two percent of mice injected with TS/A-CIITA rejected the tumor and were completely resistant to challenge with parental TS/A. Only CD4+ and CD8+ cells were required for rejection. The tumor microenvironment in TS/A-CIITA-injected mice changed dramatically when compared with the TS/A parental-injected mice. Rapid infiltration with CD4+ T cells followed by dendritic cells, CD8+ T cells, and granulocytes was observed. Importantly, TS/A-CIITA cells could act as antigen-presenting cells because they process and present nominal antigens to CD4+ T cells. Tumor-specific CD4+ T cells of TS/A-CIITA-injected mice had the functional characteristics of Th1 cells and produced IFN-γ and this was relevant for generation and maintenance of protective antitumor response, because IFN-γ knockout mice were no longer rejecting TS/A-CIITA tumor cells. Conclusion: CIITA-dependent MHC class II expression confers to TS/A tumor cells the capacity to act as a protective vaccine against the tumor by triggering tumor antigen presentation to T helper cells, antitumor polarization of cellular and soluble components of the tumor microenvironment, and establishment of antitumor immune memory.

APC gene mutations and allelic losses in sporadic ampullary tumours: Evidence of genetic difference from tumours associated with familial adenomatous polyposis

We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early‐stage tumours (3 adenomas, 3 carcinomas) and 12 advanced‐stage cancers. Eleven PCR‐amplified polymorphic sequences were used to analyse 5qLOH. APC mutations were investigated both by an in vitro APC‐protein truncation test and by single‐strand conformation polymorphism analysis. Mutations in the Ki‐ras, N‐ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including 1 adenoma, 3 early‐ and 4 advanced‐stage cancers; APC mutations in 2 adenomas and 1 advanced‐stage carcinoma; Ki‐ or N‐ras mutations in 3 adenomas and 3 advanced‐stage cancers; p53 mutations in 2 early‐stage and 7 advanced‐stage adenocarcinomas. Our results suggest that 5qLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (17% vs. 64%) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions.

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.

Ia-negative B-cell variants reveal a coordinate regulation in the transcription of the HLA Class II gene family

Class lI genes of the human major histocompatibility complex (HLA) encode a heterogeneous group of highly polymorphic cell surface glycoproteins, the Ia antigens. These molecules consist of two noncovalently linked subunits, the ~ and/~ chains (Shackelford et al. 1982). A third chain, termed the p33 invariant chain, has been found intracellularly in association with Ia antigens. It has been postulated to be involved in the assembly and/or the transport of the c~-/¢ heterodimer to the cell surface (Owen et al. 1981, Kvist et al. 1982). Ia antigens are primarily expressed on B lymphocytes, macrophages and activated T cells. They act as restriction elements for a correct cooperation between macrophage and T cells and between T and B cells (Benacerraf 1981). At least three subsets of polymorphic Ia antigens have been distinguished at the structural level: the DR, DC, and SB molecules (Corte et al. 1981, Hurley et al. 1982). Further subsets have been defined, for instance the NG1 and NG2 molecules (Accolla et al. 1981a) which are structurally related to the serologically defined DR antigens. The structural heterogeneity of the human Ia antigens may correlate with the various functions in which they are involved (Accolla et al. 198 lb, Shaw et al. 1980, Corte et al. 1982). Ia-negative B-cell variants were derived from Raji cells (DR 3,W6-DC1) after Yray irradiation and immunoselection with Ia-specific monoclonal antibodies (Mabs) and complement (Accolla 1983). Among them, the variants RJ 2.2.5 and RJ D1.2 were derived after immunoselection with two distinct Ia-specific Mabs, the BT 2.2 (anti-NG2 subset) and the Dl-12 (anti-NG1 subset), respectively. Although the immunoselection was performed with monomorphic Ia-specific Mabs directed

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

Different levels of control prevent interferon-γ-inducible HLA-class II expression in human neuroblastoma cells

The HLA class II expression is controlled by the transcriptional activator CIITA. The transcription of CIITA is controlled by different promoters, among which promoter-IV is inducible by IFN-γ. We analysed the regulation of HLA class II molecules by IFN-γ in a large series of human neuroblastoma cell lines. No induction of surface or intracellular HLA class II molecules and of specific mRNA was observed, in all neuroblastomas, with the exception of a nonprototypic cell line, ACN. In a large subset of neuroblastomas IFN-γ induced expression of CIITA mRNA, derived from promoter-IV, which was not methylated. In contrast, in another subset of neuroblastomas, CIITA was not inducible by IFN-γ and CIITA promoter-IV was either completely or partially methylated. Interestingly, the use of DNA demethylating agents restored CIITA gene transcriptional activation by IFN-γ, but not HLA class II expression. The defect of HLA class II was not related to alterations in RFX or NF-Y transcription factors, as suggested by EMSA or RFX gene transfection experiments. In addition, the transfection of a functional CIITA cDNA failed to induce HLA class II expression in typical neuroblastoma cells. Confocal microscopy and Western blot analysis suggested a defective nuclear translocation and/or reduced protein synthesis in CIITA-transfected NB cells. Altogether, these data point to multiple mechanisms preventing HLA class II expression in the neuroblastoma, either involving CIITA promoter-IV silencing, or acting at the CIITA post-transcriptional level.

MHC: orchestrating the immune response

The major histocompatibility complex (MHC) is one of the most interesting and intriguing genetic systems, and continues to amaze geneticists, biochemists and particularly, immunologists and pathologists. The latest achievements on the structure and function of MHC genes and their products were discussed at a recent workshop.*

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.