Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.

Dendritic cells, the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In three different well-characterized tumour models, naive mice injected with bone marrow-derived dendritic cells prepulsed with tumour-associated peptides previously characterized as being recognized by class I major histocompatibility complex-restricted cytotoxic T lymphocytes, developed a specific T-lymphocyte response and were protected against a subsequent lethal tumour challenge. Moreover, in the C3 sarcoma and the 3LL lung carcinoma murine models, treatment of animals bearing established macroscopic tumours (up to 1 cm2 in size) with tumour peptide-pulsed dendritic cells resulted in sustained tumour regression and tumour-free status in more than 80% of cases. These results support the clinical use of tumour peptide-pulsed dendritic cells as components in developing effective cancer vaccines and therapies.

G(AKSL2): a new cell surface antigen of the mouse related to the dualtropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody.

Normal mouse sera were tested for cytotoxic antibody to surface antigens of cultured monolayer cells infected with AKR-derived ecotropic MuLV, xentropic MuLV, or dualtropic MCF 247 MuLV. Antibody to ecotropic MuLV-infected cells was found in a proportion of C57BL/6, C3Hf/Bi, AKR-Fv-1b, and (C3Hf/Bi X AKR)F1 mice, but not AKR or (AKR X C3Hf/Bi)F1 mice. Antibody to xenotropic MuLV-infected cells was virtually restricted to C57BL/6 mice. Antibody to MCF 247-infected cells was found in all strains tested, including AKR mice. Absorption analysis of (C3Hf/Bi x akr)f1 and AKR-Fv-1b sera with selective reactivity for MCF 247-infected cells showed that these sera recognize distinctive antigens on MCF 247-infected cells that are not present on ecotropic or xenotropic MuLV-infected cells. The transplantable AKR spontaneous leukemia AKSL2 was found to be uniquely sensitive to the cytotoxic action of naturally occurring antibody to MCF 247-related antigens and absorption tests with AKSL2 as the target cell and sera from a single AKR-Fv-1b mouse have permitted the definition of a new MuLV-related cell surface antigen, which has been designated G(AKSL2). Thymocytes from young mice of high leukemia-incidence strains (AKR, C58, and PL) express G(AKSL2), whereas thymocytes from 12 other strains do not. In AKR mice, the antigen is expressed in higher amounts on cells from thymus and bone marrow than on spleen cells. All AKR spontaneous leukemias tested express G(AKSL2), as did three MuLV-induced leukemias arising in G(AKSL2)- strains. Five X-ray-induced leukemias of G(AKSL2)- strains were G(AKSL2)-, as were MuLV+ and MuLV- chemically induced sarcomas. In the limited survey conducted to date, natural antibody to G(AKSL2) has been restricted to strains expressing G(AKSL2) in their normal tissues: AKR, AKR congenic mice AKR-Fv-1b and AKR hybrid mice (C3Hf/Bi x akr)f1 and (C57BL/6 X AKR)F1. In vitro G(AKSL2) induction tests involving MuLV infection of cultured monolayer cells showed that 8 of 12 newly isolated dualtropic MuLV shared the property of G(AKSL2) induction with the prototype MCF MuLV, MCF 247. Of the 12 ecotropic MuLV tested, only the N-tropic MuLV isolated from a leukemia originally induced by Passage A Gross virus induced G(AKSL2). The xenotropic and amphotropic MuLV isolates tested lacked G(AKSL2) inducing activity. Recognition of the g(aksl2) system provides a way to trace the origin and natural history of a class of dualtropic MCF MuLV in the mouse and to determine whether natural antibody to G(AKSL2) plays a role in AKR leukemogenesis.

Chromosome assignment of the tumor-specific antigen of a 3-methylcholanthrene-induced mouse sarcoma.

Chemically induced sarcomas of inbred mice express antigens that are distinct and specific for each individual tumor. Chromosome assignment of tumor-specific antigens would help to elucidate the genetic basis of their diversity. Expression of the serologically defined Meth A sarcoma antigen is not suppressed in hybrid cells containing a complete foreign (Chinese hamster) genome. Chromosome and serologic analysis of microcell hybrids between Meth A sarcoma cells and Chinese hamster cells shows a clear correlation of Meth A antigen expression with the presence of the distal region of chromosome 12 (bands F1 and F2). Chromosome 16 may also be implicated. The significance of the close linkage of genes determining Meth A antigen expression with the immunoglobulin heavy chain gene cluster (on chromosome 12) and the lambda light chain genes (on chromosome 16) is discussed.

Cell surface antigens of chemically induced sarcomas of the mouse. I. Murine leukemia virus-related antigens and alloantigens on cultured fibroblasts and sarcoma cells: description of a unique antigen on BALB/c Meth A sarcoma.

As background for a serological definition of the unique antigens of chemically induced sarcomas, we have typed a series of fibroblast and sarcoma cell lines of BALB/c and C57BL/6 origin by cytoxicity and absorption tests for murine leukemia virus (MuLV)-related cell surface antigens and known alloantigens. 7 of the 17 cultured lines expressed the range of cell surface antigens associated with MuLV (GIX, GCSA, gp70, p30), and this was invariably associated with MuLV production. In nonproducer lines of C57BL/6 (but not BALB/c) origin, a MuLV-gp70-like molecule was found on the surface of fibroblasts and sarcoma cells. The alloantigenic phenotype of these MuLV+ and MuLV- cell lines was H-2D+, H-2K+, Thy-1.2+ or -, PC.1+ or -, Lyt-1.2-, Lyt-2.2-, Ia.7-, and TL.2-. A unique antigen was defined on the BALB/c ascites sarcoma Meth A with antisera prepared in BALB/c or (BALB/c X C57BL/6)F1 mice. Tissue culture lines derived from this tumor were MuLV-, which facilitated serological study of the antigen. Absorption analysis indicated that the antigen was restricted to Meth A; it could not be detected in normal or fetal BALB/c tissue MuLV+ or MuLV- fibroblast lines, 12 syngeneic or allogeneic sarcomas, or normal lymphoid cells from 13 different inbred mouse strains.

Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines.

Antigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic murine tumor models that bone marrow (BM)-derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J.I., T. Zorina, W.J. Storkus, C. Celluzzi, L.D. Falo, C.J. Melief, T. Ildstad, W.M. Kast, A.B. DeLeo, and M.T. Lotze. 1995. Nature Med. 1:1297-1302). However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony-stimulating factor-stimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W.J., H.J. Zeh III, R.D. Salter, and M.T. Lotze. 1993. J. Immunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 x 10(5) tumor peptide-pulsed DC dramatically suppressed the growth of weakly immunogenic tumors in day 4 to day 8 established MCA205 (H-2b) and TS/A (H-2d) tumor models, when applied in three biweekly intravenous injections. Using the immunogenic C3 (H-2b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4+ or CD8+ T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor-derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) gamma production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DC-induced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1-associated cytokines (such as IL-12, tumor necrosis factor alpha, IFN-gamma), and eventually, but not initially, blocked by anti-mIL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weakly immunogenic tumors, and serves as a basis for designing clinical trials in cancer patients.

Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells.

To study the cellular structures involved in NK and lymphokine-activated killer (LAK) cell function, we have produced a panel of mAbs that modulate the cytolytic function of a population of cells with LAK activity that derive from large granular lymphocyte (LGL)/NK cells (adherent LAK [A-LAK] cells). In this report, we describe an mAb (3.2.3; IgG1k) that recognizes a triggering structure that is expressed on rat LGL/NK cells and A-LAK cells. This epitope is also expressed on polymorphonuclear leukocytes (PMN). The expression of the epitope identified by mAb 3.2.3 increased progressively on A-LAK cells after culture in the presence of rIL-2. mAb 3.2.3 enhanced the cytolytic activity of NK and A-LAK cells against FcR+ target cells, but not FcR- target cells. However, this effect was not induced by F(ab')2 fragments of 3.2.3. This antibody also induced the release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzy esteresterase by A-LAK cells. These data suggest that the epitope identified by mAb 3.2.3 is on a triggering structure expressed on rat NK cells and A-LAK cells. The expression of the epitope recognized by mAb 3.2.3 on LGL/NK cells and PMN suggests that this structure may be analogous to that identified by the anti-CD16 (-FcR) mAbs. However, the molecule immunoprecipitated by mAb 3.2.3 was a 60-kD dimer composed of two 30-kD chains. These data suggest that mAb 3.2.3 recognizes a unique triggering structure. As mAb 3.2.3 is the first antibody recognizing a determinant with functional significance, selectively expressed on both rat NK cells and A-LAK cells, it will be a useful tool for the study of NK cell ontogeny and function, and the development of cells with LAK activity from the NK cell compartment.

A cell surface antigen of the mouse related to xenotropic MuLv defined by naturally occurring antibody and monoclonal antibody. Relation to Gix G(rada1), G(aksl2) systems of MuLV-related antigens.

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

Elevated levels of p53 antigen in cultured skin fibroblasts from patients with hereditary adenocarcinoma of the colon and rectum and its relevance to oncogenic mechanisms.

Skin fibroblasts (SFs) from patients with adenomatosis of the colon and rectum (ACR) were shown, in general, to express elevated amounts of the p53 antigen as determined by immunoprecipitation with the monoclonal antibody PAb421. Infection with simian virus 40 (SV40) induced expression of the p53 antigen in SFs from normal individuals and further amplified its expression in ACR cells. The half-lives of the p53 antigen in mock-infected ACR cells and in SV40-transformed normal or ACR cells were about 2 and 15 hours, respectively. No immediate differences were detected in the coding segment of the human p53 gene between normal and ACR cells by either Southern or Northern blot analysis. These results suggest that over-expression of the p53 antigen is due, in part, to the increased stability of the p53 antigen in ACR cells. Over-expression represents an early event that is possibly associated with initiation and promotion of inherited colon cancer.

Expression of a shared tumor-specific antigen by two chemically induced BALB/c sarcomas.

A tumor-specific antigen (TSA) expressed on the chemically induced BALB/c Meth A sarcoma and one of 22 other BALB/c sarcomas tested, CMS13, was detected in in vitro cellular and humoral assays. The distribution pattern of the TSA defined in a complement-dependent microcytoxicity assay by cytotoxic antibodies present in CMS13 antisera was similar to that detected by a cytotoxic T-cell clone, designated CTLL-MA10B, in an 18-h cell-mediated cytotoxicity assay. The serologically defined TSA was shown to be expressed on gp96, a Mr 96,000 glycoprotein isolated from Meth A cytosol with immunoprotective activity in in vivo tumor rejection assays. Immunization of BALB/c mice with Meth A, CMS13, or preparations of gp96 isolated from these sarcomas induced tumor resistance in these mice to Meth A and CMS13 but not CMS5, an antigenically unrelated sarcoma. These results suggest that the shared TSA is expressed on gp96 and is functional in tumor rejection assays.

Immunogenic properties of soluble cytosol fractions on Meth A sarcoma cells.

Tumor-associated transplantation antigen (TATA) was found to be present in fractions derived from the cytosol of the Meth A cell. Meth A ascites cells were disrupted nuclei and membranes were removed by low- and high-speed centrifugation, and the soluble protein was fractionated by ammonium sulfate precipitation and gel filtration chromatography. The TATA of the soluble cytosol fractions appears to be identical with the TATA solubilized from plasma membranes. The TATA of the cytosol fractions was found to be associated with proteins of an approximate apparent molecular weight of 60,000, specific for the Meth A tumor, and as immunogenic as the membrane-derived TATA. In addition, the most enriched TATA cytosol fraction shows inhibition of an antiserum capable of detecting a tumor-specific surface antigen of Meth A. These results suggest that Meth A TATA is not an integral membrane protein and may be related to the tumor-specific surface antigen detected serologically.

Identification of Meth A sarcoma-derived class I major histocompatibility complex-associated peptides recognized by a specific CD8+ cytotoxic T lymphocyte.

The finding that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) recognize peptide antigens (epitopes) bound to class I MHC molecules has accelerated efforts to identify CTL-defined tumor peptides for the development of peptide-based cancer immunotherapy. The Meth A sarcoma is probably one of the best studied of all murine tumors. It is extremely lethal unless protective immunity is induced. We recently reported the characterization of a cloned H-2Kd-restricted, CD8+ anti-Meth A CTL line (CTLMA-9C; Frassanito et al., Cancer Res., 54: 4424-4429, 1994). The cytotoxic reactivity of this CTL was shown to be restricted to Meth A sarcoma, and the results of the analysis of the immunogenicity of the CTL-resistant variant of Meth A, designated Meth A4R, indicate that the CTL-defined epitope is functional in tumor rejection. Here we have isolated class I MHC-associated peptides from Meth A sarcoma by mild acid treatment and resolved them into sixty fractions by reverse phase-HPLC. These fractions were then tested for their ability to sensitize the DBA/2 mastocytoma P815 to cytolysis by the anti-Meth A CTL. A single fraction, fraction 27, has been repeatedly identified as containing the CTL-defined epitope. Peptides eluted from the CTL-resistant variant, Meth A4R, failed to sensitize P815 to cytolysis by the anti-Meth A CTL, while fraction 27 derived from Meth A sensitized Meth A4R to lysis by the CTL. These findings confirm the peptide nature of the epitope recognized by CTL on the surface of Meth A. Our future efforts will focus on the identification and sequence analysis of the tumor peptides and the development of a tumor peptide-based vaccine model for immunotherapy.

p53-based immunotherapy of cancer.

Immunotherapy targeting p53 missense mutations, which occur in nearly half of all human tumors, is limited by several factors, including the constraints of antigen processing and presentation. Due to the accumulation of mutated p53 molecules in tumors expressing p53 mutations, an alternative approach would be to target wild-type sequence, CTL-defined p53 epitopes. Obviously, the possibility of an autoimmune response is a major potential drawback to this therapy. Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC) generated in the presence of GM-CSF/IL-4 and prepulsed with the H-2Kd-binding wild-type p53(232-240) peptide has been shown to induce anti-peptide CTL. These effectors were cross-reactive against sarcomas expressing p53 missense mutations outside of the p53(232-240) epitope, but not within it. Mitogen-activated splenocytes, which express elevated levels of p53, were not sensitive to these CTL. The p53 peptide-pulsed DC-based vaccine was shown to be effective in inducing tumor rejection in immunization and therapy models in the absence of any observable deleterious effect on naive mice. The murine model has now been extended to include the use of genetically modified DC-based vaccines as well.

A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck.

Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125-134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients.

Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells.

CTLs recognizing the HLA-A2.1-restricted, wild-type sequence p53 epitopes p53(149-157) and p53(264-272) were generated from CD8-enriched populations of nonadherent peripheral blood lymphocytes (PBLs) obtained from healthy donors. The PBLs were restimulated in vitro with peptide-pulsed granulocyte macrophage colony-stimulating factor- and interleukin (IL)-4-induced autologous dendritic cells in the presence of IL-6 and IL-12 and subsequently cultivated with IL-1alpha, IL-2, IL-4, IL-6, and IL-7. Bulk anti-p53(264-272) CTL populations were generated from PBLs obtained from two of five donors. Both CTL populations were cytotoxic against peptide-pulsed HLA-A2+ target cells, but not against untreated target cells. A CD8+ anti-p53 CTL clone designated p264#2 was isolated from one of the bulk populations. It was found to have an intermediate affinity of approximately 10(-9) M for the epitope and to mediate cytotoxicity against several human tumor cell lines, including the squamous cell carcinoma of the head and neck cell line SCC-9, which is known to present the wild-type sequence p53(264-272) epitope. In addition, CTLs reactive against p53(149-157)-pulsed targets as well as a HLA-A2+ tumor cell line were cloned from a bulk population of antitumor CTLs obtained from one of the five normal PBLs restimulated with this epitope. The results indicate that CTLs recognizing wild-type sequence epitopes can be generated from precursors present in PBLs obtained from some normal individuals using autologous dendritic cells as antigen-presenting cells and suggest that vaccine strategies targeting these epitopes can lead to antitumor CTL generation, thereby emphasizing the therapeutic potential of p53-based cancer vaccines.

Expression of diverse and functional TCR gamma and Ig heavy chain transcripts in fetal liver cells cultured with interleukin-7.

We have previously shown that specific T cell receptor (TCR) gamma V regions genes (V gamma 4 and V gamma 6) are rearranged and expressed by murine fetal liver (FL) cells cultured with IL-7. The present studies determined that the sequences of the TCR V region gene transcripts expressed in response to IL-7 included diverse and functional sequences expressed by thymocyte and peripheral V gamma 4+ and V gamma 6+ T cells, indicating that the IL-7-induced expression of these genes is functionally relevant and mimics normal in vivo developmental events of gamma delta T cells. We found that more than 50% of these TCR transcripts had N region diversity. The presence of N region diversity indicates that these TCR rearrangements took place in vitro, presumably in response to IL-7, because fresh (uncultured) FL cells do not produce detectable terminal deoxynucleotidyl transferase (TdT) mRNA or protein. We also found that 100% of immunoglobulin (Ig) VH7183-JH4 transcripts from FL cells cultured with IL-7 had N region diversity at the V-DJ region, while only 40% of Ig VH7183-JH4 transcripts from FL cells cultured in the absence of IL-7 had N region diversity at this region. FL cell cultures supplemented for 7 days with IL-7 had increased TdT mRNA and protein levels. However, since 1-day culture of FL cells with or without IL-7 resulted in induction of expression of TdT, IL-7 probably does not directly stimulate TdT expression, but increases the development and expansion of TdT+ lymphoid cells. These findings implicate IL-7 as a regulator of the molecular signals involved in controlling TCR gamma rearrangement and diversity, and provide an in vitro system for studying the regulation of TdT and N region diversity in B and T lymphoid progenitors by environmental signals.

Tumor-specific antigens of mouse tumors.

This article reviews the serological analysis of cell surface antigens of chemically induced sarcomas of murine origin. This analysis, which was aimed at molecular characterization of the highly restricted tumor specific transplantation antigens of sarcomas, led to the initial biochemical identification and characterization of p53. The results of this early study clearly pointed to p53 as playing a key role in cellular proliferation and transformation. Current research of the tumor-specific or -associated antigens of chemically-induced sarcomas and melanomas of murine origin is also discussed.

p53 content in relation to cell growth and proliferation in murine L1210 leukemia and normal lymphocytes.

The protein p53 has been reported to be associated with cell transformation and/or proliferation. Using p53 monoclonal antibodies we estimated by flow cytometry the relative content of this protein in individual L1210 leukemic cells from exponentially growing and plateau-phase cultures and compared it with that in normal thymocytes of parental DBA/2 mice and in mitogen-stimulation and nonstimulated human lymphocytes. Simultaneous differential staining of p53 vs DNA and p53 vs RNA, followed by bivariate analysis, made it possible to estimate p53 with respect to cell position in the cell cycle and correlate it with RNA (predominantly rRNA) content. The data show that in exponentially growing L1210 cells p53 is being progressively accumulated during the G1, S and G2 phases and that the content of p53 and RNA are highly correlated. In plateau L1210 cultures most cells are arrested in G1, some cells, however, still continue to progress through S and G2. In these cultures the p53 content of all cells, regardless of the phase of the cell cycle, is diminished and the decrease in p53 is more pronounced than that of RNA or total protein content. The normal thymocytes as well as the stimulated lymphocytes show bimodal distribution with respect to p53 expression, compatible with the assumption that the cycling cells have increased expression of this protein related to the G0 cells. Some cycling cells, however, have minimal p53. The quantitative p53 immunofluorescence data were confirmed by the immunoprecipitation and gel electrophoresis. The results suggest that expression of p53 in leukemic and normal cells is more correlated with cell growth than with entrance to the cell cycle or progression through particular phases of the cycle.

Similarities between transplantation antigens on methylcholanthrene-induced sarcomas and T-cell regulatory molecules.

Successful interaction among T-cell subsets requires, among other things, homology at certain genetic loci that code for cellular interaction molecules (CIM). One such interaction, the induction of an acceptor-cell population by an Ly-1 T-suppressor-inducer cell, is controlled by genes that map to the variable region of the immunoglobulin heavy chain (Igh) complex. If the suppressor-induced cells (or their cell-free products) do not share Igh-V polymorphisms with their acceptor cells, the induction event fails to take place. Recently, structural genes of a transplantation antigen on the methylcholanthrene-induced sarcoma Meth A were mapped to the same region of chromosome 12 as the Igh gene complex. We tested whether there was any relationship between the Meth A transplantation antigen and T-cell regulatory molecules by using antisera against the Meth A antigen to block this particular Igh-linked T-T interaction. We found that isoantisera against a large number of methylcholanthrene-induced sarcomas tested were capable of blocking the induction of T-suppressor cells so long as the inducer and acceptor cells bore the Igha polymorphism. Further, we found a structural gene on MC-induced tumors that could absorb out this activity, and the structural gene for this antigen is coded for the same region as the Igh gene loci. The antisera binds to the I-J+ portion of a T-cell regulatory molecule Ly-1 TsiF, the portion of the molecule that has no specificity for antigen and imparts the Igh-linked genetic restriction. The implications of these findings for both oncology and immunology were discussed.

Serological characterization of a shared melanoma-associated antigen of mouse melanomas: relationship to the B700 glycoprotein.

A group of murine melanomas, consisting of the C57BL/6 melanomas JB/RH and B16 F10, and the C3H/He melanoma K1735, have been shown to be cross-immunogenic in tumour rejection assays, and to be antigenically distinct from the DBA/2 melanoma S91. In addition, the M(r) 65,000 melanoma-associated glycoprotein, B700, isolated from the B16F10 melanoma, was shown to induce a pattern of cross-immunity in semi-syngeneic mice, which was identical to that obtained with the melanomas. An antigen expressed by the JB/RH melanoma has been serologically defined by complement-dependent cytotoxic antibodies present in the sera of semi-syngeneic mice hyperimmunized against this melanoma. This antigen, designate JB/RH antigen, also was detected on JB/MS, B16 F10 and K1735 melanomas, but not on S91 melanoma. The cytotoxic antibodies defining the JB/RH antigen could be absorbed by the B700 glycoprotein isolated from B16 F10 melanoma, but not from S91 melanoma. Monoclonal antibodies were generated and shown to recognize a M(r) 65,000 antigen expressed by B16 F10 melanoma, but not S91 melanoma, suggesting that they have a specificity similar to that of the anti-JB/RH serum.