The expression of CD70 and CD80 by gene-modified tumor cells induces an antitumor response depending on the MHC status.

The expression of costimulatory molecules such as CD70 or CD80 by gene-modified tumor cells has been shown to enhance the antitumor immune response based mainly on T lymphocytes. However, most human tumors show defects of major histocompatibility complex (MHC) expression, preventing them from being recognized by MHC-restricted T cells. To investigate if coexpression of CD70 and CD80 costimulatory molecules induces comparable antitumor responses in low and high MHC-expressing tumor cells, we used two low immunogenic murine tumor models, the B16.F10 melanoma and the TS/A mammary adenocarcinoma cell lines expressing, respectively, low and high levels of MHC class I molecules. Transfection of both CD70 and CD80 genes resulted in an increased capacity of gene-modified tumor cells to costimulate in vitro the proliferation and cytokine production of optimally activated lymphoid cells. Coexpression of CD70 and CD80 by the two tumor cell lines, TS/A and B16.F10, resulted in both cases in partial regression of subcutaneous tumors. Immunochemical analysis and studies in nude mice showed that, even in the B16.F10 model, T cells had a significant role in the antitumor response induced by combining CD70 and CD80. However, rejection of the CD70/CD80-transfected tumor cells appeared more effective in the MHC class I high TS/A model, leading to a protection against parental tumor cells. B16.F10 and TS/A transfectants were then tested with fibroblasts genetically modified to secrete interleukin-12 (IL-12) as a therapeutic vaccine in mice bearing parental tumors. In the two models tested, the injections of irradiated IL-12 and CD70/CD80 gene-modified cells generated an antitumor response to established tumors leading to the slowing down of the tumor growth rate. Although the mechanisms remain to be defined, these findings suggest that the combination of several immuno-modulatory molecules could provide additional strategies for cancer immuno-gene therapy, even for MHC expression-deficient tumors.

Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. There are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.

Mutations in residue 61 of H-Ras p21 protein influence MHC class II presentation.

We have investigated the influence of mutations in the Ras 61 codon on the immunogenicity of synthetic peptides and H-Ras p21 proteins. H-2k mice produced Th responses when immunized with mutated peptides in which the Gln at position 61 in the wild type sequence was replaced by Leu (L) or His (H). T cell hybridomas specific for the 61L and 61H peptides were then produced. The responses of both were I-Ak restricted. Competition experiments indicated that the wild type peptide did not bind to the I-Ak molecule whereas the two mutations generated a site on the peptides that was agretopic for the I-Ak molecule. Nevertheless the recognition of the corresponding Ras proteins was highly dependent upon the nature of the substitution. The H-Ras p21 protein with the 61L mutation (61L) was processed by syngeneic splenocytes and the epitope dependent on 61L was recognized as efficiently as the corresponding peptide by the T cell hybridoma specific for 61L. In contrast, the processing of H-Ras p21 with the 61H mutation (61H) was probably inefficient in producing the epitope recognized by the hybridoma specific for 61H. Furthermore, immunization studies with the two mutated H-Ras p21 proteins suggest that only the 61L substitution can be exploited for immunotherapy. Thus this work demonstrates that any peptide immunotherapy must be undertaken with the reservation that not all oncogenic mutations at codon 61 will be amenable to immune therapy.

Autoreactive HLA-DR-specific autoreactive T-cell clones: possible regulatory function for B lymphocytes and hematopoietic precursors.

The physiological significance of autoreactive T cells derived from normal individuals and activated in the absence of any identifiable foreign antigen by class II MHC-syngeneic molecules remains unexplained. Here we report that autoreactive T-cell clones (Tilkin et al. 1987) proliferate and are able to kill autologous or syngeneic EBV-cell lines but not autologous or syngeneic HLA-Class II-positive PHA-activated T-cell blasts. Furthermore, they are able to efficiently inhibit in vitro the differentiation of CFU-GM and BFU-E colonies, in agreement with the well-known observation that hematopoietic precursors express HLA-DR molecules (Cannistra et al. 1986). The reasons why the autoreactive clones do not recognize T-cell blasts, as well as their possible implications in regulatory mechanisms involving HLA-class II molecules are discussed.

Reconstitution of cytotoxic T lymphocyte activity following allogeneic bone marrow transplantation in man.

Longitudinal studies over an eight-month period have been performed to follow the T killer response restoration after allogeneic bone marrow transplantation (BMT). The capacity of the patient's peripheral blood mononuclear cells (PBM) to develop cytotoxic effector cells directed either against allogeneic cells or against Epstein-Barr virus (EBV) or Herpes-simplex virus-1 (HSV-1)-infected syngeneic cells was tested monthly. The data suggest that in most cases the cytotoxic T lymphocyte (CTL) activity, either allospecific or directed against EBV and the HSV-1 specific killer (HNK) responses, is quickly reconstituted in BMT patients. Roughly, these activities seem to be normal after two months following classic nondepleted grafting. However, in the two patients who had received a T-cell-depleted graft, the restoration was delayed. Furthermore, in the majority of the patients, once reconstituted the killer responses were relatively stable and the occurrence or absence of graft-versus-host diseases (GVHD) did not significantly modify these responses.

Autoreactive T cell clones of MHC class II specificities are produced during responses against foreign antigens in man.

Although the existence of autoreactive T cells has been widely reported in mice and in guinea pigs, a similar phenomenon is poorly documented in man. Here we report the study of three human autoreactive T cell clones isolated during immunization of HLA-DRw13 donors either against influenza A/Texas virus or against allogeneic cells. These clones are specific for autologous HLA-class II specificities either common to all HLA-DRw13 molecules or restricted to the HLA-DR products specific for the DW19 subtype of HLA-DRw13. They are also cytotoxic and they have the same specificity when tested for lytic activity or in proliferation assays. Furthermore, they are also able to help autologous B cells to polyclonally produce Ig. The possible implication of such clones in regulatory mechanisms involving HLA-class II molecules is discussed.

A human autoreactive T cell line specific for minor histocompatibility antigen(s) isolated from a bone marrow-grafted patient.

A human autoreactive T cell line named Bur-1 has been obtained from a woman 4 mo after an allogeneic bone marrow transplantation (BMT) from one of her HLA-identical brothers. The phenotype of the cell line is 100% T11+ and over 90% T4+, and the karyotype confirms its donor (male) origin. These donor T cells proliferate specifically in the presence of donor's peripheral blood monocytes (PBM) but not recipient's cells, and they kill specifically donor's but not recipient's Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines (LCL). PBM from another HLA-identical brother and from several unrelated donors also stimulate Bur-1 cells, and EBV-induced LCL from the same donors are killed in cytotoxicity assays. All of these donors share HLA-DR5 or HLA-DRw11 (the major split of HLA-DR5) with Bur-1 cells. However, some but not all of the PBM sharing HLA-DR5 with Bur-1 cells are recognized. Therefore, in contrast with the previously described autoreactive T cells, Bur-1 cells are not directed against self-MHC antigens but rather recognize autologous minor histocompatibility (mH) antigens in the context of autologous HLA class II molecules. Because both male and female cells can be recognized, the reacting minor antigen could not be the male-specific HY antigen. It is suggested that autoreactivity against mH antigens can be observed in bone marrow-grafted patients due to the education of bone marrow donor precursors in the recipient thymus not allowing tolerance to autologous (donor) mH antigens not shared by the recipient.

A possible predictive test for graft-versus-host disease in bone marrow graft recipients: the mixed-epidermal cell-lymphocyte reaction.

The possible ability of the mixed-epidermal-cell-lymphocyte reaction (MECLR) to detect alloreactivities in HLA-identical MLR negative siblings has been investigated before grafting in 30 donor-recipients pairs and their families. Results indicate that recipients' epidermal cells (EC) can induce proliferative responses of HLA-identical MLR-negative donors' lymphocytes in 55% of the pairs tested. Moreover, further evaluation of 21 patients shows that the positivity of the MECLR before the graft is correlated with later appearance of acute and chronic GVHD, and especially with the severity of cutaneous injury. EC have been shown to be more efficient antigen presenting cells than peripheral blood lymphocytes for in vitro primary proliferative responses, so these reactions could be directed against some minor histocompatibility antigens, thus leading to possible improvement in selecting bone marrow graft donors and to the detection of donor-recipient pairs with a high risk of GVHD.

B cells from leukemic patients are relatively resistant to in-vitro EBV transformation.

Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation.

Natural suppressor cell inhibiting T killer responses against retroviruses: a model for self tolerance.

We previously reported the characterization of a spontaneous suppressor T cell population (NSC) present in naive mice and able to suppress the cytotoxic response (CTL) against tumor cells induced only by endogenous Gross virus (GLV). In this study we demonstrate the existence of such NSC inhibiting the CTL activity against tumor cells induced by the normally exogenous Moloney virus (M-MLV) in mice of the Mov-13 (V+) strain in which the M-MLV has been artificially endogenized and which express the virus during the embryonal life. These NSC are not found in other Mov strains in which the endogenized M-MLV is not expressed during fetal life. The implication of these data in the mechanism of self tolerance is discussed.

T cells from naive mice suppress the in vitro cytotoxic response against endogenous Gross virus-induced tumor cells.

Syngeneic normal lymphoid cells added in co-culture of immune lymphocytes and tumor cells reveal a suppressive activity inhibiting the generation of cytolytic T lymphocytes. The suppression was specific for the response directed against endogenous virus-induced or x-ray-induced tumor cells expressing endogenous C type virus antigens. Thymocytes, spleen cells, or lymph node cells from naive mice were able to express this suppressive activity. The same cells displayed no suppressive activity on killer cells directed against exogenous C type virus-induced tumor cells. The suppressor cells were Thy-1+, Lyt-1- 2+. Our results strongly suggested that the spontaneous suppressor cells exert their activity by interacting with an early step on the CTL response, probably at the level of the helper T cell function. The suppressive activity was mediated by soluble factor(s) that were antigen specific and possibly H-2 restricted. The possible implications of these spontaneous suppressor T lymphocytes in the development of endogenous virus-induced tumors and their possible implications in tolerance to self antigens are discussed.

Quantitative variations in the expression of H-2 antigens on murine leukemia virus-induced tumor cells can affect the H-2 restriction patterns of tumor-specific cytolytic T lymphocytes.

Comparative quantitative experiments were designed to study the expression of H-2Kd and H-2Dd antigens on three different leukemia cell lines induced by Gross murine leukemia virus (MuLV)in BALB/c (H-2d) mice. The H-2 restriction patterns of syngeneic cytolytic T lymphocytes (CTL) directed against Gross MuLV-induced tumors were correlated with these quantitations of H-2Kd and H-2Dd antigens, Our results obtained by quantitative absorption of monospecific antisera indicated that the three BALB/c tumor cell lines expressed different amounts of H-2Kd and H-2Dd antigens, with H-2Dd antigen showing the greatest variability in expression because it ranged from barely detectable levels to one-eighth the amount of H-2Dd antigen expressed on normal BALB/c spleen cells. The H-2 restriction patterns of Gross MuLV-specific CTL were directly affected by these quantitative modulations in the expression of H-2Kd and H-2Dd antigens, as revealed by three independent approaches: (a) inhibition of CTL activity by monospecific anti-H-2 sera in the absence of complement; (b)competitive inhibition of CTL-mediated cytotoxicity by the addition of excess tumor cells into the reaction mixture; and (c) analysis of CTL specificities using cloned CTL populations. Our results thus indicate that H-2 restriction of tumor-specific CTL activity can be directed at the target cell level by variations in the expression of H-2 antigens.

Reduced tumor growth after low-dose irradiation or immunization against blastic suppressor T cells.

Suppressor T cells have been shown to be much more radiosensitive than other lymphoïd cells, and we have tried to reduce tumor growth by low-dose irradiation. Syngeneic DBA/2 mice received whole-body irradiation (150 rads; 1 rad = 0.01 J/kg) 6 days after P815 tumor inoculation. Tumor growth is significantly reduced in mildly irradiated mice. We also attempted to reduce syngeneic tumor growth by raising immunity against suppressor T cells in two different systems. DBA/2 mice were immunized against splenic T cells collected after disappearance of cytotoxicity and then injected with P815 tumor cells. These mice develop a very high primary cytotoxicity against P815 cells. C57BL/6 mice were immunized against blastic suppressor T cells, before injection of T2 tumor cells. Some of these mice reject the tumor and other develop smaller tumors than control mice. These results could be explained by the induction of antiidiotypic activity directed against the immunological receptors of suppressor T lymphocytes, because immunization with blastic suppressor T cells from mice bearing the T2 tumor does not modify the growth of another tumor, T10.

Transplantable IgD immunoglobulin-secreting tumors in rat.

The LOU/C/Wsl rat inbred strain presents a high incidence of spontaneous malignant ileocecal immunocytomas or monoclonal immunoglobulin-secreting tumors. Some tumors have been transplanted in histocompatible animals over years without any change in their secretion products. Among approximately 600 different monoclonal proteins we have studied so far, we recognized six showing properties different from those of rat IgM, IgA, IgE, or IgG classes, and characteristic of the IgD class.