Control of pathogenic CD8+ T cell migration to the brain by IFN-gamma during experimental cerebral malaria.

Previous studies have shown that IFN-gamma is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN-gamma in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post-infection with PbA. In this strain, both CD4(+) and CD8(+) T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN-gamma receptor alpha chain (IFN-gammaR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ-specific pattern of expression. Up-regulation of RANTES/CCL5, IP-10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP-1/CCL2, IP-10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN-gamma controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ-specific pathology induced by PbA infection.

Regression of primary hepatocarcinoma in cancer-prone transgenic mice by local interferon-gamma delivery is associated with macrophages recruitment and nitric oxide production.

The clinical potential of tumor therapies must be evaluated using animal models closely resembling human cancers. We investigated the impact of locally delivered interferon-gamma (IFN-gamma) on primary hepatocarcinoma spontaneously developed by T-SV40 transgenic mice. A single intratumor injection of adenovirus IFN-gamma was sufficient enough to induce in vivo production of biologically active IFN-gamma, as assessed by STAT1 activation. IFN-gamma secretion led to the regression of primary tumor, principally by apoptosis of tumor hepatocytes. The lack of T-cells infiltrates in the liver upon treatment excluded a role of a specific immune response. In contrast, indirect pathways may include tumoricidal function of macrophages. Indeed, they were massively recruited in the entire liver under IFN-gamma treatment; transmigration through hepatic blood vessels could be observed and co-localization with damaged hepatocytes was obvious. This correlated with nonparenchymal liver cell iNOS expression and high level of NO in hepatic extracts. Moreover, in vitro experiments showed that NO releasing agents induced cell death of freshly isolated tumor hepatocytes, suggesting that NO could be one of the major effector molecules. Altogether, these observations defined an important role of IFN-gamma in controlling tumor development in a model of primary hepatocarcinoma.

Passive but not active CD8+ T cell-based immunotherapy interferes with liver tumor progression in a transgenic mouse model.

To evaluate tumor immunotherapies, we used transgenic mice that harbor a progressive liver tumor associated with the expression of the SV40 large tumor T oncoprotein (SV40-T). To induce "self" tumor Ag-specific CD8+ T cells, mice were injected with an immunodominant SV40-T CTL epitope mixed with a heterologous helper peptide. Despite repeated injections, this vaccine failed to raise a tumor-specific CD8+ T cell response that was efficient enough to counteract tumors. Although coimmunization with SV40-T CTL epitope and heterologous helper peptide efficiently recruited the respective Th cells, only low-avidity SV40-T-specific CD8+ T cells were activated. Furthermore, major alterations in SV40-T-specific B and Th cell responses were characterized. In contrast, transfers of higher-avidity CTLs specific for the same SV40-T epitope were effective in counteracting tumors. These results suggest that passive therapies targeted to self tumor Ag may be more suitable than active immunization in the treatment of spontaneous tumors.

IFN-gamma-secreting Th cells regulate both the frequency and avidity of epitope-specific CD8+ T lymphocytes induced by peptide immunization: an ex vivo analysis.

CD8+ T lymphocytes are involved in protective immune responses to infected or tumor cells. In this report, we examined the regulation of antigen-specific CD8+ T cell frequency and avidity by distinct Th cell subsets. Peptide-specific CD8+ T cells were induced by immunization of mice with a MHC class I-restricted epitope, co-injected with a MHC class II-restricted epitope to recruit Th cells. CD8+ T cell responses were assessed directly ex vivo for lytic activity and IFN-gamma secretion using the enzyme-linked immunospot (ELISPOT) assay. Co-immunization in incomplete Freund's adjuvant (IFA) with three different helper peptides induced IFN-gamma- and IL-2-secreting Th cells, in the absence of IL-4 secretion, suggesting preferential Th1 profiles. Such immunization resulted in the increase of antigen-specific CD8+ T cell frequency, which was detected in blood as efficiently as in lymph nodes and spleen, and elicited high-avidity CD8+ T cells. We investigated whether these effects were dependent upon a particular Th profile. When alum was used instead of IFA, the production of IL-2 by Th cells was still significant, while the production of IFN-gamma was undetectable. Such Th cell activation failed to support an increase of antigen-specific CD8+ T cell frequency. Altogether, these results document in vivo the regulatory role played by Th cells in CD8+ T cell activation and may be relevant for the design of efficient vaccination schedules.

Differential presentation of endogenously processed cytotoxic T lymphocyte epitopes by mouse hepatocarcinoma cell lines induced by SV40 large T antigen.

Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40-transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.

Critical stages of tumor growth regulation in transgenic mice harboring a hepatocellular carcinoma revealed by distinct patterns of tumor necrosis factor-alpha and transforming growth factor-beta mRNA production.

There is now good evidence that cytokines contribute to the regulation of tumor growth. The cytokine-driven modulation of tumor growth was investigated during the progression of a hepatocellular carcinoma (HCC) in SV40 large T tumor antigen transgenic mice. In vivo, an increased rate of liver growth correlated with increased transforming growth factor (TGF)-beta 1 mRNA expression, while the greatest amounts of tumor necrosis factor (TNF)-alpha mRNA were detected earlier during tumor development. Conversely, no particular alteration of IL-1 alpha, IL-1 beta, IL-6, IL-2, IL-4 and IFN-gamma mRNA production could be reported. In vitro, hepatocyte-like tumor cell lines established at two stages, either before or after HCC differentiation, were characterized. The early-stage-derived cell line produced TNF-alpha mRNA, but had barely detectable expression of TGF-beta 1 mRNA, while later-stage-derived cell lines showed the reciprocal pattern. All cell lines displayed a lack of sensitivity to TNF-alpha, although some degree of sensitivity to TNF-alpha could be observed in the presence of actinomycin-D or after treatment with IFN-gamma. The early-stage-derived cell line was sensitive to the growth inhibitory effects of TGF-beta 1, but late-stage-derived tumor cell lines displayed a loss of sensitivity to TGF-beta 1 which correlated with the increased expression of TGF-beta 1 mRNA. Altogether, this suggests that tumor cells contribute to the discrete TNF-alpha and TGF-beta 1 expression patterns during HCC progression. This model of HCC could be of valuable interest to assess the impact of various immunotherapeutic strategies on modulation of tumor growth.

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.

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.

Modulation of CALLA and HLA-DR on a non T non B leukemic cell line by lipid treatment.

The relationship between membrane lipid fluidity and expression of HLA-DR and cALL (CALLA) antigens was studied in a human non T non B acute lymphoblastic leukemia cell line (Reh). The membrane fluidity was modulated by treatment with cholesteryl hemisuccinate or phospholipids (e.g. egg lecithin) and monitored by fluorescence polarization. HLA-DR and CALLA expression was measured in an indirect immunofluorescence test with a Fluorescence Activated Cell Sorter (FACS 440), on 24, 48, 72 and 96 hour-cultured cells. Significant antigenic modulation was obtained with cholesteryl hemisuccinate treatment on 48 hour-cells where a slight increase in HLA-DR and a marked decrease in CALLA were observed. In contrast no antigenic modification was observed on lecithin-treated cells.

[Ultrastructural peroxidases and immunological markers in acute adult leukaemia: therapeutic applications (author's transl)]. / Peroxydases ultrastructurales et marqueurs immunologiques dans les leucémies aiguës de l'adulte: incidence thérapeutique.

In a retrospective study of 112 adult patients with acute leukaemia (AL), the percentage of undifferentiated leukaemia was reduced to almost 1% by an analysis of routine cytochemical reactions (PAS, peroxidase, esterases and esterase inhibition), B and T lymphocyte markers (IgS, E-rosettes, HuTLA) cAll antigen and ultrastructural detection of peroxidase activity (PO-ME). A simultaneous study of cALL antigen and PO-ME showed reciprocal exclusion of these markers, except in one case of mixed leukaemia. Therapeutically, the value of these tests lies in that patients with typical PAS reaction and cALL antigen consistently respond to vincristine- corticosteroid treatment, whereas patients without cALL and with PO-ME are frequently resistant to that combination of drugs.

Determination of ultrastructural peroxidases and immunologic membrane markers in the diagnosis of acute leukemias.

Detection of membrane markers and ultrastructural peroxidase activity was carried out on the blasts of 16 apparently nonmyeloid adult acute leukemias. These patients were selected from 73 adult leukemic patients by the negativity of their routine cytochemical myeloid markers: i.e., myeloperoxidase, chloresterase activity, and Sudan Black B staining. B and T acute lymphoid leukemias (ALL) were excluded from the study. After concurrent testing from human T lymphocyte antigen (HuTLA), common ALL antigen (cALL), la-like antigens, and peroxidase activity at the electron microscopic level (POEM), only two patients remained undifferentiated (cALL-, POEM-). The other cases were classified as following : 6 common ALL (cALL+, POEM-), 1 pre-T-ALL (cALL+, HuTLA+, POEM-), 5 very poorly differentiated acute myelogenous leukemia (AML) (cALL-, POEM+), and 2 mixed leukemias (cALL+, POEM+). Terminal deoxynucleotidyl transferase activity (TdT) was measured in 7 cases and was found to be present at high levels in 4 cases of cALL and in the 2 cases of acute undifferentiated leukemias (AUL): it was absent in two cases of AML. Cytogenetic analysis had showed that 2 of the cALLs, 3 of the AMLs, and the 2 mixed leukemias were PH1+. We conclude that POEM detection is useful in apparently nonmyeloid leukemias with negative immunologic lymphoid markers, and that the existence of a Ph1 chromosome should be investigated, particularly in the unusual case of mixed (lymphoid-myeloid) acute leukemia.