B7-1, IFN gamma and anti-CTLA-4 co-operate to prevent T-cell tolerization during immunotherapy against a murine T-lymphoma.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Attenuation of Trypanosoma brucei is associated with reduced immunosuppression and concomitant production of Th2 lymphokines.

Mechanisms regulating resistance to African trypanosomes were addressed by comparing the immune responses of mice infected with attenuated Trypanosoma brucei brucei lacking the phospholipase C gene (PLC-/-) and those of mice infected with wild-type (WT) parasites. Inhibition of concanavalin A (ConA)-induced T cell proliferation occurred in spleen and lymph nodes of PLC-/-- and WT-infected mice. Although suppressive cells were elicited in spleen and lymph nodes of WT-infected animals, such cells were not detected in lymph nodes of PLC-/--infected mice. PLC-/--infected mice had more interleukin-4 and -10 in their blood than did WT-infected mice. Correspondingly, PLC-/--infected mice had higher IgG1 antibody levels against variant surface glycoprotein than did WT-infected mice. These data indicate that attenuation of T. b. brucei correlates with the absence of cells suppressing ConA-induced T cell proliferation in the lymph nodes, with increased production of Th2 cytokines and a stronger IgG1 antibody response to trypanosome antigens.

Increased IL-12 P40 homodimer secretion by spleen cells during in vivo growth of the BW-19 T cell hybridoma accompanies suppression of natural immunity.

The high capacity of the T cell hybridoma BW-19 to metastasize to the spleen, despite its high and moderate sensitivity to lysis by macrophages and natural killer (NK) cells, respectively, appears to be linked to its capacity to suppress local resident NK cell and macrophage activity. Such suppression of splenic NK cell and macrophage activity is accompanied by an increased production of the p40 subunit of interleukin-12 (IL-12) by spleen cells. Closer examination revealed that most of the p40 subunit is present under the form of the homodimer (p40)2, whereas the heterodimeric form of IL-12 is present only in small amounts. Since (p40)2 is known to be a strong antagonist of IL-12-mediated effects, i.e., NK cell activation and interferon-gamma (IFN-gamma) secretion, the increased production of (p40)2 after BW-19 cell inoculation may contribute to the suppression of NK cell and macrophage activity. In addition, we found that the high production of (p40)2 in our tumor model was accompanied by a drastic decrease in IL-2 and IFN-gamma production by spleen cells, further favoring the possibility that (p40)2 plays a role in the suppression of NK cell and macrophage cytotoxicity. Our results show that normal spleen cells can produce (p40)2 in response to cancer cell growth in vivo and are highly suggestive of a role for (p40)2 in the suppression of natural immunity.

Metastasis of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Dk antigens.

In vivo inoculation of a low metastatic BW 5147 derived T-cell lymphoma variant into syngeneic mice, had led to the generation of a highly metastatic variant. The shift towards a more metastatic phenotype is accompanied by an increase in major histocompatibility class I H-2Dk antigen expression. This suggests that H-2Dk antigens may control the metastatic potential of BW T lymphoma cells. Our present findings indicate that H-2Dk expression is directly correlated with the metastatic potential of BW cells. We have confirmed such correlation by specifically altering the level of H-2Dk expression by: 1) FACS analysis, 2) IFN-gamma treatment, 3) H-2Dk gene transfection. Cells sorted for low H-2Dk expression had a significantly reduced metastatic potential. Induction of H-2Dk expression on these cells by either IFN-gamma treatment or H-2Dk gene transfection concomitantly led to increased metastasis. We also assessed metastatic potential of BW cells in irradiated, immunocompromised recipients. Our results show that the immune system is implicated and we further tested which immune effectors are involved. In vivo depletion of natural killer (NK) and CD8+ T-cells revealed that the difference in metastatic potential of the H-2Dk variants relies upon an NK-dependent mechanism, whereas CD8+ T-cells are not implicated. Our observations suggest that highly metastatic cells, expressing a high level of H-2Dk antigens are more resistant to NK-cell-mediated cytotoxicity in vivo. We have confirmed our in vivo results by in vitro cytotoxicity assays using poly I:C induced NK and IL-2 activated LAK cells. We conclude that a NK-dependent mechanism accounts for the association between differential H-2Dk antigen expression and metastasis.

A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis.

A series of monoclonal antibodies (MoAb) produced against excretory and secretory products from 10- and 20-week-old Taenia saginata cysticerci were tested for their ability to detect circulating antigen in a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Two MoAb, 12G5 and 2H8, proved to be highly reactive with the tegument of viable T. saginata cysticerci and recognized antigenic components of 65, 87 and 100 kDa in immunoblotting. The detection limit of the assay using 12G5 as trapping antibody and 2H8 as a biotinylated indicator antibody was 0.1 ng protein per ml. Although the sensitivity of the test varied from one animal to another, the minimum number of living cysticerci, which could be detected by the ELISA, was 88. Animals harbouring only dead cysticerci gave similar reactions as non-infected control animals. Cross-reactions were only observed with taeniid parasites. The test was able to detect circulating antigen also in sheep and pigs, respectively infected with T. ovis and T. solium and in the serum samples of confirmed cases of human T. solium cysticercosis.

Mycobacterial proliferation in macrophages is prevented by incubation with lymphocytes activated in vitro with a mycobacterial antigen complex.

Antigen A60 from Mycobacterium bovis bacillus Calmette Guérin was shown to trigger both humoral and cellular immune reactions. We explored the ability of A60 to block intracellular proliferation of phagocytosed mycobacteria with a model system involving peritoneal murine macrophages infected with Mycobacterium avium. Mixed lymphocytes from lymph nodes of mice inoculated with A60 hindered intracellular proliferation of this mycobacterium, owing to A60-specific cells, proliferation of which was induced in vitro in an antigen concentration-dependent manner. The lymphokines released by A60-stimulated T lymphocytes in vitro were identified as interleukin 2 (IL2) and interferon-gamma (IFN-gamma): their production showed a clear A60 dose dependence. When supernatants of such induced lymphocyte cultures were incubated with anti-IFN-gamma antibodies, macrophage activation was prevented, whereas anti-IL 2 immunoglobulin had little effect. Treatment of infected macrophages with recombinant IFN-gamma reduced intracellular proliferation of mycobacteria, while exogenous IL 2 and tumor necrosis factor were ineffective. Therefore, A60 elicits in vitro proliferation of T lymphocytes responding specifically to this antigen with production of IFN-gamma, which in turn activates macrophages and prevents multiplication of phagocytosed mycobacteria.

Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chabaudi IP-PC1.

The complete nucleotide sequence of the gene encoding the precursor to the major merozoite surface antigens of Plasmodium chabaudi chabaudi strain IP-PC1 has been determined. A single open reading frame was detected, that coded for a protein of 199 kDa. The encoded protein (p199) contains putative signal and membrane anchor sequences and shows a clustering of Cys residues in the last 120 amino acids. Incompletely conserved tandem repeat oligopeptides are present at different positions in the molecule. P199 shows 69% overall homology to the analogous antigen in Plasmodium yoelii yoelii strain YM. The divergence between these antigens is largely confined to 4 areas where a number of insertions and/or deletions have occurred. All repeats occur in these divergent regions. The overall homology with both alleles of Plasmodium falciparum PMMSA is 33%.

Evidence for circulating activated cytotoxic T cells in HIV-infected subjects before the onset of opportunistic infections.

The activity of both cytotoxic T lymphocyte (CTL) and natural killer (NK) cells were measured cross-sectionally in 43 subjects seropositive for HIV, in 27 HIV- blood donors and in 24 HIV- persons from the Outpatients Clinic for sexually transmitted diseases. CTL activity was evaluated using the HL-60 cells coated with OKT3 as the targets and freshly separated peripheral blood lymphocytes as the effectors. In 20 out of 43 HIV+ subjects, CTL activity was significantly enhanced in comparison to the HIV- subjects. This lytic activity correlated positively with the percentages of CD3+ HLA-DR+, of CD8+ CR3- and of CD57+ CD16- lymphocytes, and was greatly reduced after elimination of CD8+, of HLA-DR+ or of CD57+ cells. The median CTL activity seemed to increase from CDC group II to CDC group IV (Centers for Disease Control classification), but to return back to control levels in those patients with a history of opportunistic infections. NK function in HIV+ subjects was not significantly different from that in the blood donors. In seropositive patients, NK activity correlated positively with the percentages of both CD16+ CD57+ and of CD8+ CR3+ cells and was strongly diminished after elimination of CD16+ or of CD57+ cells. There was no significant change in NK function according to the clinical stage. The data show that circulating CD8+ HLA-DR+ CD57+ T cells in HIV+ subjects are activated cytotoxic T cells and point to progressive (over) activation of this T cell compartment until the onset of opportunistic infections.