Nancy Craighead

Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes.

Background. Genetic variations in cytokine genes are thought to regulate cytokine protein production. However, studies using T cell mitogens have not always demonstrated a significant relationship between cytokine polymorphisms and in vitro protein production. Furthermore, the functional consequence of a polymorphism at position −330 in the IL-2 gene has not been described. We associated in vitro protein production with cytokine gene polymorphic genotypes after costimulation of cultured peripheral blood lymphocytes. Methods. PBL were isolated from forty healthy volunteers. Cytokine protein production was assessed by enzyme-linked immunosorbent assay. Polymorphisms in interleukin- (IL) 2, IL-6, IL-10, tumor necrosis factor (TNF-&agr;), tumor growth factor (TGF-&bgr;), and interferon (IFN-&ggr;) were determined by polymerase chain reaction (PCR). Results. Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-&ggr;, and TNF-&agr; genes was not evident after 48-hour stimulation with concanavalin-A. In contrast, after anti-CD3/CD28 stimulation significant differences (P <0.05) were found among high and low producers for IL-2, IL-6, and among high, intermediate, and low producers for IFN-&ggr;, and IL-10. Augmented levels of IL-2 in individuals that were homozygous for the polymorphic IL-2 allele were due to an early and sustained enhancement of IL-2 production. No association was found among TNF-&agr; and TGF-&bgr; genotypes and protein production. Conclusion. Polymorphisms in IL-2, IL-6, IL-10, and IFN-&ggr; genes are associated with their protein production after anti-CD3/CD28 stimulation. The profound effect of the IL-2 gene polymorphism in homozygous individuals may serve as a marker for those that could mount the most vigorous allo- or autoimmune responses, or perhaps become tolerant more easily.

Targeted T-cell depletion or CD154 blockade generates mixed hemopoietic chimerism and donor-specific tolerance in mice treated with sirolimus and donor bone marrow

Background. The administration of donor specific bone marrow (DSBM) to mice conditioned with antilymphocyte serum (ALS) and sirolimus can result in stable multilineage mixed chimerism and long-term graft survival. This study seeks to determine if either the targeted depletion of CD4 and/or CD8 pos T cells or costimulation blockade can substitute for ALS and preserve the efficacy of this regimen. Methods. C57BL/6 recipients of BALB/c skin allografts were treated with DSBM (150× 106 cells), sirolimus (24 mg/kg intraperitonealy), and either ALS or various monoclonal antibodies (&agr;CD4, &agr;CD8, &agr;CD154 alone or in combination). Recipient peripheral blood mononuclear cell (PBMC) depletion, donor chimerism, and deletion of donor reactive T cells were assessed using flow cytometry. The specificity of immunologic nonreactivity and the presence of immunoregulatory activity were assessed through a mixed lymphocyte reaction assay. Results. The administration of ALS, sirolimus, and DSBM resulted in sustained recipient PBMC depletion, transient chimerism, and prolonged graft survival. The substitution of an equivalent degree and duration of targeted depletion of either CD4 or CD8 pos T cells alone for ALS failed to produce chimerism or prolonged graft survival. In contrast, depletion of both CD4 and CD8 pos T cells resulted in durable multilineage chimerism, indefinite allograft acceptance (>350 days), and donor-specific tolerance to secondary skin grafts. Substitution of &agr;CD154 monoclonal antibody for ALS also resulted in a state of mixed chimerism and donor specific tolerance. This tolerant state appears to be maintained at least partially through clonal deletion and suppression. Conclusion. Either combined CD4 and CD8 T-cell depletion or &agr;CD154 blockade can effectively substitute for ALS in producing chimerism and tolerance in this model.