A peptide of the human 60-kDa heat-shock protein (hsp60), designated p277, was found to be useful as a therapeutic agent to arrest the autoimmune process responsible for diabetes in nonobese diabetic (NOD) mice. The effectiveness of peptide treatment was associated with the induction of peptide-specific antibodies of the IgG1 but not of the IgG2a isotype, suggesting the possibility that a Th2-type response may have been induced. We now report that the effectiveness of p277 treatment is associated with the transient activation of anti-p277 splenic T-cells that produce the Th2 cytokines interleukin-4 (IL-4) and IL-10. The Th2 response to p277 was associated with reduced Th1-type autoimmunity to hsp60 and to two other target antigens associated with diabetes: GAD and insulin. The Th2 shift appeared to be relatively specific; spontaneous T-cell reactivity to a bacterial antigen peptide remained in the Th1 mode in the p277-treated mice. Moreover, treatment with the bacterial peptide did not induce a change in cytokine profile, and it did not affect progression of the disease. Thus, effective peptide treatment of the diabetogenic process associated with the induction of antibodies may be explained by selective and transient activation of Th2 autoimmune reactivity.
The basic tenet underlying the present work and supported by recent studies is that there is a dialogue between developing thymocytes and thymic stromal cells. One direction in this dialogue, i.e. thymic stromal cell role in shaping thymocyte maturation, has been extensively studied. The other direction, thymocyte effect on stromal cell development and function, started to emerge only recently on the basis of in vivo observations in SCID and knockout mice. An in vitro approach to the analysis of this interaction may add substantial insight into the process, as demonstrated by the present work. We made use of a culture system of either murine thymic epithelial cells (TEC line) cultured alone or cocultured with thymocytes. Unstimulated thymocytes or their supernatant caused 40-80% inhibition of TEC cell proliferation, as measured by 3H-thymidine incorporation. Cell cycle analysis by flow cytometry indicated that this inhibition can be attributed to reduction in G2/M phase cell number pari passu with an increase in Go/G1 cell number. This inhibitory effect was found to be partially mediated by TGF-beta produced by thymocytes. On the other hand, thymocytes augmented IL-6 production by TEC cells in coculture, an effect which could not be reproduced by thymocyte culture supernatant and was not inhibited by thymocyte pretreatment with formaldehyde or emetine. Furthermore, antibodies against thymocyte adhesion molecules (CD2, LFA-1) blocked the thymocyte-induced IL-6 secretion. IL-6 was found to be an autocrine growth factor of TEC in culture, since a combination of anti IL-6 and anti IL-6 receptor antibodies caused 70% inhibition of TEC proliferation and addition of exogenous recombinant IL-6 doubled the rate of proliferation. These results suggest that thymocytes regulate thymic epithelial cell growth by a complex set of inhibitory and enhancing signals mediated through either soluble factors or direct contact. The ultimate effect is dependent on the balance between different signals and may be different in different microenvironmental settings in vivo. In coculture in vitro the dominant effect was growth inhibition of the epithelial cells by thymocytes.
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