William Hendrickson

Associate Professor,

Ph. D., Tufts University, 1981.

Room: E706C MSB, Tel: 312-996-5600

Email: whend@uic.edu

Molecular Pathogenesis of Autoimmune Diseases

The IG-20 gene is essential for survival of the animal. It is over-expressed in human tumors and cancer cell lines, and can encode 4 different splice variants. The DENN-SV splice variant is constitutively expressed in all cells and tissues, and is highly-expressed in human tumors and cancer cell lines relative to normal tissues and other splice variants. Cells transfected with a cDNA encoding DENN-SV proliferate more aggressively, form larger colonies in soft agar and become resistant to TNF-á, TRAIL, gamma-irradiation, etoposide and vinblastine treatment. In contrast, cells transfected with a cDNA encoding IG20 splice variant grow slowly in culture and become more susceptible to cell death induced by the above treatments. The increased susceptibility of IG20 transfected cells to TRAIL induced death is mediated by the activation of caspases-8 and -3 resulting from enhanced recruitment of caspase 8 and FADD to the Death Inducing Signaling Complex (DISC). Reduced proliferation of IG20 transfected cells appears to be largely due to cell-cycle arrest. The other two splice variants, MADD and IG20-SV2, exhibit little or no effect. Several ovarian carcinoma cell lines tested to date have shown little or no expression of IG20 but have shown very high levels of expression of DENN-SV. PA-1 ovarian carcinoma cells that lack endogenous IG20 proliferate rapidly and are resistant to TRAIL induced apoptosis. However, once IG20 is introduced, they become more susceptible to TRAIL induced apoptosis. IG20 can interact with DR4/DR5 and enhance apoptosis by allowing preferential recruitment of FADD and caspase 8 to the death inducing signaling complex. Moreover, IG20 dramatically reduces the ability of PA-1 cells to grow both in vitro and in vivo in nude mice, presumably through cell cycle arrest. In contrast, DENN-SV appears to mediate cell proliferation and resistance to apoptosis by enhancing activation of NFêB. These observations clearly show that DENN-SV and IG20 are biologically very important. Currently, efforts are underway to 1. exploit the pro-apoptotic properties of IG20 variants to render cancer cells that are otherwise resistant to become susceptible to killing by TRAIL and/or chemotherapeutic agents; 2. understand the mechanism of action of IG20 and its splice variants; 3. develop novel strategies to manipulate the levels of expression of various splice variants to regulate cell growth/death.

Experimental Autoimmune Thyroiditis (EAT) is characterized by eventual destruction of thyroid by infiltrating lymphocytes resulting in hypothyroidism. CD4+ T cells that produce IFN-gamma, TNF-alpha and IL-12 are critical for the pathogenesis. We carried out experiments to test the efficacy of thyroid targeted engagement of CTLA-4 using a bispecific Ab (BiAb), which is prepared using an anti-CTLA-4 and an inert anti-TSHR Ab, in modulating immune response to mouse thyroglobulin (mTg) in CBA/J mice. Treatment with BiAb resulted in down-regulation of mTg specific immune responses as well as lymphocytic infiltration and associated thyroid destruction. This immune down-modulation was found to be Ag specific as indicated by a lack of suppressive effect on immune response to a non-thyroid Ag, ovalbumin (OVA). The BiAb targeted to the thyroid most likely facilitated engagement of CTLA-4, resulting in an increase in the number of CD4+CD25+ T cells. These Treg cells suppressed in vitro mTg-specific T cell responses, which were associated with an enhanced transforming growth factor (TGF)-â1 production. Neutralization of TGF-â1 increased mTg-specific in vitro proliferation of, and IL-2 production by, T cells from BiAb-treated mice. Further, a significant reduction in Th1 cytokine response to mTg was observed in these tolerant mice, while Th2 cytokine responses were found to be unaffected. These results again suggested the importance of Th1 type cytokines in the pathogenesis of EAT, and Th2 type cytokines in conferring protection against the development of EAT. Encouraged by these results, we initiated experiments to directly modulate DCs and study the effects on the pathogenesis of EAT. In recent studies, we showed that relative to thyroglobulin (mTg) immunized control mice treated with Flt3-L developed a more severe EAT characterized by massive thyroid lymphocytic infiltration, and IL-2 and IFN-gamma production. In contrast, GM-CSF treated mice failed to develop EAT, and showed a significant increase in CD4+CD25+ regulatory T cells (Tregs). Activation of lymphocytes from these mice with mTg in vitro yielded higher levels of IL-4 and IL-10. Neutralization of IL-10, but not IL-4, and depletion of Tregs from these cultures restored mTg specific T cell proliferation, and IL-2 and IFN-gamma production. Further, adoptive transfer of Tregs to mTg immunized mice suppressed EAT while, inoculation of anti-IL-10 receptor Ab reversed GM-CSF induced suppression resulting in EAT. These results showed that suppression of EAT was mediated by Tregs most likely through enhanced production of IL-10.

Based on these results we are currently testing 1. the efficacy of GM-CSF and targeted CTLA-4 treatment to confer long-term antigen specific protection upon re-exposure to mTg; 2. the ability of CD8a- and CD8a+ DCs to undergo maturation, capture and present the antigen, and produce cytokines, and determine the mTg specific T cell phenotype and cytokine profiles; 3. both DCs and T cells for their ability to suppress in vitro and in vivo mTg specific responses; 4. the effects of GM-CSF, DCs and Tregs on the expression of MHC and B7 molecules, APC function, and sensitivity to Fas induced apoptosis of thyrocytes from WT and SCID mice.

These studies are expected to provide significant insights into the mode of action of GM-CSF and CTLA-4 mediated signalling in the generation of regulatory T cells and their mode of action in suppressing EAT, and perhaps other autoimmune diseases.