Medical Microbe Immunology (MMI) - Faculty: Stanislaw Stepkowski, DVM, PhD, DSc.

Faculty: Stanislaw Stepkowski, DVM, PhD., DSc.

Summary: My lab’s overall work is focused on: 1) development of novel non-toxic immunosuppressive agents; 2) induction of permanent acceptance of allografts (transplantation tolerance); 3) improving survival of pancreatic islets; 4) cytokine signaling in T cells. Special efforts are made to better understand cytokine-induced signaling through Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) pathways in T cells. Undergoing work aims to identify novel regulatory phosphotyrosine sites in function of Jak3, using knock-in mice with mutated Jak3 sites. The role of Stat3 and Stat 5a/b transcription factors are explored in Stat3 and Stat5 conditional knockouts, respectively.

Figure 1

My lab’s interests are in cell and organ transplantation, with research focused on improvement of long-term allograft survival and development of new immunosuppressive modalities. Activation of T lymphocytes requires three overlapping and sequential signals, with signal 3 delivered by multiple cytokines that regulate T cell proliferation, differentiation, and survival/death. Cytokines binding to their receptors engages two key molecular families, namely, Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Among Jaks, gamma-cytokines bind to cytokine receptors consisting gamma-chain, thereby engaging Jak3/Stat5 cascade leading to proliferation and differentiation of T cells.

Recently, novel tyrosine (Y904 and Y935) phosphorylation sites have been identified as important for human Jak3 function using an in vitro assay measuring Jak3 kinase activity. Localization of Y904 in the N lobe of the kinase domain flanking by glutamine at 903 position and leucine at 905 position may suggest the role of Y904 in ATP binding during catalytic reaction (Fig. 1A). Furthermore, since Y939 is localized in a-helix in the C lobe of the kinase domain, which typically promotes substrate access to the catalytic cleft, suggests that Y939 may regulate interaction with Stat5 transcription factor (Fig. 1B). The importance of these two sites is also supported by their conserved alignment in Jak1, Tyk2, Jak3 and Jak2 (Fig. 1C). My lab plans to investigate the exact in vivo role played by these two phosphorylation sites on Jak3 in T cell functions, such as response to common gamma (gc)-cytokines (interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, IL-21), proliferation and differentiation. Although all these cytokines use gc/Jak3 signaling, each has an individual alpha chain (whereas IL-2 and IL-15 have also a common b chain) to deliver distinct quality signals resulting in major functional consequences of proliferation, differentiation, and survival/apoptosis. Our hypothesis is that some of the functional differences in T cells may be dependent of the Y904 and/or Y939 phosphorylation. To test this possibility we have designed lentivector constructs with wild-type Jak3, or Jak3 with mutation of tyrosine (Y) to phenylalanine (F) producing Jak3Y904F or Jak3Y939F mutants. These two lentivector mutants and a wild-type Jak3 control are planed to be used for transfection of Jak3-deficient T cells. Using these transfection systems, we plan to define the in vivo role of Y904 and Y939 in the function of human Jak3 in T cells. The equivalents of the human Y905 and Y939 are localized on the mouse Jak3 as Y900 and Y935. Consequently, we have produced knock-in mouse constructs with Jak3Y900F or Jak3Y935F mutation of Y900 or Y935. These mice will be tested for the impact of each mutation on development and generation of functionally different T cell populations. In another project using conditional knockout mice, we plan to examine the role of Stat3 and Stat5 transcription factors in regulating T cells. Binding of cytokines to cytokine receptors attracts and activates one or more of the Jaks, which are tyrosine phosphorylated themselves and then Jaks phosphorylate tyrosines on the cytokine receptor sites, thereby creating active docking sites for Stats. Among Stats, recent evidence suggests that Stat3 is involved in the generation of a new population of T helper 17 (Th17) cells, and in the regulation of dendritic cell function and the process of angiogenesis. We are investigating whether Stat3 deficiency may affect the regulation of Th17 cells and other functions. Since survival of T cells is critically dependent on Stat5. Based on screening of multiple compounds from NIH data base, novel and selective inhibitor of Jak3, NC1153, has been developed. It has been shown that NC1153 may inhibit kidney allograft rejection in rats and cynomolgus monkeys. Most of this work has been published in the Journal of Immunology, Blood, and Transplantation.

For the last 5 years, my lab has investigated a sphingosin-1-phosphate receptor (S1P) agonist, FTY720 (2-amino-2-2-[4[octylphenyl]ethyl)propane-1,3-diol hygrochloride), but this promising compound was abandoned following clinical kidney trials because of its side effects. Physiologically, S1P (a bioreactive lysolipid) acts through the family of G protein-coupled receptors, namely S1P1, S1P2, S1P3, S1P4 and S1P5. While S1P1 expression was shown to be restricted to the vascular endothelium, S1P3 was abundantly expressed in cardiomyocytes of perivascular smooth muscle cells. Consequently, activation of S1P1 receptor correlated with lymphopenia and increased entrapment of lymphocytes in the lymphoid compartment, while activation of S1P3 correlated with bradycardia and hypertension. The latter symptoms occurred because of poor selectivity as FTY720 was able to engage S1P1, S1P3, S1P4 and S1P5. Over the last 3 years we have tested a novel compound, KRP203 (2-amino-2-propanediol hydrochloride), that was shown to have selective agonist activity on S1P1 but not on S1P3. Our results showed that KRP203 not only extended allograft survival of kidney allgrafts but also induced transplantation tolerance to islet allografts when combined with local infusion of T regulatory cells. At present this work is in preparation for publication.

Dr. Stepkowski was educated in Warsaw, Poland (Veterinary Medicine), trained at Radium Hospital in Oslo, Norway (PhD), Dalhousie University in Halifax, Canada (postdoctoral), and at the University of Texas in Houston, USA (DSc). He considers the United States his home and the best place to perform the most advanced research.

Current Grant Funding:

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NIH-development new methods for tolerance induction

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Novartis Inc-development of Jak3 inhibitors

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Icagen Inc-development new immunosuppressive drugs