Immunomodulatory Activity of Glycodelin : Implications in Allograft Rejection

Abstract

Glycodelin, a homodimeric glycoprotein belonging to the lipocalin superfamily, is synthesised predominantly by the cells of the reproductive system of certain primates including humans. Of the four different known glycoforms of the molecule, glycodelin A (GdA), secreted by the glandular epithelial cells of the endometrium in response to progesterone, is involved in the immunosuppression of the maternal immune response to the semi-allograft fetus. GdA secretion onsets few days after ovulation. In the absence of fertilization, GdA levels drop, but subsequent to a successful fertilization, the concentrations peak till the 12th week of pregnancy and fall steadily to low levels. The importance of GdA has been implicated in implantation, endometrial receptivity, trophoblast invasion and differentiation, and modulating the functions of almost all immune cells. GdA has profound influence on the activity of T cells. It inhibits the proliferation of T cells, induces apoptosis in activated T cells, inhibits the IL-2 production and leads to skewing of the Th-1/Th-2 balance towards Th-2 type of immune response. Cytotoxic T lymphocytes are more resistant to the induction of apoptosis by GdA, but, it suppresses their cytolytic activity Additionally, GdA induces apoptosis in monocytes and natural killer (NK) cells, inhibits the proliferation of B cells and induces tolerogenic phenotype in dendritic cells. Clinical studies showing that women undergoing recurring spontaneous abortions have low levels of GdA supports its role in prevention of fetus rejection. The immunomodulatory activity of Gd resides in the protein backbone, however, apart from GdA and GdF which have similar oligosaccharide chains, other glycoforms do not possess this activity. Glycosylation seems to dictate the stability, folding and activity of Gd. In absence of glycosylation, the expression of the recombinant Gd is compromised and the protein is improperly folded while over-mannosylation of Gd impairs its immunomodulatory function. Additionally, sialylation seen on the glycan chain regulates the activity. Therefore, in order to obtain adequate amounts of active recombinant Gd (rGd), expression of the protein was attempted in three different systems, insect, yeast and bacteria (Chapter 1). In all of the described systems, the rGd protein was found apoptotically active. The protein expressed in the Sf21 insect cells was demonstrated to be differentially glycosylated compromising the activity. Hence, a genetically modified yeast strain, Pichia pastoris SuperMAN5 was explored for expression. Though presence of a single glycosylated protein species was observed in small-scale cultures, similar to the case of Sf21 cell expression, differentially glycosylated proteins were detected in large-scale fermentation and even the yield was low. Eventually, mutant Gd, modified to increase the stability and aid in proper protein folding, was expressed in E.coli and demonstrated to be able to induce apoptosis in Jurkat cells (T cell leukemia cell line). This active rGd was used for further studies. The immunomodulatory function of GdA during pregnancy protects the semi-allograft fetus from rejection by the maternal immune system. In the process, GdA tweaks the T cell immune response from pro-inflammatory to anti-inflammatory in a specific and localized manner. Allograft rejection seen during mis-match transplantations is basically a pro-inflammatory condition which is mediated by the activation of cellular immune response, NK cell cytotoxicity and antibody-dependent immune response, the same processes that are suppressed for a successful pregnancy. Chapter 2 discusses whether it is feasible to use Gd to prevent allograft rejection. Killing of target graft cells by the cytotoxic T lymphocytes (CTLs) predominantly presides acute graft rejection. GdA treatment has been shown to suppress the cytotoxicity of in vitro generated CTLs. On this basis, the earlier study was translated to in vivo conditions by establishing an allograft nude mouse model. The tumor rejection mediated by the action of in vitro generated cytotoxic alloactivated PBMCs in the nude mouse imitated the allograft rejection. A heterogenous population of immune cells with the predominance of CTLs was chosen to accommodate a more interactive immune response in the tumor microenvironment and enabled the study of other cells which may contribute to the rejection. Reactivation and proliferation of CD4+ and CD8+ T cells following their infiltration in the tumor validated our hypothesis. On treatment with rGd, the cytotoxicity of the alloactivated PBMCs was suppressed, thereby inhibiting the tumor rejection in the nude mouse. Real time PCR analysis showed that rGd treatment was able to affect the functions of the immune cells in vivo. It decreased the T cell population most probably by inducing apoptosis. As expected, the reduction was more prominent in case of CD4+ T cells than CD8+ T cells. The their expression of key molecules responsible for the cytotoxicity such as IL-2, granzyme B and EOMES, was observed to be downregulated by rGd. Concomitantly, decreased levels of pro-inflammatory cytokines, TNFα and IL-6 were also seen. Expression of Foxp3, marker for regulatory T cells, was upregulated in the tumor infiltrating immune cells suggesting an expansion of the concerned population upon rGd treatment. Overall, rGd seems to suppress the cellular immune response to the tumor by modulating the T cell population and their functions. Since, T cell-dependent immune response is central to allograft rejection, the ability of rGd to regulate it could be of therapeutic use in the management of allograft rejection. NK cells are essential for the maintenance of pregnancy, evident from their abundance (70% of total leukocytes) at the first trimester decidua. The third chapter focuses on how Gd regulates the NK cell function. The cytokine production from CD56bright subset of NK cells and their interaction with the HLA antigens expressed by the trophoblast cells helps in creating a favourable environment for the growth of the fetus. It is important to note that the NK cell population present in the decidua exclusively express Gd, implicating a role of Gd in their differentiation from the peripheral CD56bright cells. However, an increased number of CD56dimCD16+ cells in the peripheral blood dictates a negative outcome for the pregnancy. The study, presented in Chapter 3, demonstrated that rGd treatment induces caspase-dependent apoptosis in the activated CD56dimCD16+ cells and reduces their cytotoxicity by downregulating granzyme B and IFNγ production. Similar effect of rGd is also seen on the NKT cells characterised as CD3+CD56dimCD16-. Furthermore, in YT-Indy cells, an activated NK cell line, it was shown that the induction of apoptosis by rGd involves Ca2+ signalling which could explain why Gd affects activated immune cells only. This study therefore reinforces the role of Gd in modulating the NK cell activity during pregnancy. Cytotoxicity of NK and NKT cells also plays an important role during allograft rejection. Decrease in the mRNA levels of CD56 upon rGd treatment in the allograft mouse model indicates that the effect of Gd on NK cells observed in cell culture system can be translated to in vivo conditions. In conclusion, suppression of the cellular immune response and NK cell mediated cytotoxicity by rGd could potentiate its’ probable use in the management of allograft rejection.

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