Glycodelin is a 162 amino acid secreted glycoprotein classified as a member of the lipocalin (carriers of small hydrophobic molecules) superfamily based on the presence of lipocalin signature motifs in its primary sequence. The protein has several isoforms which are expressed by various primate tissues, predominantly reproductive tissues. These isoforms are products of the same gene and hence have the same primary sequence; however, they are differentially glycosylated depending on tissue origin. The individual glycodelin isoforms perform very varied functions, which are largely dictated or modulated by the specific glycans on the molecule. Glycodelin A (GdA) is the major glycodelin isoform of the female reproductive tract; and is subclassified as an immunocalin (lipocalins with immunological function) due to its ability to modulate immune responses. Diverse activities have been associated with GdA; pertaining to determination of cell fate, tissue differentiation and significantly, immunomodulation towards fetal-allograft tolerance. The fetus expresses paternal allo-antigens and would be regarded as non-self or foreign by the maternal immune system. However, several synergistic mechanisms of immunomodulation at the fetal-maternal interface establish tolerance towards fetal antigens, protecting it from rejection. GdA is secreted by the uterine endometrium under progesterone induction, and is therefore the most abundant progesterone-regulated secretory glycoprotein of the uterus at the time of implantation and early pregnancy. GdA has been shown to have immunomodulatory activity targeting innate, humoral and cellular responses. It is inhibitory to T cell and B cell proliferation, and NK cell activity. It stimulates the Th2-type cytokine profile, and inhibits interleukins IL-2 and IL-1 production from mitogenically stimulated lymphocytes and mononuclear cell cultures. It has been reported from our laboratory that GdA induces apoptosis in activated T cells. GdA has also been shown to be inhibitory to B cells and monocytes. Clinical studies correlate subnormal levels of GdA with implantation Synopsis failure, habitual abortion and recurrent miscarriage. Due to its pleiotropic nature namely its diverse activities on different immune cell types; its spatio-temporal restriction of expression by progesterone; and its indispensable requirement for successful pregnancy; GdA is being increasingly recognized as a mechanism towards fetal allograft tolerance. Our laboratory has focused on the T cell inhibitory activity of GdA, with particular emphasis on T cell apoptosis. This study was aimed at delineating the molecular mechanism of GdA-induced apoptosis in activated T cells. Previous results from our laboratory have revealed that GdA-induced apoptosis is caspase dependent; and is not initiated by the extrinsic pathway involving Fas/death receptor signailing or initiator caspase 8. In this thesis, we present evidence that GdA triggers the intrinsic apoptotic program in T cells. Characterization of the apoptotic program initiated by GdA is presented in Chapter 1. We observe that GdA treatment triggers a stress response leading to decrease in mitochondrial transmembrane potential, which indicates mitochondrial membrane permeabilization (MMP). GdA-induced apoptosis can also be blocked by inhibition of caspase 9, the initiator caspase for the intrinsic program. The kinetics of mitochondrial depolarization precede onset of DNA fragmentation in both peripheral blood T cells and Jurkat cells treated with GdA. We also observe caspase 2 activation downstream of the mitochondria. Overexpression of the antiapoptotic protein Bcl-2 is sufficient to protect from GdA-induced cellular stress indicating that the apoptotic program can be reversed upstream of the mitochondria. Further, our studies reveal that stress signaling by GdA is not mediated by any of the canonical second messengers of stress signaling, namely, reactive oxygen species; the stress activated protein kinases JNK, p38 MAPK and ERK; intracellular calcium or ceramide. It has been reported that GdA desensitizes T cell receptor (TCR) signaling by decreasing the stability of TCR-triggered phosphoproteins, probably by its association ith the transmembrane tyrosine phosphatase CD45. TCR-desensitization would result in decreased proliferation and cytokine secretion, and has been postulated as the mechanism of T cell-inhibition by GdA. We have tested this theory and Chapter 2 provides evidence that the apoptogenic activity of GdA is not a consequence of its ability to blunt TCR-signaling. Further, GdA-induced apoptosis does not depend on components of the TCR signal cascade namely CD45, the kinase Lck and CTLA4, molecules that are proven transducers of apoptotic signals to the mitochondria in response to diverse stress stimuli. GdA triggers apoptosis in the CD45 deficient cell line J45.01 with similar kinetics of MMP and DNA fragmentation as with Synopsis wildtype cells, demonstrating that CD45 is not the determinant receptor for apoptosis on cells. We also observe that GdA is inhibitory to T cells stimulated with phorbol ester and calcium ionophore, which bypasses TCR-proximal signaling events; and that GdA treatment does not interfere with early T cell activation as evidenced from induction of the activation marker CD69. Thus, GdA initiates mitochondrial stress mediated apoptosis in T cells by a pathway that is distinct and independent from the TCR-coupled signaling pathway. This study presents a novel mode of immunosuppression for GdA and highlights the ability of GdA to suppress the immune response by more than one mechanism. Cell surface glycoproteins undergo alterations in their carbohydrate profiles upon T cell activation and differentiation, and this has a significant role to play in lymphocyte fate and function. One such global alteration in cell surface glycans is a difference in sialylation upon T cell activation and differentiation. While activated T cell have a lesser degree of sialylated surface glycoproteins as compared to naïve T cells, memory T cells are sialylated to a higher extent, and Th2 cells have more cell surface sialic acids than Th1 cells. As GdA is capable of triggering apoptosis in activated T cells, we investigated the requirement of cell surface glycans for differential recognition of T cell subsets by GdA, the results for which are detailed in Chapter 3. We observe that the activity of GdA could be competed out by asialofetuin and not fetuin, suggesting that GdA recognizes terminal galactose residues on asialofetuin glycans, which would be masked by sialic acids in case of fetuin glycans. This assumption was confirmed as the free sugars lactose and galactose, but not annose, could also competitively inhibit GdA activity. We also demonstrate that the lectin-activity of dA is calcium independent, typical of mammalian galectins. Thus, our results reveal GdA to be a novel galactose-specific lectin of the female reproductive tract. This carbohydrate specificity of GdA is responsible for its apoptotic activity on T cells. The selectivity of GdA towards activated T cells is a result of increased exposure of terminal galactose residues on activated T cell surface receptors, as demonstrated by staining of naïve and stimulated T cells with Fluorescent lectin-conjugates of different carbohydrate specificities. We also demonstrate hat GdA shows specificity towards N-liked glycans on cell surface glycoproteins. This is evident from the use of glycan processing inhibitors, which prevent addition of galactose to the core glycan on the nascent polypeptide chain. We observe that inhibition of processing of N-glycans, and not O-glycans, render cells resistant to GdA. Incidentally, we observe that another property of GdA, namely its ability to induce Synopsis epithelial differentiation and apoptosis in the breast cancer cell line MCF-7, is also due to ts galactose-specific lectin activity. It is therefore probable that the diverse functions ssociated with GdA are a consequence of its ability to recognize different glycoprotein receptors on different cell types. We can thus draw a comparison for GdA with the galectins, which are the prototype beta-galactoside binding mammalian lectins with diverse roles in determining cell fate and apoptosis, especially in the immune system. In fact, the immune-related activities of GdA are almost identical to the effects of galectin-1 on the immune system. Galectin-1 has also very recently been shown to play a significant role in fetal-tolerance. This raises a strong possibility of shared receptors for GdA and galectin-1 on the T cell surface, resulting from a shared calcium-independent recognition property for complex glycans with terminal galactose residues. Two predominant galectin-1 receptors on T cells are the glycoproteins CD45 and CD7. We have already observed that though GdA may recognize CD45, this association does not mediate its apoptotic activity. We therefore examined the possibility of the activation-induced glycoprotein CD7 as receptor for GdA. Our experiments reveal that the apoptotic activity of GdA on different T cell lines is dependent on the degree of CD7 expression by these cell lines. Notably, the CD7 negative lymphoma cell line HuT78 was completely resistant to GdA. To confirm CD7 as receptor, we obtained a cell line HuT78.7 in which CD7 expression has been restored by stable transfection. We observed that these CD7 positive cells now responded to GdA comparable to Jurkat cells, and GdA-induced apoptosis in these cells could be completely competed out with asialofetuin, not fetuin. To summarize, our study identifies GdA as a novel pregnancy-related galectin-like lectin of the female reproductive tract, which triggers mitochondrial stress and apoptosis in activated T cells. GdA shares receptors on T cells with galectin-1 due a common carbohydrate recognition property. We identify CD7 as a molecular target for GdA on activated T cells, capable of mediating the apoptotic signal. However, it is likely that GdA also recognizes other galectin receptors on T cells, as it is capable of inhibition by more than one mechanism. This underscores the requirement for redundant mechanisms indispensable for establishment and maintenance of successful pregnancy.

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Approved for public release; distribution in unlimited. / In recent years there has been an increased interest in effective individual control and enhanced security measures, and face recognition schemes play an important role in this increasing market. In the past, most face recognition research studies have been conducted with visible imaging data. Only recently have IR imaging face recognition studies been reported for wide use applications, as uncooled IR imaging technology has improved to the point where the resolution of these much cheaper cameras closely approaches that of cooled counterparts. This study is part of an on-going research conducted at the Naval Postgraduate School which investigates the feasibility of applying a low cost uncooled IR camera for face recognition applications. This specific study investigates whether nonlinear kernel-based classifiers may improve overall classification rates over those obtained with linear classification schemes. The study is applied to a 50 subject IR database developed in house with a low resolution uncooled IR camera. Results show best overall mean classification performances around 98.55% which represents a 5% performance improvement over the best linear classifier results obtained previously on the same database. This study also considers several metrics to evaluate the impacts variations in various user-specified parameters have on the resulting classification performances. These results show that a low-cost, low-resolution IR camera combined with an efficient classifier can play an effective role in security related applications. / Captain, Hellenic Air Force

Glycodelin is a 162 amino acid dimeric, glycosylated, secretory protein of the lipocalin superfamily. Its classification as a lipocalin(carriers of small hydrophobic molecules) is based mainly on the presence of lipocalin signature motifs in its primary sequence and no ligand for this protein has been identified till date. Glycodelin has 40-55% sequence identity with β-lactoglobulin which is the second type member of the lipocalin superfamily (the first being retinol binding protein (RBP). Glycodelin is primarily a primate specific protein (though there have been isolated reports of mRNA in mice and rats) with many isoforms secreted by various tissues, predominantly of the reproductive tract. These isoforms, being the product of the same gene, are identical in primary sequence and differ only in their glycosylation due to differences in tissue origin; hence they may be better addressed as glycoforms of glycodelin. The main glycoforms of glycodelin reported till now are GdA, GdS, GdM, GdF and GdC. Each glycoform of the protein has a varied function, dictated or modulated largely by the complex glycans on its surface. GdA, the most well studied glycoform of glycodelin, is secreted by the endometrium under progesterone control and accumulates in the amniotic fluid (from where it is isolated.). GdA has been subclassifed as an immunocalin (immuno-modulatory lipocalins) due to the many immuno-modulatory functions pertaining to tissue differentiation, implantation and angiogenesis and most sifnificantly, modulation of immune responses at ehe feto-maternal interface. The fetus expresses paternal allo-antigens on its surface and would be regarded as foreign or non-self by the maternal immune system. Yet the fetus is not rejected, and is in fact protected from attack by the maternal immune system by a variety of tolerogenic mechanisms. GdA is the most abundant secretary glycoprotein of the primate uterine compartment during implantation and early pregnancy. It has been shown to have inhibitory effect on innate as well as adaptive and humoral immune responses. It inhibits the proliferation of T and B cells, Nk cytoxocity and suppresses monocyte chemotaxis. It also skews the cytokine profile from Th1 to Th2 and inhibits IL1 and IL2 secretion from mitogenically stimulated lymphocyte and mononuclear cell cultures. In our laboratory, we have demonstrated earlier that the inhibitory effect of GdA on T cell proliferation is due to apoptosis being induced. The apoptotic signaling induced by GdA was found to be caspase dependent and follows the intrinsic mitochondrial stress induced pathway of apoptosis. Having determined the effect of glycodelin A on T cells, we wanted to look at its effect on other cells playing a role in immune responses. We decided to look at its effect, if any, on the innate immune system. Chapter 1 of the thesis describes our studies on the effect of GdA on monocytes. We have looked at the effect of GdA on primary monocytes isolated from blood of healthy human volunteers and found that GdA induces apoptosis of primary monocytes and this appears to be mediated through a caspase independent pathway. The mitochondrial membrane potential of primary monocytes was lost upon GdA treatment therefore the mitochondria seem to be involved in the apoptotic cascade. As the yield of monocytes from peripheral blood is very low, further studies on the effect of GdA on monocytes were carried out using a human monocytic cell line, THP1, as a model system. We have demonstrated the GdA is able to inhibit the proliferation of these cells and also induce apoptosis in them. We also found that this signaling is partially caspase-dependent and involvement of other caspase independent pathways is possible. Further, we have shown that there is no effect of GdA on the phagocytic ability of these cells after differentiation into the macrophage lineage. However, when added before differentiation, glycodelin is able to inhibit the phagocytic ability of THP1 cells. We also found that THP1 cells were relatively resistant to GdA-induced apoptosis post differentiation into macrophages. We have also looked at the effect of GdA on B cells using primary B cells as well as a B cell line U266B1 as our model system. GdA was shown to inhibit the proliferation of primary B cells as well as of the cell line. The protein was not able to induce apoptosis in the primary cells (both activated as well as unactivated cells) as well as in the cell line. Treatment of the cells with MAP kinase inhibitors also did not render them susceptible to GdA induced apoptosis(as has been seen in the case of U937 cells). U266B1 cells remained relatively resistant to GdA-induced apoptosis even when treated for long periods. They did not undergo significant necrosis uponGdA treatment even though the proliferation of these cells was inhibited by the protein. We were surprised to find that there was loss of mitochondrial membrane potential of the cells upon GdA treatment even when there was no cell death. The reason for this is not clear. The inhibition of proliferation of B cells by GdA does not involve caspases and the signalilng induced by GdA in these cells seems to be different to that induced in T cells atleast downstream of the mitochondria as the cells cannot proliferate in presence of GdA but seem immune to further damage or apoptosis. These studies have been described in chapter 2 of the thesis. The third and final chapter of the thesis deals with our investigation into the effect of GdA on Nk cells. GdA, in an earlier report, has been shown to inhibit the activity of circulatory NK cells. However, the mechanism of this action has not been delineated. We made attempts to determine the effect of GdA on NK cells using a human NK cell line YT Indy as our model system, as isolation and culture of primary Nk cells in good numbers is difficult. Preliminary studies revealed that GdA triggered apoptosis in these cells. However, the process was found to be caspase independent. Another surprising finding was that GdA did not bring about significant loss of mitochondrial membrane potential of these cells, implying that the involvement of mitochondria in the apoptotic signaling in these cells may be at the later stages, as amplifiers rather than initiators, as has been seen in the case of T cells and monocytes.

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. .

The ability of our immune system to mount a response against non-self-antigens legitimates the semi-allogenic fetus as a target for maternal immune attack. Yet, in a normal pregnancy the fetus stays well protected due to the concerted action of several diverse mechanisms which either suppress the fetal allogenicity or spatio-temporally inhibit maternal immune cells’ growth and functions. One such factor which aids in the establishment, progression and maintenance of pregnancy is the 28 kDa dimeric sialylated glycoprotein Glycodelin-A (GdA). Synthesized by the endometrium and decidua, this protein has myriad functions, the most important being that of immunosuppression. GdA is inhibitory to all hematopoietic cells and also induces programmed cell death in activated T cells and monocytes via the intrinsic mitochondrial pathway. In the Introductory chapter of this thesis, details about GdA and the other isoforms of the glycodelin family of proteins have been presented which highlight the involvement of glycodelins in primate pregnancy, with emphasis on GdA and its pleiotropic functions associated with reproduction in females. Activated T-lymphocytes against paternal antigens are found in the uterine compartment and in the maternal circulation throughout pregnancy. Activated CD8+ T-lymphocytes have been reported to pre-dominate the uterine T-lymphocyte population during pregnancy and unlike the CD4+ T cells, are retained until term. Studies show that activated CD8+ T-lymphocytes are necessary for the establishment and progression of early pregnancy. However, how these lymphocytes harbouring cytotoxic activity are regulated at the later stages of pregnancy is poorly defined. We attempted to uncover a possible mechanism of regulation of CTL (cytotoxic T lymphocyte) activity (if any) during primate pregnancy by GdA. In the absence of established human CD8+ T cell lines, we first standardized the generation of CTLs in-vitro from hPBMCs (human peripheral blood mononuclear cells) by alloactivating them with an ovarian carcinoma cell line OVCAR-3 utilized as a mimic of an allograft. The details of the rationale behind using this method for generating CTLs and the alloactivation methodology have been put together in the Chapter 1 of this thesis. The activation of hPBMCs was confirmed by the surface expression of an early activation marker CD69 and tritiated thymidine incorporation. Differentiation of CD8+ T cells into effector cells was confirmed by the upregulation of perforin and granzyme transcripts by real time RT-PCR analysis. Target-cell specific cytolytic activity of the CTLs was assessed by using a cytotoxicity measurement assay- JAM test, details of which also form a part of chapter 1. Having generated effective CTLs in vitro, we tested the effect of GdA on CTL activity. Our findings, on the effect of GdA on CTLs have also been discussed in the Chapter 1. We observed that the cytolytic activity of CTLs was significantly reduced by GdA treatment albeit at a dose three to four times higher than that required for inhibiting CD8+ T cell proliferation, implying that a mechanism of temporal regulation of CTL activity operated at the feto-maternal interface, thereby contributing to the establishment and progression of pregnancy. Interestingly, in our quest to uncover the mechanism of inhibition of CTL activity by GdA, we found that the inhibition of proliferation was comparable in both CD4+ and CD8+ T-lymphocytes at all dosages of GdA, but unlike CD4 + T cells CD8 + T cells were resistant to GdA-induced apoptosis even at high dosage of GdA. Hence we could rule out that the loss of CTL activity upon GdA treatment was due to CD8+ T cell death. Further, we assessed the functional competence of alloactivated CTLs by quantitating the mRNA transcripts of key cytolytic molecules; perforin and granzyme B, in GdA treated alloactivated hPBMCs and found that there was a significant reduction in the mRNA of these cytolytic molecules. Additionally, we also found that GdA treated CD8+ T cells exhibited impaired release of the cytolytic molecules by the process of degranulation, measured by the surface exposure of LAMPs (Lysosome associated membrane proteins) on the surface of cells by flow cytometry and as seen by the retention of perforin protein in them assessed by intracellular staining and flow cytometry. Intrigued by the observations, we probed for the regulators of perforin and granzymes in CTLs. EOMES (Eomesodermin) and T- Bet are well known transcription factors which control the differentiation of CD8+ T cells into effector and memory cell CD8+ T cell type. Interestingly we found that the expression of EOMES was significantly reduced in activated GdA treated hPBMCs, both at the transcriptional and translational level, however T-Bet did not show any variation in expression upon GdA treatment. All the above findings have been compiled in Chapter 2 along with our studies on the possibility of GdA to induce a tolerogenic phenotype in T cells. We found there was no difference in the mRNA level and surface expression of CD103 and CD28 in alloactivated PBMCs, while FOXP3 mRNA did not show any variation upon GdA treatment, indicating that GdA does not induce a tolerogenic phenotype in T-lymphocytes, further confirming our data that the decreased cytolytic activity of CTLs upon GdA treatment was not due to tolerance but due to impaired function Interestingly, IL-2/IL-2R signaling is known to directly regulate perforin and granzyme expression as well as it plays a role in the expression of T-Bet and EOMES. Therefore, as a read out of IL-2 signaling we checked for the surface expression of the high affinity IL-2R subunit, CD25. As expected, CD25 expression was more pronounced in CD4+ T cells and consistent with published reports in literature that GdA suppresses IL-2 synthesis, we also observed a significant reduction in the CD25bright population in both the T cell subsets (CD4+ and CD8+) upon GdA treatment (addressed in Chapter 3). This finding supports a mechanism of action of GdA, wherein the cytolytic activity of CTLs is compromised by the downregulation of EOMES, triggered by the low IL-2 levels. This translates to aberrant synthesis of key cytolytic molecules perforin and granzyme B, leading to low efficiency CTLs, which are further disabled by defective degranulation machinery induced by GdA. We did not look into the mechanistic aspects of how GdA suppresses degranulation, which can be addressed later as a part of another study. Building up on our observations, and taking cues from existing literature, that IL-2 regulates the expression of pro and anti-apoptotic protein levels within activated cells, we looked at the expression profile of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in activated PBMCs upon GdA treatment. There was a significant reduction in the total mRNA and protein level of Bcl-2, while a very significant increase in Bax mRNA and protein was observed. Chapter 3 of the thesis also presents this data and explains a plausible mechanism of the inhibitory effect of GdA on T-lymphocytes. In Chapter 2, we have also addressed the probable reasons for the differences in the responses of CD4+ and CD8+ T-lymphocytes to GdA. Interestingly, surface glycan profile of CD4+ and CD8+ T-lymphocytes upon activation and the surface expression of the most probable receptor for GdA i.e. CD7 was comparable in both the T cell subsets, indicating that possibly the downstream signaling events leading to GdA-induced apoptosis and not the surface binding of GdA may vary in CD4+ and CD8+ T-lymphocytes, due to which we observed a difference in the extent of apoptosis induced in these cell types by GdA although the inhibition of proliferation in both the subsets was comparable. In summary, this study is the first to provide evidence for a possible mechanism of temporal regulation of CTL activity at the feto-maternal interface, where activated CD8+ T cells are abundantly present. We can say with much confidence that binding of GdA to T-lymphocytes causes sub-optimal IL-2 signaling which translates into reduced expression of EOMES and hence downregulation of perforin and granzyme B, leading to impaired CTL activity in CD8+ T-lymphocytes, which is further weakened by the impaired release of the cytolytic molecules from them. Insufficient IL-2 signaling in the presence of GdA can also be a cause of inhibition of proliferation in T-lymphocytes, while the resulting decrease in anti-apoptotic protein Bcl-2 and increase in pro-apoptotic protein Bax seem to contribute to the induction of apoptosis in CD4+ T cell. It will be interesting to explore the mediators involved in the IL-2 signaling pathway that are differentially regulated in CD4+ and CD8+ T cells which confer resistance in CD8+ T cells to GdA-induced apoptosis and also the mechanism by which GdA regulates the degranulation of cytolytic vesicles in CTLs needs to be worked out.

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