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This thesis aims to place corneal allograft rejection in the context of general transplantation immunology, examine the role of lymphocyte subsets in the rejection process and consider the potential application of monoclonal antibody therapy in clinical corneal graft rejection. The literature relating to the current clinical practice of corneal grafting, with particular reference to corneal allograft rejection, is reviewed in chapter 1 to present the extent of the problem. Chapter 2 then reviews the mechanisms of allograft rejection from the literature of transplantation immunology, much of which has arisen from studies of kidney, heart, pancreatic islets and liver in animal models. The materials and methods are described in detail in chapter 3, and only the relevant experimental design is detailed in the Materials and Methods sections of the succeeding chapters. The experimental mouse model of transplanting corneal tissue into the renal subcapsular is evaluated in chapter 4, demonstrating that isografts survive indefinitely whereas allografts are rejected typically by 30 days. Pretransplant sensitisation decreased allograft survival time to 10 days. Immunohistochemistry demonstrated the presence of CD4⁺ and CD8⁺ lymphocytes and macrophages at the rejection site. Heterotopic corneal graft recipients were then treated with various monoclonal antibody regimes. Chapter 5 demonstrates that allograft survival can be increased by either anti-CD4 or anti-CD8 therapy, providing near total depletion of the respective lymphocyte subset is achieved. Xenograft rejection is shown to depend on mainly CD4⁺ lymphocytes in chapter 6, with no benefit being found of depleting the CD8⁺ subset in addition. A mild immunosuppressive effect of anti-Vβ8 monoclonal antibody is demonstrated and discussed in chapter 7. The final chapter discusses these results in the light of recent, related work in other transplant systems, and presents a case for a trial of intracameral pan-T-cell monoclonal antibody treatment.

Macrophages (Mφ) have previously been implicated in both acute and chronic renal allograft rejection however the mechanisms remain unclear. In this thesis I set out to explore the effect of the Mφ on the endothelium in the context of renal graft rejection. Initial studies focussed upon human renal allograft tissue from transplant nephrectomies performed because of chronic allograft nephropathy (CAN). Immunostaining was carried out on these tissues (n=29) and control kidney tissue obtained from nephrectomies performed for renal cell carcinoma (n=19). An increased interstitial Mφ infiltrate was found compared to control tissue. Immunostaining for the T cell marker CD3 and the B cell marker CD20 demonstrated that both lymphocyte populations were present in the CAN tissue with almost negligible numbers seen in control tissue. Previous work in the group had demonstrated a reduced number of CD31 positive peritubular capillaries in the tissues used in these studies. In the work undertaken in this thesis, additional analysis was performed to study lymphatic vessels. Immunostaining of control tissue with the lymphatic endothelial cell (LEC) marker podoplanin demonstrated a normal distribution of lymphatic vessels around large interlobular arteries. CAN tissue, however, exhibited an increased lymphatic density with lymphatic vessels evident within the interstitium; a finding verified with two additional LEC markers (LYVE-1 and VEGFR-3). Further investigations examined possible mediators that could be responsible for the reduced microvascular peritubular capillary network and increased lymphatic vessels present in tissues affected by CAN. Previous work had implicated nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) in cardiac allograft rejection. Double immunolabelling for iNOS and the Mφ marker CD68 revealed evidence of Mφ expression of iNOS. No obvious reduction in vascular endothelial growth factor (VEGF)-A was evident although marked expression of VEGF-A was found in CD20 positive B cells within CAN tissue. Occasional interstitial cells expressed the lymphangiogenic growth factor VEGF-C, with double labelling studies indicating occasional CD68 +ve Mø that were positive for VEGF-C. In vitro studies were undertaken to dissect the interaction between Mø and microvascular endothelial cells (MCEC-1) using well established in vitro co-culture techniques. Co-culture of cytokine activated bone marrow derived Mø with MCEC-1 cells (a murine cardiac microvascular endothelial cell line) resulted in increasing levels of MCEC-1 apoptosis and a reduced cell number over a 24-hour time course. Non-activated Mø or cytokines alone were not cytotoxic. Co-cultures were performed in the presence of L-Nimino- ethyl lysine (L-Nil), a specific inhibitor of iNOS (control D-N6- (1-iminoethyl)-lysine (D-Nil)). L-Nil significantly inhibited MCEC-1 apoptosis and preserved cell number implicating a major role for NO in Mø-mediated MCEC-1 death. Importantly, L-Nil treatment did not affect TNFα production by cytokines suggesting that TNFα is not involved in MCEC-1 death in this in vitro experimental system. Experiments were then undertaken involving the depletion of Mø in a murine model of acute renal allograft rejection. Renal transplants were performed between donor Balb/c mice and either FVB/N CD11b-DTR mice transgenic for the diphtheria toxin receptor (DTR) under the CD11b promoter or control non-transgenic FVB/N mice. Diphtheria toxin (DT) was administered on days 3 and 5 to induce Mø depletion and mice sacrificed at day 7. Isograft controls were also performed between FVB/N mice. Murine allografts exhibited marked interstitial F4/80 positive Mø infiltration with expression of iNOS in the allografts. There was significant loss of peritubular capillaries (PTC) in allografts compared to isografts, indicating microvascular injury. DT treated CD11b-DTR mice exhibited 75% reduction in Mø infiltration and this was associated with dramatic microvascular protection. B and T cells were not evident in the isograft but significant accumulation of B and T cells was present in the allograft and not affect by Mø depletion. Interestingly, there was an increase in the number of podoplanin positive lymphatic vessels in the allograft compared to the isograft, which was significantly inhibited following Mø depletion. The final area of study focussed upon attempts to isolate lymphatic endothelial cells in vitro. Two types of vascular cells (HUVECs and HDMECs) were analysed by flow cytometry for LEC markers and immunofluorescence to phenotype the cells. Magnetic bead sorting was then undertaken to isolate discrete populations of endothelial cells expressing LEC markers. The murine studies reinforce the cytotoxic potential of Mø and supports a role for Mø in the deleterious rarefaction of microvascular interstitial vessels with resultant tissue hypoxia and ischaemia. Furthermore, these data support the involvement of Mø in the interstitial lymphangiogenesis that may occur in renal allografts. Furthermore, the study of human allograft tissue indicates that microvascular rarefaction and an increase in intrarenal lymphatic vessels occurs in human disease. Lastly, Mø expression of iNOS and VEGF-C suggests that Mø are involved in key processes that may adversely affect graft outcome.

The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pre-transplant donor-specific antibody (DSA), with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA post-transplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years post-transplant. Independent predictors of dnDSA were HLA-DRβ1 MM > 0 (OR 5.66, p < 0.006); and non-adherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p=0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury occurred and progressed in patients with dnDSA in the absence of graft dysfunction. Furthermore, non-adherence and cellular rejection contributed to both dnDSA development and the risk of progression to graft loss. (Human leukocyte antigen) HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for a subset of 286 donor–recipient pairs in which samples were available for high-resolution HLA-typing. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p<0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p<0.001). An optimal threshold for epitope mismatch (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA using a receiver operating characteristic analysis. Applying these thresholds, 0% and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years follow-up. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA when mismatched between the donor and recipient. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

Solid organ transplantation is now an established and effective treatment option for end-stage organ failure. Whilst early outcomes have improved significantly over recent decades, longer-term outcomes have changed little. Despite advances in immunosuppression, most transplanted organs suffer an inevitable decline in function attributed to chronic rejection. It is evident that the alloimmune response remains incompletely characterised. Crucially, despite description several decades ago, the precise contribution that the direct (recognition of intact allogeneic MHC) and indirect (recognition of self-MHC restricted allopeptide) pathways make to allograft rejection remains incompletely understood. In this thesis, murine models of heterotopic cardiac transplantation have been utilised to analyse these pathways. The key findings of this work are as follows: 1) If able to evade NK cell killing, passenger donor CD4 T cells can make cognate, direct-pathway, interactions with recipient B cells. This interaction results in augmentation of all arms of the alloimmune response and acceleration of allograft rejection. 2) Direct-pathway CD4 T cell allorecognition is restricted to the immediate post transplantation period. Donor APCs are the major source of MHC class II for direct-pathway priming, and these are cleared rapidly by both innate and adaptive responses of the recipient, effectively limiting the longevity of direct allorecognition. 3) The duration of indirect-pathway responses against different alloantigens is variable, limited by availability of donor antigen. Expression of donor MHC class II is restricted to APCs and possibly endothelium (where expression is transient) limiting the duration of indirect-pathway allorecognition against MHC class II alloantigen. Indirect-pathway CD4 T cell responses targeted against parenchymal alloantigen are long-lived, and can provide help for generating alloantibody against different MHC alloantigens. 4) In response to continual presentation of target epitope indirect-pathway CD4 T cell responses against parenchymal expressed alloantigen are long-lived. The continual division of these cells results in greatly increased numbers of alloantigen-specific CD4 T cells in the chronic phase of the response, but despite this, memory responses are impaired. 5) Generating indirect-pathway regulatory T cells specific for parenchymal expressed alloantigen appears to be the most effective strategy to ameliorating chronic rejection.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12958号 / 論医博第2100号 / 新制||医||1011(附属図書館) / 32357 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三嶋 理晃, 教授 三森 経世, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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