Yide Jin

Heterotopic Uterus Transplantation in a Swine Model

Background. The aim of our study was to examine the feasibility of allogeneic uterine transplantation in a large animal model. Methods. We performed heterotopic uterine transplants in genetically defined mini-pigs. Immunosuppression was tacrolimus administered intravenously for the first 12 days posttransplantation followed by oral cyclosporine maintenance immunosuppression. The graft was transplanted heterotopically in the lower abdominal cavity of the recipient. The vaginal vault was exteriorized as a stoma in the lower right abdominal wall. The uterine grafts were followed with endoscopies and biopsies. Results. Ten transplants were performed. Follow-up was until July 2008. At the end of the follow-up period, 5 animals were alive and healthy, 0.5 to 12 months posttransplantation. There were 5 deaths due to pneumonia (n=1), intussusception of the graft (n=1), cardiorespiratory arrest during anesthesia (n=1), and complications of the stoma (n=2). Acute rejections of the graft presented during the 2nd and 3rd month posttransplantation were treated successfully with increase of the maintenance immunosuppression and steroids. Other complications included prolapse and infections of the graft stoma. Pathological changes seen in the endometrial biopsies included acute rejection and acute endometritis. Conclusion. These findings demonstrate that successful uterus transplantation in a large animal model (miniature swine) is feasible using this heterotopic model, and it can be useful for the study of these transplants.

The human bone marrow as an immunoregulatory organ.

It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1–3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define “acquired specific immunological tolerance” as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11–15), to date, specific immunological tolerance in organ transplantation in humans has not been possible.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34(+) stem cells and the CD34(-) early progeny like CD38(+), CD2(+), CD5(+) and CD1(+) lymphoid cells as well as CD33(+) (but CD15(-)) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell-cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34(+) cells were found to give rise both to CD3(-) TCRalphabeta(+), as well as CD3(+) TCRalphabeta(+) cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (−) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV− patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCRαβ+, CD4−CD8−, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ “intermediate” T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV− livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV− patients produced low levels of cytokines IL-1β, IL-2, IL-6, TNFα, and IFN-γ but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.

The regulation of phenotype and function of human liver CD3+/CD56+ lymphocytes, and cells that also co-express CD8 by IL-2, IL-12, and anti-CD3 monoclonal antibody

The regulation of phenotype and function of human liver infiltrating lymphocytes (LIL) by in vitro culture with IL-2, IL-12 and anti-CD3 monoclonal antibodies (mAb) was investigated. The CD3+ LIL which express 50% less CD3 molecules per cell than peripheral blood T lymphocytes, exhibited a 6-fold reduction in proliferation when stimulated through the CD3 complex by anti-CD3 mAb. LIL freshly isolated or cultured in medium did not suppress MLR response, nor were they cytotoxic. However, treatment of the LIL cells with IL-2, IL-12 and anti-CD3 induced these cells to suppress autologous responding cells in MLR (ca. 70%) and to kill autologous or allogeneic cells. Low level cytotoxicity could be induced by cytokines IL-2, IL-12 or anti-CD3 alone. However, the development of optimum MLR suppression and cytotoxicity induction was dependent upon stimulation of the LIL cells through the CD3 complex. The co-expression of CD3 and CD56 on LIL was also up-regulated by anti-CD3 stimulation in the combination of IL-2 and IL-12. Most of the CD3+/CD56+ cells, also expressed CD8. After the magnetic bead separation procedure, the cytotoxic activity was found mainly in the CD3+/CD56+/CD8+ population. These results suggest that CD3+/CD56+/CD8+ cells can be expanded by stimulation through the TCR/CD3 complex in the presence of IL-2 and IL-12, which results in the suppression of autologous responding cells by a cytotoxic mechanism. The proliferative response of the CD3+/CD56+/CD8+ population was enhanced by the induction of CD1 molecules on the stimulating cells, and anti-CD1 mAb were able to block the response in a dose-dependent manner. The CD3+/CD56+/CD8+ cells were examined for cytokine production by flow cytometry. Cytokines IL-4, TNF-alpha, and IFN-gamma were produced by 91.7%, 29.2%, and 27.4% of the cells, respectively.

Bone marrow cells inhibit the generation of autologous EBV-specific CTL

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 +/- 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 +/- 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3(+) cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E(2) (PGE(2)), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE(2) in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.

Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation

This laboratory recently reported that human bone marrow cells (BMC) inhibit the generation of virus-specific CTL in culture. The culture supernatants contained increased levels of prostaglandin E(2) (PGE(2)) (shown to favor TH2 cell development) and also inhibited EBV-CTL effector cell development. In this study, we obtained PBL from Epstein-Barr virus (EBV) IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) one year after renal transplantation. EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells (EBV-B) in the presence or absence of autologous BMC. The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4,-5,-6,-10, and -13. These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3. Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B. It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. Protein Kinase A (PKA), a cAMP kinase that is involved in the upregulation of TH2 cytokine activity, was increased in EBV-CTL cultures by the presence of BMC. Additionally, IL-4-mediated signal transduction and activation of transcription (STAT-6) phosphorylation was slightly increased. These results show that the BMC inhibition is mediated by soluble factors (cytokines) and that cell-cell contact in this autologous system is not required, so that BMC (at least partially, via cytokine production) promote TH2 polarization in culture. Moreover, TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation, and TH2 associated PKA, CCR3, and STAT-6 phosphorylation are enhanced by BMC.

Induction of Auto-reactive Regulatory T Cells by Stimulation with Immature Autologous Dendritic Cells

We have shown in ex vivo studies in donor bone marrow-infused kidney transplant recipients, that chimeric cells of either donor or recipient origin taken from the recipients bone marrow down-regulated the recipients cellular immune responses. In the present study, we have now induced regulatory T cells from peripheral blood mononuclear cells (PBMC) of renal transplant recipients or laboratory volunteers by multi-stimulation with autologous immature dendritic cell (iDC) enriched populations derived from either bone marrow cells (BMC) of the (immunosuppressed) kidney transplant recipients or PBMC of the laboratory volunteers (i.e., ibDC and ipDC, respectively). These regulatory T cells, induced by ibDC and ipDC, were autoreactive and designated as TAb and TAp with similar phenotypes and functional profiles. They were largely CD4 + CD25high, CD45RA low and CD45RO high, and uniformly expressed intracellular CTLA-4, and message of IL-4, IL-10, Foxp3, and differentially expressed TGFβ. Their proliferative responses to autologous mature dendritic stimulating cells (mDC) were ∼two-fold stronger than to allogeneic mDC, and to allogeneic mDC were significantly lower than those of (control) autologous TPBL, suggesting an anergic state. TAb and TAp were not cytotoxic to autologous cells expressing Epstein-Barr virus (EBV) antigens, but were able to inhibit (regulate) the effector phase of this TPBL response to both autologous and allogeneic EBV lymphoblasts. This regulation appeared to require cell-to-cell contact.

Transplant-associated autoimmune mechanisms in human hepatitis C virus infection.

In order to define factors which are important for the development of hepatitis C virus (HCV) infection and disease in transplant patients, we examined the role of class II MHC antigen restriction in viral antigen presentation to support a hypothesis of the association of this disease with an autoimmune pathogenesis. A greater degree of histocompatibility match between these donors and their HCV-negative recipients was associated with a greater predisposition to recipient HCV liver disease (ALT elevation) posttransplant. The HCV carrier state could be identified with significant amplification of autologous mixed lymphocyte reactivity (AMLR) in both long-term hemodialysis and long-term renal transplant patients, but the AMLR was absent in end-stage liver disease patients with HCV-associated cirrhosis and was insignificantly elevated in these patients with persistent infection in the first 2 years after a new liver was transplanted. There was also a moderate reduction in autologous reactivity as well as serum HCV titers among renal transplant patients who displayed biochemical evidence of chronic liver disease as opposed to those who did not. This appeared later in the course of the disease. HCV RNA could be detected in peripheral blood mononuclear cells (PBMC) of only a portion of HCV-infected renal transplant patients and these showed significantly higher autologous reactivity. In contrast, despite the fact that observations were earlier after de novo liver transplantation, HCV RNA (i.e., earlier in the course of a new or recurrent disease process) was found in PBMC of all liver transplant recipients tested. The AMLR of noninfected laboratory volunteers could be amplified by preincubating their stimulating cells (APCs) with enriched HCV possibly in immune complex (pHCV-IC). This amplification appeared only with specific combinations of HCV strains with HLA DR serotypes. In addition, HCV-primed T cells could be generated to the virus which displayed accelerated activation kinetics. Liver infiltrating lymphocytes extracted from HCV-positive end-stage diseased livers had significantly higher proliferative and cytotoxic reactivity to autologous (HCV-infected) hepatocytes than the extracted lymphocytes responding to autologous hepatocytes from HCV-negative livers. These findings offer evidence of dynamic autoimmune mechanisms in the spectrum of progression of HCV disease and may help to predict the effect of intervention at various intervals in this progression in organ transplant recipients.

Molecular Genetic Analysis of Interleukin-1 Promoter and Receptor Antagonist Tandem Repeat Polymorphisms Among HLA-Identical Renal Transplant Recipient and Donor Pairs

The cytokine gene polymorphisms as well as the minor histocompatibility antigen (mHAg) disparities have been thought to result in transplant graft rejection and graft-versus-host reaction between HLA-identical donor/recipient pairs. We detected interleukin (IL)-1 promoter and receptor antagonist tandem repeat polymorphisms among 21 HLA identical renal transplant recipient and donor pairs; of 21 pairs, 15 (71%) showed at least one locus difference between donor and recipient, and 73% of the pairs with the disparities at these loci showed either recipient to donor (R-->D) or donor to recipient (D-->R) positive CD4+ cell response measured by a modified Cylex assay developed in our laboratory. Allele 2 of IL-1 receptor antagonist genotype (IL-1ra VNTR), a gene associated with an increasing IL-1beta production, was detected in the three recipients who developed rejections. One HLA-identical pair that had variations on all four loci of IL-1 gene cluster consistently showed positive CD4+ cell immune responses between the donor and the recipient. This study may provide information of the role of non-HLA genes participating in allograft rejection; this demonstrates that in addition to the disparities of the mHAgs, the non-HLA antigens have to be considered as risk factors in HLA-identical solid organ transplantation.