Role of anti-donor antibody in the rejection of pig proislet xenografts in mice

Abstract: CBA/H mice produced serum anti-pig IgG₁, IgG_2a, and IgG_2b following xenotransplantation of pig proislets beneath the kidney capsule; anti-pig IgM was present as pre-existing antibody in the serum of normal CBA/H mice and was also produced in response to pig proislet xenografts. Serum anti-pig IgG₃was not detected post-xenotransplantation. Rejection of pig proislet xenografts and the production of anti-pig IgG₁, IgG_2a, and IgG_2b isotypes were CD4 T cell-dependent. The capacity for adoptively transferred hyperimmune CBA/H mouse anti-pig PBL serum to induce the rejection of intact pig proislet xenografts in CD4 T cell-depleted mice was dose dependent and correlated with markedly elevated levels of serum anti-pig IgG₃. Levels of anti-pig IgG₁, IgG_2a, IgG_2b, and IgM comparable to control mice acutely rejecting pig proislet xenografts and achieved following adoptive transfer of hyperimmune serum did not correlate with induced xenograft rejection. These findings suggest that anti-pig Ig isotypes produced during the conventional process of acute proislet xenograft rejection do not play a major role in mediating graft damage. The acute rejection of pig proislet xenografts in the absence of serum anti-pig Ig in μMT-/- hosts confirmed that anti-pig antibody is not essential for proislet xenograft destruction.     

Evidence that T-helper type 2 cell-derived cytokines and eosinophils contribute to acute rejection of orthotopic corneal xenografts in mice

BACKGROUND: Because guinea pig corneal xenografts are rejected acutely (within 16 days) in mouse eyes by a T-cell-dependent mechanism, the authors wished to determine the functional phenotype of CD4+ effector T cells. METHODS: Orthotopic corneal xenotransplantation was performed from strain 13 guinea pigs to BALB/c mice. Grafted eyes were removed at specified times and examined histologically or subjected to cytokine and chemokine mRNA analysis using a multi-probe ribonuclease protection assay. Draining cervical lymph node cells were harvested at specified times and stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells. Supernatants were assayed by enzyme-linked immunosorbent assay for content of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, and IL-10 and cells were used for mRNA analysis. RESULTS: Rejected corneal xenografts were heavily infiltrated with polymorphonuclear leukocytes, the majority of which were eosinophils. These eyes contained mRNA for IL-4, IL-6, IL-10, IL-13, IL-15, and IFN-gamma. When stimulated with guinea pig spleen cells, T cells from draining cervical lymph nodes secreted primarily IL-4, IL-5, IL-10, and IFN-gamma. Eotaxin was overexpressed in eyes with rejected corneal xenografts. CONCLUSIONS: Acute rejection of corneal xenografts in mice is mediated by T cells that display a mixed T-helper (Th) type 2/Th1 phenotype and secrete eotaxin, an eosinophil chemoattractant. Eosinophil-dependent xenograft rejection bears similarities to immune elimination of parasites.     

Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD4+ T cell-depleted mice

Treatment of CBA/H mice with 5 injections of anti-CD4 (GK1.5 mAb) terminating on day 10 posttransplant resulted in long-term survival (greater than or equal to 6 weeks) of fetal pig proislet (pancreatic islet precursor) xenografts. The GK1.5 mAb dose determined the duration of CD4+ T cell depletion and the extent to which the survival of pig proislet xenografts was prolonged. Sustained depletion of CD4+ T cells (0%, 1%, and 9% of total T cells in peripheral lymph nodes at 2, 4, and 6 weeks, respectively) and survival of proislet xenografts at 6 weeks posttransplant was observed when transplant recipients were treated with 5.4 mg GK1.5 mAb/injection. Treatment of transplanted mice with a suboptimal dose of GK1.5 mAb (0.2 mg/injection) resulted in the same level of depletion at 2 weeks posttransplant but a more rapid recovery of CD4+ T cells in the periphery (24% of total T cells at 4 weeks) and only temporary prolongation in xenograft survival (less than or equal to 4 weeks). Control xenografts showed evidence of graft destruction by as early as 6-7 days posttransplant and were completely rejected by 2 weeks. The rejection reaction consisted predominantly of CD4+ T cells, eosinophils and F4/80-positive macrophages. Only small numbers of CD8+ T cells were identified. CD4+ T cells therefore represented the major T cell component of the cellular infiltrate. In contrast, surviving xenografts in GK1.5 mAb-treated recipient mice showed essentially an absence of CD4+ T cells but presence of CD8+ T cells. This finding may be attributable to the increase (1.7-3.1-fold) in the absolute size of the population of CD8+ T cells in the periphery following GK1.5 mAb treatment in vivo. Compared with isolated fetal pig proislets, which contained only a small population of insulin-producing cells in addition to glucagon- and somatostatin-positive cells, surviving pig proislet xenografts contained mainly insulin-positive beta cells with smaller populations of glucagon- and somatostatin-positive cells. Fetal pig proislets therefore differentiate into insulin-producing islet tissue posttransplant and thus show evidence of normal development of endocrine function.     

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.     

Antibody-induced rejection of pig proislet xenografts in CD4+ T cell-depleted diabetic mice

Reversal of diabetes in mice was achieved following in vivo depletion of host CD4+ T cells and transplantation of xenogeneic fetal pig proislets (pancreatic islet precursors). These procedures resulted in xenograft tolerance since established pig proislet xenografts were not rejected by antipig antibodies produced in the host, and rejection was not induced following the administration of donor major histocompatibility complex--specific pig lymphocytes. Proislet xenografts were rejected following the administration of donor MHC-specific hyper-immune antipig PBL serum raised in normal mice. Although established proislet xenografts in anti-CD4-treated mice are sensitive to antibody-mediated destruction, such hosts are unable to produce an antibody response that leads to graft rejection. The study indicates that the mechanism of preventing xenograft rejection by anti-CD4 treatment in vivo involves not only initial CD4+ T cell depletion but also quantitative and/or qualitative modulation of a CD4+ T cell-dependent antibody response. As a consequence, an apparent state of xenograft tolerance is produced.     

CD8+ T cell subsets TC1 and TC2 cause different histopathologic forms of murine cardiac allograft rejection

BACKGROUND: CD4+ T cell effector function is sufficient to mediate allograft rejection, and it is suggested that CD8+ T cell-mediated effects are dependent on CD4+ T cell help. CD8+ T cells can be classified into at least two functional subsets: Tc1, producing high amounts of interferon (IFN)-gamma and Tc2, producing interleukin (IL)-4, -5, -10, and -13 and low levels of IFN-gamma. Because these subsets express different chemokine receptors, they may have different capabilities of migrating into grafts. Once in the graft, each subset may perform different effector functions dependent on the cytokines it produces. We asked whether allospecific CD8+ T cells, in the absence of CD4+ T cells, are capable of mediating rejection of a primarily vascularized allograft, and if Tcl and Tc2 cells differ in their ability to mediate rejection. METHODS: Hearts from H-2d mice were transplanted into H-2b RAG 1-/- recipients. Without manipulation, these fully mismatched allografts would survive indefinitely due to the absence of mature T and B cells. We adoptively transferred allo-(H-2d)-reactive Tcl or Tc2 cells from H-2b mice into each recipient. Grafts were harvested and analyzed on predefined timepoints, rejection was graded on a modified ISHLT scale. RESULTS: On day 7, grafts from Tc1- or Tc2-injected animals showed grade 1-2 parenchymal rejection with stable phenotype and comparable distribution of graft infiltrating CD8+ T cells. Adoptive transfer of IFN-gammahigh Tc1, but not of IFN-gammalow Tc2 cells was followed by the development of graft vasculitis, as well as graft arteriopathy. Adoptive transfer of IL-4high IL-5high Tc2, but not of IL-4low IL-5low Tc1 cells lead to extensive infiltration of eosinophils and formation of giant cells. CONCLUSIONS: Both Tc1 and Tc2 cells can mediate murine cardiac allograft rejection in the absence of CD4+ T cell help, although each subset elicits a different type of inflammatory response. In this model, cytokine secretion of either functional CD8+ T effector cell subset is an important effector mechanism in the process of allograft rejection: IFN-gammahigh Tc1 cells are important in early graft vasculitis, although IL-4high IL-5high Tc2 cells promote recruitment of secondary effectors like eosinophils.     

Acute Xenograft Rejection Mediated by Antibodies Produced Independently of TH1/TH2 Cytokine Profiles

There is substantial support for the hypothesis that T(H)1 cytokine responses are critical for the normal elaboration of allograft rejection. Recent studies by Wang et al. (1) underscore the importance of T(H)2 responses in xenograft rejection and revealed that T(H)1 cytokines, IL-12 and interferon-gamma (IFN-gamma), can negatively regulate the development of humoral responses necessary for xenograft rejection. Their exceptional studies prompted us to test whether the ability of allografts to elicit cellular rejection and xenografts to induce humoral rejection also result from the differential ability to induce T(H)1 and T(H)2 responses. We compared the kinetics of antibody and cytokine (IFN-gamma and IL-4) production in C57BL/6 mice following allograft transplantation with BALB/c hearts and in C57BL/6 and BALB/c mice following transplantation with Lewis rat hearts. We also compared the ability of BALB/c mice, deficient in the ability to produce IL-4 or IFN-gamma, to reject xenografts and produce xenoantibodies. We observed that T(H)1/T(H)2 cytokine production minimally affected the kinetics of graft rejection but regulated the magnitude of IgG subclass production. Anti-graft IgM played a critical role in initiating acute antibody-mediated xenograft rejection, and the production antigraft IgM was unaffected by IL-4 or IFN-gamma deficiency. In contrast to the report by Wang et al. (1), we conclude that antibody-mediated xenograft rejection in the concordant Lewis rat heart-to-C57BL/6 mouse xenotransplantation model is dependent on anti-IgM production but independent of T(H) cytokine profiles.     

Interleukin-9 promotes eosinophilic rejection of mouse heart allografts

BACKGROUND: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined. METHODS: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft. RESULTS: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5. CONCLUSION: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.     

Cytokine and T cell receptor gene expression at the site of allograft rejection.

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.     

Contribution of CD4+ and CD8+ T cells and interferon-gamma to the progress of chronic rejection of kidney allografts: the Th1 response mediates both acute and chronic rejection

T cells mediating chronic rejection (CR) of human kidney allografts were characterized by comparing them with those mediating acute rejection (AR). Two lines of analysis were performed using biopsy specimens (23 CR and 8 AR). First, the extent of infiltration of CD4+ and CD8+ T cells into allografts was assessed from mRNA expression of CD4 and CD8. The group of CR specimens was not significantly different from the group of AR specimens in terms of the extent of CD4+ and CD8+ T cell infiltration, underlining the importance of the immunological contribution to the progress of CR. Second, Th1/Th2 polarization in infiltrating T cells was investigated by measuring mRNA expression of interferon gamma (IFN-gamma; a Th1 cytokine) and interleukin 4 (IL-4; a Th2 cytokine). IFN-gamma expression was detected in most CR specimens, and was not significantly different between the group of CR specimens and the group of AR specimens. On the other hand, IL-4 expression was detected in only two CR specimens and one AR specimen; from its pathological features, the AR in this last case was concomitant with CR. These results suggest that most cases of CR and of AR are mediated by Th1 mechanisms, although some cases of CR show features of both Th1 and Th2.     

Cellular rejection of fetal pancreas grafts: Differences between alio- and xenograft rejection

Abstract: Hyperacute rejection (HAR) is the major immunologic problem with vascularized xenografts between discordant donor/recipient combinations but does not occur in neovascularized grafts of organ-cultured fetal pig pancreas in either mice or cynomolgus monkeys. However, a form of cell-mediated acute rejection with quite different histopathologic features does occur with kinetics that are similar to acute cellular rejection of fetal pancreas allografts in non-immunosuppressed MHC-mismatched mice. Xenograft rejection is dominated by non-lymphoid cells, mostly eosinophils, that appear some days after transplantation. In contrast, in mouse allografts, mononuclear cells are the dominant population throughout the rejection process^. The rejected allograft site rapidly resolves to form a mature non-irifiltrated scar whereas the infiltrate in the xenograft site remains for weeks and forms a large granuloma before its eventual resolution. There are also differences in the intra-graft cytokine profile in the graft site between alio- and xenografts during acute rejection with an early predominance of IL-5 and TNF-α and an absence of TNF-γ in the xenografts. Immunosuppression with a depleting anti-CD4 mAb shows that xenograft rejection is more dependent on CD4+ve T cells but xenografts are more difficult to maintain with conventional immunosuppression that is often effective for allografts. Limited studies in primates have shown that the histopathology of fetal pig pancreas rejection is similar to that seen in mice but occurs at a faster tempo. Thus, although HAR may not be a problem in rejection of neovascularised xenografts, a vigorous form of cellular rejection is present that may require different immunosuppression than is usually used for the I control of allograft rejection.     

IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts

BACKGROUND: Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection. METHODS: We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4) or C57BL/6 IL-4 deficient mice (IL-4). Graft survival, in vitro T cell reactivity, and histology were compared. RESULTS: We observed that 50% of IL-4 mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4 mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4 mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival. CONCLUSIONS: Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.     

Interleukin-22 deficiency accelerates the rejection of full major histocompatibility complex-disparate heart allografts

Interleukin-22 (IL-22) was recently described as an effector cytokine produced by TH17 CD4(+) T lymphocytes that, cooperatively with IL-17, mediates IL-23-driven inflammation. Because there was experimental evidence for the role of IL-17 in acute rejection of vascularized allografts, we undertook the present study to assess the function of IL-22 in the process. There was an early transient expression of IL-22 in C57BL/6 mouse cardiac allografts (2-4 days posttransplantation) transplanted to BALB/c recipients. The main source of IL-22 among infiltrating leukocytes was cells expressing the macrophage/monocyte markers Mac3 and CD11b. T cells and granulocytes present in the rejected graft did not express IL-22. Surprisingly, the absence of IL-22 accelerated the rejection of fully histoincompatible hearts. Histology of rejected organs revealed the presence of intensive intragraft thrombosis and disseminated hemorrhagic necrosis. Taken together, these results demonstrated that IL-22 was not an effector lymphokine in cardiac allograft rejection, but early intragraft expression of the cytokine protected it from rejection.     

Mechanisms of survival prolongation of murine cardiac allografts using the treatment of CTLA4-Ig and MR1

BACKGROUND: [corrected] The present study was undertaken to determine the role of costimulatory blockade in a murine cardiac transplant model. MATERIALS AND METHODS: We blocked the CD28/B7 and CD154/CD40 costimulatory pathways by transient administration of CTLA4-Ig and MR1 antibody to study the effects on allograft survival time, deviation of Th1 and Th2 cytokine secretion, and other mechanisms related to prolonged survival. RESULTS: Costimulatory blockade prolonged the mean survival time (MST) of cardiac allografts to 43 days for the treated group vs 8 days for the untreated group (P < .01). The costimulatory blockade down-regulated the expression of 2 Th1 cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and 2 Th2 cytokines (IL-4 and IL-10), reduced the numbers of graft-infiltrating CD4+ and CD8+ lymphocytes, and inhibited the expression of both perforin/GrB and FasL in allografts. CONCLUSIONS: Combined administration of CTLA4-Ig/MR1 inhibited acute rejection reactions in murine cardiac allografts, prolonging the survival of cardiac grafts through several mechanisms, including inhibition of Th1 and Th2 cytokine expression, graft infiltration by CD4+ and CD8+ T lymphocytes, and reduced both perforin/GrB and Fas-FasL.