Acute rejection of marrow grafts in patients transplanted from a partially mismatched related donor: clinical and immunologic characteristics

Bone marrow transplantation (BMT) from a partially mismatched related donor (PMRD) provides a treatment option for patients lacking a matched sibling donor. T lymphocyte depletion of the graft reduces the risk of severe graft-versus-host disease, but may increase the risk of graft failure. We evaluated the pattern of acute graft rejection in eight patients receiving PMRD BMT with respect to the conditioning therapy, diagnosis, age and sex of donor and recipient, degree of HLA mismatch, and peripheral blood immunophenotype at the time of graft failure. All grafts were partially depleted of T lymphocytes. Marrow grafts infused into patients who experienced acute rejection did not differ significantly in nucleated cell dose, degree of T lymphocyte depletion, T cell dose, or CFU-GM/kg infused, from those received by 31 patients who showed durable engraftment. In three of four patients who rejected their grafts, and had sufficient peripheral blood cells for immunophenotyping, a CD3+CD8+ T lymphocyte phenotype was predominant at the time of acute rejection. In one patient rejection was associated with a predominant population of CD3+CD4+ T lymphocytes. Rejection was significantly associated with chronic myelogeneous leukemia and in patients mismatched by more than two antigens.     

Soluble and cell surface ICAM-1 as markers for disease activity in multiple sclerosis

Infiltration of a transplanted organ by host lymphoid cells is the hallmark of acute rejection. However, after intestinal transplantation, physiological lymphocyte migration may lead to host cell infiltration of the graft even in the absence of rejection. It is unclear whether this lymphocyte migration also involves the intraepithelial compartment of the graft or whether infiltration there is indicative of acute rejection. We demonstrate here that host cell infiltration of the intestinal mucosa occurs both during acute rejection of a small bowel allograft and, to a lesser extent, when rejection is prevented by immunosuppression with FK506. The infiltrating host cells consisted of CD3+ T cells with a predominant CD4-CD8+ phenotype resembling intraepithelial lymphocytes (IELs). Functional studies showed that the nonspecific cytolytic activity of IELs was not affected by acute rejection or by immunosuppression with FK506. These findings indicate that host cell infiltration of the intestinal mucosa does not connote an ongoing acute rejection. Furthermore, the decreased mucosal barrier function during acute rejection of intestinal allgrafts is probably not due to impaired cytolytic activity of IELs.     

Donor resting B cells induce indefinite prolongation of fully allogeneic cardiac grafts when delivered with anti-immunoglobulin-D monoclonal antibody: evidence for tolerogenicity of donor resting B cells in vivo

BACKGROUND: Resting B (rB) cells have been shown to induce T-cell anergy in vitro and to prolong the survival of skin and cardiac grafts mismatched for minor histocompatibility antigens. However, rB cells were unable to modulate the rejection response when grafts mismatched for major histocompatibility complex antigens were transplanted. We reasoned that donor antigens, which presented via the indirect pathway by recipient antigen-presenting cells, in particular B cells, might influence the ability of rB cells to induce unresponsiveness. To explore this hypothesis, we used an anti-immunoglobulin (Ig)-D monoclonal antibody (mAb) specific for recipient B cells to deplete these cells, thereby decreasing the potential for indirect presentation in vivo. METHODS: CBA mice were pretreated with 1 x 10(7) donor rB or activated B (aB) cells 7 days before transplantation of a C57BL/10 cardiac graft in the absence or presence of anti-IgD mAb. RESULTS: Naive CBA mice rejected C57BL/10 grafts acutely (median survival time [MST]=8 days). Pretreatment with rB cells alone resulted in a modest prolongation of graft survival (MST=11.5 days). In marked contrast, when rB cells were delivered with anti-IgD mAb, indefinite graft prolongation (MST>100 days) was observed in all recipients. Interestingly, aB cells produced only a small prolongation of graft survival when delivered with anti-IgD mAb (MST=15 days). Recipients treated with anti-IgD mAb alone rejected C57BL/10 cardiac allografts acutely (MST=8 days). CONCLUSION: These data suggest that depletion of recipient B cells in vivo can augment the ability of donor rB cells to induce indefinite prolongation of fully allogeneic cardiac grafts. Thus, IgD+ B cells in the recipient may influence the development of unresponsiveness in vivo.     

Effect of major histocompatibility complex expression on murine intestinal graft survival

BACKGROUND: Clinical intestinal transplantation has been plagued by frequent and severe graft rejection. It has been proposed that the major histocompatibility complex (MHC) antigens might play a critical role in this process owing to their extensive expression on enterocytes and mucosa-associated immune cells. METHODS: The present study examined the role of MHC antigens in intestinal graft rejection using MHC class I-deficient and MHC class II-deficient donors. RESULTS: Grafts with normal MHC expression were rejected by 9 days, whereas survival was prolonged to 14 days in the MHC class II-deficient grafts (P=NS) and to 20 days in the MHC I-deficient grafts (P<0.002). In all groups, early rejection was characterized by (1) increased crypt cell apoptosis, as detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique of in situ labeling; and (2) the increased expression of perforin and a CD8 phenotype in the graft-infiltrating cells. CONCLUSIONS: These data suggest that MHC antigens, CD8-positive T cells, and perforin-expressing cells contribute to intestinal graft rejection. Apoptosis of the progenitor epithelial crypt cells during early intestinal rejection may impair the gut's ability to regenerate and repair mucosal damage.     

Reconstitution of the extrathymic intestinal T cell compartment in the absence of irradiation

Extrathymic development of intestinal intraepithelial lymphocytes was studied using a reconstitution model that does not require irradiation. WBB6F1/J-Kit(W)/Kit(W-v) mice were reconstituted with normal fetal liver cells. In this system, reduced c-Kit activity in host hemopoietic progenitors imparts normal precursors with a growth advantage and, thus, chimerism can be established without irradiation. In control mice, TCR gammadelta and TCR alphabeta intraepithelial lymphocytes (IEL) developed efficiently from fetal liver cells, with a predominance of TCR alphabeta over TCR gammadelta IEL. In contrast, development in reconstituted thymectomized mice was heavily skewed toward TCR gammadelta IEL generation. In thymectomized mice, development of CD4+ 8- and CD4+ 8+ TCR alphabeta IEL did not occur, while TCR alphabeta CD8 alphabeta development was nearly absent. The results indicated that without irradiation the majority of TCR alphabeta IEL were thymus dependent, whereas TCR gammadelta IEL developed extrathymically. Thus, the discrepancies observed between different models of athymic development may be explained by the induction of T cell development as a result of irradiation.     

The role of "indirect" recognition in initiating rejection of skin grafts from major histocompatibility complex class II-deficient mice

In vitro studies have revealed several pathways by which T cells can respond to alloantigens, including CD4+ direct responses to allogeneic class II antigens, CD8+ direct responses to allogeneic class I antigens, and CD4+ "indirect" responses to peptides of alloantigens presented in association with responder class II molecules. In vivo studies of skin graft rejection, however, have so far provided clear evidence for the contribution of only the two direct pathways and not for indirect recognition. We have used major histocompatibility complex class II-deficient mice as donors to test the role of indirect recognition in rejection of skin grafts. Class II-deficient skin was always rejected without delay by normal recipients. Removal of recipient CD8+ cells (to leave the animals dependent on CD4+ function) or depletion of recipient CD4+ cells revealed that CD4+ cells were usually involved and sometimes absolutely required in this rapid rejection. Since the donor grafts lacked class II antigens, the CD4+ cells must have recognized donor antigens presented in association with recipient class II molecules. These results therefore indicate that indirect recognition can initiate rapid skin graft rejection.     

The effect of dietary polyunsaturated fatty acids (PUFA) on acute rejection and cardiac allograft blood flow in rats

For six weeks, recipient (Lewis RT11) and donor rats (LBNF11/n) were fed three diets that varied only in their lipid content. Diet A (MO) contained 19.5% menhaden oil and 0.5% safflower oil and was rich in omega 3 PUFA; diet B (SO) was 20% safflower oil rich in omega 6 PUFA; and diet C (BT) was 20% beef tallow rich in omega 9 monounsaturated fatty acids and saturated fat. In the first set of graft survival studies a group fed laboratory chow was included (CHOW). Heterotopic cardiac transplantation from donor to recipient animals was performed after the six-week feeding period. The effect of these diets on cardiac allograft survival, mixed lymphocyte response, and blood flow in the rejecting grafts was investigated. The median graft survival in days was significantly prolonged in the rats maintained on either MO (12 days) or SO (14.5 days) compared with the BT (8 days)-or lab chow (7.5 days)-fed animals (P < 0.05). Cyclosporine (CsA) administered at subtherapeutic levels further increased the differences between the PUFA-fed animals and the BT-fed group. The myocardial blood flow of the rejecting allografts was measured using an 85Sr-labeled microsphere technique on the fifth posttransplant day. Flow was greatest in the MO-fed group, and both MO and SO groups had significantly higher myocardial blood flow than BT-fed rats (P < 0.05) or those bearing isografts. The allogenic mixed lymphocyte responses of peripheral blood mononuclear cells (PBMC) and splenic lymphocytes were suppressed in MO- and SO-fed groups compared with BT-fed animals. The immunosuppressive effect of dietary PUFA warrants further investigation, and their use as a possible adjunctive treatment in organ transplantation should be considered.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.     

The relation between arterial oxygen tension and cerebral blood flow during cardiopulmonary bypass

BACKGROUND: The precise mechanisms involved in islet xenograft rejection remain unknown. The purpose of the present study was to determine cellular mechanisms responsible for islet xenograft rejection in the liver to facilitate finding a procedure for prevention of immune rejection. METHODS: Hepatic mononuclear cells (MNC) as well as splenocytes, peripheral blood MNC, and thymocytes from streptozotocin-induced diabetic mice (BALB/c) rejecting the intrahepatic rat (Lewis) islet xenografts were isolated and examined by two-color FACS analysis. RESULTS: The characteristic finding of the hepatic MNC from the mice rejecting islet xenografts compared with mice receiving isografts was a significant increase in the yield as well as in the percentage of the cells expressing CD3+ interleukin-2 receptor (IL-2R) alpha- beta+, CD3+ CD8alpha+ beta+, and T cell receptor (TCR) alphabeta+ lymphocyte function-associated antigen-1+. The expression of CD3 and TCR alphabeta of these T cells was found to be of intermediate intensity (TCR(int) cells). The expansion of these TCR(int) cells occurred predominantly in the liver. There was no significant difference in the cells expressing CD3+ IL-2R alpha+, CD3+ CD4+, CD3+ TCRgammadelta+, CD3- IL-2Rbeta+ (natural killer cells), and B220+ (B cells). In vivo administration of anti-IL-2Rbeta monoclonal antibody directed to the expanded cells produced a prevention of rejection. CONCLUSIONS: These findings suggest that islet xenograft rejection in the liver from rat to mouse is an event for which the TCR(int) cells are responsible.     

Pitx2 participates in the late phase of the pathway controlling left-right asymmetry

BACKGROUND: We have studied the role of the different MHC (RT1) subregions in acute natural killer (NK) cell-mediated bone marrow allograft rejection in lethally irradiated, bone marrow cell (BMC) reconstituted rats. METHODS: We employed a series of MHC congenic and intra-MHC recombinant rat strains so that effects of mismatches in defined RT1 subregions could be studied systematically. BMC allograft survival was measured as 125IUdR uptake in the spleen between day 5 and day 7 after irradiation and BMC reconstitution. RESULTS: We found that in certain RT1 haplotype combinations, nonclassical RT1.C disparities by themselves could determine graft rejection (i.e., in the u/av1 recombinant haplotypes), whereas in another combination (between the av1 and c haplotypes) a mismatch for an isolated classical RT1.A region was decisive for engraftment. Thus, PVG.R1 BMC failed to proliferate in PVG rats, differing in the RT1.A region only, whereas in PVG.1U rats rejection could be determined by isolated differences in the RT1.C region (LEW.1WR1). Also, RT1 homozygous rats (RT1.U) rejected semi-allogeneic F1 hybrid BMC. The acute rejection of BMC was mediated by NK cells, as athymic nude rats, lacking alloreactive T cells but with normal alloreactive NK cells, showed the same patterns of rejection as did normal rats. Nude rats also rejected allogeneic lymphocytes, a previously documented NK-mediated phenomenon, with identical requirements of MHC disparity. CONCLUSIONS: This investigation shows that rat effector NK cells are radioresistant, independent of the thymus, and capable of recognizing and rejecting MHC mismatched transplanted BMC on the basis of mismatches in both classical and nonclassical class I regions in vivo. The studies underline the importance also of NK cells in determining BMC allograft survival.     

Small-intestinal transplantation

The management of patients with intestinal failure has benefited from progress in parenteral nutrition (PN), especially home-based PN. Intestinal transplantation is therefore possible and is now, in some conditions, the logical therapeutic option. Since 1985, more than 180 small-bowel grafts have been done, involving the isolated small bowel with or without the colon (38%), the liver-small bowel (46%) or several organs (16%). Two-thirds of recipients were under 20 years of age, and indications were short-bowel syndrome (64%), severe intractable diarrhoea (13%), abdominal cancer (13%) or chronic intestinal pseudo-obstruction syndrome (8%). Of the patients, 51% survived > 2 years after the graft. Patient and graft survival depends on the type of immunosuppression, i.e. cyclosporine or FK506. The results must be interpreted carefully as they represent the first experience in numerous centres using different immunosuppressive protocols, without any randomization. The results from the largest of these centres reflect the current situation more closely. Functional grafts lead to gastrointestinal autonomy (weaning of PN) while maintaining satisfactory nutritional status and normal growth in childhood. Intestinal transplantation is theoretically indicated for all patients permanently or dependent for a long time on PN. However, as PN is generally well tolerated, even for long periods, each indication for transplantation must be carefully weighed up in terms of the iatrogenic risk and quality of life. When PN has reached its limits, especially in those associated with vascular, infectious, hepatic or metabolic complications, intestinal transplantation must be undertaken. Transplantation of the small bowel alone remains the first option, as combined liver-small bowel grafting is only indicated in the case of life-threatening progressive cirrhogenic liver disease.     

The intragraft CD8+ T cell response in renal allograft rejection in the mouse

To identify the role of donor class I alloantigens in regulating the CD8+ T cell response to a kidney allograft, we analyzed and compared the CD8+ infiltrate in kidney transplants from MHC class I-deficient (class I-) mouse donors and class I+ controls. One week after transplantation, there was a prominent CD8+ infiltrate in control allografts, whereas CD8+ T cells were virtually absent in grafts from class I- donors. In class I+ allografts, infiltrating CD8+ cells utilized a wide range of T cell receptor (TCR) Vbeta families and their Vbeta usage was similar to that of the systemic CD8+ population. However, there was a modest but significant overrepresentation of cells bearing Vbeta8 in the graft compared with the spleen due to an expansion of CD8+ Vbeta8.3+ cells. This could be detected as early as 1 week and became more pronounced by 3 weeks after transplantation. In 3-week allografts, only 52% of CD8+ cells expressed alphabetaTCR. Among T cells isolated from class I+ grafts, the CD8+ Vbeta8+ cells demonstrated allospecific responses that were numerically larger than responses of the CD8+ Vbeta8- population. In contrast to the early (1 week) time point, significant numbers of CD8+ cells could be isolated from class I- grafts by 3 weeks after transplantation and their Vbeta repertoire resembled that seen in controls. While increasing numbers of CD8+ Vbeta8+ were present in the class I- grafts at 3 weeks, this increase was not statistically significant. Thus, expression of class I alloantigens on a kidney graft plays an important role in regulating the rate of accumulation of CD8+ T cells in rejecting kidney grafts. However, the TCR Vbeta repertoire of the CD8+ T cell infiltrate is largely determined by factors that are independent of normal class I expression on the graft.     

New method to measure the retention of viscoelastic agents on a rabbit corneal endothelial cell line after irrigation and aspiration

BACKGROUND/AIMS: Murine and human studies have documented the existence of subpopulations of lymphocytes in particular tissues that differ phenotypically and functionally from those in peripheral blood and may mature locally. Since little is known about lymphocyte subpopulations in the normal human liver, we have analysed the surface phenotypes of lymphocytes isolated from liver specimens taken from 15 donors at the time of liver transplantation, and compared these with those of peripheral blood lymphocytes. METHODS: Hepatic lymphocytes were prepared by mechanical dissociation and enzymatic digestion of liver tissue. The cells were stained with a panel of monoclonal antibodies (CD3, CD4, CD8, CD19, CD56, gammadeltaTCR, alphabetaTCR, CD8alpha-chain, CD8alphabeta dimer), and analysed by flow cytometry. In situ characterisation of hepatic lymphocytes was by haematoxylin and eosin staining of fixed liver sections and by immunohistochemical staining for common leukocyte antigen and CD3. RESULTS: Significant numbers of hepatic T lymphocytes were localised to the portal tracts and parenchyma of normal liver specimens. Flow cytometry revealed that the CD4/CD8 ratio (1:3.5) was consistently reversed compared with that in peripheral blood (2:1). Other lymphocyte populations identified include double positive CD3+CD4+CD8+ cells which accounted for a mean of 5.5% (range 3-11.6%) of hepatic CD3+ cells compared with 1.3% in blood (range 0.7-3.6%; p < 0.007), and double negative CD3+ CD4-8- cells (14.5%; range 2.7-29% compared with 5.0%; range 2.1-10.8%, p < 0.02). Over 15% (range 6.8-34%) of all hepatic CD3+ cells expressed a gammadeltaTCR compared to 2.7% (range 0.9-4.7%) of CD3+ peripheral blood lymphocytes (p < 0.004) and almost 50% of these coexpressed CD8. The CD8 alpha-chain was expressed without the beta-chain (CD8alpha+beta-) by 15.4% (range 4-29.1%) of hepatic T cells, but this phenotype was undetectable among peripheral blood lymphocytes (p < 0.009). Cells expressing both the T cell marker CD3 and the natural killer cell marker CD56 constituted 31.6% (range 14-54%) of all hepatic CD3+ lymphocytes but were rarely present amongst peripheral blood lymphocytes (0-6%; p < 0.0001). CONCLUSIONS: These data are the first to describe and quantify unconventional T lymphocyte subpopulations in the normal adult human liver which may have specialised functions in regional immune responses and which may differentiate locally. These findings have important implications for our understanding of hepatic immunoregulation and the pathogenic mechanisms involved in viral and immune-mediated liver disease and allograft rejection.     

Heterotopic liver transplantation in rats: effect of intrahepatic islet isografts and split portal blood flow on liver integrity after auxiliary liver isotransplantation

BACKGROUND: Auxiliary heterotopic liver grafts atrophy in the absence of portal venous inflow; evidence suggests that an islet-derived hepatotrophic factor may exist in the portal drainage. Here we examine the effects of intrahepatic islet isografts in maintaining hepatocyte integrity in Wistar Furth rats with one of several types of arterialized auxiliary liver isografts. METHODS: In type 1 procedures the auxiliary liver was interposed into the recipient infrarenal vena cava and perfused through the graft portal vein with caval blood. In type 2 procedures the donor infrahepatic vena cava was anastomosed end-to-side to the recipient vena cava and the recipient portal vein was diverted to the graft portal vein. Both types of auxiliary grafts were arterialized; bile duct drainage was through the duodenum. Syngeneic islets were isolated and embolized into the portal veins of one half of the donor type 1 or native type 2 livers (1500 to 1700 islets). Finally, we performed six type 3 procedures in which a type 2 procedure was performed except that the portal blood flow was split so that the portal vein receiving the splenic, gastric, pancreatic, and duodenal drainage supplied the native liver and that the common mesenteric vein supplied the auxiliary graft with equivalent portal blood flow. Atrophy in heterotopic and native livers were compared for the three models after 3 months. RESULTS: Intrahepatic islets in type 1 auxiliary liver isografts without portal venous inflow did not prevent graft atrophy. Conversely, native livers deprived of portal venous inflow in our type 2 procedures, regardless of the presence of intrahepatic islet isografts, atrophied relative to auxiliary liver grafts in which portal venous inflow was provided by diverting the recipient's portal vein to the graft. In type 3 recipients atrophy was greater in the native livers than in the grafts. CONCLUSIONS: The results of our study suggest that islet-derived factors are not sufficient to prevent hepatocellular atrophy in auxiliary rat liver transplantation models and that a potent hepatotrophic factor may exist in the venous drainage of the bowel distal to the duodenum.     

Solitary syringoma. Report of five cases and clinicopathologic comparison with microcystic adnexal carcinoma of the skin

Interleukin-1 receptor antagonist (IL-1ra) competes with IL-1 for binding of the IL-1 receptor, but does not elicit a cellular immune response. This study was designed to evaluate the effectiveness of IL-1ra in the immune and inflammatory responses to rat heart allografts. Experimental design was as follows: Group I HTx was syngeneic, BN to BN. The remaining groups were DA (RT 1a) to BN (RT 1n) allogeneic HTx. Group II was transplanted without immunosuppression. Group III received a low-dose IL-1ra regimen via osmotic pump into the peritoneum. Group IV recipients were similarly treated with a higher dose IL-1ra regimen. Group V rats received subtherapeutic cyclosporine (CsA) therapy while Group VI was treated with both CsA and low-dose IL-1ra. Group I rats survived indefinitely. Group II rats rejected their grafts at 5.33 +/- 1.37 days. Group III grafts survived for 7.16 +/- 0.48 days, and Group IV grafts for 8.16 +/- 0.75 days, both significantly longer than in Group II (P < 0.01). Group V animals treated with low-dose CsA had graft survival of 7.7 +/- 1.6 days, but combined therapy with CsA and IL-1ra in Group VI yielded significantly prolonged graft survival of 17.2 +/- 1.3 days (P < 0.0001). Histologic examination in treated recipients revealed delayed appearance of mononuclear cell infiltration. IL-1ra-treated recipients all demonstrated significantly reduced numbers of graft-infiltrating leukocytes; all phenotype subsets were equally affected. This study demonstrates the effectiveness of IL-1ra, in combination with low-dose CsA, in reducing the inflammatory response and rejection in the transplant setting.     

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.     

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.