Search

Donor-specific blood transfusion (DSBT) given 1–2 weeks prior to transplantation prolongs the survival of fully allogeneic ACI (RT1n) renal allografts in PVG (RT1c) recipients from 6–8 days to >100 days. We have previously demonstrated that ACI kidneys transplanted to autologous blood transfusion (ABT)- or DSBT-pretreated PVG recipients stimulated an increase in CD8(OX8) cells in the peripheral blood by 6 days after transplantation. To determine whether this increase represents a general expansion of the entire CD8population or only a subpopulation of CD8cells, subset analysis was performed on peripheral blood lymphocytes depleted of cells reactive with monoclonal antibodies against rat αβ T cell receptor (TCR), CD8, or NK cells (R7.3, OX8, or 3.2.3, respectively). Phenotypic studies of PBL depleted of CD8cells demonstrated that all 3.2.3 NK cells coexpressed CD8; depletion of 3.2.3PBL revealed a second subpopulation of CD83.2.3-cells comprised predominantly of αβTCRT cells. In naive PVG rats the prevelence of these two CD8sub-populations was approximately equal. Both ABT- and DSBT-pretreated renal allograft recipients demonstrated a significant and equivalent expansion of the CD8cell subpopulation that coexpresses the 3.2.3 NK marker. In contrast, the second subpopulation of CD83.2.3-cells did not change significantly after allografting. There were also no differences between DSBT and ABT pretreated rats in activity of PBL against the NK targets YAC-1 and Doxie at 6 days after renal transplantation, though the level of activity was modestly increased compared with naive controls. These findings indicate that renal transplantation in the rat is associated with a significant increase in PBL with the NK phenotype (CD83.2.3) and a modest increase of NK activity, but that DSBT enhancement does not affect this NK cell response.

The effects of preoperative donor-specific blood transfusion (DSBT) on the physiologic, morphologic, and immunologic aspects of aspects of allograft responsiveness were evaluated in a rat renal transplant model, using the ACI (RT1a) into PVG (RT1c) high-responder strain combination. Indefinite graft survival (mean >63 days) could be induced by DSBT administration alone. In comparison, animals receiving autolgous blood transfusion (ABT) all died within 7 days posttransplantation. As assessed by clearance of inulin and paraaminohippurate, renal allograft function in DSBT-pretreated recipients at 6 days was equivalent to that of isograft recipients, and in contrast to the significant reduction seen in ABT treated rats. Likewise, thromboxane B2(TXB2) production by ex-vivo-perfused allografts from DSBT-treated recipients was comparable to that of isografts, and significantly lower than that of allografts from ABT-treated rats. A significant inverse correlation was found between renal TXB2production and inulin clearance. Despite these substantial differences in renal function and eicosanoid metabolism, morphologic evaluation of renal allografts from DSBT-enhanced and ABT-rejecting recipients at comparable time points showed equivalent histologic manifestations of rejection. In addition, immunohistologic labeling of renal allograft sections and fluorescence-activated cell sorter analysis of cells eluted from allografts showed the same phenotype and pattern of infiltrating T cell subsets in both groups. Specific antidonor cytotoxic T lymphocyte precursor (pCTL) frequencies of cells eluted from kidney grafts were equivalent in DSBT and ABT-pretreated animals, and both groups expressed significantly higher (but equivalent) pCTL frequencies in the kidneys than spleens. Comparisons of the lysis of PVG.R1 (RT1.Aaon a PVG background) and ACI targets indicated that cytotoxic responses from effector cells freshly eluted from DSBT and ABT kidneys were primarily directed against allogeneic class I major histocompatibility complex (MHC) specificities, whereas several long term T cell lines generated from 6-day kidney transplants of both groups expressed a predominant W3/25+(T helper) phenotype and cytotoxic activity against donor specificites other than RT1.Aaclass I MHC. Specific antidonor proliferative T lymphocyte (pPTL) precursor frequencies of cells eluted from renal allografts were also equivalent for both DSBT- and ABT-treated recipients, and the range of pPTL frequencies for allograft cell eluates was similar to that in spleens, regardless of the source of the transfusion. Thus, in the rat, the prolonged renal allograft survival seen with DSBT pretreatment is associatated with reduced thromboxane production, and does not appear to involve early depletion of alloreactive cytotoxic or proliferative T cell clones or activity. Moreover, these findings suggest that the presence of significant alloreactive cytotoxic and helper T cell populations within the spleen and allograft itself does not necessarily result in renal transplant dysfunction or rejection.

Hyperacute rejection is a major obstacle to successful transplantation of vascularized xenogeneic organs and is believed to be mediated at least in part by preformed xenoreactive “natural antibodies” (NAb). In this study, human NAb that could be involved in hyperacute rejection of pig heart xenografts were identified and characterized using an ex vivo model in which pig hearts were perfused with whole blood from individual human or pig donors. This ex vivo perfusion model allows for the continuous monitoring of physiologic parameters of cardiac function as well as sequential sampling of tissue and blood. Pig hearts perfused with allogeneic pig blood maintained normal function for at least 4 hr, whereas those perfused with xenogeneic AB+human blood never achieved normal function and rejected completely after 30 min. In three separate experiments involving different human blood donors and pig hearts, sequential samples of perfused blood revealed a progressive depletion of antiporcine NAb. Samples of all three rejected cardiac xenografts were homogenized, and the specifically bound human anti-porcine antibodies were eluted with citric acid. The eluted antibodies were enriched approximately 50–120-fold for anti-porcine reactivity compared with serum from the corresponding donor. Eluates contained NAb of predominately IgM and IgG2 isotypes. Immunofluorescence histology confirmed the deposition of IgM and IgG2 but not other IgG subclasses in the rejected pig hearts. Since IgG2 utilized predominantly in response to bacterial polysaccharide antigens, our findings are consistent with the possibility that some NAb arise via crossreactivity with microbial antigens and are predominantly directed against carbohydrate rather than protein antigens.

Donor-specific blood transfusion prolongs survival of fully allogeneic ACI (RT1a) renal grafts in PVG (RT1c) recipients from 6–8 days to >100 days. To determine how DSBT alters effector cytotoxic cell responses, we tested freshly isolated peripheral blood lymphocytes, spleen cells, and graft infiltrating cells (GIC) from pairs of PVG recipients of ACI kidneys pretreated with DSBT or autologous blood transfusion (ABT) for cell-mediated lympholysis, antibody-dependent cellular cytotoxicity (ADCC), and natural killer activity at the day of transplantation (day 0) and days 3 and 6 posttransplantation. PBL and GIC from the same pairs of animals were examined for their phenotypic profile (CD4, CD8, 3.2.3 NK cell marker, IL-2 receptor). CML, ADCC, and NK activity were higher in PBL than splenocytes or GIC of both ABT and DSBT groups at all time points examined. CML activity of PBL at day 3 was significantly higher in DSBT vs. ABT recipients (P<0.01), while at day 6 both groups were equally elevated. Splenocytes demonstrated significantly lower CML activity in DSBT vs. ABT recipients at day 6 (P<0.05). CML activity of GIC eluted from ABT and DSBT kidneys was not detected at day 3, but was significantly elevated and equivalent in both groups at day 6. ADCC and NK activities of PBL did not differ between ABT and DSBT groups, and were negligible in splenocytes. GIC demonstrated higher NK activity in DSBT vs. ABT recipients at day 3 (P<0.05), while ADCC activity was not detectable at any time in either group. Phenotypic analysis of PBL and GIC at day 3 showed no significant differences between ABT and DSBT groups in the percentage of CD4 or CD8 cells. However, in both ABT and DSBT groups the ratio of CD4:CD8 cells was markedly lower in GIC than PBL. By day 6, PBL from both ABT and DSBT groups showed equivalent and significant decreases in CD4+cells and increases in CD8+and 3.2.3+(NK) cells. The percentage of IL-2R+cells remained low (5) in both groups at day 3. In contrast, at day 6 there was a significant increase in IL-2R+(and to a lesser extent CD4+) GIC in ABT- but not DSBT-treated recipients, while CD8+cells were significantly increased in both groups. These findings indicate that DSBT enhancement of rat renal allo-grafts does not result in the functional deletion or depletion of CML, ADCC, or NK cytotoxic effector cells in PBL, spleen, or allograft at early times after transplantation (day 3), and only a modest reduction in CML activity of splenocytes at day 6. Furthermore, CD8+and 3.2.3+cells in PBL and the graft do not appear to be affected by DSBT enhancement, whereas the significant increase in IL-2R+(and CD4+) infiltrating cells seen in rejecting ABT controls is not observed in DSBT animals. These results suggest that DSBT enhancement in the rat is more likely to be the result of diminished alloimmune responsiveness involving noncytolytic lymphocyte functions, such as alloantibody production and delayed-type hypersensitivity.

Donor-specific blood transfusion (DSBT) in animals and humans can either promote subsequent renal graft survival or lead to sensitization and graft rejection. Using a rat renal allograft model, we have examined whether the effects of DSBT on renal allograft outcome and IgG alloantibody responses are linked to the host MHC. In F1rats produced by mating PVG (RT1c), a low IgG alloantibody responder to transfused ACI (RT1a) blood, with 3 different high-IgG responders [W/F (RT1u), LOU (RT1u), and LEW (RT11)], high IgG alloantibody production was found to be inherited as a dominant trait and associated with acute rejection of ACI renal allografts. DSBT given to offspring of (PVG±W/F)F1rats backcrossed to W/F with either RT1u//e(u/c) or RT1u/u) (u/u) phenotype induced high-IgG-alloantibody responses that were associated with acute renal allograft rejection. Likewise, offspring of (PVG±W/F)F1rats backcrossed to PVG expressing the u/c phenotype had high IgG responses to ACI DSBT associated with acute renal allograft rejection. In contrast, DSBT given to backcrossed recipients expressing the RT1c/c(c/c) phenotype elicited a transient IgM response that switched to a very low IgG response and was associated with renal allograft acceptance. Analysis of IgG isotypes demonstrated that DSBT prevented production of IgGl and IgG2a, and to a lesser extent IgG2b and IgG2c alloantibodies in c/c but not u/c renal allograft recipients. The differences in the level and isotype of IgG alloantibody responses found in sera of DSBT-pretreated backcross rats of u/c and c/c phenotypes were also present in allograft eluates and splenocyte cultures. Likewise, DSBT-pretreated renal allograft recipients of the c/c phenotype produced lower levels of alloantibodies directed to class I RT1.Aaantigens compared with their u/c counterparts; in contrast, no difference was found in alloantibody responses to class II RT1.Baantigens. These findings demonstrate that the variable ability of DSBT to enhance renal allograft survival correlates with the inhibition of antidonor class I alloantibody responses of all IgG subclasses by a mechanism that is linked to host MHC.