Nancy L. Reinsmoen

Five HLA-D clusters associated with HLA-DR4☆

In order to investigate the HLA-D clusters associated with DR4, 54 DR4-positive, Dw4- and Dw10-negative responders, together with selected Dw4- or Dw10-positive responders, were tested with 22 HTCs that define DR4-associated D specificities. The results are consistent with previous data defining four distinct D clusters--Dw4, Dw10, DB3, and DYT--and have identified a new cluster provisionally termed LD40. In addition, the DB3 cluster is complex and appears to give typing response patterns overlapping those of the KT2 cluster originally defined as being associated with DR4 in Japanese populations. Of 116 DR4-positive haplotypes tested, 44% typed as Dw4, 18% were LD40, 16% were Dw10, 9% were DB3, 3% were DYT, and 10% gave no typing response to the HTCs defining any of these clusters. These studies are informative not only in defining the DR4-associated D clusters and in supporting the concept that D and DR cannot be considered identical but also in emphasizing the complexity of the D region.

Evidence that improved late renal transplant outcome correlates with the development of in vitro donor antigen-specific hyporeactivity

Previous studies suggest stable renal transplant recipients can have either prednisone (P) or cyclosporine withdrawn; however, 30% of these patients undergo rejection requiring reinstitution of P or CsA. Some patients return to baseline creatinine levels, while others either stabilize at a higher creatinine level or lose their graft. It would be ideal to establish immunologically based criteria for selecting patients who can be successfully withdrawn or tapered from immunosuppression. We have investigated the development of donor antigen-specific hyporeactivity by using donor cells and/or homozygous typing cells defining the HLA-Dw specificities of the donor cells as stimulator cells in the mixed lymphocyte culture (MLC) and comparing the pre-and posttransplant responses of peripheral blood mononuclear cells from 199 kidney transplant recipients. Of these, 27% of the haploidentical living-related donor and 25% of the cadaver recipients developed in vitro donor antigen-specific hyporeactivity. The LRD recipients who did so have lower mean creatinine levels at 6, 12, and 24 months posttransplant (1.3, 1.3, and 1.2, respectively) than those who remained responsive to the donor antigens (1.6, 1.7, and 1.8) (P<0.05). However, no differences in the mean creatinine levels were observed between CAD recipients who developed donor antigen-specific hyporeactivity and those who remained responsive. Rejection episodes were common in all groups in the first 3 months posttransplant; however, recipients who developed donor antigen-specific hypo-reactivity tended to experience fewer rejection episodes

Beyond hyperacute rejection : accelerated rejection in a discordant xenograft model by adoptive transfer of specific cell subsets

If hyperacute rejection is prevented in the guineapig (GP)-to-Lewis rat (Lew) cardiac xenograft (CXg) model, an accelerated rejection involving cellular infiltration occurs in 3 to 4 days. In previous work using an adoptive transfer model, we found that this accelerated rejection was facilitated by either sensitized splenocytes or sensitized serum. In the current study, in an attempt to determine which splenocyte subset(s) facilitated this process, sensitized splenocytes, with or without subset depletion were injected, into complement- and natural antibody-depleted Lew recipients of GP CXgs. Graft survival was 4.18 +/- 0.75 days with no injection (n = 11), 4.13 +/- 0.99 days with naive splenocytes (n = 8), 1.80 +/- 0.45 days with sensitized splenocytes (n = 5), 2.67 +/- 1.03 days with CD4(W3/25+) depletion of the sensitized splenocytes (n = 6), 3.13 +/- 0.84 days with CD8(OX8+) cell depletion (n = 8), 4.70 +/- 0.68 days with macrophage depletion (n = 10), and 4.22 +/- 0.41 days with B cell depletion (n = 9). Cellular infiltrates, hemorrhage, myocyte necrosis, and endothelial deposition of IgG, IgM, and fibrin were seen in rejected grafts. In most groups, infiltrating cells consisted of CD4 (W3/25+), CD8 (OX8+), IL2R+ cells, macrophages, and natural killer (NK) cells. However, in the macrophages-depleted group, activated (ED2+) macrophages and NK cells were significantly reduced. Total IgM, anti-GP IgM, and anti-GP IgG rebounded in all groups over several days but were not consistent at the time of rejection. Lewis rats rejecting GP CXgs early had lower final titers than those rejecting later. Total IgG titers rebounded to baseline by posttransplant day 1 and were therefore similar in all groups at the time of rejection. These findings suggest that this accelerated rejection requires interaction between macrophages and B cells, since depletion of either significantly alters the rejection tempo. A possible explanation is that xenoreactive IgG antibodies, synthesized by sensitized B cells, bind their target antigens--but also bind sensitized macrophages through their Fc region, thus causing rejection by antibody-dependent cell-mediated cytotoxicity.

Are multiple immunopathogenetic events occurring during the development of obliterative bronchiolitis and acute rejection

The primed lymphocyte test (PLT) has been used to detect donor antigen-specific reactivity of bronchoalveolar lavage (BAL) lymphocytes associated with acute lung rejection and obliterative bronchiolitis (OB). To identify more precisely the immunopathogenetic events related to these processes, we have determined the PLT alloreactivity of 162 BAL specimens from 40 recipients as being directed against individual class I or class II antigens. We used selected homozygous typing cells representing the specific HLA class I and II antigens expressed by the recipient and donor cells. Our previous studies demonstrated a predominant CD8+ cell population mediating class I donor antigen-specific reactivity, correlating with OB in 3 out of 3 recipients tested, and a predominant CD4+ cell population mediating class II donor antigen-specific reactivity, correlating with acute rejection episodes in 13 out of 15 recipients tested. The obstructive airway disease OB, which is frequently fatal, is identified histologically by the presence of small airway inflammation and fibrosis of the lamina propria and lumen, and is characterized clinically by rapidly progressive airflow obstruction. However, a subgroup of patients with histologically proven OB demonstrates stabilization or only minimal progression of airflow limitation after augmentation of their immuno-suppressive regimen, usually with high-dose methyl-prednisolone. To further characterize this subgroup, we tested the BAL-derived lymphocytes from 4 of these patients and observed PLT reactivity that correlated with the class II antigens of the donor, in contrast to the predominant donor class I antigen-specific PLT reactivity of patients with progressive OB. The distinct patterns of PLT reactivity observed for the BAL-derived lymphocytes from patients with the progressive versus the less progressive form of OB suggest that more than one immune process or perhaps different cell targets are involved. Alternatively, these clinical and in vitro findings may represent different stages of the same disease process. Taken together, these results suggest that distinct immunopathogenetic events may be occurring during acute lung rejection and OB.

Organ-specific patterns of donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in lung, kidney, and liver transplant recipients

Although their relative importance and interaction are unclear, donor antigen(Ag)*-specific hyporeactivity and allogeneic microchimerism have been associated with improved long-term graft outcome and a lower incidence of chronic rejection in solid organ transplant recipients. We have postulated that a critical level of donor antigen, for a critical time period, is necessary to develop and maintain donor antigen-specific hyporeactivity; both the level and the time may differ by organ transplanted. In our current study, we tested donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in liver and kidney recipients and compared these values with our previous findings in lung recipients. We tested 25 liver recipients at 12 to 29 months posttransplant: 10 (40%) had developed donor antigen-specific hyporeactivity; 5 (20%), peripheral blood allogeneic microchimerism. For all but 1 of the chimeric and hyporeactive recipients, the level of donor cells was very low (< 1:20,000). Five hyporeactive recipients and all 15 donor antigen-responsive recipients did not have detectable levels of peripheral blood microchimerism. No chronic rejection has developed in any of these recipients to date--however, a lower incidence of acute rejection was observed for those recipients with donor antigen-specific hyporeactivity (30% versus 60% without) or with peripheral blood allogeneic microchimerism (20% versus 55% without) (P = ns). These results differ from our previous findings in 19 lung recipients: at 12 to 18 months posttransplant, 35% of them had developed donor antigen-specific hyporeactivity; 47%, peripheral blood allogeneic microchimerism. All donor antigen-specific hyporeactivity recipients as well as some donor antigen-responsive recipients had peripheral blood allogeneic microchimerism. We expanded our current study to include 26 recipients and a quantitative estimate of the level of allogeneic microchimerism. We observed that the hyporesponsive recipients tended to have higher levels of donor cells in their peripheral blood (> 1:6,000) than did the responsive recipients. We previously reported that 22% of kidney recipients had developed donor antigen-specific hyporeactivity at 12 to 18 months posttransplant. In our current study of 33 kidney recipients, we observed peripheral blood allogeneic microchimerism in 7 (21%) at 12 to 18 months posttransplant. The level of donor cells was very low (approximately 1:75,000), with no correlation between donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism at the time point tested. These studies emphasize the organ-specific nature of the development of donor antigen-specific hyporeactivity and the persistence of peripheral blood allogeneic microchimerism. Donor antigen-specific hyporeactivity correlates with very low levels of donor cells in liver recipients, while a higher critical level of donor cells is important in lung recipients. Additional sequential early posttransplant studies are necessary to further define the possible interrelationship between donor antigen and the development and maintenance of donor antigen-specific hyporeactivity.

Cellular rejection in discordant xenografts when hyperacute rejection is prevented: analysis using adoptive and passive transfer

Hyperacute rejection of discordant xenografts occurs rapidly, precluding cellular infiltration. Thus the role of cellular rejection in discordant xenografts is debated. Using adoptive transfer of sensitized splenocytes and passive transfer of sensitized serum, we evaluated the influence of cellular and humoral elements on cellular infiltration and rejection in the guinea-pig-to-rat discordant xenograft model. Guinea-pig hearts were transplanted into Lewis rats. Pretransplant, rats underwent splenectomy and plasma exchange and were started on daily cobra venom factor injections. Xenografts rejected faster after adoptive (1, 2, 2 and 2 days) or passive (1, 1, 2 and 2 days) transfer than controls (4, 4, 4 and 4 days; p < 0.05). Macrophages and neutrophils were predominant in early prerejection specimens. Over time, cellular infiltrates were dominated by mononuclear cells. Natural killer cells were present in all groups, as were interleukin 2 receptor positive cells. Our data suggest that either sensitized serum or sensitized cells are capable of initiating an accelerated rejection characterized by cellular infiltration. Despite subtle differences, the population of infiltrating cells was similar in each group. Thus, although rejection may be initiated by either cellular or humoral influences, the ultimate result is the same. We have, therefore, established a small animal model to study cellular rejection in discordant xenografts. This model will help evaluate the role of cell subsets and xenoantibodies in xenograft rejection and will help determine the precise relationship between the two when hyperacute rejection is prevented.

A study of HLA-DR2 associated HLA-Dw/LD specificities☆☆☆

HLA-Dw2 and Dw12 are both associated with HLA-DR2; however, these specificities accounts for only 86% (161/188) of the DR2+ haplotypes in our North American Caucasian panel. In an attempt to identify new DR2 associated antigenic clusters, we have generated four primed lymphocyte (LD) typing (PLT) reagents in haploidentical familial combination against DR2+ Dw blank haplotypes. These reagents were positively restimulated by 11 of 16 DR2+ Dw blank cells tested, with good discrimination from Dw2 and Dw12+ cells, thus identifying a new antigenic cluster provisionally termed LD-MN2. We have compared the LD-MN2 specificity with the specificity LD-5a defined by two DR2+ HTCs, BAS and REM, (Layrisse, Caracas) which have been included in the pre-1984 Workshop Cluster DB9. Although none of our DR2+ cells gave typing responses to these two HTCs defining LD-5a, PLT studies did indicate an interrelationship between these specificities and with the specificity tb24 defined with the HTC, FJO (Betuel). The LD-5a HTCs, four LD-5a heterozygous cells, and two additional HTCs (WJR-Hansen, Seattle and FJO/tb24--Betuel, Lyon) significantly restimulated the anti-MN2 PLT reagents, though usually not as strongly as the MN2+ cells. MN2+ cells primed against the LD-5a HTCs were restimulated by only the LD-5a+ cells. Dw2+ cells primed against FJO were restimulated by some, but not all MN2+ cells. These results suggest that MN2, tb24, and LD-5a share some determinants, not shared with most cells which type as Dw2 and Dw12, though differing by other stimulatory determinants. These studies emphasize the necessity of studying new antigenic clusters by both PLT and HTC methodologies as well as testing different ethnic groups.

Detection of HLA restricted anti-minor histocompatibility antigen(s) reactive cells from skin GVHD lesions☆

Recipients of marrow grafts from HLA-identical, (including MLC non-reactive) sibling donors have a 40-50% incidence of acute graft vs. host disease (GVHD); the involvement of non-HLA linked, minor histocompatibility antigens (miHA) has been implicated. One of the target tissues of GVHD is the skin where morphological analysis of biopsy specimens is often used to support this diagnosis. We have obtained skin biopsy specimens from the site of GVHD lesions, grown the cells in the presence of T cell growth factor (TCGF) and feeder cells, and tested these cultured cells in the primed lymphocyte test (PLT) and cell mediated lympholysis (CML) assay. Five of six cell cultures tested demonstrated secondary proliferative but not cytolytic reactivity; cells from one culture demonstrated both reactivities. The cell culture populations generated are presumably directed against non-HLA antigens, i.e., miHA whose expression is restricted by an HLA antigen of the recipient. The data are consistent with the suggestion that in the majority of these cultures, the restriction element may be a determinant encoded within the HLA-D region: DQ, DR, and possibly DP based on panel testing. Although the number of cases is small, these preliminary data demonstrate the feasibility of this type of culturing system and also suggest that the cellular immunological events leading to the manifestation of skin GVHD lesions may be predominantly a delayed type hypersensitivity reaction.

The impact of HLA matching on graft survival and on sensitization after a failed transplant--evidence that failure of poorly matched renal transplants does not result in increased sensitization.

There are costs (both financial and ethical) to distributing kidneys by HLA-match (time, transportation, repeat crossmatch; possibly bypassing a more deserving recipient). Arguments favoring matching include better short- and long-term survival, and decreased panel-reactive antibody (PRA) if a well-matched vs. poorly matched transplant fails. We studied these phenomena in a single institution. Since 1970, 1329 patients received cadaver (CAD) transplants; for those with defined antigens (n = 1316) there was no difference in 10-year graft survival in those with a less than or equal to 1 AB match vs. those with greater than 1 AB match or those with less than or equal to 1 AB mismatch (mm) vs. greater than 1 AB mm. Similarly there was no difference in those with less than or equal to 2 BDR mm vs. greater than 2 BDR mm. In fact, those with less than ABDR mm had worse 10-year graft survival (55%) than those with greater than or equal to 3 ABDR mm (61%) (P = .001). For patients with function greater than 6 months there was no difference in long-term outcome based on HLA match or mm. These findings were similar for patients both with and without CsA immunosuppression, and for primary and retransplants. A total of 382 patients transplanted since 1980 have lost their grafts (146 died with function). All had received pretransplant transfusions. Of 236 alive after graft loss, 64 had no postgraft failure PRAs (22 out of state, 23 chose to remain on dialysis, 19 died less than 3 months after graft loss); 172 had PRAs after failure; 106 (62%) have been retransplanted. Mean peak PRA in those retransplanted was 23 +/- 31 (range 0-100) vs. 46 +/- 39 (range 0-100) in those not retransplanted (P less than .05). Patients were stratified by PRA prior to first transplant (0%, 1-20%, greater than 20%). For recipients with 0% PRA, failure of a CAD transplant (n = 58) was no more likely to result in an increase of PRA than failure of a living-related donor (LRD) transplant (n = 49) (NS). For those with an increase, mean increase was 45% +/- 34 after LRD transplant and 41% +/- 28 after CAD transplant (NS). The proportion developing PRA greater than or equal to 60% was not different after a failed LRD (7/49) or CAD (11/58) transplant (NS). Other subgroups had similar results. AZA or CsA immunosuppression did not affect development of increased PRA after a failed graft.(ABSTRACT TRUNCATED AT 400 WORDS)

Early tolerance in pediatric liver allograft recipients.

The authors report on six pediatric liver transplant recipients for whom allograft tolerance occurred shortly after transplantation (ie, less than 1.5 years). All the patients had associated life-threatening viral complications. They are currently immunocompetent. The tolerant state may be related to the development of a TH2 cytokine pattern.