Bernd M. Spriewald

CD40-CD40 ligand-independent activation of CD8+ T cells can trigger allograft rejection.

In experimental transplantation, blockade of CD40-CD40 ligand (CD40L) interactions has proved effective at permitting long-term graft survival and has recently been approved for clinical evaluation. We show that CD4+ T cell-mediated rejection is prevented by anti-CD40L mAb therapy but that CD8+ T cells remain fully functional. Furthermore, blocking CD40L interactions has no effect on CD8+ T cell activation, proliferation, differentiation, homing to the target allograft, or cytokine production. We conclude that CD40L is not an important costimulatory molecule for CD8+ T cell activation and that following transplantation donor APC can activate recipient CD8+ T cells directly without first being primed by CD4+ T cells.

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade.

BACKGROUNDnThe CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade.nnnMETHODSnBALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation.nnnRESULTSnOnly combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups.nnnCONCLUSIONSnOur data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.

Pretransplant IgG subclasses of donor-specific human leukocyte antigen antibodies and development of antibody-mediated rejection.

Background. The subclass of IgG antibodies contributes to their capability to activate complement. It is currently unknown whether the pretransplant IgG subclass composition allows distinguishing harmful from presumably irrelevant donor-specific human leukocyte antigen (HLA) antibodies (HLA-DSA) detected by single-antigen flow beads (SAFB). Methods. Seventy-four patients transplanted in the presence of HLA-DSA were investigated. HLA-DSA characteristics were not different between patients experiencing antibody-mediated rejection (AMR) (n=40) and patients who did not (n=34) experience AMR. Sera were reanalyzed using SAFB with IgG subclass-specific reporter antibodies. Results. The 74 patients had in total 141 HLA-DSA. IgG1 was the predominant subclass (78%), followed by IgG2 (49%), IgG3 (36%), and IgG4 (20%). When grouped according to the complement-activating capability, only 4 of 74 patients (5%) had exclusively weak/no complement-activating HLA-DSA (i.e., IgG2 and IgG4), 21 of 74 patients (28%) had isolated strong complement-activating HLA-DSA (i.e., IgG1 and IgG3), and 46 of 74 patients (62%) had a mixture of both. There was no difference between the strong complement-activating and the mixture group regarding incidence of AMR (57% vs. 54%; P=0.81), phenotypes of AMR (P=0.70), and death-censored allograft survival at 5 years (78% vs. 78%; P=0.74). Interestingly, patients with exclusively weak/no complement-activating HLA-DSA (n=4) had a numerically lower incidence of AMR (25%) and no allograft loss has occurred yet. Conclusion. In 90% of patients, pretransplant HLA-DSA are composed of isolated strong or a mixture of strong and weak/no complement-activating IgG subclasses. Because outcomes in these two groups were similar, pretransplant IgG subclass analysis is likely not providing substantial value beyond the standard IgG SAFB assay for pretransplant risk stratification.

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4+ T cell activation but is unable to prevent immune responses mediated by CD8+ T cells. However, even in the absence of CD8+ T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40−/− (H2b) recipients were transplanted with MHC-mismatched BALB/c (H2d) aortas. Transplant arteriosclerosis was evident in both CD40−/− and CD40+/− mice (intimal proliferation was 59 ± 5% for CD40−/− mice vs 58 ± 4% for CD40+/− mice) in the presence or absence of CD8+ T cells (intimal proliferation was 46 ± 7% for CD40−/− anti-CD8-treated mice vs 50 ± 10% for CD40+/− anti-CD8-treated mice), confirming that CD8+ T cells are not essential effector cells for the development of this disease. In CD40−/− recipients depleted of CD8+ T cells, the number of eosinophils infiltrating the graft was markedly increased (109 ± 24 eosinophils/grid for CD40−/− anti-CD8-treated mice vs 28 ± 7 for CD40+/− anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40−/− recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 ± 5 eosinophils/grid for CD40−/− anti-CD8+, anti-IL-4-treated mice vs 109 ± 24 for CD40−/− anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 ± 5% for CD40−/− anti-CD8+-, anti-IL-4-treated mice vs 46 ± 7% for CD40−/− anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8+ T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Islet allograft rejection can be mediated by CD4+, alloantigen experienced, direct pathway T cells of TH1 and TH2 cytokine phenotype

Background. It is widely believed that Th1 cells that secrete interferon-&ggr; are primarily involved in the rejection of allografts whereas Th2 cells [interleukin(IL) 4 and IL-10] are thought to be protective of this process. However, the exact role and specificity of these helper T lymphocytes in mediating allograft damage is presently unknown. Methods. Th0, Th1, and Th2 cell lines specific for the class II MHC molecule H2IAb were adoptively transferred into T cell deficient, syngeneic, diabetic mice before transplantation of fully allogeneic C57BL/10 (H2b) or (CBKxBALB/c)F1 (H2k/d+Kb) islet grafts. T cells were 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester- (CFSE) labeled to allow detection, immunohistochemistry was performed, and IL-4 transcripts within the rejected islet grafts were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). Results. Adoptive transfer (IV) of Th0-, Th1-, and Th2 IAb-specific T cells resulted in rejection of H2b islet allografts. CFSE-labeling demonstrated that these T cells were able to home to the graft site. CD4+ T cells and CD11b+ macrophages were present within the graft after adoptive transfer of both Th1 and Th2 cells. Interestingly, CD8+ T cells and B cells were absent from these rejecting grafts. Even when Th2 cells were introduced directly at the graft site, prompt rejection was still observed despite the presence of increased IL-4 mRNA expression within the islet allografts. Conclusions. Th2 and Th0 alloreactive CD4+ T helper cells can reject islet grafts with similar efficiency to Th1 cells. These results suggest that deviation of the immune response from a Th1 to Th2 phenotype will not be sufficient to allow successful engraftment of allogeneic organs or tissues.

Accelerated kidney transplant rejection and hypertensive encephalopathy in a pediatric patient associated with antibodies against angiotensin type 1 receptor and HLA class II.

This work was supported by the University of Nebraska Medical Center Research Support Fund. J.T.L. has received money for support from Merck & Co., Inc. to study sitagliptin in a subsequent study. All other authors declare no conflicts of interest. Address correspondence to: James T. Lane, M.D., Division of Endocrinology and Diabetes, Department of Internal Medicine, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, 1000 N. Lincoln Blvd., Suite 2900, Oklahoma City, OK 73104-3252. E-mail: james-lane@ouhsc.edu J.T.L. participated in the design, and implementation of the study, participated in the recruitment and clinical care of patients, completed data acquisition, statistical analysis, and manuscript preparation. D.E.O. participated in statistical analysis, data acquisition, and review of the manuscript. C.E.H participated in the recruitment of patients, clinical follow-up within the clinical research center, data acquisition, and review of the manuscript. D.S.C. participated in the design of the study and review of the manuscript. L.E.W. performed renal transplants and administered clinical care. R.B.S. performed renal transplants, administered clinical care, participated in the design and implementation of the study, recruited patients, and reviewed the manuscript. Received 21 July 2011. Accepted 24 August 2011. Copyright

Attenuation of transplant arteriosclerosis with clopidogrel is associated with a reduction of infiltrating dendritic cells and macrophages in murine aortic allografts.

Background. Monotherapy with clopidogrel reduced the formation of transplant arteriosclerosis in a murine aortic allograft model. However, the underlying immunologic mechanisms are still unknown. Methods. Fully major histocompatibility complex-mismatched C57BL/6 (H2b) donor aortas were transplanted into CBA.J (H2k) recipients and mice received different doses (1, 10, and 20 mg/kg) of clopidogrel, an antagonist of the P2Y12 ADP receptor on platelets, or control saline for 30 days. Blood was analyzed for changes in adhesion molecule and sCD40L concentrations by ELISA. Grafts were analyzed by histology, morphometry, and immunofluorescence on day 30 after transplantation. Intragraft cytokine mRNA production was analyzed by reverse-transcriptase polymerase chain reaction on day 14 after transplantation. Results. Treatment with clopidogrel resulted in significantly decreased blood concentrations of sCD40L and P-selectin after transplantation. Cellular analysis of the aortic transplant revealed fewer numbers of infiltrating dendritic cells (CD205+) and macrophages (F4/80+) after application of clopidogrel, whereas T-cells within the graft were unaltered. In addition cellular P-/E-selectin, ICAM-1, and platelet-derived-growth-factor (PDGF)-&bgr; surface expression were significantly reduced as compared with untreated controls. Intragraft mRNA expression confirmed these results and showed significant lower production of P-/E-selectin, ICAM-1, and PDGF-&bgr; after treatment with clopidogrel. Antiglycoprotein-Ib and antiglycoprotein VI had no beneficial effect on the development of transplant arteriosclerosis. Conclusion. This report shows that application of clopidogrel after transplantation results in a reduction in adhesion molecule expression within the blood and transplant tissue and is associated with reduced transendothelial migration of dendritic cells and macrophages within the vascular wall.

Microchimerism after liver transplantation : Prevalence and methodological aspects of detection

BACKGROUNDnMicrochimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail.nnnMETHODSnTherefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP.nnnRESULTSnAlthough at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases.nnnCONCLUSIONnThe optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.

Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies.

Human leukocyte antigen alloantibodies have a multitude of damaging effects on the allograft, both complement (C′) activation and Fc‐independent ones. To date, the clinical significance of non‐C′ fixing (NCF) HLA donor‐specific antibodies (DSA) is still unclear. In this study, we investigated whether renal transplant recipients with NCF‐DSA subclasses (IgG2/IgG4, IgA1/IgA2) are at higher risk of graft loss compared to patients with exclusively C′ fixing (IgG1/IgG3). Blood samples from 274 patients were analyzed for HLA IgG and IgA subclasses using a modified single‐antigen bead assay. We identified 50 (18.2%) patients with circulating NCF antibodies either DSA (n = 17) or against third‐party HLA (n = 33). NCF‐DSAs were preferentially of IgG2/IgG4 isotype (11/17) and were mainly directed against HLA class II (13/17). NCF DSA were present as a mixture with strong C′ fixing IgG1/IgG3. Graft survival was similar between patients with exclusively C′ fixing antibodies and those with a mixture panel (log rang test P = 0.162), and also among patients with different immunoglobulin isotype and subclasses (long‐rank test, P = 0.732). We conclude that expansion of DSA to NCF subclasses postrenal transplantation does not seem to be associated with worse graft survival as compared to the presence of exclusive C′ fixing subclasses.

Intragraft interleukin-4 mRNA expression after short-term CD154 blockade may trigger delayed development of transplant arteriosclerosis in the absence of CD8+ T cells

Background. It has recently been shown that, although anti-CD154 induces CD4+ T-cell tolerance, it is unable to prevent allograft rejection mediated by CD8+ T cells. We have also shown that anti-CD154 monotherapy does not protect the graft from the development of transplant arteriosclerosis even in the absence of CD8+ T cells. This study was designed to investigate and characterize possible mechanisms responsible for the development of transplant arteriosclerosis after CD154 blockade in the absence of CD8+ T cells. Methods. C57BL/6 (H2b) recipients received a fully MHC-mismatched BALB/c donor aorta (H2d). Animals were either treated with anti-CD154 monoclonal antibody (mAb) in the presence or absence of CD8 T cells. Histology, morphometric measurements, immunohistochemistry, and the production of alloantibodies (IgM, IgG1, IgG2a) were analyzed on days 14, 30, and 50 after transplantation. Cytokine production within the graft was investigated by competitive reverse transcription-polymerase chain reaction on day 14. Results. Combined treatment with anti-CD154 and a depleting CD8 mAb resulted in a delay in the development of transplant arteriosclerosis (intimal proliferation: 33±10% vs. 67±11% untreated control, day 30) but ultimately did not prevent its progression (intimal proliferation: 55±10% vs. 78±9% untreated control, day 50). Although there was a significant decrease in the number of CD4+, CD11b+, and CD40+ graft-infiltrating cells and a reduction in the formation of donor-specific IgG1 alloantibodies in recipients treated with anti-CD154 and anti-CD8 mAbs, mRNA for interleukin (IL)-4 was increased, suggesting a shift in the intragraft cytokine profile towards a Th2-like pattern. Conclusions. Our data provide evidence that short-term CD154 blockade is insufficient to prevent transplant arteriosclerosis, even in combination with CD8+ T-cell depletion. Moreover, the increased expression of the Th2 cytokine interleukin-4 within the graft may be responsible for the development of transplant arteriosclerosis in the long term.