Chethan Ashokkumar

Allospecific CD154+ T Cells Associate with Rejection Risk After Pediatric Liver Transplantation

Antigen‐specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16‐h MLR of 58 identically‐immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy‐proven rejection within 60 days posttransplantation). Thirty‐one children were sampled once, cross‐sectionally. Twenty‐seven children were sampled longitudinally, pre‐LTx, and at 1–60 and 61–200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE‐dye dilution (n = 23), and CTLA4, a negative T‐cell costimulator, which antagonizes CD154‐mediated effects (n = 31). In cross‐sectional observations, logistic regression and leave‐one‐out cross‐validation identified donor‐specific, CD154 + T‐cytotoxic (Tc)‐memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc‐memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1–60 days, and (2) elevated pre‐LTx CD154 + Tc‐memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e‐07), and inversely with CTLA4 expression of allospecific CD154 + Tc‐memory cells (r =−0.706, p = 3.0e‐05). Allospecific CD154 + T‐helper‐memory cells, not CD154 + Tc‐memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

NOD2 gene polymorphism rs2066844 associates with need for combined liver-intestine transplantation in children with short-gut syndrome.

OBJECTIVES:The nucleotide-binding oligomerization protein 2 (NOD2) gene single nucleotide polymorphisms (SNPs) associated with Crohns disease were recently associated with severe rejection after small-bowel transplantation (SBTx). The purpose of this study was to re-test this association and explore whether deficient innate immunity suggested by the NOD2 SNPs predisposes to intestine failure requiring isolated SBTx or combined liver–intestine failure requiring combined liver–SBTx (LSBTx).METHODS:Archived DNA from 85 children with primary isolated SBTx or LSBTx was genotyped with Taqman biallelic discrimination assays. To minimize confounding effects of racial differences in minor allele frequencies (MAFs), allelic associations were tested in 60 Caucasian recipients (discovery cohort). Replication was sought in an independent cohort of 39 Caucasian pediatric and adult SBTx patients.RESULTS:MAF for rs2066845 and rs2066847 was similar to that seen in 538 healthy North American Caucasians. In the discovery cohort, MAF for rs2066844 was significantly higher in LSBTx (13.5 vs. 3.6%, P=0.0007, Fishers exact test), but not in isolated SBTx recipients (2.2 vs. 3.6%, P=NS), when compared with 538 healthy Caucasians. In addition, among LSBTx recipients who received identical immunosuppression, the minor allele of rs2066844 associated with early rejection in linear regression analysis (P=0.028) (all but one of the risk alleles were found in rejectors), decreased survival (P=0.015, log-rank, Kaplan–Meier analysis), and a 20-fold greater hazard of septic death in proportional hazard analysis (P=0.030). Steroid-resistant (severe) rejection and graft loss were associated with isolated SBTx (P=0.036 and 0.082, respectively), but not with NOD2 SNPs. The association between rs2066844 and combined liver–intestine failure requiring LSBTx was significant in the replication cohort (P=0.014), and achieved greater significance in the combined cohort (P=0.00006).CONCLUSIONS:The NOD2 SNP rs2066844 associates with combined liver and intestinal failure in subjects with short-gut syndrome, who require combined liver–intestine transplantation, and secondarily with early rejection and septic deaths.

Allospecific CD154+ T cells identify rejection-prone recipients after pediatric small-bowel transplantation.

BACKGROUND Up to 70% of children with small bowel transplantation (SBTx) experience acute cellular rejection (ACR). Allospecific CD154+ T cells predict liver ACR in children in a novel, 16-hour mixed leukocyte response (MLR) assay, but remain untested in SBTx. METHODS The expression of CD154 was measured in 4 subsets-naive (N) and memory (M) CD154+ T-helper (Th) and T-cytotoxic (Tc) cells (ie, CD154+ ThN, CD154+ ThM, CD154+ TcN, and CD154+ TcM, respectively)-in the MLR of single blood samples obtained from 32 children with SBTx within 60 days of SBTx biopsy. Children showing ACR in these biopsies were termed Rejectors. The ratio of donor-induced to third-party-induced CD154+ T cells was called the immunoreactivity index (IR). We hypothesized that IR >1 denoted increased donor-specific alloreactivity and increased risk of rejection; in contrast, IR <1 implied decreased risk. CD154 expression was correlated with the expression of CTLA4, a negative T-cell costimulator that antagonizes and is inversely related to CD154 (n = 18). RESULTS Rejectors showed significantly greater numbers of donor-specific CD154+ T-cell subsets. Logistic regression analysis and leave-one-out cross validation followed by receiver operating characteristic analysis showed that, among the 4 subsets, IR > or =1.23 for CD154+ TcM identified Rejectors with a sensitivity and specificity of 93% and 88%. Also, a significant negative correlation was observed between CD154 expression and CTLA4 expression in allospecific Tc (Spearmans rho = -0.616, P = .006) but not in Th. CONCLUSION Allospecific CD154+ TcM identify rejection-prone children with SBTx.

Allospecific CD154 + T-cytotoxic memory cells as potential surrogate for rejection risk in pediatric intestine transplantation

Sindhi R, Ashokkumar C, Higgs BW, Gilbert PB, Sun Q, Ranganathan S, Jaffe R, Snyder S, Ningappa M, Soltys KA, Bond GJ, Mazariegos GV, Abu‐Elmagd K, Zeevi A. Allospecific CD154 + T‐cytotoxic memory cells as potential surrogate for rejection risk in pediatric intestine transplantation. 
Pediatr Transplantation 2012: 16: 83–91.

Elevated myeloid: plasmacytoid dendritic cell ratio associates with late, but not early, liver rejection in children induced with rabbit anti-human thymocyte globulin.

Background. Dendritic cells (DC) play an important role in the induction and regulation of immune responses. Methods. Myeloid CD11c+ DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, cross-sectionally, once, in 48 children, and longitudinally (pretransplant and at days 1–60, 61–200, and 201–400 posttransplant) in 30 children after liver transplantation (LTx). All children received cadaveric (n=53) or live donor (n=25) liver allografts with rabbit anti-human thymocyte globulin induction and steroid-free tacrolimus therapy. Rejectors in both groups were those children (n=35), who experienced biopsy-proven acute cellular rejection (ACR) within 60 days of DC monitoring. Results. Among rejectors in the longitudinal and cross-sectional cohorts, the MDC:PDC ratio was higher and was associated with decreased PDC frequencies. Logistic regression analysis, leave-one-out cross-validation, and receiver operating characteristic analysis applied to 30 cross-sectional subjects revealed that an MDC:PDC ratio more than or equal to 1.78 was associated with rejector status with sensitivity and specificity of 76.9% and 88.2%, respectively. Sensitivity and specificity were replicated in the 18 remaining cross-sectional subjects (88.8% and 78.8%, respectively), but not in longitudinally monitored subjects, during the early 60-day period after LTx (30.76% and 62.50%, respectively). A significant negative correlation was observed between tacrolimus whole blood concentrations and PDC frequencies (Spearman r=−0.370, P=0.005) in 48 cross-sectional subjects in whom DC subsets were monitored 1 to 3 years after LTx, but not during the early post-LTx period. Conclusions. We conclude that an elevated MDC:PDC ratio associates with liver graft rejection, which occurs after first year in children induced with rabbit anti-human thymocyte globulin.

Allospecific CD154+ B cells associate with intestine allograft rejection in children.

Background. As a significant determinant of T- and B-cell cooperation, CD154 has been used to identify allospecific T-cytotoxic memory cells (TcM) for rejection risk assessment with high sensitivity or specificity but not for alloreactive B-cells, especially among recipients predisposed to acute cellular and humoral rejection, that is, children with intestinal transplantation (ITx). Methods. Single blood samples from 32 pediatric ITx after lymphocyte depleting induction therapy were obtained within 30 days of protocol biopsies. Samples were assayed for allospecific CD154+CD19+ B cells and allospecific CD154+ TcM in 16-hr live-cell mixed leukocyte reaction using multiparametric flow cytometry. Results were expressed as the immunoreactivity index (IR) or the ratio of donor- to third-party-induced CD154+ B cells or TcM. The rejection threshold IR of B cells was determined by logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses. Results. Biopsy-proven acute cellular rejection was present in 15 subjects (rejectors) and absent in 17 (nonrejectors). In archived serum samples from 16 of 32 subjects donor-specific anti-HLA antibodies (DSA) were assayed by Luminex bead array. DSA were absent in all 7 nonrejectors but present in 7 of 9 rejectors. The IR of allospecific CD154+CD19+ B cells more than or equal to 1.351 was associated with rejector status and was present in 13 of 15 rejectors (sensitivity 87%) and absent in 15 of 17 nonrejectors (specificity 88%). Excellent correlations were seen between CD154+CD19+ B cells and CD154+ TcM (Spearman &rgr;=0.647, P=0.0001) but could not be tested independently for DSA, which was highly correlated with rejector status and with CD154+ TcM. Conclusions. Allospecific CD154+CD19+ B cells identify rejection-prone children with ITx and can likely substitute for T-cell alloreactivity in estimating rejection risk in this rare subject population.

Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection.

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.

Allospecific CD154+ T-cytotoxic memory cells identify recipients experiencing acute cellular rejection after renal transplantation.

Background. The novel, recently described allo (antigen)-specific CD154+T cells were evaluated for their association with acute cellular rejection (ACR) in 43 adult renal transplant recipients receiving steroid-free tacrolimus after alemtuzumab induction. Methods. Single blood samples corresponding to “for cause” allograft biopsies were assayed for CD154+naive or memory T-helper or T-cytotoxic cells in 16-hr mixed leukocyte reaction. Results. Intra- and interassay variation was less than 10% for a variety of conditions. In logistic regression, leave-one-out cross-validation, and receiver-operating characteristic analyses, the rejection-risk threshold of allospecific CD154+T-cytotoxic memory cells (TcMs) associated best with biopsy-proven ACR with a sensitivity/specificity of 88% in 32 of 43 subjects. Sensitivity/specificity of 100%/88% was replicated in blinded prediction in the remaining 11 subjects. Allospecific CD154+TcM correlated inversely with CTLA4+TcM (Spearman r=−0.358, P=0.029) and increased significantly with increasing histological severity of ACR (P=2.99E-05, Kruskall-Wallis). Conclusions. The strong association between ACR and allospecific CD154+TcM may be useful in minimizing protocol biopsies among recipients at reduced rejection risk.

Elevated Myeloid: Plasmacytoid Dendritic Cell Ratio Associates With Early Acute Cellular Rejection in Pediatric Small Bowel Transplantation

Background. Acute cellular rejection affects more than 60% of children after small bowel transplantation (SBTx). Dendritic cells (DCs) are potent antigen-presenting cells, modulate immune responses to gut microbes, and may serve as markers of rejection-prone small bowel transplantation (SBTx). Methods. Myeloid CD11c+ DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, longitudinally (pretransplant, and at days 1 to 60, 61 to 200 posttransplant) in 23 children after SBTx. All children received cadaveric allografts with rabbit anti-human thymocyte globulin induction and steroid-free tacrolimus maintenance therapy. Rejectors were those children (n=16), who experienced biopsy-proven acute cellular rejection within 60 days of SBTx. Results. Of 69 maximum possible observations, 62 were available for analysis. Among rejectors, a significantly higher MDC:PDC ratio (P=0.004) was associated with numerically higher MDC counts and significantly lower PDC frequencies (P=0.017) during the 1- to 60-day time period, compared with nonrejectors. Logistic regression analysis, leave-one-out cross-validation, and receiver operating characteristic analysis revealed that MDC:PDC ratio more than or equal to 1.52 was associated with rejector status with sensitivity/specificity of 86/67% during the 1- to 60-day risk period for early SBTx rejection. Repeated measures analysis showed a significantly higher MDC:PDC ratio (P=0.043, F-test) among rejectors, compared with nonrejectors in cumulative data for pre-SBTx and 1- to 60-day time points. No correlation was seen between DC subsets and tacrolimus blood concentration at any time point. Conclusions. We conclude that an elevated MDC:PDC ratio associates with early small bowel allograft rejection and may, therefore, identify at-risk recipients in the clinic.

Genetic Variants in Major Histocompatibility Complex-Linked Genes Associate With Pediatric Liver Transplant Rejection

BACKGROUND & AIMS Limited access to large samples precludes genome-wide association studies of rare but complex traits. To localize candidate genes with family-based genome-wide association, a novel exploratory analysis was first tested on 1774 major histocompatibility complex single nucleotide polymorphisms (SNPs) in 240 DNA samples from 80 children with primary liver transplantation and their biologic parents. METHODS Initially, 57 SNPs with large differences (P < .05) in minor allele frequencies were selected when parents of children with early rejection (rejectors) were compared with parents of nonrejectors. RESULTS In hypothesis testing of selected SNPs, the gamete competition statistic identified the minor allele G of the SNP rs9296068, near HLA-DOA, as being significantly different (P = .018) between outcome groups in parent-to-child transmission. Subsequent simple association testing confirmed over- and undertransmission of rs9296068 based on the most significant differences between outcome groups, of 1774 SNPs tested (P = .002), and allele (G) frequencies that were greater among rejectors (51.4% vs 36.8%, respectively, P = .015) and lower among nonrejectors (26.8% vs 36.8%, respectively, P = .074) compared with 400 normal control Caucasian children. In early functional validation, rejectors demonstrated significant repression of the first HLA-DOA exon closest to rs9296068. Also, intragraft B lymphocytes, whose antigen-presenting function is selectively inhibited by HLA-DOA were 3-fold more numerous during rejection among rejectors with the risk allele, than those without. CONCLUSIONS The minor allele of the SNP rs9296068 is significantly associated with liver transplantation rejection and with enhanced B-lymphocyte participation in rejection, likely because of a dysfunctional HLA-DOA gene product.