Allospecific CD154+ T cells associate with rejection risk after pediatric liver transplantation

Abstract

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.

Figure 1

Shows gating strategy used for flow cytomteric analysis. Live responder T-cells (CD3+) are identified after exclusion of donor or third-party stimulators, which are prestained with anti-CD45− allophycocyanin, and after exclusion of dead cells. T-helper (Th, CD3+CD4+) and T-cytotoxic (Tc, CD3+CD4−) families are each divided into memory (CD45RO+), and naive (CD45RO−).

Figure 2

Scatter plots in panel a) show significantly higher proportions of donor-specific CD154+ (uppermost) and CFSElow (lowermost) T-cytotoxic cells in a Rejector, compared with corresponding third-party-induced alloresponses. In the middle subpanel, donor-induced expression of the negative costimulator, CTLA4, is less than third-party-induced CTLA expression. For the Non-Rejector shown in panel b) the reverse features are seen in a Non-Rejector. Donor-induced markers of T-cell activation, CD154 and CFSElow cells, are less than those induced with third-party stimulation. On the other hand, donor-induced cells expressing the negative costimulator, CTLA4, exceed third-party induced CTLA4+ cells.

Figure 3

Representative liver biopsies from a Rejector and Non-Rejector. Two stains are presented for each biopsy-hematoxylin and eosin, and immunostain for T-cytotoxic cells (aminoethylcarbazol, AEC). Corresponding immunoreactivity indices (IR) of CD154+ Tc-memory cells are also shown. a) Allograft liver with no cellular infiltration of portal areas. No Acute cellular rejection (HEx200) Fig. 3b as in 3a, with few red-stained Tc (CD8+) cells (AEC) 3c. Allograft liver-central vein inflammation, hepatocyte dropout, and centrilobular extravasation–moderate centrilobular ACR (HEx200). Fig. 3d as in 3c, with a large number of red-stained Tc (AEC).

Figure 4

IR for CD154+Tc-memory cells obtained from 27 children in the longitudinal cohort are shown. The cohort consisted of 13 Rejectors and 14 Non-Rejectors. Twenty of 27 children were sampled before LTx, 26 of 27 during the 1–60-day time period, and 27 of 27 at the 61–200-day time period. Figure 4a. IR<1.13 (below dotted line) was seen in 9 of 11 Non-Rejectors, sampled before LTx, in 11 of 13 sampled during the 1–60-day period, and all 14 children sampled at the 61–200-day period. One of two Non-Rejectors showing pre-LTx IR≥1.13 (red line) went on to experience late ACR. Figure 4b. IR>1.13 (above dotted line) was seen in 7 of 9 Rejectors sampled pre-LTx, and in 12 of 13 Rejectors sampled during the 1–60-day period. All but four of 13 Rejectors achieved IR<1.13, or reduced risk of rejection at 61–200 days. Two of these four children experienced late biopsy-proven ACR.

Figure 5

A) Rejection-free survival during the first 200 days after LTx for children whose CD154+Tc-memory cell showed high pre-LTx rejection risk, or IR≥1.13 is significantly lower, compared with children with low pre-LTx risk or IR<1.13. (B) Time in days required to achieve low rejection risk, or IR<1.13 after LTx was significantly longer in Rejectors, compared with Non-Rejectors (median 167 vs 56 days, p=0.016).

Figure 6

a) Highly significant correlations exist between IR for CFSE^low Tc-memory, and allospecific CD154+ Tc-memory cells in 23 children (Spearman rho=0.835, p=7.1e-07). b) The negative correlation between IR for allospecific CD154 Tc-memory and IR for allospecific CTLA4+Tc-memory cells is highly significant in 31 of 58 recipients. Ranked placement shows Non-Rejectors clustered in the lower right hand side of the graph, while Rejectors are clustered in the upper left hand side (Spearman rho −0.706, p=3.0e-05).

Figure 7

This figure summarizes experiments demonstrating properties of allospecific CD154+ Tc-memory cells as markers of rejection-prone recipients. a) Summary data for six allostimulated normal control MLR show that CD154 expression peaks at 16 hours, is 2.5 fold greater in Th cells, compared with Tc, and is inhibited by 80% in Th cells, pretreated with Tacrolimus. Tc cells are minimally inhibited by Tacrolimus. B) Post-dose IR (Mean±SD) for Th-memory cells obtained from 7 children with LTx who are Rejectors are lower than pre-dose IR. Post-dose IR for Tc-memory decreases numerically compared with pre-dose IR, but the change is not statistically significant. Changes in pre- and post-dose IR for each subject are connected by lines.