Antithymocyte globulin facilitates alloreactive T-cell apoptosis by means of caspase-3: potential implications for monitoring rejection-free outcomes

Abstract

Background: Alloreactive T-cell apoptosis may explain reduced immunosuppression requirements with proapoptotic immunosuppression and among rejection-free recipients. This possibility remains unproven.

Methods: Apoptotic (caspase-3+, cathepsin B+) and inflammatory (CD154+) T-cell subsets were evaluated before and after adding rabbit antithymocyte globulin (rATG) to mixed lymphocyte co-cultures between human leukocyte antigen-mismatched peripheral blood lymphocytes from healthy adults. In random samples from children with liver (LTx-20) and intestine (ITx-13) transplantation, apoptotic T cells were evaluated for association with rejection-free outcomes using the caspase-3 substrate, phiphilux.

Results: In mixed lymphocyte co-cultures between normal human peripheral blood lymphocytes, (1) frequencies of memory (M) and naive (N) Th and Tc, which expressed activated caspase-3, were enhanced most by the combination of allostimulation and rATG, than either stimulus alone. These findings were confirmed with antibody to activated caspase-3, phiphilux, and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay; (2) frequencies of Th subsets, which expressed activated cathepsin B, were similarly increased with combined stimulation. Tc seemed resistant to cathepsin B activation; (3) with increasing rATG concentrations, proportionately more allospecific CD154+T-cytotoxic memory cells (TcM) survived than TcM, resulting in relative enrichment of allospecific CD154+TcM. In random blood samples, phiphilux+T-cell subset frequencies were higher among 14 rejection-free LTx and ITx recipients and demonstrated a greater increase with ex vivo rATG pretreatment than 19 rejectors. In logistic regression analysis, phiphilux+TcM associated best with rejection-free outcomes with a sensitivity of 57% and a specificity of 89%.

Conclusion: Rabbit antithymocyte globulin facilitates apoptosis of alloreactive T cells by means of caspase-3 activation, which may explain its steroid-sparing effect in pediatric liver and intestine recipients. Apoptotic susceptibility of T-cytotoxic memory cells, which resist cathepsin B activation, may distinguish rejection-free and rejection-prone liver recipients.

Trial registration: ClinicalTrials.gov NCT01163578.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Frequencies of naive (N) and memory (M) Th and Tc which express activated caspase-3 (panels on left) and activated cathepsin-B (panels on right) are shown after no treatment, rATG-pretreatment, allostimulation, and combined allostimulation and rATG pre-treatment of normal human responder PBL. Data are shown as representative histograms for ThM and TcM from one replicate (upper two panels), summary of data from four replicates for a single rATG concentration of 20 μg/ml (bar diagram), and summary of data for four replicates for each of four rATG concentrations. Activated caspase-3 and cathepsin-B are measured with specific fluorochrome-labeled antibodies and ICS.

Figure 2

Frequencies of phiphilux+ThM and TcM subsets (upper and middle panels) are shown after no treatment, rATG-pretreatment, allostimulation, and combined allostimulation and rATG pre-treatment of normal human responder PBL. Phiphilux measures activated caspase-3 without ICS. Bar diagram in lower panel shows summary of data from six replicates for a single rATG concentration of 20 μg/ml.

Figure 3

a. Representative histograms show DNA damage in flow-sorted CD3+T-cells by TUNEL assay after no treatment, rATG-pretreatment, allostimulation, and combined allostimulation and rATG pre-treatment of normal human responder PBL from one of the four replicate.b. Summary data (n=4) for frequencies of phiphilux+TcM, and CD3+T-cells manifesting DNA damage in normal human PBL pretreated with increasing rATG concentrations. c. Summary data (n=3) showing increased production of the cytokines IL6, IL1β and TNFα by allostimulated T-cell with increasing rATG concentrations. d. Summary data from MLC showing that (97%) decrease in allostimulated TcM with increasing rATG concentrations is accompanied by a numerically smaller (75%) decrease in allospecific CD154+TcM (n=6). e. Summary data from MLC (n=6) showing increased frequency of allospecific CD154+TcM among surviving TcM at increasing rATG concentrations.

Figure 4

Summary data for apoptotic memory (M) (upper row) and naïve (N) (bottom row) Tc (column on left) and Th (column on right) among PBL from 20 children with LTx, after ex viso pre-treatment with 0-200 μg/ml rATG. Apoptotic subsets are measured with the caspase-3 substrate, phiphilux. The population consists of 8 non-rejectors (NR, black squares) and 12 rejectors (R, grey triangles). P-values for between-group comparisons using 2-way repeated measures ANOVA are shown.