title: 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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1 Thomas E. Starzl Transplantation Institute, Hillman Center for Pediatric Transplantation, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Department of Transplant Surgery, Pittsburgh, PA. 2 Tissue Typing Laboratory, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA.
authors.forename: Chethan
authors.initials: C
authors.lastname: Ashokkumar
authors.forename: Qing
authors.initials: Q
authors.lastname: Sun
authors.forename: Mylarappa
authors.initials: M
authors.lastname: Ningappa
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Higgs
authors.forename: George
authors.initials: G
authors.lastname: Mazariegos
authors.forename: Adriana
authors.initials: A
authors.lastname: Zeevi
authors.forename: Rakesh
authors.initials: R
authors.lastname: Sindhi
mesh_terms: D000961:Antilymphocyte Serum / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D002401:Cathepsin B / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005260:Female; D006084:Graft Rejection / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D006085:Graft Survival / Q000187:drug effects; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory / Q000187:drug effects; D007166:Immunosuppressive Agents / Q000627:therapeutic use*; D007422:Intestines / Q000637:transplantation*; D016031:Liver Transplantation / Q000009:adverse effects; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D015398:Signal Transduction / Q000187:drug effects; D016176:T-Lymphocyte Subsets / Q000187:drug effects* / Q000276:immunology / Q000473:pathology; D013997:Time Factors; D016896:Treatment Outcome
chemical_list: D000961:Antilymphocyte Serum; D015415:Biomarkers; D016207:Cytokines; D006680:HLA Antigens; D007166:Immunosuppressive Agents; D023201:CD40 Ligand; C505407:CASP3 protein, human; D053148:Caspase 3; C533912:CTSB protein, human; D002401:Cathepsin B