title: Correlations of lymphocyte subset infiltrates with donor-specific antibodies and acute antibody-mediated rejection in endomyocardial biopsies.
abstract: BACKGROUND
  Acute antibody-mediated rejection (AMR) is a major complication after heart transplantation, posing a significant risk for allograft failure, cardiac allograft vasculopathy, and poor survival. While the inflammatory milieu of cellular rejection and Quilty lesions is well known, the immunologic components of AMR are not well understood. Our aim was to better define the immunophenotype of infiltrating lymphocytes in biopsies with AMR, specifically in relation to donor-specific antibodies to human leukocyte antigen (HLA) class I, II, or both.
  
  
  METHODS
  We performed a retrospective analysis of cardiac transplant patients with concurrent endomyocardial biopsies (EMB), donor-specific antibody (DSA) measurements, and immunofluorescence for C4d at our institution (2005-2011). DSA was evaluated against HLA class I and class II specificities pre- and posttransplant using flow cytometry and/or Luminex bead assays. Acute cellular rejection (ACR) and pathologic AMR (pAMR) were based on the International Society for Heart and Lung Transplantation 2005/2013 reports. Immunohistochemical analysis for CD3, CD4, CD8, and CD79a was performed using standard immunohistochemical protocols on one formalin-fixed, paraffin-embedded EMB from each patient. The number of lymphocytes expressing each protein was enumerated microscopically at 400×. Ratios of T:B cells and CD4:CD8 T cells were then calculated for each EMB.
  
  
  RESULTS
  Seventy-nine cardiac transplant patients who had pre- and posttransplant DSA measurements were analyzed. Of these 79 patients, 37 had DSA against HLA class I, HLA class II, or both. Of patients with DSA, the average CD4:CD8 ratio in the EMB was 0.80, while those with only ACR had a CD4:CD8 ratio of 1.49. Interestingly, the T:B cell ratio in patients with and without DSA was 5.7 and 5.5, respectively.
  
  
  CONCLUSIONS
  Cardiac transplant patients with DSA against HLA have more CD8 cytotoxic T cells than CD4 helper T cells in the EMB lymphocytic infiltrate compared with patients without DSA against HLA. The inflammatory infiltrate T:B cell ratio was similar in patients both with and without DSA. The relative increase of cytotoxic T cells in EMB while the patient has DSA suggests a possible pathogenic role of these cells and may aid in the diagnosis and treatment of AMR.
authors.affiliation: Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States. Electronic address: renee.s.frank@gmail.com.
authors.forename: Renee
authors.initials: R
authors.lastname: Frank
authors.affiliation: Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Dean
authors.affiliation: Heart Failure and Cardiac Transplant Program, Division of Cardiovascular Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Molina
authors.affiliation: Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Malek
authors.initials: M
authors.lastname: Kamoun
authors.affiliation: Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Priti
authors.initials: P
authors.lastname: Lal
mesh_terms: D000328:Adult; D000368:Aged; D064591:Allografts / Q000276:immunology; D000906:Antibodies / Q000276:immunology*; D001706:Biopsy; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D006084:Graft Rejection / Q000276:immunology* / Q000473:pathology; D016027:Heart Transplantation / Q000009:adverse effects; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000276:immunology; D012189:Retrospective Studies; D014019:Tissue Donors; D055815:Young Adult
chemical_list: D000906:Antibodies
keywords: Antibody-mediated rejection; Cardiac transplantation; Donor-specific antibody; Endomyocardial biopsy