Rapid Detection and Isolation of Viable Virus-Reactive CD4+ and CD8+ T Cells Based on Activation-Induced CD137 Expression.
Authors: Wehler Thomas C. , Karg Michael , Konur Abdo , Nonn Marion , Meyer Ralf G. , Huber Christoph , Hartwig Udo F. , Herr Wolfgang 
Published: October 2019

Abstract
Current methods for the detection and isolation of antigen-specific CD4+ and CD8+ T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We have recently reported that de novo cell surface expression of the TNF receptor family member CD137 (4-1BB) identifies currently activated, but not resting, human alloreactive CD8+ T cells. This observation allowed us to develop a CD137-based technology for the depletion of alloreactive CD8+ T cells in vitro (Wehler et al. Blood2007;109:365–373). More recently, a similar approach has been described that uses activation-induced CD137 expression for the detection and enrichment of antigen-specific CD8+ T cells (Wolfl et al. Blood2007;110:201–210). In the current study we complement this work and demonstrate the transient up-regulation of CD137 directly on activated cytomegalovirus (CMV) or Epstein-Barr virus specific CD8+ T cells using peptide/HLA tetramer staining of PBMC from seropositive healthy individuals. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. Most importantly, we also observed a similar CD137 expression kinetics (i.e. low baseline, maximum at 24h) on virus-specific CD4+ T cells upon activation with CMVpp65 peptides. The median frequencies of CMVpp65-reactive CD137+ cells measured ex vivo in 4 different CMV+ healthy donors after 24h of stimulation were 3.6% (range, 1.1–6.7) in CD8+ T cells and 2.7% (range, 0.8–6.4) in CD4+ T cells, respectively. We also analyzed PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors (n=3) stimulated with CMV peptides. None of these samples contained more than 0.3% CD137+ cells per total CD4+ and CD8+ T cells, thereby confirming the specificity of antigen-induced CD137 expression. We next established a two-step in vitro approach allowing the activation and subsequent CD137-based immunomagnetic cell sorting of virus-reactive CD4+ and CD8+ T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65-reactive CD4+ and CD8+ T cells from PBMC of 6 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 69.4% (range 12.7–94.1) among CD4+ T cells and 70.6% (range, 28.5–93.4) among CD8+ T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4+ and CD8+ T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-γ secretion assay to isolate CMVpp65-specific CD4+ and CD8+ T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-γ+ cells (n=5), suggesting that CD137 might detect a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid detection and isolation of viable virus-reactive CD4+ and CD8+ T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials.