New tools to monitor the impact of viral infection on the alloreactive T‐cell repertoire

Abstract

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.

Stimulation of Human EBV- and CMV-Specific Cytolytic Effector Function Using Allogeneic HLA Molecules

“…For example, EBV infection in a HLA-B8 + individual always selects for a dominant "public" Vb6S2 TCR (13), which cross-reacts against allo-HLA-B*44:02 (14). We confirmed the previously described alloreactivity of this EBV EBNA3A-specific T cell (HLA-B8/FLRGRAYGL restricted) against allogeneic HLA-B*44:02 (12,15). Allo-HLA cross-reactivity was also shown for CMV, varicella-zoster virus (VZV) and influenza virus-specific T cells, which express nonpublic TCRs (12).…”

“…Although not all virus-specific immune responses give rise to a public TCR, the allo-HLA cross-reactivity of virusspecific T cells from a given individual can be detected easily in vitro using techniques we have described here and elsewhere (12,15). Indeed, successful stimulation of cytolytic effector function against CMV Ag reveals that this technique could potentially be useful to elicit T cell cytolytic effector function against any virus or specificity.…”

“…In these experiments, we have used healthy blood donors as responder PBMCs, not cells from immunosuppressed patients. Whereas infusion of irradiated leukocytes should not be associated with chimerism or engraftment, this possibility should be considered Positive control EBNA3A T cell clone is described previously (15), and responder PBMCs used in this assay are also obtained from the same donor. HLA typing of responder PBMCs, autologous target PBMCs, and EBNA3A T cell clone HLA-A*01, 02; B*08:01, 2; DRB1*03, 2.…”

“…A high level of cross-reactivity against allo-HLA molecules is therefore an essential feature of the virus-specific memory TCR (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). This allo-HLA cross-reactivity by virus-specific T cells can be reproducibly detected in vitro.…”

Allo-HLA reactivity of virus-specific memory T cells is common

“…22 Furthermore, we observed alloreactivity against EBV-LCLs expressing HLA-B*5501 as was recently described by us. 33 Allo-HLA reactivity against HLA-B*4402 and HLA-B*5501 was confirmed by recognition of K562 transfected with either HLA-B*4402 or HLA-B*5501 by this T-cell clone ( Figure 2E). In addition to CD8 ϩ T-cell lines and clones, we also analyzed the cross-reactive potential of CD4 ϩ T-cell clones to allo-HLA molecules.…”