Recovery of Functional EBV Specific T Cells Is Not Impaired In Patients with Chronic Gvhd.
Authors: Hoegh-Petersen Mette , Liu Yiping , Liu Stephanie , Ugarte-Torres Alejandra , Fonseca Kevin , Khan Faisal , Storek Jan 
Published: October 2019

Abstract
Abstract 1286

Introduction:
T cell expression of PD-1, a marker of functional exhaustion manifested by inability to produce cytokines upon stimulation, is upregulated in patients with acute GHVD. This is thought to explain at least in part why patients with acute GVHD have frequent infections (Gallez-Hawkins et al, BBMT15:872, 2009). Here we wished to evaluate whether this is true also for chronic GVHD.

Patients and Methods:
We studied 17 allogeneic HCT recipients for AML who have not developed acute or chronic GVHD by day 84. Their blood was drawn on day 84, 180 and 365. Between day 84 and 365, 7 patients did and 10 patients did not develop chronic GVHD needing systemic immunosuppressive therapy (de novo, ie, without preceding acute GVHD). Onset of the chronic GVHD was on median day 103 (range, 90–147). We studied total CD4 and CD8 T cells as well as Epstein-Barr virus (EBV)-specific CD4 and CD8 T cells, as patients with chronic GVHD are at risk of EBV disease (Landgren et al, Blood,14:4992, 2009). Blood mononuclear cells were stimulated with Epstein-Barr virus (EBV) lysate, EBNA3A+B+C overlapping peptides, no or irrelevant stimulus as negative control, or Staphylococcal enterotoxin B as positive control. After overnight incubation, expression of IFNγ, TNFα, IL2 and PD-1 on CD3+CD4+CD8- or CD3+CD4-CD8+ cells was determined by flow cytometry. Cells expressing IFNγ, TNFα, IL2 or their combinations were enumerated. PD-1 expression was quantified using beads coated with anti-mouse antibody (Quantum Simply Cellular, Bangs Laboratories) and expressed as antibody binding capacity units (ABC) (dynamic range, approximately 300 to 500,000 ABC units per cell).

Results:
PD-1 expression on total, EBV lysate-specific or EBNA3-specific CD4 or CD8 T cells was not significantly higher among patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward lower PD-1 expression on EBV lysate-specific CD4 and CD8 T cells and EBNA3-specific CD4 T cells in patients who developed chronic GVHD. This was significant (p<.05, Mann-Whitney test) for EBV lysate-specific CD4 T cells on day 84, EBV lysate-specific CD8 T cells on day 180, EBV lysate-specific CD4 and CD8 T cells on day 365, EBNA3-specific CD4 T cells on day 84 and EBNA3-specific CD4 T cells on day 365. Consistent with that, absolute counts of total, EBV lysate-specific or EBNA3-specific T cells were not significantly lower in patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward higher EBV lysate-specific and EBNA3-specific CD4 or CD8 T cell counts in patients who developed chronic GVHD. This was significant on day 84 for total EBV lysate-specific CD4 and CD8 cells, EBV lysate-specific CD4+IFNγ+ cells and CD8+IFNγ+ cells, and total EBNA3-specific CD4 cells, EBNA3-specific CD4+IFNγ+ cells, CD4+IL2+ cells, CD4+IFNγ+TNFα+IL2+ cells and CD8+IFNγ+ cells.

Conclusion:
De novo chronic GvHD and its treatment do not adversely affect the counts of functional EBV specific T cells.

Disclosures:
No relevant conflicts of interest to declare.