Increased NKT-like Cells and Markers of T Cell Activation in Long Term Survivors of Allogeneic Hematopoietic Cell Transplantation

Background: Both impaired immune reconstitution (IR) of selected cellular subsets and abnormally high lymphocyte expression of activation markers, such as HLA-DR, CD25, and CD69, have been observed in recipients of HCT experiencing graft-versus-host disease (GVHD). Whether such alterations in cell subsets and expression of activation markers occurs normally in long term survivors of allogeneic HCT, including those without any prior GVHD, has not been well described.

Patients and methods: 122 consecutive adult allogeneic HCT survivors who were at least 2 years beyond HCT and returning for long-term follow up were included if they had an immune reconstitution panel drawn between 9-14-11 (date of current panel antibody choice and gating strategy) and 9-5-13. Sixty-four were male, 52 underwent reduced intensity conditioning, 69 received grafts from matched unrelated donors (MUD), 60 received GVHD prophylaxis that included prednisone, 74 had prior acute GVHD, and 111 had chronic GVHD. Transplant factors considered in analyzing differences in IR and lymphocyte activation included age, sex, conditioning intensity, donor, GVHD prophylaxis, prior acute GVHD, and prior chronic GVHD. IR parameters analyzed included the absolute neutrophil, lymphocyte, monocyte, and eosinophil counts (ANC, ALC, AMC, AEC), platelet counts, and percentages of the following lymphocyte phenotypes: CD3+, CD3+/4+, CD3+/8+, double negative CD3, double positive CD4, naïve T, NK, NKT-like (CD3+56+), total B, naïve B, marginal zone-like B, switched memory B, CD4/8 ratio, and lymphocytes expressing activation markers, including CD3+HLA-DR+, CD4+25+, CD4+25bright+, CD8+CD69+.

Results: CD4/8 ratio was <1 in 54 patients (44%) in this cohort of long-term survivors. Several determinants of IR showed variability with respect to different transplant-related factors. Total CD4% and CD4%CD25bright+% but not absolute counts were increased in those receiving myeloablative conditioning. CD4% was also increased in those receiving grafts from HLA-matched siblings compared to MUD. Prednisone-based GVHD prophylaxis was associated with a decreased percentage of naïve B cells compared to others. Absolute monocyte count was increased in those with a history of acute GVHD. CD3+HLA-DR+% was increased in those with chronic GVHD. As dichotomized variables below or above the normal range, increases in NKT-like cells were observed in 63% of patients (median 3.1%, IQR 1.3 – 6.8%, typically 1-2% in healthy controls) and increased T cell subsets expressing activation markers, (e.g. increased CD25 in 43% and CD69 in 25% of patients), could be observed in even in those who had never experienced GVHD. B cells were negatively correlated (r= -0.44, p<0.001) and CD69-expressing CD8+ T cells positively correlated (r=0.33, p=0.005) with NKT-like cells. History of acute GVHD was associated with increased CD4+25bright+ T cells during long term follow up (increased in 10% of patients with a history of acute GVHD, compared to 1.6% of those without a history of acute GVHD, p=0.046), otherwise no significant differences in those who had acute or chronic GVHD versus those who never had GVHD were identified.

Conclusions: Over half of long-term allogeneic HCT survivors have an increased percentage of circulating NKT-like cells. In addition, many survivors, including those who have never had GVHD, have abnormally high expression of T cell activation markers. Despite most of this cohort exhibiting clinical tolerance with treated chronic GVHD, subclinical abnormalities in lymphocyte activation remained detectable. Further longitudinal studies are needed to determine whether these abnormalities predict future flares of chronic GVHD, susceptibility to infections, or other post-HCT complications.