The Role of CMV Serostatus of the Donor in the Graft Lymphoid Composition and Prediction of Gvhd and CMV Reactivation
Authors: Kreutzman Anna , Portero Sainz Itxaso , Gomez Garcia De Soria Valle , Fernandez Carlos , Mercedes Royg , Marcos Ana , Colom Fernandez Beatriz , Ramirez Ana , Cardeñoso Laura , De La Camara Rafael , Muñoz Calleja Cecilia 
Published: November 2019

Abstract
Background. Cytomegalovirus (CMV) is a widespread persistent β–herpesvirus, which can cause severe complications during primary infection or reactivation in immunocompromised patients, such as after allogeneic stem cell transplantation (alloSCT). Another major complication associated with alloSCT is graft-versus-host disease (GVHD). The pathogenesis of GVHD involves migration of the transplanted donor naïve T-cells into the secondary lymphoid organs in the recipient, which is mainly steered by CD62L and CCR7. As these homing molecules have been associated with both acute GVHD (aGVHD) and chronic GVHD (cGVHD), we studied whether the CMV serostatus of the donor affects the lymphoid composition of the graft product and whether this phenotype can predict CMV reactivation and GVHD.
Methods. This single-center study included 77 donor-recipient pairs who underwent alloSCT. 64 pairs were HLA identical, 12 had 1 mismatch and 3 had 2 mismatch. 36 donors were related to their recipients. All recipients were followed at least for 100 (aGVHD) or 360 days (cGVHD) after transplantation. 43 donors were CMV-seropositive (CMVpos) and 34 were CMV-seronegative (CMVneg). 62 recipients were CMVpos, and 32 of them developed CMV reactivation, 25 aGVHD and 30 cGVHD. Samples from the graft product (donor) were phenotyped by flow cytometry (CD45, CD3, CD8, CD4, CD62L, CCR7) and both frequency (freq) and absolute number (abs) of each T-cell subpopulation were analyzed.
Results. When the donors were divided based on their CMV serostatus, we observed that the grafts from CMVpos donors had a lower freq of naïve (CCR7+CD62L+) CD4+ T-cells (of lymphocytes p=0.06, of CD3 p=0.06, of CD4 p=0.07) and naïve CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.041, of CD3 p=0.011, of CD8 p=0.012) compared to CMVneg donors. Further, the abs of transplanted naïve CD8+ T-cells was significantly lower in the grafts from CMVpos donors (p=0.048). No differences were observed in T-cells (CD3+, CD4+, CD8+).
We next studied if the CMV-serostatus and T-cell phenotype of the graft associates with GVHD. CMVpos donors whose recipients developed aGVHD had higher abs (p=0.05) and freq of naïve CD8+ T-cells (of lymphocytes p=0.08, of CD3 p=0.08, of CD8 p=0.11) compared to those without aGVHD. The same trend was observed with abs (p=0.11) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.15). Similarly, those CMVpos donors whose recipients developed cGVHD had higher abs (p=0.05) and freq of CCR7+CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.06). Further, cGVHD patients who received the transplant from CMVpos donors were infused with a higher freq of CD3+ (of leukocytes p=0.03) and CD4+ T cells (of leukocytes p=0.04) than patients who received a graft from CMVpos donors but did not develop cGVHD. In contrast, CMVneg donors who developed aGVHD had a higher freq of CD3+ (p=0.018) and CD4+ T-cells (p=0.09), whereas no differences were seen in the T-cell subpopulations. Conversely, the abs (p=0.08) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.11) were higher in those who later developed cGVHD.
To study whether the graft lymphoid composition can be used to predict CMV reactivation, we analyzed the lymphoid composition in the graft product of those donors (both CMVpos and CMVneg) whose recipients were CMV seropositive but did not develop any form of GVHD (to avoid the influence of GVHD in the reactivation of CMV). Despite the low number of patients (CMV reactivation n=9, and no CMV reactivation n=13), we observed trends of higher portion of CD4+ T-cells (p=0.09 of lymphocytes, of CD3 p=0.20) and CCR7+CD4+ T-cells (of lymphocytes p=0.18, of CD4 p=0.16) in those grafts that were transplanted into CMV seropositive recipients who did not reactivate CMV.
Conclusions. CMVpos donors whose recipient developed either aGVHD or cGVHD had a higher abs and freq of naïve CD8+ T-cells, which was not seen with CMVneg donors. This suggests that seropositivity sets the abs and freq of CD8 subpopulations near to a decisive cutoff for the development of GVHD. Conversely, other factors influences the development of GVHD in those patients whose donors were seronegative. In other words, seropositivity of the donor affects the graft composition and thus the risk of GVHD. Finally, our data indicate that a higher proportion of naïve or central memory CCR7+ CD4+ T cells in the donor graft could prevent CMV reactivation suggesting that graft composition affects also CMV reactivation.

Disclosures
No relevant conflicts of interest to declare.