ALBERT

Description: Acute GVHD (aGVHD) remains a major cause of morbidity in patients who undergo allogeneic hematopoietic stem cell transplantation (HSCT). Although numerous studies have investigated potential immunologic parameters that could predict for aGVHD, there are no validated immune biomarkers presently used in clinical practice. We are currently performing a prospective analysis of immune reconstitution after allogeneic HSCT in different patient cohorts. The goal of the present study is to detect abnormalities of immune reconstitution associated with an inability to maintain T cell tolerance after allogeneic HSCT and to identify specific immunologic biomarkers predictive of aGVHD. We have thus far accrued 36 unrelated HSCT recipients with a median age of 48 years old (16 females; 20 males). The degree of HLA-matching at the allele level between patients and donors was: 10/10 (n=20), 9/10 (n=15), 8/10 (n=1). Graft source consisted of unmanipulated bone marrow (n=18) or peripheral blood stem cells (n=18). All patients received a reduced-intensity conditioning regimen with fludarabine, melphalan and thymoglobulin (dose dependent on the degree of HLA matching), in association with cyclosporine and mycophenolate mofetil for GVHD prophylaxis. A total of 21 patients (58%) developed grades II-IV aGVHD in the first six months post-transplant. Whole blood and serum samples were collected from HSCT recipients 1, 2, 3 and 6 months post-HSCT. To assess the impact of different immune markers on the occurrence of aGVHD, patients were categorized into 2 groups based on the development or not of grade II-IV aGVHD in the first six months post-HSCT. Our comprehensive 7-colour flow cytometry panel encompasses phenotypic markers for relevant T, B, NK, NKT and dendritic cell subsets, further including intracellular markers for the assessment of proliferation and susceptibility to apoptosis within regulatory (Treg), conventional (Tcon) CD4 T cells and CD8 T cell subsets. Serum concentrations of IL-2, IL-7, IL-10, IL-15, IFN-γ and TNF-α were evaluated with a multiplex cytokine assay. Absolute counts of lymphocytes, total T cells, CD4 Tcon, CD4 Treg and CD8 T cells display similar reconstitution patterns in unrelated HSCT recipients with and without aGVHD. However, we have observed a clear trend for higher levels of γδ T cells, both in terms of frequency and absolute counts, in patients who develop aGVHD, particularly at 1 month post-HSCT (Mann Whitney test, P = 0.0699). Furthermore, we found a significantly higher frequency of proliferating cells, as assessed by Ki67 expression, within the CD4 T cell population in patients who do not develop aGVHD, both at 2 and 3 months (Mann Whitney test, P = 0.0400 and P = 0.0177, respectively) post-HSCT. When dissected into proliferating CD4 Tcon and Treg, the frequency of both Ki67+ Tcon and Ki67+ Treg within the CD4 T cell population is higher in patients who do not develop aGVHD in all time-points analysed, reaching statistical significance at months 2 and 3 for Tcon (Mann Whitney test: P = 0.0280 and P = 0.0051, respectively) and at month 6 for Treg (Mann Whitney test, P = 0.0043). We are also quantifying the serum levels of relevant pro-inflammatory, suppressive and homeostatic cytokines in the two patient groups. We have observed that IL-7 serum levels (pg/ml) in the first 3 months post-transplant are significantly lower in patients who develop aGVHD compared to those who do not (Mann Whitney test: Month 1 P = 0.0051; Month 2 P = 0.0177; Month 3 P = 0.0303), whereas IL-10 levels are significantly higher in aGVHD patients (Mann Whitney test: Month 1 P = 0.0442; Month 2 P = 0.0051; Month 3 P = 0.0480). None of the other cytokines analyzed has revealed significant differences between the two groups of patients, although there seems to be a trend for higher IL-15 serum levels in patients who develop aGVHD, particularly in the first two months post-HSCT. These results indicate that in our patient population sensitive methods for the detection of IL-7 and IL-10 serum levels can identify patients at high risk for grades II-IV aGVHD early post-transplant. The accrual of more patients will clarify whether the levels of γδ T cells post-transplant, as well as the frequency of proliferating CD4 Tcon and Treg populations, can also be used as an early indicator for the development of aGVHD. Disclosures No relevant conflicts of interest to declare.

Description: Chronic Graft versus Host disease (cGVHD) is a major limiting factor for the success of allo-HSCT. In this prospective study we aimed to evaluate the association between the kinetics of Regulatory T cells (TREG) and Conventional CD4+T cells (TCON) reconstitution and the emergence of cGVHD. We performed a detailed phenotypic analysis by multiparametric flow cytometry using fresh blood from 39 patients undergoing unrelated donor HSCT after a reduced intensity conditioning regimen containing ATG over a 2 year period, representing a total of 213 samples analyzed. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil. 11 patients were excluded due to disease relapse and/or death due to infection or acute GVHD in the first 9 months post-HSCT. We observed indiscriminately low numbers of TREG (CD4+CD127lowFoxp3+CD25bright) until mo 6 after HSCT and reduced TREG numbers in patients developing cGVHD (GVHD+) versus those who did not (GVHD-) (p=0.02 at mo 9). We further studied the dynamics of TREG subset reconstitution. CM TREG (CD45RA-CD62L+) was the predominant population in both patient groups. EM (CD45RA-CD62L-) was the second most abundant TREG subset. CM and EM TREG numbers were similar between patient groups. EMRA (CD45RA+CD62L-) TREG remained very low throughout the follow-up but were significantly increased at mo 9 in GVHD+ (p= 0.03). Interestingly, Naïve TREG (CD45RA+CD62L+CD95-) started to emerge at mo 9 and were significantly reduced in GVHD+ patients at mo 12 (0.71 vs 0.14 cells/µl; p=0.02) The Stem Cell Memory subset (SCM), identified as CD45RA+CD62L+CD95+, is thought to be self renewing and multipotent, being able to differentiate into CM, EM and TEMRA memory subsets. While in GVHD- SCM TREG started to emerge at mo 9, this subset remained low in GVHD+. Statistically significant differences were observed at mo 18 (0.91 vs 0.15 cells/µl; p=0.02). To ascertain the role of thymic output in TREG reconstitution we quantified CD31+ naïve TREG. Recent Thymic Emigrant cells (RTE) TREG were significantly reduced in GVHD+ at mo 12 (0.8 vs 0.16cells/µl; p=0.02) and at mo 18 (1.93 vs 0.28 cells/µl; p=0.02). In order to clarify whether both thymic output and peripheral expansion were contributing factors to decreased Naïve TREG, we quantified proliferation using Ki-67. TREG from GVHD+ patients proliferated less from months 2 to 18 reaching statistical significance at mo 9 and 12 (p= 0.003 and 0.02, respectively), suggesting that decreased TREG numbers are at least partly due to reduced peripheral expansion. Taken together, these observations suggest a compromised peripheral expansion and de novo generation of TREG through thymic output in cGVHD patients. Of note, susceptibility to apoptosis was not increased in TREG from GVHD+ patients, as Bcl-2 levels tended to be higher in relation to GVHD- patients. In the CD4+Foxp3- TCON population, we observed a clear predominance of EM in all patients. These cells, together with CM, are the first to appear at similar levels in both patient groups. EMRA emerged at higher numbers in GVHD+, although the results did not reach statistical significance. Naïve and RTE TCON started to emerge after mo 6 in GVHD-. Interestingly, these cells remained much lower in GVHD+ vs GVHD- patients throughout the follow-up, reaching statistical significance at mo 12 (p=0.03 for naïve; p=0.03 for RTE TCON). SCM TCON remained very low in all patients showing a tendency to be reduced in GVHD+ when compared to GVHD-. This difference reached statistical significance at mo 18 (6.85 vs 2.13 cells/µl; p=0.02). The small number of TREG and TCON subsets in GVHD+ patients were unlikely due to increased susceptibility to apoptosis, as assessed by Bcl-2 expression. The low numbers of cells within naïve and SCM TREG gates precluded the analysis of Bcl-2 expression in these subsets. Bcl-2 tended to be increased in total TREG CM and EM in GVHD+. In TCONs, Bcl-2 levels were similar in both patient groups. In summary, our data in patients developing cGVHD suggest that decreased thymic output and increased differentiation into terminally differentiated effector cells may have a negative impact on the number of naïve and SCM TCON and TREG. We speculate that the inability to generate and maintain the more immature TREG subsets may lead to decreased TREG numbers after 6 months post transplant, potentially resulting in decreased control of effector T cells contributing to the development of cGVHD. Disclosures Ritz: Kiadis: Membership on an entity's Board of Directors or advisory committees.