PB2328 CMV‐SPECIFIC T‐CELL RECONSTITUTION AS A KEY FACTOR FOR IDENTIFICATION OF CANDIDATES FOR CMV‐SPECIFIC LYMPHOCYTES INFUSION

Abstract

Background:Cytomegalovirus (CMV) is the opportunistic infection that persists in healthy individuals asymptomatically and reactivates in immunodeficient host, e.g. after allo‐HSCT. The main cause of viral reactivation is the lack of CMV‐specific T cells. The adoptive cellular therapy with CMV‐specific T cells seems very attractive clinical option to accelerate virus‐specific T‐cell reconstitution after allo‐HSCT. Here we report the preliminary data about CMV‐specific T‐cell reconstitution in allo‐HSCT patients.Aims:To identify a group of patients after allo‐HSCT who are in high‐risk of CMV reactivation and could be eligible for prophylaxis with CMV‐specific T cells.Methods:To detect CMV‐specific CD8+ cells we utilized MHC I tetramers loaded with the two immunodominant epitopes of viral pp65 protein: NLVPMVATV (NLV) and TPRVTGGGAM (TPR) presented in HLA‐A∗02 and ‐B∗07 respectively.We analyzed CD3+CD8+ cytotoxic T‐lymphocytes (CTLs) in peripheral blood and bone marrow of 7 patients on day +30 after allo‐HSCT. All patients were CMV‐seropositive. 5 patients were HLA‐A∗02 positive and 2 HLA‐B∗07 positive.Leukocyte cell suspension (5∗106 white blood cells per test) after preliminary red blood cells lysing with Lysing Buffer (BD Pharm Lyse™) were incubated with the protein tyrosine kinase inhibitor dasatinib for 1 hr at 37°С to increase surface expression of both TCR and CD8. NLV/TPR‐ specific cytotoxic T‐lymphocytes (CTLs) were identified by flow cytometry using PE‐Cy7 labeled anti‐CD45 antibody (Ab), Alexa Fluor 700 labeled anti‐CD3 Ab and PerCP‐Cy5.5 labeled anti‐CD8 Ab (Sony Biotechnology Inc.) and in house produced MHC class I tetramers conjugated with Phycoerythrin and loaded with NLV or TPR peptides. Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) was used (Invitrogen™) to exclude dead cells. Flow cytometry was performed on a BD FACSCanto™ II Flow Cytometer using BD FACSDiva™ software. Two‐platform method was used to calculate absolute CMV‐specific CD3+CD8+ count in peripheral blood.Results:According to our data, the lowest rates of CMV‐specific CTLs are presented in patients who underwent allo‐HSCT with post‐transplant high‐dose cyclophosphamide (PT‐Cy, 50 mg/kg on +3, +4 day) as GVHD prophylaxis.Results are presented in Table 1.Summary/Conclusion:Clinical option to accelerate virus‐specific T‐cell reconstitution using adoptive cellular therapy with CMV‐specific T cells seems very attractive. Modern technologies make this possible but first we should identify the patients after allo‐HSCT who benefit from this effective but very expensive therapy. Patients with using PT‐Cy as GVHD prophylaxis are the possible candidates for this treatment. Further studies CMV‐specific T‐cell reconstitution will help to expand the group of patients in high risk.image