Publication Only: Stem cell transplantation - Clinical
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.
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.
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° 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.
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.
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.