Reconstitution of CASTs after alloHCT has previously been quantified using CMV tetramers. We developed an assay for quantifying CASTs using CMV specific MHC multimers (Dextramers, Immudex, Denmark). CMV Dextramers consist of dextran polymers conjugated to optimized numbers of MHC-CMV peptide and fluorochrome molecules allowing higher avidity binding to CMV specific T cells. Dextramers were restricted to HLA-A01, -A02, -A03, -A24 -B07, -B08, and -B35, and were informative in 93% of our

more »

... tly White (94%) population. Patient blood samples were prospectively collected pre-alloHCT and day +30, 100, and 365, and analyzed by flow cytometry for CASTs with CMV Dextramers. More than 300 CMV Dextramer measurements from 89 consecutive alloHCT patients treated from 2005 -2011 with . 1 post-alloHCT CAST measurement were analyzed. CMV reactivation was defined as the presence of CMV antigen on . 2/50,000 cells, . 1/100,000 cells twice consecutively, or . 1/100,000 cells followed by preemptive CMV therapy. Absence of CMV reactivation was defined as 0 cells or 1/100,000 cells positive for CMV no more than once with no anti-CMV treatment. 30/89 (34%) patients reactivated CMV a median of 40 days (range 23-341) post-alloHCT. None of 37 recipients with undetectable CASTs before allo-HCT reactivated CMV by day +30 vs. 10/52 (19%) of recipients with detectable CASTs (p 5 0.005), recapitulating CMV reactivation patterns seen in recipients with positive CMV serologies. We correlated the number of CASTs with recipient and donor CMV serology as a surrogate for anti-CMV immunity. In samples with CD3+ T cells $ 400/mL, CASTs correlated with recipient CMV serology with 67% sensitivity and 90% specificity (see Table) . Sensitivity may have been lower than expected due to decreased anti-CMV IgG production secondary to chemotherapy and/or increased granularity of flow cytometry measurements for CASTs. In another analysis, CASTs at day +30 corresponded to positive CMV donor, but not recipient, serology, and not with CMV reactivation up to day +30, suggesting that the adoptive transfer of CASTs occurs with alloHCT. Preliminary analyses confirm prior reports of decreased relapse of underlying cancers after CMV reactivation. Our study suggests that Dextramers may be used to monitor reconstitution of CMV immunity post-alloHCT, that they may be a more sensitive test than CMV serology, and that adoptive transfer of anti-CMV immunity can be quantified.