Detection and Ex Vivo Expansion of Anti-Viral T Cells Isolated From Recipients of Unrelated Umbilical Cord Blood Transplant.

Viral infection is commonly detected within the first weeks after umbilical cord blood transplant (UCBT), likely triggering T cell activation and in vivo priming of infused naïve T cells. This hypothesis is supported by the observation that despite the continuous exposure to immunosuppressive (IS) drugs, viremia may resolve even in the absence of antiviral agents. However, IS may delay or even prevent effective T cell priming and the development of protective immunity, therefore causing significant morbidity and mortality.

Our laboratory has a long standing interest in defining pre-requisites of protective immunity after UCBT. In this study we focused on two of the most common potentially fatal viruses, CMV and Adenovirus to assess antigen-specific immune reconstitution. We hypothesized that 1) threshold numbers of CTL precursors can be identified for each virus that will impact clinical outcome 2) there may be circulating CTL precursors even if below the level of detection in lymphopenic patients that receive IS drugs, however, ex vivo restimulation could lead to expansion and subsequent quantitation.

We have established in vitro assays to test the impact of cytokines and new culture techniques to promote anti-viral precursor survival and expansion. Monocytes in bulk cultures, infected with replication incompetent adenovirus vectors encoding the pp65 CMV matrix protein (Ad5f35pp65), served as APC. Antiviral responses were quantitated by ELISPOT, utilizing computerized enumeration of interferon gamma (IFNg) producing spot forming cells (SFC) in response to overlapping peptide spanning immunodominant viral antigens. Hexon and penton for adenovirus, and pp65 and EI-1 for CMV.

There were 8 patients enrolled on this IRB approved study transplanted at a median age of 9.9 years (range 2-17). Blood samples was obtained at a median of 70 days (range 34-470). Only 1 of 3 patients with adenovirus infection ( stool x1, urine x1, respiratory tract x 1) had previous viremia while all five (5) patients with CMV had viremia. Only 1 of the 3 adeno patients had detectable SFC from freshly drawn peripheral blood, 11 SFC/1×10e5 cells against hexon and penton respectively. None of the 5 CMV+ patients had detectable pp65 or EI-1-specific T cell responses from freshly drawn blood. However, after 9-12 days of ex vivo culture, in the presence of IL7 (10ng/ml), we were able to enumerate significantly amplified CTL responses in 7 of the 8 patients, with one CMV viremic the exception. In the remaining patients the median adenovirus hexon-specific response was 52 SFC/1×10e5 cells (range 20-90). the median penton-specific CTLp frequency was 13 SFC/1×10e5 (range 1 - 25). The median CMV pp65-specific response was 6.7 SFC/1×10e5 (range 0-14) while no responses were recorded against EI-1 demonstrating the critical role of antigenic restimulation provided by the Ad5f35pp65 construct lacking IE. The amplified immune responses were highly restricted; in the CMV+ patients (all adeno-) we were able to detect SFC only against pp65 and to neither IE or penton/hexon, while in the adenovirus+/CMV- patients (n=-3) we were able to detect SFC solely in those wells that were stimulated by hexon and penton peptides. All patients have survived at a median of 220 days after transplant (range 81-532).

To our knowledge these ongoing experiments are the first to demonstrate that dormant virus-specific immune responses are present already in the first 100 days in most UCBT recipients (7/8 in this dataset) if infected with either adenovirus or CMV despite failure to detect these extremely rare events by standard ELISPOT assays from freshly isolated blood. Moreover, these data suggests that anti-viral CTLp can be expanded ex vivo by antigen specific restimulation that lends support to further efforts aimed at developing adoptive anti-viral T cell therapies.

No relevant conflicts of interest to declare.