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S. Vasileiou et al.
Table 1. Reactivity of expanded virus-specific T cells lines against individual stimulating antigens.
Functional characterization of multi-respiratory virus-targeted cells
The production of multiple proinflammatory cytokines and expression of effector molecules has been shown to correlate with enhanced cytolytic function and improved in vivo T-cell activity. Hence, we next examined the cytokine profile of our multi-R-VST following antigen exposure. The majority of IFNγ-producing cells also pro- duced TNFα (see Figure 3A for detailed ICS results from 1 donor, and Figure 3B, for summary results for 9 donors), in addition to GM-CSF, as measured by Luminex array (Figure 3C, left panel) with baseline levels of prototypic Th2/suppressive cytokines (Figure 3C, right panel). Furthermore, upon antigenic stimulation our cells pro- duced the effector molecule Granzyme B, suggesting the cytolytic potential of these expanded cells (Figure 3D, n=9). Taken together, these data demonstrate the Th1- polarized and polyfunctional characteristics of our multi- R-VST.
Multi-respiratory virus-targeted cells are cytolytic and kill virus-loaded targets
To investigate the cytolytic potential of these expanded cells in vitro, we co-cultured multi-R-VST with autologous Cr51-labeled PHA blasts, which were loaded with viral pepmixes with unloaded PHA blasts serving as a control. Viral antigen-loaded targets were specifically recognized and lysed by our expanded multi-R-VST (40:1 E:T - Influenza: 13±5%, RSV: 36±8%, hMPV: 26±7%, PIV-3: 22±5%, n=8) (Figure 4A and Online Supplementary Figure S6). Finally, even though these VST had received only a single stimulation, there was no evidence of activity against non-infected autologous targets nor of alloreactiv- ity (graft-versus-host potential) using HLA-mismatched PHA blasts as targets (Figure 4B), an important considera- tion if these cells are to be administered to allogeneic HSCT recipients.
Detection of CARV-specific T cells in hematopoietic stem cell transplant recipients
Finally, to assess the potential clinical relevance of
multi-R-VST we investigated whether allogeneic HSCT recipients with active/recent CARV infections exhibited elevated levels of reactive T cells during/following an active viral episode. Figure 5A shows the results of Patient #1, a 64-year old male with acute myeloid leukemia (AML) who received a matched related donor (MRD) transplant with reduced intensity conditioning. The patient developed a severe upper respiratory tract infec- tion (URTI) nine months post-HSCT that was confirmed to be RSV-related by polymerase chain reaction (PCR) analysis. He was not on any immunosuppression at the time of infection but was placed on prednisone the day of infection diagnosis to control pulmonary inflammation. Within four weeks his symptoms resolved without spe- cific antiviral treatment. To assess whether T-cell immu- nity contributed to viral clearance, we analyzed the circu- lating frequency of RSV-specific T cells over the course of his infection. Immediately prior to infection this patient exhibited a very weak response to the RSV antigens N and F (6.5 SFC/5x105 PBMC). However, within a month of viral exposure, RSV-specific T cells had expanded in vivo (527 SFC/5x105 PBMC), representing an 81-fold increase in reactive cells (Figure 5A) which declined thereafter, coincident with viral clearance. Of note, the observed RSV-specific responses did not follow the over- all increase in lymphocyte/CD4+ counts, thus indicating that T-cell expansion was virus-driven and not due to general immune reconstitution. Similarly, Patient #2, a 23- year old male with acute lymphoblastic leukemia (ALL) who received a matched unrelated donor (MUD) trans- plant with myeloablative conditioning, and developed a severe RSV-related URTI five months post HSCT while on tapering doses of tacrolimus. His infection sympto- matically resolved within one week, coincident with the administration of ribavirin. To investigate whether endogenous immunity also played a role in viral clearance we monitored reactive T-cell numbers over time. Viral clearance was accompanied by an increase in the circulat- ing frequency of RSV-specific T cells (peak 93 SFC/5x105 PBMC) with subsequent return to baseline levels (Figure 5B). The same patient was hospitalized seven months
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haematologica | 2020; 105(1)