Towards Functional Immune Monitoring in Allogeneic Hematopoietic Stem Cell Transpant Recipients

Introduction: Serious viral infections that occur as a consequence of poor or delayed endogenous immune reconstitution are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). In the post HSCT setting immune reconstitution is currently evaluated by phenotypic profiling to assess lymphocytes subset recovery over time. However, these studies fail to assess either the functional capacity or antigen specificity of the reconstituting cells. We hypothesized that utilizing functional assays in combination with phenotypic assessments would enable the rapid and accurate risk-stratification of allogeneic HSCT recipients into those whose endogenous immunity was sufficient to deal with viral challenge versus those requiring clinical intervention with targeted antiviral therapies.

Methods: To evaluate phenotypic and functional immune response post-HSCT, we performed a prospective study in pediatric HSCT recipients where we monitored the frequency (by flow cytometry) and function of T cells directed against immunogenic antigens for the most common viruses detected post-transplant (CMV, EBV, HHV-6, Adenovirus, BK virus) using IFNg ELIspot assays. Assessments were performed at least monthly for up to 6 months post-transplant and weekly in patients who developed a viral reactivation post-HSCT.

Results: To date we have enrolled 23 allogeneic HSCT recipients, ages 3 to 16 years. Of these, 9 received a matched related donor graft (MRD), 13 received a matched/mismatched unrelated donor graft (MUD/MMUD) and 1 patient received a cord blood (CB) donor graft. All patients underwent myeloablative conditioning for a spectrum of underlying malignant (ALL - 10, AML - 4, MDS - 2) and non-malignant diagnoses (Hemoglobinopathy - 3, SAA/Marrow failure - 4, immune deficiency - 1). Based on donor type used for transplant and underlying diagnosis, 15 of these patients received T-cell depleting conditioning regimens using Alemtuzumab for in-vivo T-cell depletion while 8 patients did not receive T-cell depletion as part of their conditioning regimen. In patients who received a non-T-cell depleting conditioning regimen immune reconstitution was rapid with recovery to a median of 524 CD3+ T cells counts/µL by day 30 post-transplant (range 333-1711) [months 2, 3, 4, 5, 6 of 1090, 1382, 2184, 2077, 2481 cells/µL, respectively; n=8]. In contrast, for patients that received T-cell depleting conditioning regimens, reconstitution was significantly slower with median CD3 T-cell counts/µL of 0 (range: 0-249) by day 30 and recovery to 500 cells/µL occurring on or after month 5 post-transplant (months 2, 3, 4, 5, 6 of 19, 138, 147, 516, 836 cells/µL, respectively; n=15). In total, we detected 32 episodes of viral infection/reactivation [CMV (n=12), EBV (n=10), HHV-6 (n=5), Adenovirus (n=3) and BK (n=2)]. Twenty four occurred in our T-cell depleted transplant recipients, while 8 in non-T-cell depleted transplant recipients. By measuring functional virus-specific T cell recovery by IFNg ELIspot assays, we were able to identify infections that could be endogenously controlled (n=23) and those that could not (n=9). This allowed for rapid intervention with additional antiviral agents in patients that could not control their infections. Of these, 3 received 'off the shelf' CMV-specific T cells to treat persistent and drug refractory CMV viremia.

Conclusion: Screening for virus-specific T-cell responses with IFNg ELIspot assays allowed us to risk-stratify and tailor therapy for 23 pediatric HSCT recipients with viral infection/reactivation. Our encouraging results warrant further exploration in a larger cohort of patients.