High Expression of Granzymes and Perforin along with Increased T cell Cycling In Vivo Predicts the Development of Opportunistic Infections (OI) Following Unrelated Cord Blood Transplantation (UCBT).
Authors: Szabolcs Paul , Lee Young-Ah , Marti Luciana , Reese Melissa , Kurtzberg Joanne 
Published: October 2019

Introduction: Unrelated umbilical cord blood transplantation (UUCBT) is a viable option for those who lack HLA-matched sibling donors. However, opportunistic infections (OI) occurring in the first 100 days, remain the major cause of morbidity and mortality. Viral infections are the primary cause of OI death. As previously shown, cord blood T cells have significantly less preformed effector molecules available intracellularly to kill virally infected cells via the perforin-granzyme pathway than adult PB T cells. Since several virally infected patients control their infections in the absence of specific antiviral therapy ( e.g adenoviral enteris, polyoma cystitis) we postulated that the T cell compartment of those UUCBT recipients who experience early viral infections maty upregulate expression of the perforin exocytosis pathway. In parallel the impact of viral infection on T cell turnover would also be appreciable. Here we report on 19 prospectively studied pediatric patients, all full donor chimera, following myeloablative therapy.

Methods: On day +50 we determined by 4-color FACS the expression of intracellular Granzyme, A, B, along with perforin. To monitor T cell turnover proliferating cells were identified by monitoring for the KI-67 nuclear antigen. The expression of the antiapoptosis gene BCL-2 was similarly monitored in both CD4+ and CD8+ T cells. We analyzed their association with the development of de novo OI up to day +100 employing Student’s t-test.

Results: Mean age of patients was 6.2 years. 10 of 19 patients developed OI (adenovirus x 4, CMV x 7, EBVx1, parainfluenza x 1) with 5/10 patients experiencing more than one viral infections simultaneously) at a median of 29 days after UUCBT. Of those with OI 6/10 died due to their infections while 8/9 without OI are alive at a median of 15.8 months after UUCBT with one death due to leukemic relapse. Table I presents the correlation between the tested parameters with the development of OI. Patients experiencing viral infections had significantly higher % of their T cells in particular CD8+ T cells equipped with effectors of cytotoxicity and were proliferating in higher percentage compared to those with no active infections. However, the anti-apoptotic protein BCL-2 expression was significantly lower in patients experiencing OI that may lead to their shorter life span and overall T cell lymphopenia observed in OI patietns that we have previously detected in a larger cohort of 102 patients (ASBMT 2004 abstract#48). Conclusion: Correlating with active viral infections significant maturation of cord blood T cells is evident as early as 50 days after UUCBT towards acquiring effector molecules of the perforin pathway. Enhanced T cell proliferation is counteracted by reduced expression of BCL-2 that may lead to the lymphopenia in patients with OI. Future strategies aiming to enhance the longevity of antiviral T cells may protect from death due to viral infections.

Univariate analysis

VARIABLE MEDIAN VALUE FOR PATIENTS WITH OI MEDIAN VALUE FOR PATIENTS WITHOUT OI t-Test p value % Granzyme A+ T cells 52% 9% 0.006 % Granzyme A+ CD8+ T cells 91% 47% <0.001 % Granzyme B+ T cells 36% 6% 0.036 % Granzyme B+ T cells 87% 39% <0.001 % Perforin+ T cells 38% 4% 0.009 % Perforin+ CD8+ T cells 61% 21% <0.001 % Ki-67+ T cells 27% 16% 0.0041 % Ki-67+ CD8+ T cells 35% 16% 0.0037 BCL-2 expression level (MFI) 87 117 0.028