CMV Specific T Cell Polyfunctionality after HCT: A Detailed Analysis of the Impact of Steroid Exposure, Immunosuppressive Regimens and Viral Reactivation

Introduction

A detailed understanding of factors impacting CMV-specific T-cell polyfunctionality after allogeneic hematopoietic cell transplantation (HCT) is critical for rational study design and, ultimately, the development of effective T-cell therapies. Clinical trials of pathogen-specific T-cells have excluded patients who received glucocorticoids (GC) at different dose levels or time frames prior to enrollment, based largely on data from earlier studies. Also, limited data exist on the impact of other immunosuppressive drugs and conditions. Here we comprehensively assessed the impact of GC exposure (dose, timing), mycophenolate mofetil (MMF), posttransplant cyclophosphamide (PTCy), and CMV reactivation on CMV T-cell responses after HCT.

Methods

Flow cytometry was used to assess T-cell polyfunctionality 90 days after HCT following stimulation with CMV pp65 antigen in seropositive HCT recipients. T-cell depletion was not commonly used in this population. Polyfunctional CMV specific T-cell subsets were defined as those expressing IFN-ϒ plus ≥1 functional marker (CD107a, TNFα or IL-2) with the frequencies greater than 0.05% after background subtraction and at least threefold greater than DMSO response at the same cell population. A clinically significant CMV-specific T response was defined as an absolute polyfunctional cell count of ≥0.5 cell/µL. Data on the use of MMF and GC on the day of sample collection and within 2 and 4 weeks of collection were obtained from medical records. Prednisone-equivalent doses were categorized as follows: low (>0 - <0.5 mg/kg) intermediate (≥0.5-<1 mg/kg), or high (≥1 mg/kg). Multivariable (MV) logistic and linear regression models were used to analyze associations with clinically significant responses and polyfunctionality levels, respectively.

Results

Day-90 samples from 243 CMV seropositive HCT recipients (83% peripheral blood stem cells, 12% bone marrow, 5% cord blood) were analyzed. Antiviral prophylaxis for CMV was used in all recipients of cord blood grafts (high-dose valacyclovir) and those transplanted after 2018 (letermovir, 21%); all patients underwent weekly CMV PCR monitoring. At the time of day-90 sample collection, 22% received MMF and the proportions of patients treated with low, intermediate and high doses of GC were 21%, 11%, 8%; and 26%, 15% and 19% within 2-4 weeks before day-90 sample collection. In univariable models, use of PTCy showed a trend towards reduced day-90 CMV specific T-cell responses. While low-dose GC did not affect the proportion and strength of CD4 and CD8 CMV-specific responses, treatment with intermediate and high doses of GC inhibited responses, especially in CD4 populations. Seventy-seven percent of the study population had CMV reactivation within the first 90 days after HCT, and both CD4 and CD8 CMV-specific responses were more robust in reactivators. The concomitant use of GC and MMF had enhanced suppressive effect on CMV-specific CD4 and CD8 polyfunctional T-cell responses. In multivariable logistic regression models including CMV viremia, MMF, GC and letermovir use, intermediate- and high-dose GC treatment decreased adjusted odds ratio (aOR) to develop CD4 or CD8 responses (aOR 0.28, 95% CI 0.09-0.86, p=0.02 and aOR 0.3, 0.1-0.95, p=0.04). CMV-specific CD8 responses were also significantly impaired by letermovir (aOR 0.41, 0.17-0.99, p=0.04) and MMF use (aOR 0.49, 0.23-1.02, p=0.06). CD8 responses were significantly more pronounced in patients with CMV reactivation (aOR 2.46, 1.02-5.91, p=0.04).In both MV linear and logistic regression models, there was greater impairment of responses when GC treatment (≥0.5mg/kg) was administered earlier, i.e. within 2-4 weeks before sample collection, with a higher loss of polyfunctional CD4 and CD8 cells (aOR -0.89 cells/ul [-1.59, -0.18, p=0.01] and aOR -2.55 cells/ul [-4.23, -8.85, p=0.003], respectively; logistic regression showed similar results, data not shown).

Conclusions

Among CMV seropositive HCT recipients, day-90 polyfunctional CMV-specific T-cell responses were observed despite treatment with low doses of GC or MMF. However, intermediate or high doses of GC, especially when given within 2-4 weeks before testing, and concomitant treatment with MMF had significant inhibitory effect on both CD4 and CD8 polyfunctional CMV-specific responses. These data will be critical to design effective T-cell therapy trials.