Senescent CD8+ T Cells in Myeloma Patients: Implications for Cellular Therapies
Authors: Lee Lisa Xingguang , Toskic Denis , Ma Xun , Zhou Ping , Kugelmass Adin , Fogaren Teresa , Comenzo Raymond L 
Published: October 2019

Abstract

Background: For autologous engineered T cell therapies such as CAR-T cells, the characteristics of the input donor-derived T cells impact the final T cell product. Early less-experienced T cells are thought to generate a T cell product with optimal functions whereas, terminally differentiated T cells, displaying features of cellular senescence, are unlikely to generate an ineffective product. An increase in senescence within the CD8+ T cell population has been described in multiple myeloma (MM) (Leukemia 2016;30:1716). To better understand the challenges that may be encountered in the production of engineered T cells in MM, we evaluated the overall T cell frequency and features of senescence in the CD8+ T cells in the peripheral blood of 14 MM patients. For those patients who underwent stem cell mobilization (n = 11) we assessed the impact of this procedure on the characteristics of the T cells collected.
Methods: Peripheral blood samples were collected during treatment from patients in steady-state. Patients who subsequently underwent stem cell mobilization (G-CSF/plerixafor or Cyclophosphamide) as part of their clinical care and provided a second blood sample on the day of stem cell collection (mobilized sample).
PBMCs were isolated and characterized by flow cytometry using the BD ACCURI C6 flow cytometer. Staining of surface antigens was performed by using fluorescently labeled CD3, CD8, CD28, CD57, and KLRG. Senescent T cells were defined as CD3+/CD8+CD28-/CD57+, and also as CD28-/KLRG+. Statistical analyses were performed with MedCalc.
Summary of Results: Steady-state samples were collected from 14 MM patients (10M, 4F) with a median age of 66.5 years (range, 52-82) and a median time from diagnosis of 7.5 months (3-72). All patients had been treated with a proteasome inhibitor, an IMiD, and dexamethasone, and 4 were receiving daratumumab. At the time of sample collection, disease response was variable with 5 PR, 7 VPGR, 1 CR, and 1 refractory disease.
In the steady state PBMC, CD3+ T cells were 49.1% and CD3+/CD8+ 5.1%. Median numbers of CD28-/CD57+ and CD28-/KLRG+ T cells within the CD8+ population were 24.1% (2.5-74.4) and 31% (2.9-73%) respectively. Two subgroups of MM patients were observed. Four patients had a significantly increased frequency of senescence within the CD8+ T cells (53-74% CD28-) and 10 had a much lower frequency of senescence (2.5%-25.9%). The group with elevated senescence within the CD8+ T cells included the oldest patient in the cohort (82 years), the patient with the longest duration of disease (6 years), a heavily pre-treated patient on daratumumab with severe lymphopenia, and a patient whose myeloma was a post-transplant lymphoproliferative disorder occurring a year after heart transplant on chronic immunosuppression with tacrolimus and prednisone.
Following mobilization, the percentage of T cells within the PBMCs dropped significantly from 49.1% to 8.3%. Mobilization particularly impacted the CD8+ T cell population which dropped from a median 5.4% of PBMCs in the steady-state sample to 0.8%. In the paired samples, we did not observe a consistent impact on frequency of CD28- or CD57+ (senescent) cells within the CD8+ T cells (19.1% vs 21.1%).
Conclusions: A subset of MM patients exhibited a significant increase in markers of senescence within the peripheral CD8+ T cells. Older patients, those on chronic immunosuppression, and those treated for an extended time appeared to be at increased risk. T cells from such patients may be suboptimal for manufacture of engineered T cell therapies. Further, stem cell mobilization resulted in a significant decrease in T cell frequency, particularly in CD8+ T cells although senescence markers were not consistently changed. Thus, mobilized blood collections may not be optimal starting material either.

Disclosures
Lee: Unum Therapeutics: Research Funding. Ma:Tufts Medical Center: Patents & Royalties: Patent: 9593332.