Evaluation of T Cell and Monocyte Immune Subsets in the Stem Cell Product of Patients Developing Engraftment Syndrome Following Autologous Stem Cell Transplantation for Multiple Myeloma and Lymphoma
Authors: Descalzi-Montoya Dante B. , Yang Zheng , Goldgirsh Kira , Feinman Rena , Siegel David S , Biran Noa , Vesole David H. , Goy Andre , Feldman Tatyana A. , Leslie Lori A. , Rowley Scott D. , Skarbnik Alan P , McKiernan Phyllis , Baker Melissa , Donato Michele L. , Korngold Robert 
Published: October 2019

Abstract
BACKGROUND: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is now standard of care for newly diagnosed patients with multiple myeloma (MM) and is used for some forms of non-Hodgkin lymphoma, providing improved outcomes. ASCT has been associated with a high incidence of engraftment syndrome (ES), which clinically presents as skin rashes, diarrhea, non-infectious fevers, and capillary leak syndrome in the peri-engraftment period. ES can be a severe and potentially lethal complication in patients who do not respond to corticosteroid therapy.
RATIONALE: Prior to stem cell collection, MM and lymphoma patients are typically exposed to chemotherapy and immunomodulatory agents in order to reduce the cancer cell burden and other drugs to mobilize stem cells. It has been hypothesized that these agents may be involved with ES development in that they can disrupt the balance of immune regulatory and effector cell subsets.
OBJECTIVE: To analyze T cell activation/memory and Treg cell compartments as well as classical, intermediate, and non-classical monocytes in the autologous apheresis product of MM and lymphoma patients undergoing ASCT.
STUDY DESIGN: Samples were collected under IRB approval from 28 patients undergoing ASCT for the treatment of MM (n=21) or lymphoma (n=7) at the time of peripheral blood stem cell collection (the apheresis product); Peripheral blood mononuclear cells (PBMC) were isolated by sucrose gradient centrifugation and frozen until needed for phenotyping by multi-parametric flow cytometry. MM patients were conditioned for ASCT with melphalan alone (n=14) or in combination with bortezomib (n=7); lymphoma patients received conditioning with BEAM (carmustine, etoposide, cytarabine, and melphalan) chemotherapy.
RESULTS: Of the 28 patients undergoing ASCT, 10 (35.7%) developed ES within the first four weeks post-transplantation. We analyzed by flow cytometric phenotyping the following immune cell subsets: overall CD4/CD8 T cells, CD4/CD8 naïve and memory cells, HLA-DR expression on memory T Cells, memory CD4 Tregs, as well as overall classical, intermediate, and non-classical monocytes. The absolute numbers (PBMC conc. (cell/ml of blood) x (% of cells in PBMC/100) and percentage population (# of events in population gate/# of total PBMC events x 100) data were transformed with log and square root functions, respectively. All data were tested for normality with a Shapiro-Wilks online test and p-values were obtained by performing an unpaired Student T-test. From our analysis of the apheresis product, the main cell compartments that were significantly increased in the ES+ group were the % CD8+ T cells [5.41 mean ± 0.51 s.e. vs. 3.34 ± 0.36 (ES-), p=0.003] and naïve CD8+ T cells [3.76 ± 0.56 vs. 1.61 ± 0.18 (ES-), p<0.001; In addition, although the memory CD8 T cell subset was not significantly increased in the apheresis product, the % of those memory CD8 T cells expressing HLA-DR significantly increased [2.0 ± 0.22 (ES+) vs. 1.2 ± 0.15 (ES-); p<0.01], suggesting an increase in CD8 T cell activation in the ES+ group. No major differences were observed in the CD4 memory Treg compartment or CD25 expression on CD4 T cells.
In the monocyte compartment, the main subset that was significantly increased was the non-classical CD16+CD14low cells in the ES+ group [0.40 ± 0.08 vs. 0.25 ± 0.03 (ES-), p=0.02]. In addition, the % of CD25+ and CD163+ non-classical and intermediate monocytes were favorably increased in the ES+ group (non-classical monocytes, CD25+ [0.14 ± 0.03 vs. 0.07 ± 0.02 (ES-), p=0.03] and CD163+ [0.33 ± 0.06 vs. 0.18 ± 0.02 (ES-), p=0.03]; and for intermediate monocytes, CD25+ [0.44 ± 0.1 vs. 0.22 ± 0.04 (ES-), p=0.02] and CD163+ (p=0.02). No major differences were observed between ES+ and ES- groups for CD64 and PDL-1 expression.
CONCLUSIONS: The development of ES correlated with the observation of a significant increase of naïve and activated CD8 T cells in the autologous apheresis product. In contrast, no significant differences were found between the ES+ and ES- groups in the CD4 T cell or memory Treg subsets, suggesting that they do not contribute to the etiology of the syndrome. Moreover, an increased presence of non-classical monocytes with higher expression of both CD163 and CD25 was found, suggesting increased potential for both M2 and M1 activity. Further investigation is needed to determine the implications of these findings for the development of ES.

Disclosures
Siegel: Merck: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Novartis: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Biran:BMS: Research Funding; Merck: Research Funding; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Feldman:Johnson and Johnson: Speakers Bureau; KITE: Speakers Bureau; Seattle Genetics: Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau; Janssen: Speakers Bureau; Portola: Research Funding; Celgene: Speakers Bureau. Skarbnik:Gilead Sciences: Honoraria, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.