Tracking Immune Reconstitution in the Bone Marrow Following Allogeneic Hematopoietic Transplantation in Humans Reveals Early Dominance of Innate Lymphoid Populations

Introduction: T cell immune reconstitution is an important correlate with long-term successful outcomes after allogeneic hematopoietic cell transplantation (allo-HCT) for hematologic malignancies. However, most present knowledge of immune reconstitution in humans arises from analyses of peripheral blood (PB), even though the bone marrow (BM) compartment generally represents the tumor microenvironment in myeloid hematologic diseases. Furthermore, emerging studies suggest important roles for innate immune populations, such as natural killer (NK) cells and innate lymphoid cells (ILCs), in graft versus leukemia (GVL) responses and graft versus host disease (GVHD), and knowledge of their recovery in the BM is limited as well. We therefore sought to characterize the immunologic reconstitution of both adaptive and innate lymphoid populations in BM, a potentially critical site for alloimmunity following allo-HCT, and to compare this with their patterns in circulation.

Methods: We prospectively profiled serial BM aspirate (n = 102) and PB (n = 109) samples from pre-transplant (recipient) to one-year post-transplant in a cohort of 30 adults undergoing allo-HCT at Memorial Sloan Kettering Cancer Center between 2019 and 2021 for hematologic malignancies; n = 25 myeloid diseases (AML, MDS, CMML, MPAL) and n= 5 lymphoid with bone marrow involvement (ALL T-PLL, FL). Allograft sources included unmodified peripheral blood stem cells (PBSCs, n =13), T-cell depleted grafts (TCDs, n = 8), cord blood (n = 5), and haploidentical donors (n = 4). To optimize identification of low frequency populations such as ILCs in samples with low lymphocyte content early post-transplant, we performed multicolor flow cytometry on fresh samples. Analyses quantified the distribution of CD4, CD8, and regulatory (CD25⁺⁺CD127^-) T cells (CD3⁺), NK cells (CD56⁺), and ILCs (lineage^-CD127⁺CD161⁺; stratified by cKit, CRTH2, and NKp44 to define ILC1, ILC2, and ILC3 subsets) along with selected phenotypic markers (PD1, CD69, and CD25).

Results: We measured immune reconstitution of major lymphocyte subsets as a fraction of CD45⁺ cells in all profiled samples (Figure 1). An early burst of NK cells, similar to previous observations in PB, was found in both the BM and PB compartments, particularly in TCD recipients. In PB by day 100, T cell frequencies were comparable to those of NK cells, and T cells ultimately surpassed them as a greater proportion of PB lymphocytes (Day 365 p = 0.002, unpaired Mann Whitney). In contrast, in the BM at day 100, NK cells continued to outnumber T cells, and T and NK cell frequencies subsequently remained similar throughout the first-year post-transplant (Day 365: p = 0.3), underscoring the NK cell contribution to the BM lymphocyte compartment early post-transplant. Of note, T cell frequencies were more similar than anticipated in the BM of patients receiving PBSC and TCD allografts.

Analyses of both PB and BM indicated an increase in CD56^bright NKs cells at day 30 post-transplant compared to pre-transplant (PB mean 51.32%, SD 26.1 versus 15.3%, SD 17.5; BM mean 34.1%, SD 27.5 versus 12.4%, SD 7.6), consistent with a shift in both circulating and tissue-derived NK populations post-transplant. Furthermore, CD69 expression was significantly increased in BM compared to PB for CD4 (p = 0.03, unpaired Mann Whitney) and CD8 (p = 0.01) T cells and for CD56^bright NK cells (p=0.01) at Day 100, suggesting potential acquisition of a tissue-resident phenotype. Investigating innate lymphocytes beyond NK cells, we also identified ILC1s, ILC2s, and ILC3s in PB and BM post allo-HCT at low frequency (<1%), with distinct phenotypes by tissue source (increased CD69 in BM, increased CD25 in PB). Across allograft sources, ILC2s were rare compared to ILC1s and ILC3s in both the PB and BM, with a trend toward relatively higher frequencies of ILC1s in PB and ILC3s in BM, respectively.

Conclusions: These findings highlight tissue-specific features of innate and adaptive lymphocyte compartments in BM, the tumor immune environment in leukemia, compared to PB over-time following allo-HCT. The disparate phenotypes observed among BM lymphoid populations compared to those in circulation and the relative dominance of NK cells within the BM lymphocyte compartment early post-transplant highlight the importance of tissue-level evaluations and support further investigation of innate lymphocyte populations in the transplant setting.

Vinci:ClearView Healthcare Partners: Current Employment. Slingerland:Seres Therapeutics: Other: salary support through a sponsored agreement. Giardina:Merck: Current Employment. Gyurkocza:Actinium Pharmaceuticals, Inc.: Research Funding. Shaffer:Miltenyi Biotec: Research Funding; Gamida Cell: Consultancy; Hansa Biopharma: Consultancy. Tallman:UpToDate: Patents & Royalties; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Innate Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Kura: Membership on an entity's Board of Directors or advisory committees; Ipsen Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; KAHR-Adv Bd: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Rafael Pharmaceuticals: Research Funding; Glycomimetics: Research Funding; Biosight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Giralt:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinuum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; MILTENYI: Research Funding; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Research Funding; OMEROS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; SPECTRUM Pharma: Membership on an entity's Board of Directors or advisory committees. Politikos:ExcelThera: Membership on an entity's Board of Directors or advisory committees; PrecicionHeor: Honoraria; Merck: Research Funding. Barker:Merck: Research Funding; New York Blood Center: Consultancy; Gamida Cell: Consultancy. Perales:Orca Bio: Consultancy; Abbvie: Honoraria; VectivBio AG: Honoraria; Takeda: Honoraria; Medigene: Consultancy; Servier: Consultancy; Cidara Therapeutics: Consultancy; Vor Biopharma: Honoraria; DSMB: Other; Bellicum: Honoraria; Celgene: Honoraria; Sellas Life Sciences: Consultancy; Miltenyi Biotec: Consultancy, Honoraria; Kite, a Gilead Company: Honoraria, Research Funding; Merck: Consultancy; Omeros: Consultancy; Karyopharm: Honoraria; Incyte: Honoraria, Research Funding; Novartis: Honoraria; Nektar Therapeutics: Consultancy, Honoraria; Bristol-Mysers Squibb: Honoraria; MorphoSys: Consultancy, Honoraria; Astellas: Honoraria. van den Brink:Wolters Kluwer: Patents & Royalties; Seres Therapeutics: Current holder of stock options in a privately-held company, Honoraria, Other: IP Licensing , Research Funding; Notch Therapeutics: Current holder of stock options in a privately-held company, Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Honoraria; Vor Biopharma: Honoraria; Rheos Medicines: Honoraria; Frazier Healthcare Partners: Honoraria; Nektar Therapeutics: Honoraria; Ceramedix: Honoraria; Lygenesis: Honoraria; GlaskoSmithKline: Honoraria; Da Volterra: Honoraria; Thymofox: Honoraria; Garuda: Honoraria; Novartis (Spouse): Honoraria; Synthekine (Spouse): Honoraria; Beigene (Spouse): Honoraria; Kite (Spouse): Honoraria; Juno Therapeutics: Other: IP Licensing ; DKMS: Other: fiduciary role on the Foundation Board . Hanash:Intellectual Property: Other: Holds intellectual property related to Interleukin-22 and GVHD; Evive Biotech: Other: Served as Co-PI of a clinical trial supported by Evive Biotech, Research Funding.