Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT

Abstract

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients.

Keywords: Allogeneic hematopoietic stem cell transplantation; Graft-versus-host disease; IL-2; Natural killer cell; Post-transplantation cyclophosphamide; Regulatory T cell; TL1A.

Fig. 1.:. NK cell recovery in recipients treated with expanded donor Tregs or PTCy is improved following aHSCT.

A HSCT utilizing a C3H.SW → B6 donor/recipient mouse model involving a minor MHC mismatch was performed on day 0. Lethally irradiated (10.5 Gy on day 0) B6 mice received 7×10⁶ TCD C3H.SW BM cells and spleen+LN cells from expanded (TL1A-Ig/IL-2; TrED group) or untreated C3H.SW (GVHD and PTCy group) donor mice adjusted to contain 2×10⁶ CD8 T cells. Cyclophosphamide was given on day 3 and 4 post-HSCT at 50 mg/kg ip. For the syngeneic HSCT 10×10⁶ non-TCD BM cells plus 5×10⁶ spleen cells from B6 mice were transplanted. (A) Percent (%) donor derived NK cells (CD3⁻CD19⁻NKp46⁺CD122⁺) on day 14 and 30 post HSCT in spleens are depicted. (B) Donor derived NK cell number (#) per spleen is shown on day 14 and 30 post HSCT. (C) Proliferation of donor NK cells (% Ki67) in spleen on day 14 and 30. n = 5–12 per group per time point. Data in column graphs are expressed as means ± SD and were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons. Data in kinetics graphs were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Data are pooled from 3 independent experiments, respectively.

Fig. 2.:. Donor NK cell subset recovery.

A HSCT was performed as in Fig 1. Donor NK cell subsets (immature [imm.] NK cells = CD27⁺/CD11b⁻; M1 NK cells = CD27⁺/CD11b⁺; M2 NK cells = CD27⁻/CD11b⁺) were examined in recipient spleens 14 days post HSCT (C3H.SW → B6). (A) Percent (%) and (B) Numbers (#) per spleen are shown (C) Kinetics of the M2 subset over time (% and #). Data are pooled from 3 independent experiments with n = 7–13 per group per time point. Data in column graphs are expressed as means ± SD and were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons. Data in kinetics graphs were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 3.:. Functional assessment of NK cells in vitro and in vivo following minor mismatch aHSCT.

The (C3H.SW → B6 experimental transplant model was performed as in Fig 1. (A) Kinetics of IFNγ – producing NK cells on day 14 and day 30 is shown after 5 h of in vitro stimulation with PMA (50 ng/ml) and Ionomycin (500 ng/ml) in the presence of 1 μl/ml BD GolgiStop. n = 5 per group. (B,C) Expression of the degranulation marker CD107 after in vitro and in vivo stimulation. (B) CD107 expression on unstimulated (dotted line) and in vitro stimulated NK cells (full line; 5 h, PMA+Ionomycin stimulation as in A) on day 14 and 30. n = 4–5 per group. (C) CD107 expression on NK cells on day 30 after in vivo (1×10⁶ iv injected RMA-S cell for 18 h), in vitro (5 h PMA + Ionomycin as in A) or double stimulation (in vivo + in vitro). n = 3–5 per group. Data are pooled from 2 independent experiments (A-C). (D) In vivo cytotoxicity assay. On day 30 post HSCT, 10×10⁶ NK resistant RMA and 10×10⁶ NK sensitive RMA-S cells, respectively, were labeled with low (0.4 uM) and high (4 uM) amounts of CellTrace Violet (CTV) and injected iv into recipients. 5 h later, the killing of the NK sensitive RMA-S cells was quantified in blood and spleen. Representative histograms and graphs are shown from one of 2 experiments. n = 3 per group. Data in kinetics graphs are expressed as means ± SD and were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons. Data in column graphs are expressed as means ± SD and were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.