T cell and B Cell immunity can be reconstituted with mismatched hematopoietic stem cell transplantation without alkylator therapy in artemis-deficient mice using anti-natural killer cell antibody and photochemically treated sensitized donor T cells

Abstract

Children with Artemis-deficient T(-)B(-)NK(+) severe combined immunodeficiency are at high risk for graft rejection from natural killer (NK) cells and toxicity from increased sensitivity to the alkylating agents used in mismatched hematopoietic stem cell transplantation (HSCT). We evaluated the use of a nonalkylating agent regimen before HSCT in Artemis-deficient (mArt(-/-)) C57Bl/6 (B6) mice to open marrow niches and achieve long-term multilineage engraftment with full T cell and B cell immune reconstitution. We found that partial depletion of both recipient NK cells using anti-NK1.1 monoclonal antibody and donor T cells sensitized to recipient splenocytes was necessary. BALB/c-sensitized T cells (STCs) were photochemically treated (PCT) with psoralen and UVA light to inhibit proliferation, reduce the risk of graft-versus-host disease (GVHD), and target host hematopoietic stem cells (HSCs). A dose of 4 × 10(5) PCT STCs coinjected with 1 × 10(5) lineage-depleted c-kit(+) BALB/c HSCs resulted in 43.9% ± 3.3% CD4(+) and 10.9% ± 1.2% CD8(+) donor T cells in blood, 29% ± 7.8% and 21.7% ± 4.0 donor B220(+) IgM(+) in spleen and bone marrow, and 15.0% ± 3.6% donor Gran-1(+) cells in bone marrow at 6 months post-HSCT versus 0.02% ± 0.01%, 0.13% ± 0.10%, 0.53% ± 0.16%, 0.49% ± 0.09%, and 0.20% ± 0.06%, respectively, in controls who did not receive PCT STCs. We found that STCs target host HSCs and that PCT STCs are detectable only up to 24 hours after infusion, in contrast to non-photochemically treated STCs, which proliferate resulting in fatal GVHD. Increased mortality in the groups receiving 4-6 × 10(5) PCT STCs was associated with evidence of GVHD, particularly in the recipients of 6 × 10(5) cells. These results demonstrate that blocking NK cell-mediated resistance and making niches in bone marrow are both essential to achieving multilineage engraftment of mismatched donor cells and T cell and B cell reconstitution, even though GVHD is not completely eliminated.

Figure 1. Effects of anti-NK mAb 1.1 and various doses of PCT-4STC on lymphoid and myeloid long-term reconstitution after allogeneic HSCT

(A) Total number of lymphocytes in the spleen of recipient mice 6 months after HSCT compared with unmanipulated WT B6 (positive control) and B6 mArt^-/- (negative control) mice. Mean values ± SEM are showed (n=3-7); **p<0.01. (B) Donor mature B cells (B220⁺IgM⁺) in spleen and bone marrow (BM) and (C) CD4⁺, (D) CD8⁺ mature T cell phenotyping in peripheral blood (PB), spleen, lymph node (LN), and thymus from WT B6 mouse (n=3), B6 mArt^-/- untransplanted mouse (n=6), and B6 mArt^-/- mice following allogeneic HSCT with anti-NK 1.1 mAb and PCT-4STC (4×10⁵) (n=7) ≥6 months post transplant. Mean values ± SEM are showed. (E) Donor granulocyte chimerism in BM from B6 mArt^-/-mice following allogeneic HSCT with anti-NK 1.1 mAb and various PCT-4 STC (1-6 ×10⁵). Mean values ± SEM are showed (n=3-7); **p<0.01, *p<0.05.

Figure 2. T and B cell proliferative responses, specific IgM and IgG responses post immunization, and T cell receptor diversity post HSCT

(A) Proliferative responses of lymphocytes to anti-CD3 and LPS. WT B6 mice were compared to B6 mArt^-/-untransplanted mice (negative controls), recipients of allogeneic HSCT with anti-NK1.1 mAb and various doses of PCT-4 STC ≥6 months post transplant. Results (stimulated minus resting) are expressed as counts per minute (cpm) on a log scale. **p<0.01 (n>3). (B), (C) Serum IgM (B) and IgG-specific (C) antibody response to NP24-KLH after immunization (minus pre-immune response). Average response from recipients (n>3) of allogeneic HSCT with anti-NK 1.1 mAb and various dose of PCT-4 STC are compared to B6 mArt^+/+ (n=3) and B6 mArt^-/- (n=3) controls with serial dilutions. (D) TCR Vβ repertoire analysis. Four representative Vβ regions are shown. T cells were enriched from either thymus or spleen and prepared as in Materials and Methods. WT is wild type mouse and “leaky” is from representative 129/SvJ mArt^-/- mouse [17]. Examples of the repertoire from mArt^-/- recipients of allogeneic HSCT (851, 829, 627 are serial numbers) with 2, 4, 6×10⁵ PCT-4 STC also are shown.

Figure 3. PCT-4 of sensitized donor T cells limits GvHD and prolongs survival

(A) Percentage of weight change in mice after allogeneic HSCT. Transplanted mice were weighed individually on day +1 and then weekly up to the day of analysis. At 5 weeks after allogeneic HSCT, the percentage of weight loss was significant only in the group treated with 6×10⁵ non PCT-4 STC. Values are expressed as the mean ± SEM (n=4 to 10 per group, *p<0.05). (B) Clinical GvHD scores of transplanted mice 5 weeks after allogeneic HSCT. Transplanted mice were evaluated for evidence of clinical GvHD as described in the Materials and Methods. Scores are expressed as the mean ± SEM (n=4 to 10 per group, *p<0.05). (C) Kaplan-Meier survival 180 days after HSCT with 1×10⁵ HSC plus varying doses of PCT-4 STC. Control mice received 1×10⁵ HSC plus 6×10⁵ STC. Analysis by independent sample t-test of the mean survival (days) were significant only for recipients of 1 vs 4 ×10⁵ PCT-4 STC (p=0.04) and 1 vs 6×10⁵ PCT-4 STC (p=0.03). There was no significant difference in the overall comparison of survival curves between recipients of 1, 2, 4, and 6×10⁵ PCT-4 STC (p=0.5) using Log Rank or Generalized Wilcoxin analysis.

Figure 4. PCT STC makes niches in recipient bone marrow and PCT-4 decreases survival of STC

(A) Cytotoxicity of sensitized CD8a⁺T cells (BALB/c) with or without PCT-4. Killing of B6 lin^–c-kit⁺ HSC was assessed in an overnight chromium-release assay. The control was non-sensitized T cells. Each dot represents the mean value ± SEM for killing by T cells (n=3) using a ratio of 12:1, 25:1, 50:1, respectively. (B) Competitive repopulation experiments with equal amount untreated BALB/c and B6 lin^–c-kit⁺ HSC with or without pre-conditioning with donor PCT-4 STC. This panel displays a bar graph regarding engrafted granulocyte percentages for BALB/c and C57B1/6 (B6) in B6 X BALB/c F1 recipients 2 months post transplant, n=3, *p<0.05. (For details, see Materials and Methods). (C) Survival kinetics of STC in vivo. Purified STC (donor, BALB/c) with or without PCT-4 were labeled with CFSE and administered intravenously to B6 mArt^-/-mice pre-conditioned with anti-NK1.1 mAb. Bone marrow was harvested and evaluated for CFSE-positive cells at various time points. One representative result from three independent experiments. (For details, see Materials and Methods).