Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning

Abstract

In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that approximately 0.1-1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation.

Figure 1.

Donor HSCs can engraft unconditioned tolerant wild-type hosts. (A) Persistent multilineage contribution occurs only in tolerant recipients. 4,000 ckit⁺ Thy1.1^low lineage⁻ Sca-1⁺ Flk2⁻ CD34⁻ HSCs from male CD45.1 mice were transplanted into unirradiated male CD45.1 × CD45.2 (F1), CD45.2, or irradiated male CD45.1 × CD45.2 recipients. Granulocytes were pregated as side scatter^high B220⁻ TCRβ⁻ cells, B cells were pregated as Mac-1⁻ TCRβ⁻ cells, and T cells were pregated as B220⁻ Mac-1⁻ cells. Representative plots of peripheral blood at 16 wk after transplantation are presented. (B) Consistent granulocyte contribution in tolerant hosts. Granulocyte chimerism is shown at 4-wk intervals after transplantation into CD45.1 × CD45.2 and CD45.2 hosts. ⋄, F1 hosts; •, CD45.2 recipients. Data points below the dotted line represent animals with no detectable chimerism.

Figure 2.

Small numbers of engrafted wild-type HSCs can rescue lymphocyte deficiencies in unnirradiated RAG2^−/−γc^−/− mice. (A) Robust donor chimerism in unconditioned RAG2^−/−γc^−/− recipients. Unirradiated RAG2^−/−γc^−/− mice were repetitively transplanted six times with 1,750–4,000 HSCs from eGFP-transgenic mice, and peripheral blood was analyzed for donor myeloid and lymphoid contribution at 4 wk after the final transplant. Granulocytes were pregated as side scatter^high B220⁻ TCRβ⁻ cells, B cells were pregated as Mac-1⁻ TCRβ⁻ cells, and T cells were pregated as B220⁻ Mac-1⁻ cells. (B) Persistent multilineage contribution in unnirradiated RAG2^−/−γc^−/− recipients. Peripheral blood was analyzed for donor contribution periodically until 30 wk after final transplantation.

Figure 3.

Low-level HSC engraftment in primary unconditioned RAG2^−/−γc^−/− recipients. (A) Donor HSCs can be detected in primary unirradiated RAG2^−/−γc^−/− recipients. At 31 wk after the final transplantation, donor HSC contribution in the bone marrow in unconditioned primary RAG2^−/−γc^−/− recipients was quantified using the markers shown. (B) Engrafted HSCs in the bone marrow of unconditioned primary RAG2^−/−γc^−/− recipients can be serially transplanted. Unfractionated (5 × 10⁷) or c-kit–enriched (10⁶) bone marrow cells from primary RAG2^−/−γc^−/− recipients were secondarily transplanted into lethally irradiated (950 cGy) wild-type mice. GFP⁺ chimerism was assessed at 25 wk after secondary transplantation. Each secondary recipient received a transplant from a distinct nonredundant primary donor. Data points below the dotted line represent undetectable chimerism.

Figure 4.

Significant donor cell expansion is not observed until the pro–B cell stage. At 31 wk after the final transplantation, donor contribution to myeloid and lymphoid progenitor cells was analyzed. CMPs were defined as lin⁻ ckit⁺ Sca-1⁻ CD16/32^low CD34^low cells. CLPs were gated as lin⁻ Thy1.1^low IL7Rα⁺ Flk2⁺ ckit^low Sca1^low cells. Pro–B-A cells were defined as B220⁺ CD43⁺ IgM⁻ CD19⁻ NK1.1⁻, and pro–B-B cells were gated as B220⁺ CD43⁺ IgM⁻ CD19⁺ Ly51⁻ NK1.1⁻ cells. Mean values ± SEM are shown from the analysis of three mice for each subset.

Figure 5.

Stem and progenitor cell frequencies are normal in RAG2^−/−γc^−/− mice. LT-HSCs, ST-HSCs, and multipotent progenitors were stained from untransplanted mice as in Fig. 2. CLPs and CMPs were stained as in Fig. 4. MEPs and GMPs were stained according to established protocols (reference 20). Each endogenous population is presented as a percentage of total bone marrow. ○, wild-type animals; ⋄, RAG2^−/−γc^−/− mice.

Figure 6.

Reconstituted RAG2^−/−γc^−/−(mice can respond normally to antigenic challenge. RAG2^−/−γc^−/− mice were immunized with 100 μg alum-precipitated NP–chicken γ globulin 16 wk after HSC transplant. Serum was obtained 1 wk after immunization and tested for NP-specific antibodies. Mice with undetectable serum levels of NP-specific IgG are shown below the dotted line.

Figure 7.

γc expression is not directly important for HSC maintenance. (A) Purified wild-type HSCs can engraft unirradiated RAG2^−/− mice. Unconditioned RAG2^−/− and RAG2^−/−γc^−/− mice were transplanted with 3,000 HSCs from GFP-transgenic mice. Representative plots from peripheral blood obtained 4 wk after transplantation are shown. Granulocytes were pregated as side scatter^high B220⁻ CD3⁻ cells, B cells were pregated as Mac-1⁻ CD3⁻ cells, and T cells were pregated as B220⁻ Mac-1⁻ cells. (B) Persistent granulocyte chimerism but delayed lymphoid reconstitution in RAG2^−/− mice. ⋄, RAG2^−/−γc^−/− animals; •, RAG2^−/− animals.

Figure 8.

Host T cells act as barriers to productive HSC engraftment. (A) α/β T cells are required for HSC graft rejection. 1,000 purified HSCs from eGFP-transgenic mice were transplanted into TCRα^−/−β^−/−, Cμ^−/−, RAG2^−/−, and wild-type animals. Peripheral blood was analyzed for donor cell contribution at 16 wk after transplantation. Granulocytes were pregated as side scatter^high B220⁻ TCRβ⁻ cells, B cells were pregated as Mac-1⁻ TCRβ⁻ cells, and T cells were pregated as B220⁻ Mac-1⁻ cells. A representative plot for each group is shown. (B) Host CD4⁺ T cells are essential for HSC graft rejection. 800 purified HSCs from GFP-transgenic mice were transplanted into 2 I-A^−/− and 3 β₂m^−/− mice. Peripheral blood was analyzed 16 wk after transplantation for donor chimerism. Representative plots are shown. (C) LT-HSCs express MHC II. MHC II expression was analyzed on LT-HSCs from wild-type animals.

Figure 9.

Transient CD4 depletion allows productive engraftment of HSCs with minor histocompatibility mismatches. (A) Treatment of Cμ^−/− mice with a depleting antibody leads to efficient CD4⁺ T cell removal. Mice were treated consecutively for 3 d with anti-CD4 antibody, and peripheral blood was analyzed for TCRβ⁺ CD8⁻ cells to assess depletion 1 d after the final treatment. The plots are gated on Ter119⁻ TCRβ⁺ cells. (B) HSC engraftment occurs in anti-CD4–treated animals. Three Cμ^−/− mice that were treated with anti-CD4 antibody and two Cμ^−/− mice that were left untreated were transplanted with 800 GFP⁺ HSCs. Peripheral blood was analyzed at 8 wk after transplantation for granulocyte, B cell, and T cell chimerism. Granulocytes were pregated as side scatter^high B220⁻ CD3⁻ cells, B cells were pregated as Mac-1⁻ CD3⁻ cells, and T cells were pregated as B220⁻ Mac-1⁻ cells. Representative plots are shown. (C) Significant short-term multilineage engraftment in all anti-CD4(treated recipients. Donor contribution to granulocytes, T cells, and B cells at 8 wk after HSC transplant is shown. B cells are quantified as number of cells contained within 100 μl of peripheral blood. ⋄, anti-CD4–treated animals; •, untreated animals. Data points that were undetectable are shown below the dotted line.

Figure 10.

Transplanted HSC grafts are accepted more readily in aged recipients than in young mice. Purified eGFP⁺ HSCs were sorted and 1,750–4,000 cells were repetitively transplanted into 2-mo-old C57Bl6/Ka and 24-mo-old C57Bl6/Harland mice. Donor granulocyte, B cell, and T cell chimerism are shown from the first peripheral bleed taken at 6.5 wk after the first transplant (3 d after the final transplant). •, young recipients; ⋄, aged recipients. The numbers of animals with no detectable chimerism in each group are listed below the dotted line.