Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation

<div><p>Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.</p></div

At weeks 2, 4, 8, and 12 after BMT, leukocytes isolated from mesenterial lymph nodes were stained with anti-TCRβ,-CD4,-CD8,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>(A) The numbers in the dot plot show the percentage of B6-originated TCRβ⁺ T cells among the total leukocytes. (B) The bars show the ratio and absolute numbers of B6-orginated TCRβ⁺ T cells (■) and Balb/c-originated TCRβ⁺ T cells (□). (C) The bars show the ratio and absolute numbers of B6(Balb/c)-originated TCRβ⁺ CD4⁺ and TCRβ⁺ CD8⁺ T cells. Data are shown as the mean ± SD. p values compared to donor-derived cells two weeks after BMT. ***, p ≤ 0.0001 or p ≤ 0.001; **, p ≤ 0.01; and *, p ≤ 0.05.</p

At one week after BMT, leukocytes isolated from the indicated organs were stained with anti-TCRβ,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4).

<p>(A) The numbers in the dot plot show the percentage of B6-originated cells among the total leukocytes. (B) The bars show the ratio of B6- (■) and Balb/c-originated cells (□). Data are shown as the mean ± SD.</p

At weeks 1, 2, 4, 8, and 12 after BMT, leukocytes isolated from the spleen and bone marrow were stained with anti-TCRβ,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>The bars show the ratio and absolute numbers of B6-orginated TCRβ⁺ T cells (■) and Balb/c-originated TCRβ⁺ T cells (□). Data are shown as the mean ± SD. p values compared to donor-derived cells one week after BMT. ***, p ≤ 0.0001 or p ≤ 0.001; **, p ≤ 0.01; and *, p ≤ 0.05.</p

At weeks 1, 2, 4, and 12 after BMT, leukocytes isolated from the bone marrow and/or spleen were stained with anti-CD11c,-B220,-Ly6G,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>(A) The bars show the ratio and absolute numbers of B6-orginated DCs (■) and Balb/c-originated DCs (□) in the bone marrow and spleen. (B) The bars show the ratio and absolute numbers of B6-orginated B cells (■) and Balb/c-originated B cells (□) in the bone marrow and spleen. (C) The bars show the ratio and absolute numbers of B6- (■) and Balb/c-originated Ly6G⁺ cells (□) in the spleen.). Data are shown as the mean ± SD.</p

At weeks 1, 2, 4, and 12 after BMT, leukocytes isolated from the thymus were stained with anti-TCRβ,-CD4,-CD8,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>(A) The numbers in the dot plot show the percentage of B6-originated T cells among the total leukocytes. (B) The bars show the ratio and absolute numbers of B6-orginated TCRβ⁺ T cells (■) and Balb/c-originated TCRβ⁺ T cells (□). (C) The bars show the ratio and absolute numbers of B6(Balb/c)-originated TCRβ⁺CD4⁺ and TCRβ⁺CD8⁺ T cells. Data are shown as the mean ± SD. p values compared to donor-derived cells one week after BMT. **, p ≤ 0.01, and *, p ≤ 0.05.</p

At weeks 2, 4, 8, and 12 after BMT, leukocytes isolated from the thymus (A) and liver (B) were stained with αGalCer-loaded CD1d dimer or anti-TCRβ,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>The bars show the ratio and absolute numbers of B6-orginated NKT cells (■) and Balb/c-originated NKT cells (□). Data are shown as the mean ± SD. p values compared to donor-derived cells one week after BMT. *, p ≤ 0.05.</p

Quantitative evaluation of the histogram comparison.

<p>Quantitative evaluation of the histogram comparison.</p

Gamma analysis between the original CT image and resultant synthetic CT images.

<p>(a) Original CT image. (b) Water-equivalent synthetic CT (_WCT). (c) Bulk-density-assigned CT image (_BCT). (d) Gamma analysis between the original CT and _WCT with 3 mm/3% criteria. (e) Gamma analysis between the original CT and _WCT with 2 mm/2% criteria. (f) Gamma analysis between the original CT and _WCT with 1 mm/1% criteria. (g) Gamma analysis between the original CT and _BCT with 3 mm/3% criteria. (h) Gamma analysis between the original CT and _BCT with 2 mm/2% criteria. (i) Gamma analysis between the original CT and _BCT with 1 mm/1% criteria.</p

Quantitative evaluation of the gamma analysis.

<p>Quantitative evaluation of the gamma analysis.</p