Signal one and two blockade are both critical for non-myeloablative murine HSCT across a major histocompatibility complex barrier

Abstract

Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

Figure 1. Syngeneic engraftment is influenced by the early ratio of donor: recipient cells in bone marrow after transplant.

(A) Mice were conditioned with 0, 25, 75 or 125mg/kg busulfan; the concentration of SDF-1α was measured in (B) bone marrow and (C) plasma, 24 hours after the last dose of busulfan was administered (n=6 per group). (D) The SDF-1α gradient between bone marrow and plasma was calculated by dividing the bone marrow SDF-1α concentration by the plasma SDF-1α concentration. (E) Syngeneic HSCT with 20x10⁶ cells (CD45.2) was performed in recipients (CD45.1) that had received 0, 25, 75 or 125mg/kg busulfan (n=2-6 per group). (F) The number of donor and (G) recipient cells and (H) the percentage of donor cells in the bone marrow at 18 hours post-transplant were detected using flow cytometry. In B, C, D, F G and H each data point represents one recipient, horizontal bars are the mean and error bars represent the standard deviation. (I) HSCT recipients (CD45.1) were treated with 125mg/kg or 25mg/kg busulfan (BU) before receiving 2x10⁶ or 10x10⁶ syngeneic (CD45.2) donor bone marrow cells (n=5 per group). Donor chimerism in peripheral blood was monitored using flow cytometry at 2, 4, 6, and 12 weeks post-transplant. (J) The mean percentage donor chimerism over time is shown; error bars represent the standard deviation. (K) The mean percentage contribution of donor and recipient T cells, monocytes and B cells over time is also shown, as a percentage of the peripheral blood.

Figure 2. Increasing initial allogeneic chimerism is not sufficient for long-term engraftment with reduced intensity conditioning.

(A) HSCT recipients (C57BL/6) were treated with busulfan conditioning before transplant with 10x10⁶ allogeneic (CBA) cells, and received 1mg anti-CD4 and anti-CD8 mAb on days 0, 2 and 4 relative to transplant with or without additional treatments as specified (n=4-6). (B) Donor chimerism at 12 weeks post-transplant was measured using flow cytometry in mice that had received 25-125mg/kg busulfan plus 1mg anti-CD4 and anti-CD8 mAb on days 0, 2 and 4. (C) Donor chimerism over time is shown for recipients of 75mg/kg busulfan and 1mg mAb. Tolerance to donor cells at >20 weeks post-transplant, measured using the in vivo cytotoxicity assay, is presented as ratio of donor: recipient splenocytes remaining in spleen (Sp) and blood (Bl) 20 hours after injection. Tolerance is indicated by values >0.6. Additional treatments tested in combination with this regimen included (D) an increased dose of 3mg anti-CD4 and anti-CD8 mAb (n=5), (E) 1mg of each mAb on days -1, 0, 2 and 4 (n=4), (F) 1mg on days -1 to 4 (n=4), (G) 500ug ACK2 treatment on day -7 (n=5), (H) 8ug G-CSF for 4 days before HSCT (n=5), or (I) a cell dose of 100x10⁶ cells (n=5). Donor chimerism in peripheral blood at 2, 4, 6, 12 and 20 weeks post-transplant, the mean percentage contribution of donor and recipient T cells, monocytes and B cells in peripheral blood at these time points, and in vivo cytotoxicity assay results from >20 weeks post-transplant are displayed. Where some mice accepted transplant and others rejected, the contribution of different lineages and the in vivo cytotoxicity results are separated for the mice with and without donor chimerism.

Figure 3. Combined signal 1 and 2 T cell blockade is required for long-term allogeneic engraftment with non-myeloablative conditioning.

(A) HSCT recipients (C57BL/6) were treated with 25mg/kg busulfan and given allogeneic HSCT with anti-CD4/CD8, anti-CD40L, or combined mAb treatment. Donor chimerism and the mean percentage contribution of donor and recipient T cells, monocytes and B cells in peripheral blood at 2-20 weeks post-transplant, and the in vivo cytotoxicity assay results from >20 weeks post-transplant are displayed. Transplant acceptance was observed in recipients of 10x10⁶ (B) CBA donor cells (n=5), or (C) Balb/c donor cells (n=6), along with 1mg anti-CD4, anti-CD8 and anti-CD40L mAb on days 0, 2 and 4. Transplant rejection was observed in recipients of 10x10⁶ CBA donor cells with either (D) 1mg anti-CD4 and anti-CD8 mAb (n=5) or (E) 1mg anti-CD40L (n=6) on days 0, 2 and 4.

Figure 4. The success of allogeneic HSCT with combined signal 1 and 2 blockade is not due to increased homing of donor cells to the bone marrow.

(A) HSCT recipients (C57BL/6) were treated with 75mg/kg busulfan and then received syngeneic transplant (n=6), allogeneic transplant (n=6), or allogeneic transplant plus 1mg anti-CD4, anti-CD8 and anti-CD40L mAb on the day of transplant (n=6). (B) The number of donor cells, (C) number of recipient cells, and (D) percentage of donor cells in bone marrow were determined 36 hours after transplant. Each data point represents one recipient, horizontal bars are the mean and error bars represent the standard deviation.