Allogeneic T cells impair engraftment and hematopoiesis after stem cell transplantation

Abstract

Because of the perception that depleting hematopoietic grafts of T cells will result in poorer immune recovery and in increased risk of graft rejection, pure hematopoietic stem cells (HSC), which avoid the potentially lethal complication of graft-versus-host disease (GVHD), have not been used for allogeneic hematopoietic cell transplantation (HCT) in humans. Ideal grafts should contain HSC plus mature cells that confer only the benefits of protection from pathogens and suppression of malignancies. This goal requires better understanding of the effects of each blood cell type and its interactions during engraftment and immune regeneration. Here, we studied hematopoietic reconstitution post-HCT, comparing grafts of purified HSC with grafts supplemented with T cells in a minor histocompatibility antigen (mHA)-mismatched mouse model. Cell counts, composition, and chimerism of blood and lymphoid organs were evaluated and followed intensively through the first month, and then subsequently for up to 1 yr. Throughout this period, recipients of pure HSC demonstrated superior total cell recovery and lymphoid reconstitution compared with recipients of T cell-containing grafts. In the latter, rapid expansion of T cells occurred, and suppression of hematopoiesis derived from donor HSC was observed. Our findings demonstrate that even early post-HCT, T cells retard donor HSC engraftment and immune recovery. These observations contradict the postulation that mature donor T cells provide important transient immunity and facilitate HSC engraftment.

Fig. 1.

GVHD in B6 into BALB.B recipients. (A) Kaplan–Meier curves showing percent survival and (B) median percentage of baseline (BL) weight of BALB.B mice given B6 HSC alone or HSCwSP, ToTC, CD4, or CD8 cells. Data were from six independent experiments. (C) H&E staining of GVHD target tissues from representative recipients of HSC (Upper) or HSC+wSP (Lower) at 4 wk post-HCT. Liver and bowel architecture were normal in HSC recipients, but had lymphocyte infiltrates and parenchymal cell damage in mice given HSC+wSP.

Fig. 2.

White blood cell counts. (A) WBC counts of BALB.B mice given B6 HSC alone or HSC+wSP, ToTC, CD4, or CD8 measured by Coulter Counter at 4 wk and 7 wk post-HCT. Control WBC values for age adjusted (12 wk) WT BALB.B mice are shaded in gray. At 7 wk, recipients of HSC alone had higher WBC counts (median 10 × 10⁶/μL) than mice given HSC+wSP (median 5.7 × 10⁶/μL; P = 0.003), HSC+ToTC (median 3.7 × 10⁶/μL; P < 0.001), HSC+CD4 (median 7 × 10⁶/μL; P = 0.03), or HSC+CD8 T cells (median 4.6 × 10⁶/μL; P = 0.002). (B) Proportion of HSC-derived donor cells in the blood at 7 wk post-HCT. HSC recipients had a significantly higher proportion (median 93%) compared with HSC+wSP (median 29%; P < 0.001), ToTC (median 31%; P = 0.07), or CD4 (median 61%; P = 0.02), but not CD8 (median 82%; P = 0.2) groups. (C) Absolute number of donor HSC-derived WBC 7 wk post-HCT. Recipients of HSC only had significantly higher absolute numbers of WBC derived from donor HSC (9 × 10⁶/μL) compared with mice given HSC+wSP (median 1.6 × 10⁶/μL; P < 0.001), HSC+ToTC (median 1.1 × 10⁶/μL; P = 0.002), HSC+CD4 (4.2 × 10⁶/μL; P = 0.002), or HSC+CD8 T cells (median 3.2 × 10⁶/μL; P = 0.001). B and C display the mean and SEM for each respective group, derived from one experiment with five to eight mice per group.

Fig. 3.

Effects of graft composition on hematopoiesis at 1 mo post-HCT. (A) Contribution of cells by lineage determined by FACS analysis. HSC recipients had normal B cell levels (median 38%/live cells). B cell levels were significantly reduced in mice given HSC+wSP (median 6%; P < 0.0001), HSC+ToTC (median 2%; P < 0.0001), HSC+CD4 (median 0.75%; P < 0.0001), or HSC+CD8 cells (median 4%; P < 0.0001). Recovery of Mac1 cells occurred rapidly in all groups, although levels were significantly lower in recipients of HSC (median 46% of live cells) versus mice given HSC+wSP (median 73%; P < 0.0001), ToTC (median 66%; P < 0.001), CD4 (median 74%; P < 0.0001), or CD8 T cells (median 63%; P < 0.01). T-cell levels reached a median of 10%/live cells in recipients of HSC or HSC+wSP, and were slightly higher (16–17%) in recipients of HSC+ToTC, CD4 or CD8 (P < 0.01 for differences between HSC only and HSC+ToTC, CD4 or CD8). (B) Severity of GVHD was classified according to the following criteria: death from acute GVHD or weight <80%, 80–90%, or >90% of BL weight. Shown are the contributions by lineage as %/live cells for mice stratified into these groups. Weight loss correlated with B lymphopenia and predominance of Mac1 cells in the blood. There was no obvious correlation between T-cell levels and GVHD; however, higher levels of donor spleen-derived T cells corresponded with greater weight loss. Data for these figures were derived from same mice as studied in Fig. 1.

Fig. 4.

Effects of graft composition on chimerism. (A) Delineation of T-cell source by FACS analyses of blood 4 wk post-HCT from representative mice in each transplant group. T cells from BALB.B are marked by Thy1.2, CD45.2; from B6 HSC by Thy1.1, CD45.2, GFP; and from B6 splenic cells by Thy1.1 CD45.1. Recipients of HSC remained mixed donor/host T-cell chimeras. Recipients of HSC+wSP converted to full donor type, and all T cells were derived from donor splenocytes. T cells in recipients of HSC+ToTC, CD4, or CD8 cells were derived from donor splenocytes and donor HSC with residual host in some mice. (B–D) Summary of FACS results obtained from mice studied in Figs. 1 and 3. (B) Donor/host chimerism of B, Mac1, and T cells 4 wk post-HCT. All groups converted to full donor type in the B cell and myeloid lineages. T-cell origins were mixed in recipients of HSC, whereas T cells were largely donor derived in mice given lymphocyte-replete grafts. (C) Origins of T cells as percentage of T cells from each source relative to total T cells. Gray bars indicate nascent T cells from donor HSC, black bars indicate T cells cotransferred from donor spleen cells, and white bars represent host T cells, all at 1, 2, and 3 mo post-HCT. HSC recipients remained mixed T-cell chimeras and the proportion of HSC-derived T cells increased over time. In mice given HSC+ToTC or HSC+CD8 cells, the proportion of nascent HSC-derived donor T cells increased whereas spleen-derived donor T cells decreased, and residual host cells were largely eliminated. In recipients of HSC+CD4 cells, spleen-derived T cells persistently predominated, and elimination of host T cells was incomplete. (D) Origins of cells from the different lineages at 1, 2, and 3 mo post-HCT for recipients of HSC+wSP. Residual host cells were readily cleared, and spleen-derived hematopoiesis was superior compared with lymphoid and myeloid cell production from donor HSC.

Fig. 5.

Influence of CD45.1/2 allele differences on engraftment and GVHD. Blood T-cell chimerism is shown as the median percentage of T cells derived from donor HSC (gray) and spleen (black) vs. host (white) at 1, 2, and 3 mo post-HCT. (A) CD45 allele disparity of HSC and wSP B6 donors. Regenerating blood T cells were mainly donor spleen-derived (median 84% vs. 15% donor HSC derived at 1 mo post-HCT), and host T cells were eliminated (<1%). (B) CD45.2 allele identity of HSC and wSP B6 donors. HSC were from B6.Thy1.1 and wSP were from B6.Thy-1.1.GFP⁺ mice. At 1 mo post-HCT, a median 33% and 58% of T cells were donor spleen vs. HSC derived, respectively. Residual host T cells (median 12.5%) persisted. A and B show means with SEM. (C) Median weights of BALB.B (CD45.2) that received HSC+wSP grafts from CD45 disparate donors (A and B) vs. HSC+wSP from CD45.2 identical donors that also shared the CD45.2 allele with recipients (C, all CD45.2). Weight loss was more pronounced for CD45 allele disparity vs. identity between donor and host. Data for these figures were derived from same mice as studied in Fig. 1.

Fig. 6.

Lymphoid lineage and chimerism as determined by FACS analyses of BM and spleen 2 wk post-HCT. (A) Recipients of HSC only showed prompt reconstitution of the lymphoid cells (SSC/FSC plot), which were predominantly B cells (B220) derived from the GFP⁺ HSC donor. T cells in BM originated mainly from the host, whereas splenic T cells were derived equally from donor HSC and the host. (B) Recipients of HSC+wSP displayed marked lymphopenia (SSC/FSC plot) secondary to marked reduction of B cells. BM and spleen were heavily infiltrated by donor T cells (spleen derived). Little to no hematopoiesis originated from donor HSC or residual host cells. The majority of donor T cells were CD8⁺, a substantial proportion of which were tetramer reactive against H60. Representative FACS plots from mice 2 wk post-HCT are shown. Data were confirmed in three independent experiments, with three to four animals per group.