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Expansion of Allospecific Regulatory T Cells After Anergized, Mismatched Bone Marrow Transplantation

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Expansion of Allospecific Regulatory T Cells After Anergized, Mismatched Bone Marrow Transplantation

Jeffrey K Davies et al. Sci Transl Med.

Abstract

Transplantation of hematopoietic stem cells from healthy donors can cure patients with many diseases. Donor T cells can protect against recurrence of infection and disease, but some of these (alloreactive) T cells recognize patient tissues as foreign, causing graft-versus-host disease. Removing T cells from donor grafts before transplantation reduces graft-versus-host disease but increases infection and disease recurrence. Inactivation of alloreactive T cells by inducing tolerance to patient cells (anergization) before transplantation preserves beneficial donor T cell effects while reducing graft-versus-host disease. We show that this approach also results in expansion of regulatory T cells that specifically suppress alloreactive donor T cell responses in the recipient. In addition to reducing graft-versus-host disease, antigen-specific regulatory T cells generated with this strategy could suppress unwanted T cell responses that cause rejection of solid organ transplants and tissue damage in autoimmune disorders.

Figures

Fig. 1
Fig. 1
Expansion of donor-derived CD4+ Treg cells after transplantation of bone marrow containing alloanergized haploidentical donor T cells. (A) Frequency of FOXP3+ cells (expressed as percentage of CD3+CD4+ cells) in peripheral blood from patients after alloanergized haploidentical bone marrow transplantation and from their donors. (B) Absolute number of CD4+FOXP3+ cells per microliter of peripheral blood from patients after alloanergized haploidentical transplantation. (C) Cell surface and intracellular markers on FOXP3+and FOXP3–CD4+ cells from peripheral blood of patients after alloanergized haploidentical transplantation. Illustrative histograms gated on CD3+CD4+ cells from patient 2 at day 36 are shown. Black, FOXP3+ cells; gray, FOXP3– cells. (D) Cell surface expression of CD25 and CD127 on CD4+ cells in peripheral blood after alloanergized haploidentical transplantation. Left, dot plot of CD4 and intracellular FOXP3 expression (gated on CD3+ cells); right, dot plot showing expression of CD25 and CD127 on cells from the boxed regions in the left panel (black, FOXP3+ cells; gray, FOXP3– cells) from patient 2 at day 36. (E) Left, dot plot of CD25 and CD127 expression on CD3+CD4+ cells from peripheral blood of an HLA-A2– patient on day 30 after receiving an alloanergized haploidentical transplant from an HLA-A2+ donor; right, histogram showing HLA-A2 expression (black) on cells from the boxed region in the left panel and an isotype control (dotted line).
Fig. 2
Fig. 2
CD4+ Treg cells from recipients of alloanergized transplants suppressed recipient-specific alloresponses. (A) Flow cytometric sorting of patient PBMCs after transplant yielded ~98% pure populations of CD4+CD25hiCD127lo Treg cells (left) and Treg-depleted CD4+ cells with minimal (<0.2%) residual Treg cell content (right). Dot plots are gated on viable CD3+CD4+ cells. (B) Effect of addition of purified Treg cells from patient peripheral blood after transplant on mean (± SD) proliferation (thymidine incorporation) of fresh donor PBMCs stimulated with recipient (patient) or third-party allostimulators. Results are shown for patient 2 (day 43) and patient 3 (day 42). cpm, counts per minute. (C) Mean proliferation (± SD) of Treg-replete and Treg-depleted CD4+ cells from peripheral blood of patients 2 and 3 after transplant stimulated with recipient or third- party allostimulators. Results are shown for patient 2 (day 43) and patient 3 (day 42).
Fig. 3
Fig. 3
Alloanergization increased the frequency of CD4+ cells with a Treg phenotype in vitro. (A) Frequency of FOXP3+ cells, expressed as percentage of CD3+CD4+ cells in untreated and alloanergized responder PBMCs in 10 HLA-mismatched responder- stimulator pairs. Horizontal lines represent median values. P value is for a two-tailed paired t test. (B) Phenotype of CD4+ Treg cells after alloanergization of PBMCs. Dot plots of CD3+CD4+ cells are from a representative experiment (of four). (C) Cell surface expression of CD25 and CD127 on CD4+ cells in alloanergized responder PBMCs. Left, dot plot of CD4 and intracellular FOXP3 expression (gated on CD3+ cells); right, dot plot showing expression of CD25 and CD127 on cells from the boxed regions in the left panel (black, FOXP3+ cells; gray, FOXP3– cells). One representative experiment (of four) is shown. (D) Frequency of CD25hiCD127lo cells, expressed as percentage of CD3+CD4+ cells in untreated and alloanergized responder PBMCs in 20 HLA- mismatched responder-stimulator pairs. Horizontal lines represent median values. P value is for a two-tailed paired t test. (E) Frequency of CD25hiCD127lo cells, expressed as percentage of CD3+CD4+ cells in untreated and alloanergized responder PBMCs in 12 haploidentical responder-stimulator pairs. Horizontal lines represent median values. P value is for a two-tailed paired t test.
Fig. 4
Fig. 4
The effect of in vitro allorestimulation of alloanergized PBMCs on the frequency and phenotype of CD4+ Treg cells. (A) Frequency of FOXP3+ cells, expressed as percentage of CD4+ cells, in alloanergized and allorestimulated alloanergized responder PBMCs in nine HLA-mismatched responder-stimulator pairs. Horizontal lines represent median values. P value is for a two-tailed paired t test. (B) Frequency of CD25hiCD127lo cells, expressed as percentage of CD4+ cells in alloanergized and allorestimulated alloanergized responder PBMCs in 22 different HLA- mismatched responder-stimulator pairs. Horizontal lines represent median values. P value is for a two-tailed paired t test. (C) Cell surface and intracellular markers on CD4+ Treg cells after in vitro allorestimulation of alloanergized responder PBMCs. Histograms are from one representative experiment (of six). Black, FOXP3+CD4+ cells; gray, FOXP3–CD4+ cells. (D) Frequency of CD25hiCD127lo cells, expressed as percentage of CD4+ cells in untreated, alloanergized, and allorestimulated alloanergized responder PBMCs in eight HLA-mismatched responder-stimulator pairs after depletion of CD4+ Treg cells from starting populations of untreated PBMCs. (E) Histogram showing TCR Vb subfamily distribution in CD4+CD25hiCD127lo Treg cells before and after alloanergization and allorestimulation with HLA-mismatched stimulators. One representative experiment (of three) is shown.
Fig. 5
Fig. 5
Suppression by CD4+ Treg cells after in vitro alloanergization and allorestimulation of PBMCs. (A) Mean percentage suppression (± SD) of first-party stimulated alloproliferation of untreated autologous responder PBMCs by CD4+ Treg cells from untreated, alloanergized, and allorestimulated alloanergized PBMCs. Data are for 12 HLA-mismatched stimulator- responder pairs. *P < 0.05; **P <0.01 [two-tailed paired t test com- paring suppression of first-party stimulated alloproliferation by CD4+ Treg cells from allorestimulated alloanergized PBMCs with CD4+ Treg cells from untreated (left) or alloanergized (right) PBMCs]. (B) Mean percentage suppression (± SD) of cytomegalovirus- specific proliferation of untreated autologous responder PBMCs by CD4+ Treg cells purified from untreated, alloanergized, and allorestimulated alloanergized PBMCs. Data are for three different HLA-mismatched stimulator- responder pairs. *P < 0.05 [two-tailed paired t test comparing suppression of cytomegalovirus proliferation by CD4+ Treg cells from untreated PBMCs with CD4+ Treg cells from alloanergized (left) or alloanergized allorestimulated (right) PBMCs]. ns, not significant. (C) Mean percentage suppression (± SD) of first- and third-party stimulated alloproliferation of untreated autologous responder PBMCs by CD4+ Treg cells purified from allorestimulated alloanergized PBMCs. Data are for 12 HLA-mismatched stimulator-responder pairs. *P < 0.05 (two-tailed paired t test comparing suppression of first-party stimulated alloproliferation with third-party stimulated alloproliferation).
Fig. 6
Fig. 6
Specific suppression of alloresponses by unsorted alloanergized PBMCs. (A) Mean percentage suppression (± SD) of first- and third-party stimulated alloproliferation of untreated or first-party alloprimed autologous responder PBMCs by alloanergized PBMCs. Data are for 16 HLA-mismatched stimulator-responder pairs. *P <0.05 (two-tailed paired t test com- paring suppression of first- and third-party stimulated alloproliferation). (B) Mean percentage suppression (± SD) of cytomegalovirus lysate–stimulated proliferation and first-party stimulated alloproliferation of untreated autologous responder PBMCs by alloanergized PBMCs. Data are for three HLA-mismatched stimulator-responder pairs. *P < 0.05; **P < 0.01 (two-tailed paired t test comparing suppression of cytomegalovirus- stimulated proliferation and alloproliferation). (C) Mean percentage suppression (± SD) of first-party stimulated alloproliferation of untreated autologous responder PBMCs by unsorted alloanergized PBMC and alloanergized PBMCs depleted of CD4+ or CD25+ cells. Data are for six HLA-mismatched responder- stimulator pairs. *P < 0.05; **P < 0.01 [two-tailed paired t test comparing suppression by alloanergized PBMCs depleted of CD4+ (left) or CD25+ (right) cells with suppression by unsorted alloanergized PBMCs.

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