Ex vivo-expanded human regulatory T cells prevent the rejection of skin allografts in a humanized mouse model

Abstract

Background: Composite tissue transplantation effectively reconstructs the most complex defects, but its use is limited because of harmful immunosuppression and the high susceptibility of skin to rejection. Development of tolerance is an ideal solution, and protocols using regulatory T cells (Tregs) to achieve this have been promising in experimental animal models. The aim of this study was to investigate the ability of human Tregs to regulate immune responses to a human skin allograft in vivo.

Methods: We isolated and expanded naturally occurring CD127loCD25+CD4+ human Tregs from peripheral blood mononuclear cells (PBMCs) and examined their phenotype and suppressive activity in vitro. Using a clinically relevant chimeric humanized mouse system, we transplanted mice with human skin grafts followed by allogeneic populations of PBMCs with or without Tregs derived from the same PBMC donor.

Results: Ex vivo-expanded Tregs maintain the appropriate Treg markers and retain suppressive activity against allostimulated and polyclonally stimulated autologous PBMCs in vitro. Mice receiving allogeneic PBMCs alone consistently reject human skin grafts, whereas those also receiving Tregs display stable long-term human skin transplant survival along with a reduction in the CD8+ human cellular graft infiltrate.

Conclusions: We show for the first time the unique ability of human Tregs to prevent the rejection of a skin allograft in vivo, highlighting the therapeutic potential of these cells clinically.

Figure 1. Skin rejection is mediated by allogeneic human PBMCs in vivo

(a) Schematic representation of the chimeric humanised mouse model. BALB/c Rag2^−/− ;Il2rγ^−/− mice receive a 1×1cm human skin transplant which is allowed to heal for 35 days before adoptive transfer of 5×10⁶ PBMCs intraperitoneally (i.p.). Skin grafts are monitored until the point of rejection, defined as complete loss. (b) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=7 mice) or no cells (n=4 mice). Data are pooled from two separate PBMC/skin donor combinations. (c) Representative photographs of skin grafts at day 100 (no cells) or rejection (PBMCs). (d) The proportion of CD45⁺ human leukocytes of the total monocyte population (% huCD45⁺) was measured in the peripheral blood of mice by flow cytometry weekly for 6 weeks post-adoptive transfer of PBMCs (n=9 mice across three independent experiments, data are represented as mean +/− SEM). (e) At the point of rejection, the proportion of human CD45⁺ cells of the total monocyte population (%huCD45+) was measured in the spleen, peripheral blood, and draining and contralateral lymph nodes of mice receiving an adoptive transfer of human PBMCs (n=4 mice, data are represented as mean +/− SEM). (f) Representative photomicrographs (40× magnification) of sections from human skin grafts harvested from mice 21 days after adoptive transfer of human PBMCs and stained for human CD45, CD4 and CD8. (g) Representative photomicrograph of a section from a human skin graft allowed to heal for 56 days and subsequently harvested from a mouse not receiving human cells, stained for human CD45. (h) The number of graft-infiltrating CD45⁺ human leukocytes (huCD45+ cells) was quantified by immunohistochemistry in mice receiving human PBMCs (n=3 mice) or no cells (n=3 mice). (i) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=4 mice) or 5×10⁶ PBMCs syngeneic to the skin graft (n=3 mice). One mouse in the syngeneic PBMC group required euthanasia at day 76 post-cellular adoptive transfer due to the onset of GVHD (see methods section). Data are from one skin donor/PBMC combination. (j) Analysis of the number of human CD45+ cells of the total monocyte population (huCD45+) was analysed by FACS in the skin graft-draining axillary and contralateral axillary lymph nodes in mice 21 days after receiving 5×10⁶ syngeneic (n=3 mice) or allogeneic (n=4 mice) PBMCs.

Figure 1. Skin rejection is mediated by allogeneic human PBMCs in vivo

(a) Schematic representation of the chimeric humanised mouse model. BALB/c Rag2^−/− ;Il2rγ^−/− mice receive a 1×1cm human skin transplant which is allowed to heal for 35 days before adoptive transfer of 5×10⁶ PBMCs intraperitoneally (i.p.). Skin grafts are monitored until the point of rejection, defined as complete loss. (b) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=7 mice) or no cells (n=4 mice). Data are pooled from two separate PBMC/skin donor combinations. (c) Representative photographs of skin grafts at day 100 (no cells) or rejection (PBMCs). (d) The proportion of CD45⁺ human leukocytes of the total monocyte population (% huCD45⁺) was measured in the peripheral blood of mice by flow cytometry weekly for 6 weeks post-adoptive transfer of PBMCs (n=9 mice across three independent experiments, data are represented as mean +/− SEM). (e) At the point of rejection, the proportion of human CD45⁺ cells of the total monocyte population (%huCD45+) was measured in the spleen, peripheral blood, and draining and contralateral lymph nodes of mice receiving an adoptive transfer of human PBMCs (n=4 mice, data are represented as mean +/− SEM). (f) Representative photomicrographs (40× magnification) of sections from human skin grafts harvested from mice 21 days after adoptive transfer of human PBMCs and stained for human CD45, CD4 and CD8. (g) Representative photomicrograph of a section from a human skin graft allowed to heal for 56 days and subsequently harvested from a mouse not receiving human cells, stained for human CD45. (h) The number of graft-infiltrating CD45⁺ human leukocytes (huCD45+ cells) was quantified by immunohistochemistry in mice receiving human PBMCs (n=3 mice) or no cells (n=3 mice). (i) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=4 mice) or 5×10⁶ PBMCs syngeneic to the skin graft (n=3 mice). One mouse in the syngeneic PBMC group required euthanasia at day 76 post-cellular adoptive transfer due to the onset of GVHD (see methods section). Data are from one skin donor/PBMC combination. (j) Analysis of the number of human CD45+ cells of the total monocyte population (huCD45+) was analysed by FACS in the skin graft-draining axillary and contralateral axillary lymph nodes in mice 21 days after receiving 5×10⁶ syngeneic (n=3 mice) or allogeneic (n=4 mice) PBMCs.

Figure 1. Skin rejection is mediated by allogeneic human PBMCs in vivo

(a) Schematic representation of the chimeric humanised mouse model. BALB/c Rag2^−/− ;Il2rγ^−/− mice receive a 1×1cm human skin transplant which is allowed to heal for 35 days before adoptive transfer of 5×10⁶ PBMCs intraperitoneally (i.p.). Skin grafts are monitored until the point of rejection, defined as complete loss. (b) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=7 mice) or no cells (n=4 mice). Data are pooled from two separate PBMC/skin donor combinations. (c) Representative photographs of skin grafts at day 100 (no cells) or rejection (PBMCs). (d) The proportion of CD45⁺ human leukocytes of the total monocyte population (% huCD45⁺) was measured in the peripheral blood of mice by flow cytometry weekly for 6 weeks post-adoptive transfer of PBMCs (n=9 mice across three independent experiments, data are represented as mean +/− SEM). (e) At the point of rejection, the proportion of human CD45⁺ cells of the total monocyte population (%huCD45+) was measured in the spleen, peripheral blood, and draining and contralateral lymph nodes of mice receiving an adoptive transfer of human PBMCs (n=4 mice, data are represented as mean +/− SEM). (f) Representative photomicrographs (40× magnification) of sections from human skin grafts harvested from mice 21 days after adoptive transfer of human PBMCs and stained for human CD45, CD4 and CD8. (g) Representative photomicrograph of a section from a human skin graft allowed to heal for 56 days and subsequently harvested from a mouse not receiving human cells, stained for human CD45. (h) The number of graft-infiltrating CD45⁺ human leukocytes (huCD45+ cells) was quantified by immunohistochemistry in mice receiving human PBMCs (n=3 mice) or no cells (n=3 mice). (i) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=4 mice) or 5×10⁶ PBMCs syngeneic to the skin graft (n=3 mice). One mouse in the syngeneic PBMC group required euthanasia at day 76 post-cellular adoptive transfer due to the onset of GVHD (see methods section). Data are from one skin donor/PBMC combination. (j) Analysis of the number of human CD45+ cells of the total monocyte population (huCD45+) was analysed by FACS in the skin graft-draining axillary and contralateral axillary lymph nodes in mice 21 days after receiving 5×10⁶ syngeneic (n=3 mice) or allogeneic (n=4 mice) PBMCs.

Figure 1. Skin rejection is mediated by allogeneic human PBMCs in vivo

(a) Schematic representation of the chimeric humanised mouse model. BALB/c Rag2^−/− ;Il2rγ^−/− mice receive a 1×1cm human skin transplant which is allowed to heal for 35 days before adoptive transfer of 5×10⁶ PBMCs intraperitoneally (i.p.). Skin grafts are monitored until the point of rejection, defined as complete loss. (b) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=7 mice) or no cells (n=4 mice). Data are pooled from two separate PBMC/skin donor combinations. (c) Representative photographs of skin grafts at day 100 (no cells) or rejection (PBMCs). (d) The proportion of CD45⁺ human leukocytes of the total monocyte population (% huCD45⁺) was measured in the peripheral blood of mice by flow cytometry weekly for 6 weeks post-adoptive transfer of PBMCs (n=9 mice across three independent experiments, data are represented as mean +/− SEM). (e) At the point of rejection, the proportion of human CD45⁺ cells of the total monocyte population (%huCD45+) was measured in the spleen, peripheral blood, and draining and contralateral lymph nodes of mice receiving an adoptive transfer of human PBMCs (n=4 mice, data are represented as mean +/− SEM). (f) Representative photomicrographs (40× magnification) of sections from human skin grafts harvested from mice 21 days after adoptive transfer of human PBMCs and stained for human CD45, CD4 and CD8. (g) Representative photomicrograph of a section from a human skin graft allowed to heal for 56 days and subsequently harvested from a mouse not receiving human cells, stained for human CD45. (h) The number of graft-infiltrating CD45⁺ human leukocytes (huCD45+ cells) was quantified by immunohistochemistry in mice receiving human PBMCs (n=3 mice) or no cells (n=3 mice). (i) Mice received a skin graft and 5×10⁶ allogeneic PBMCs (n=4 mice) or 5×10⁶ PBMCs syngeneic to the skin graft (n=3 mice). One mouse in the syngeneic PBMC group required euthanasia at day 76 post-cellular adoptive transfer due to the onset of GVHD (see methods section). Data are from one skin donor/PBMC combination. (j) Analysis of the number of human CD45+ cells of the total monocyte population (huCD45+) was analysed by FACS in the skin graft-draining axillary and contralateral axillary lymph nodes in mice 21 days after receiving 5×10⁶ syngeneic (n=3 mice) or allogeneic (n=4 mice) PBMCs.

Figure 2. Human Tregs retain suppressive capacity after ex vivo expansion

(a) Schematic representation of the Treg sorting and expansion protocol. CD127^loCD25⁺CD4⁺ cells were FACS-sorted and expanded in vitro with recombinant human IL-2 and αCD3/αCD28-microbeads. (b) Representative flow cytometry plots of the phenotypic analysis of ex vivo-expanded human Tregs. Numbers on dot plots are representative percentages in each quadrant. (c) In vitro suppression assay of expanded human Tregs. PBMCs were stimulated with αCD3/αCD28 beads (left panel) or irradiated allogeneic PBMCs (right panel) and cultured in the presence of expanded Tregs for 7 days. 3H-thymidine was added for the last 16 hours of culture (n=4-6 independent assays). (d) In vitro 3H-thymidine incorporation assay of PBMCs polyclonally-stimulated with αCD3/αCD28 beads by autologous unexpanded or expanded human Tregs.

Figure 2. Human Tregs retain suppressive capacity after ex vivo expansion

(a) Schematic representation of the Treg sorting and expansion protocol. CD127^loCD25⁺CD4⁺ cells were FACS-sorted and expanded in vitro with recombinant human IL-2 and αCD3/αCD28-microbeads. (b) Representative flow cytometry plots of the phenotypic analysis of ex vivo-expanded human Tregs. Numbers on dot plots are representative percentages in each quadrant. (c) In vitro suppression assay of expanded human Tregs. PBMCs were stimulated with αCD3/αCD28 beads (left panel) or irradiated allogeneic PBMCs (right panel) and cultured in the presence of expanded Tregs for 7 days. 3H-thymidine was added for the last 16 hours of culture (n=4-6 independent assays). (d) In vitro 3H-thymidine incorporation assay of PBMCs polyclonally-stimulated with αCD3/αCD28 beads by autologous unexpanded or expanded human Tregs.

Figure 3. Human Tregs promote the long-term survival of allogeneic human skin grafts

(a) Mice received an adoptive transfer of 5×10⁶ PBMCs with (n=4) or without (n=5) ex vivo-expanded Tregs at a 1:1 ratio. Data shown are pooled from two separate PBMC/skin donor combinations. (b) Representative photograph of a skin graft from a mouse receiving PBMCs with Tregs at day 100 post-cellular adoptive transfer. (c) Representative photomicrograph of a section from a skin graft procured at day 100 post-adoptive transfer from a mouse receiving PBMCs with Tregs, stained for human CD45. (d) The number of graft-infiltrating human leukocytes was quantified by immunohistochemistry for CD45⁺, CD4⁺ and CD8⁺ cells in samples from mice receiving PBMCs (procured at the point of rejection, n=5) or PBMCs with Tregs (procured at 100 days post-adoptive transfer, n=4). Data are represented as mean +/− SD.

Figure 3. Human Tregs promote the long-term survival of allogeneic human skin grafts

(a) Mice received an adoptive transfer of 5×10⁶ PBMCs with (n=4) or without (n=5) ex vivo-expanded Tregs at a 1:1 ratio. Data shown are pooled from two separate PBMC/skin donor combinations. (b) Representative photograph of a skin graft from a mouse receiving PBMCs with Tregs at day 100 post-cellular adoptive transfer. (c) Representative photomicrograph of a section from a skin graft procured at day 100 post-adoptive transfer from a mouse receiving PBMCs with Tregs, stained for human CD45. (d) The number of graft-infiltrating human leukocytes was quantified by immunohistochemistry for CD45⁺, CD4⁺ and CD8⁺ cells in samples from mice receiving PBMCs (procured at the point of rejection, n=5) or PBMCs with Tregs (procured at 100 days post-adoptive transfer, n=4). Data are represented as mean +/− SD.