Bronchus-associated lymphoid tissue-resident Foxp3+ T lymphocytes prevent antibody-mediated lung rejection

Abstract

Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that Foxp3+ T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus-associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident Foxp3+ T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident Foxp3+ T lymphocytes resulted in prolonged interactions between B and CD4+ T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory Foxp3+ T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses.

Keywords: Immunology; Organ transplantation; T cells; Tolerance; Transplantation.

Conflict of interest statement

Conflict of interest: DK has a pending patent entitled “Compositions and methods for detecting CCR2 receptors” (application number 15/611,577). DK also serves on the Scientific Advisory Board of Compass Therapeutics and has received research support from Compass Therapeutics.

Figures

Figure 1. Long-term acceptance after lung transplantation is associated with induction of Foxp3+ cell–rich BALT.

(A) Gross and (B) histological appearance (H&E) of BALB/c lung graft (Tx) at least 30 days after transplantation into an immunosuppressed B6 host. Arrow depicts induced BALT (n = 8). Scale bar: 100 μm. (C) PNAd staining (brown), (D) MT staining (blue), and (E) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 host. Scale bars: 100 μm. (F) Intravital 2-photon (2P) imaging depicting aggregates of Foxp3+ cells in BALB/c lung graft at least 30 days after transplantation into an immunosuppressed B6 Foxp3-IRES GFP recipient (Foxp3+ cells, green; quantum dot–labeled vessels, red) (n = 3). Scale bar: 30 μm.

Figure 2. Depletion of graft-resident Foxp3+ T cells triggers AMR.

(A) Schematic depicting experimental model. CSB, costimulation blockade. Plots and quantification of CD4+Foxp3+ cells from (B) primary (CD45.2) or (C) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed WT B6 (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and retransplanted into DT-treated B6 (CD45.1) hosts at least 30 days later. Plots are gated on live CD45.2+CD45.1–CD90.2+ and live CD45.2–CD45.1+CD90.2+ cells. (D) Plots and quantification of distribution of CD45.1 vs. CD45.2 on live CD90.2+CD4+CD8–Foxp3+ cells without and with depletion of graft-resident (CD45.2) Foxp3+ cells. Gross and histological appearance (H&E) of BALB/c lungs, transplanted into immunosuppressed WT (E and G) or Foxp3-DTR (F and H) B6 CD45.2+ mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1+ hosts (n = 4 mice per group). (I) Airway epithelium (arrow) in human lung diagnosed with AMR (H&E) (n = 11). Staining of (J and K) MT (blue), (L and M) CCSP (red), AcT (green), and (N and O) C4d (brown) in BALB/c lungs, transplanted into immunosuppressed (J, L, and N) WT or (K, M, and O) Foxp3-DTR B6 CD45.2+ mice and, at least 30 days later, retransplanted into DT-treated B6 CD45.1+ hosts. Scale bars: 100 μm. Arrows in G, J, and L point to BALT. (P) Donor-specific IgM antibody titers 7 days after retransplantation of BALB/c lungs into DT-treated B6 CD45.1+ hosts at least 30 days after initial engraftment into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice. (Q) Reactivity of serum IgM antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen (n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.

Figure 3. Graft-resident Foxp3+ T cells maintain lung transplant tolerance.

(A and E) Gross and (B and F) histological appearance (H&E), (C and G) MT staining (blue), and (D and H) immunofluorescent staining of CCSP (red) and AcT (green) in BALB/c lungs, initially transplanted into immunosuppressed (A–D) B6 (CD45.2+) or (E–H) B6 Foxp3-DTR (CD45.2+) mice and retransplanted into DT-treated secondary B6 (CD45.1+) hosts at least 30 days after primary transplantation. Secondary hosts were examined 30 days after retransplantation. Scale bars: 100 μm. Arrows in B and C point to BALT. Serum titers of donor-specific (I) IgM and (J) IgG antibodies 30 days after retransplantation of BALB/c lungs, initially transplanted into immunosuppressed B6 WT (red) or B6 Foxp3-DTR (blue) mice and, at least 30 days later, retransplanted into B6 45.1+ hosts. (K) Reactivity of serum IgG antibodies, following depletion of graft-resident Foxp3 cells, against donor (BALB/c), recipient (B6), and third-party (CBA) antigen. Data are expressed as mean ± SEM (n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Figure 4. Graft-infiltrating B cells trigger AMR after depletion of graft-resident Foxp3+ T cells.

Activated B cells from (A and B) primary recipient (CD45.2) or (C and D) secondary recipient (CD45.1) in BALB/c lungs, transplanted into immunosuppressed (A and C) B6 (CD45.2+) (no Foxp3 depletion) or (B and D) B6 Foxp3-DTR (CD45.2+) mice (Foxp3 depletion) and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1+) hosts. Plots are gated on live CD45.2+CD45.1–B220+ (donor) and live CD45.2–CD45.1+B220+ cells (recipient). Quantification of activated (E) CD45.2 and (F) CD45.1 B cells in (circles) control and (inverted triangles) Foxp3+ T cell–depleted lungs 7 days after retransplantation. (G) Gross, (H) histological appearance (H&E), (I) MT staining (blue), and (J) CCSP (red) and AcT (green) staining in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 muMt– hosts. (K) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or muMt– (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. (L) Gross and (M) histological appearance (H&E) of BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 AID/μS knockout hosts. Scale bars: 100 μm. (N) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or AID/μS knockout (red) B6 hosts at least 30 days after engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM (n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Figure 5. AMR after depletion of graft-resident Foxp3+ T cells is dependent on graft infiltration by T cells.

Plots and histograms depicting markers characteristic of Tfh cells on CD4+ T cells derived from (A and C) primary (CD45.2) or (B and D) secondary (CD45.1) recipients in BALB/c lungs, initially transplanted into immunosuppressed (A and B) B6 (CD45.2+) or (C and D) B6 Foxp3-DTR (CD45.2+) mice and, at least 30 days later, retransplanted into DT-treated B6 (CD45.1+) hosts. Plots are gated on live CD45.2+CD45.1–CD90.2+CD4+CD8–Foxp3– and CD45.2+CD45.1+CD90.2+CD4+CD8–Foxp3– cells. Histograms are gated on CD4+ T cells that are PD-1hiCXCR5+ (bcl-6, red; isotype control, blue) (n = 4 each). Quantification of (E) CD45.2+ and (F) CD45.1+ CD4+ T cells that are PD-1hiCXCR5+ in (circles) control and (inverted triangles) Foxp3+ T cell–depleted lungs 7 days after retransplantation. (G) Gross and (H) histological appearance (H&E) and staining for (I) MT, (J) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT-treated B6 nude hosts. Scale bars: 100 μm. (K) Donor-specific IgM titers 7 days after retransplantation of BALB/c lungs into DT-treated WT (blue) or nude (red) B6 hosts at least 30 days after initial engraftment into immunosuppressed B6 Foxp3-DTR mice. Data are expressed as mean ± SEM (n = 4 mice per group). Mann-Whitney U test was used to compare the means.

Figure 6. Foxp3+ T lymphocyte depletion–triggered AMR is dependent on CXCL13-mediated chemokinesis.

(A) Foxp3+ (green), B cells (blue), and CD4+ T cells (red) in BALB/c lungs at least 30 days after transplantation into immunosuppressed B6 Foxp3-IRES GFP recipient (n = 3). Scale bar: 10 μm. CXCR5+ B cells from secondary host (recipient) in BALB/c lungs, initially transplanted into immunosuppressed (B) WT or (C) Foxp3-DTR B6 (CD45.2+) recipient and, at least 30 days later, retransplanted into DT-treated B6 CD45.1+ hosts. Plots are gated on live CD45.2–CD45.1+ cells. (D) CD45.1+CXCR5+ B cells in (circles) control and (inverted triangles) Foxp3+ T cell–depleted lungs 7 days after retransplantation (n = 4 each). CXCL13 (brown) in (E) control and (F) Foxp3+ T cell–depleted grafts 7 days after retransplantation. Scale bars: 100 μm. CD4+ T cells (green) and B cells (blue) in BALB/c lungs, initially transplanted into immunosuppressed B6 Foxp3-DTR recipients and, at least 30 days later, retransplanted into B6 hosts, treated with (G) DT/control-Ig (arrows: CD4+ T–B cell interactions) or (H) DT/anti-CXCL13 (n = 2 each) (red, quantum dots). Scale bars: 20 μm. (I) Contact duration between CD4+ T and B cells, (J) CD4+ T, and (K) B cell mean square displacements and (L) CD4+ T and (M) B cell velocities within retransplanted Foxp3+ T cell–depleted BALB/c lungs with and without CXCL13 inhibition. (N) Gross, (O) histological appearance (H&E), staining for (P) MT (blue), (Q) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT- and anti-CXCL13–treated B6 hosts. Scale bars: 100 μm. (R) Donor-specific IgM titers after retransplantation of BALB/c lungs into DT-treated control (blue) or DT/anti-CXCL13 antibody–treated (red) B6 recipients after initial engraftment into immunosuppressed B6 Foxp3-DTR mice (n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.

Figure 7. AMR of Foxp3+ T cell–depleted lung grafts can be prevented by blocking ICOS/ICOS ligand and CD40/CD40 ligand pathways.

(A) Expression levels of ICOS, ICOS ligand (ICOSL), CD40, CD40 ligand (CD40L), and IL-21 in BALB/c lung grafts, which were initially transplanted into immunosuppressed B6 WT (CD45.2) (circles) or Foxp3-DTR B6 (CD45.2) (inverted triangles) mice and at least 30 days later retransplanted into DT-treated (days 0 and 1) B6 CD45.1 hosts. Grafts were examined 7 days after retransplantation (n = 5 each). **P < 0.01. (B) Gross appearance, (C) histological appearance (H&E), (D) MT (blue) staining, (E) and immunofluorescent staining of CCSP (red) and AcT (green) after depletion of graft-resident Foxp3+ T cells and 7 days after retransplantation into anti-ICOS ligand and anti-CD40 ligand–treated secondary hosts. Scale bars: 100 μm. (F) Serum titers of donor-specific IgM 7 days after retransplantation of BALB/c lungs, initially transplanted into immunosuppressed B6 Foxp3-DTR mice and at least 30 days later retransplanted into DT-treated WT B6 hosts that were treated with anti-ICOS ligand and anti-CD40 ligand (red) or did not receive treatment (blue) (n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.