Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury

Abstract

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd^-/-) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 10³ "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2K^d MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-K^d alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2K^d-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (T_FH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a^-/- TCR75 CD4 T cells that were genetically incapable of providing T_FH cell function. The GC response was associated with affinity maturation of the anti-K^d alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by T_FH cells. Clinical strategies that target the T_FH cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

Keywords: allograft; extrafollicular B cell response; germinal center (GC); humoral allograft rejection; transplantation.

Figure 1

Development and characterization of murine model of antibody-mediated heart allograft rejection. (A) Figure is adapted from our companion paper (4). BL/6 Tcrbd^−/− recipients of BALB/c heart allografts were either unmodified (no cells group) or reconstituted the day after with either wild-type or SAP-deficient CD4 T cells from TCR transgenic Rag1^−/− TCR75 animals that recognize I-A^b-restricted H-2K${}_{54-68}^{\text{d}}$ peptide. (B) ELISA assays of serum samples obtained from BL/6 Tcrbd^−/− recipients of BALB/c hearts and reconstituted with 10³ TCR75 CD4 T cells (n = 4) shows progressive increase of anti-H-2K^d IgG alloantibody until day 100.

Figure 2

Histopathological confirmation of humoral rejection. (A) Representative photomicrographs of BALB/c hearts explanted at d50 from BL/6 Tcrbd^−/− recipients (reconstituted with 10³ wild-type [WT] TCR75 CD4 T cells) demonstrate: inflammatory infiltrates with myocyte loss and replacement fibrosis (on H&E staining); endothelial complement C4d deposition and areas of CD68⁺ (macrophage) and NK1.1⁺ (NK cell) staining, compared with grossly normal histology and negative staining in equivalent grafts from unmodified (not reconstituted) BL/6 Tcrbd^−/− recipients and BL/6 Tcrbd^−/− recipients reconstituted with Sh2d1a^−/− (SAP^−/−) TCR75 CD4 T cells. Representative photomicrographs of (B) elastin van Gieson stained sections of d50 allografts from the wild-type reconstituted group depicting fibroproliferative arterial intimal thickening, compared to control unmodified Tcrbd^−/− recipients, and (C) H&E stained sections demonstrating inflammatory infiltrates consisting predominantly of (left) polymorphs and (right) occasional plasma cells (arrows). (D) Representative photomicrographs of immunofluorescence staining showing interstitial capillary/vascular staining (arrowed) for C4d (red) and IgG deposition (green) in BALB/c cardiac allografts explanted at days 14 and 50 from BL/6 Tcrbd^−/− recipients reconstituted with 10³ WT TCR75 CD4 T cells, and at day 50 from WT reconstituted Rag2^−/− recipients. Intensity of C4d and IgG staining increased from day 14 to day 50 in Tcrbd^−/− recipients reconstituted with WT TCR75 CD4 T cells. All images are representative of at least 5 animals: scale bars–A: 50 μm (H&E, CD68, and NK1.1 images, except for the NK1.1 image of the reconstituted BL/6 Tcrbd^−/− group which is at 75 μm) or 100 μm (C4d images), B: 100 μm, C: 150 μm, and D: top row; scale bar-−50 μm; bottom row; scale bar−100 μm.

Figure 3

Germinal center responses and T_FH cell profile of adoptively transferred CD4 T cells following challenge a BALB/c heart allograft. (A) Representative confocal immunofluorescence photomicrograph of a splenic B cell follicle at d14 (left; scale bar−50 μm) and d50 (right; scale bar−250 μm) from reconstituted BL/6 Tcrbd^−/− recipients of BALB/c heart grafts stained with antibodies against B220 (B cells, blue), GL7 (GC B cells, green), and CD4 (T cells, red), demonstrating poorly defined GCs at d14 but by d50 a typical secondary follicle, with T_FH cells present within the GC (inset), readily evident. (B) Histogram of secondary follicles expressed as percentage of total follicles within d50 spleens of BL/6 Tcrbd^−/− recipients either non-reconstituted (naïve) or adoptively transferred with 1 × 10³ WT or Sh2d1a^−/− (SAP^−/−) TCR75 CD4 T cells. ^*P = 0.01, Mann-Whitney test. (C) ELISPOT assay of splenic and bone-marrow anti-K^d IgG antibody secreting cells (ASCs) 50 days after transplantation. Numbers of bone marrow ASCs in recipients reconstituted with SAP^−/− TCR75 CD4 T cells were not above background. Data represents mean ± S.E.M of a minimum of 5 animals/group, with each dot representing the biological replicate in a distinct animal; ^*1P = 0.02, ^#2P = 0.15, ^*3P = 0.02, Mann–Whitney test.

Figure 4

Characterization of allospecific B cells. (A), Splenic H-2K^d-specific B cells were identified by flow cytometric detection for binding of CD19^+ve B cells to FITC-conjugated and APC-conjugated synthetic H-2K^d tetramers in: naïve (un-reconstituted) BL/6 Tcrbd^−/− mice (n = 5); BL/6 Tcrbd^−/− mice, reconstituted with 10³ WT TCR75 CD4 T cells, 14 (n = 6) and 50 days (n = 6) after challenge with BALB/c heart allografts; and at 50 days after transplantation with BALB/c hearts and transfer of 10³ Sh2d1a^−/− TCR75 CD4 T cells (SAP^−/−; n = 5). Gated cells in middle column of representative dot plots show percentage of enriched CD19^+ve B cells binding H-2K^d tetramer; right column shows percentage of GC-specific tetramer bound CD19^+ve B cells. (B) Histogram (left)—absolute numbers of splenic H-2K^d-specific B cells (x 10⁴ cells per mouse) enumerated; histogram (right)—percentage of H-2K^d-specific B cells expressing FAS^hiGL7^+ve GC phenotype. Data represents mean ± S.E.M, with each dot representing the biological replicate in a separate animal; ^*1P = 0.01, ^*2P = 0.004, ^*3P = 0.02 ^*4P = 0.004 Mann-Whitney test. (C) Total numbers of splenic CD4 T cells (left histogram) and proportion displaying PD1⁺ CXCR5⁺ T_FH cell phenotype (right histogram) as determined by flow cytometry analysis at indicated time points following transplantation of Tcrbd^−/− recipients with a BALB/c heart graft and reconstitution with 10³ WT TCR75 CD4 T cells. Representative flow cytometry plot of unchallenged, but adoptively transferred (with 5 × 10⁵ TCR75 CD4 T cells) BL/6 Tcrbd^−/− mouse (left) and transplanted WT reconstituted recipient (right).

Figure 5

Germinal center alloantibody responses mediate chronic allograft rejection. (A) Representative low magnification confocal immunofluorescence photomicrographs of splenic B cell follicles stained with antibodies against B220 (B cells, blue) and GL7 (GC B cells, green) at d50 from BL/6 Tcrbd^−/− recipients of BALB/c heart grafts adoptively transferred with 10³ Sh2d1a^−/− (left) or wild-type (right) TCR75 CD4 T cells (scale bar – 200 μm). (B) Anti-H-2K^d alloantibody in recipients with 10³ SAP^−/− TCR75 CD4 T cells (n = 7) was markedly diminished compared to recipients given WT TCR75 CD4 T cells (n = 7); ^*P < 0.001, two-way ANOVA. (C) Whereas BALB/c heart allografts survived long-term in BL/6 Tcrbd^−/− recipients reconstituted with 10³ SAP^−/− TCR75 CD4 T cells (n = 7), allografts were rejected in recipients given 1 × 10³ WT (SAP^+/+) TCR75 CD4 T cells (n = 7, MST-50 days; P < 0.001, log-rank test). (D) Severity of vasculopathy (left) and parenchymal damage (right) in BALB/c hearts explanted: from unmodified (non-reconstituted) BL/6 Tcrbd^−/− mice (day 50, n = 4); at d14 (n = 4) and d50 (n = 8) from BL/6 Tcrbd^−/− recipients reconstituted with WT TCR75 CD4 T cells; at d50 from Rag2^−/− recipients reconstituted with WT TCR75 CD4 T cells (n = 5); at d50 from BL/6 Tcrbd^−/− recipients reconstituted with 10³ SAP^−/− TCR75 CD4 T cells (n = 5) and at day 50 from Rag2^−/− SWHEL recipients simultaneously transplanted with BALB/c heart and mHEL-Kd skin graft and reconstituted with 10³ TCR75 CD4 T cells (n = 5). Each dot represents a single animal; Left: ^*1P = 0.001, ^*2P = 0.008, ^*3P = 0.003, ^#4P = 0.29, Right: ^*1P = 0.008, ^*2P = 0.005, ^*3P = 0.003, ^#4P = 0.18; two-tailed Student's t-test for normally distributed and Mann-Whitney tests for non-parametric data. (E) Representative confocal immunofluorescence photomicrographs of splenic cryostat sections stained with antibodies against B220 (B cells, green) and CD4 (red) at day 50 after transplantation of BALB/c heart grafts into BL/6 Tcrbd^−/− recipients reconstituted with no cells (left), 10³ Sh2d1a^−/− (middle) or wild-type (right) TCR75 CD4 T cells (scale bar – 50 μm).

Figure 6

Contribution of alloantibody to allograft rejection. (A) Elastin van Gieson and H&E stained sections of d50 BALB/c heart allografts from reconstituted Rag2^−/− recipients with 10³ TCR75 CD4 T cells demonstrate focal areas of mild concentric vasculopathy and lymphocytic infiltrates (EVG) within well-preserved vasculature and myocardium (H&E), with absence of capillary C4d staining and NK cell and macrophage infiltration. Scale bars: H&E, CD68, and NK1.1–50 μm; EVG−65 μm; C4d−100 μm. (B) Model to distinguish alloantibody production from alloantigen presentation. (i) BALB/c heart graft and BL/6 mHEL-K^d skin graft which co-express membrane-bound Hen Egg Lysosyme (HEL, black triangle) and MHC class I H-2K^d (white semicircle) were engrafted to a Rag2^−/− SW_HEL recipient, simultaneously reconstituted with TCR75 CD4 T cells. Critically, on the Rag2^−/− background, VDJ recombination is not possible and all B cells express a single HEL-specific BCR. (ii) Following challenge with a BL/6 mHEL-K^d skin graft, SW_HEL B cell recognition of mHEL target antigen on the skin graft is expected to result in additional internalization and processing of the H-2K^d alloantigen co-expressed on the surface of donor cells (step 1). CD4 T cell help is limited to the adoptively-transferred TCR75 CD4 T cells that recognize processed H-2K^d allopeptide. Hence, generation of an Ig-switched anti-HEL antibody response is dependent upon presentation of co-internalized H-2K^d antigen by the SW_HEL B cell for receipt of cognate help from the TCR75 CD4 T cell (step 2). This results in generation of class-switched anti-HEL alloantibody (step 3). (C) Rag2^−/−SW_HEL recipients reconstituted with 10³ TCR75 CD4 T cells and simultaneously transplanted with a BALB/c heart and mHEL-K^d skin graft developed strong anti-HEL IgG (continuous lines), and undetectable anti-H-2K^d IgG (dotted lines). Data represent mean and S.E.M of n = 6 mice/group. [(D) Representative photomicrographs of H&E stained sections of d50 heart allografts [in (C)] depicting only mild vasculopathy and parenchymal injury (scale bar: 50 μm)].

Figure 7

Alloantibody mediates endothelial activation and proliferative vasculopathy. (A) Representative photomicrograph of scratch-wound assay (left) and histogram (right) demonstrating enhanced migration of BALB/c endothelial cells (ECs) upon incubation with day 50 serum sampled from BL/6 Tcrbd^−/− reconstituted with wild-type (WT) or SAP^−/− TCR75 CD4 T cells, whereas BL/6 EC migration was not above background (mean values, with each dot representing an average of six high power fields analyzed per biological replicate). Migration of BALB/c ECs in response to sera from d14 WT or d50 SAP^−/− was significantly weaker compared to serum from d50 WT recipients; ^*P = 0.01 and ^**P = 0.02, Mann–Whitney tests. (B) Quiescent BALB/c endothelial cells stimulated with media containing epidermal growth factor (EGF) or column-purified sera from: d50 non-reconstituted BL/6 Tcrbd^−/− recipients of BALB/c grafts (negative control), d14 WT, d50 WT, d50 SAP^−/− recipients, and pooled hyperimmune anti-H-2K^d IgG serum (BALB/c skin grafts [SG] to BL/6 mice, positive control). Cell lysates were separated and immunoblotted with anti-Akt ^Ser473, anti-phospho-Akt ^Ser473, and GAPDH (control) mAb. Displayed Western blot represents three independent experiments. (C) Representative photomicrographs of coronary arteries from allografts explanted at day 50 and double immunolabeled with endothelial marker CD31 (green) and α-smooth muscle actin (α-sm actin, red); scale bar−200 μm. Increased neointimal lesions in chronically rejecting BALB/c allografts containing a large number of α-actin–positive smooth muscle cells were observed in BL/6 Tcrbd^−/− recipients reconstituted with 10³ TCR75 T cells (middle column) but not with SAP^−/− TCR75 CD4 T cells (right); Tcrbd^−/− syngeneic grafts shown as comparison (left). (D) Representative photomicrographs of endomyocardial biopsies of human heart allografts in recipients with antibody-mediated rejection and double-immunostained with the endothelial marker CD31 (brown) and α-smooth muscle actin (red); magnification x25. Pronounced neointimal (NI) lesions, composed of dense smooth muscle cells (pink), lying between the media (M) and arterial lumen (L). Remnants of endothelial cell layer seen within neointima (arrows, right image). Images representative of staining patterns observed in three individual patients. (E) Ranking anti-H-2K^d alloantibody dissociation kinetics. Left: dot plot histogram comparing anti-H-2K^d alloantibody dissociation constants (k_off) among BL/6 Tcrbd^−/− recipients of BALB/c hearts reconstituted with either WT or SAP^−/− TCR75 CD4 T cells (at d9, 14, and 50 as depicted). Each value (mean ± SEM) represents the k_off after global fit of two serum dilutions of the same biological replicate (n = 3); ^#1P = 0.21, ^*2P = 0.01, ^*3P = 0.008, ^*4P = 0.02, ^#5P = 0.06, ^#6P = 0.05, ^#7P = 0.01; two-tailed Student's t-test. Right: graphs depict sensograms of anti-H-2K^d antibody dissociation rates in sera from BL/6 Tcrbd^−/− recipients of BALB/c hearts reconstituted as shown, with each curve representing individual recipient mice.