Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection

Abstract

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd^-/-) C57BL/6 recipients against donor H-2K^d MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2K^d allopeptide via the indirect-pathway. Transfer of large numbers (5 × 10⁵) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2K^d humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2^-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd^-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2^-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a^-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd^-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (10³), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

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

Figure 1

Pattern of de novo alloantibody development after kidney transplantation. The development of donor specific alloantibodies (DSA) against donor HLA antigen in eight human kidney transplant recipients (representative of a general cohort that were known to have DSA and whose alloantibody profile was serially assessed) was characterized by single-antigen HLA bead analysis of recipient sera. Recipient sera were analyzed for DSA until day 500 post-transplant; data is presented for the first 300 days, with cases 1 and 4 showing a mean fluorescence intensity of 733 at day 503 and 640 at day 386, respectively. Two general patterns of DSA formation were identified: very high levels of DSA which were relatively short-lived; and DSA levels which plateaued and remained at appreciable levels for a much longer period.

Figure 2

Development and characterization of murine model of antibody-mediated heart allograft rejection. (A) BL/6 Tcrbd^−/− recipients of BALB/c heart allografts were either unmodified (no cells group) or reconstituted the day after with 10², 10³ (help-limited) or 5 × 10⁵ (help-unlimited) CD4 T cells from TCR transgenic Rag1^−/− TCR75 animals, which recognize I-A^b -restricted H-2K^d ₅₄₋₆₈ peptide. (B) Development of anti-H-2K^d IgG antibody in reconstituted recipients (mean and S.E.M of n = 5 mice/group, ^*P < 0.001 for 5 × 10⁵ vs. 10³; p = 0.08 for 10³ vs. 10² T cells, two-way ANOVA). (C) Reconstitution with limiting numbers of TCR75 CD4 T cells resulted in gradual allograft failure (MST = 50 days; n = 10), whereas grafts rejected acutely in help-unlimited recipients (MST = 9 days, P < 0.001, log-rank test; n = 10). Unmodified BL/6 Tcrbd^−/− recipients that were not reconstituted (no cells, n = 6) did not reject the heart allograft.

Figure 3

Histopathological confirmation of humoral rejection in help-unlimited recipients. (A) Day 6 BALB/c hearts explanted from BL/6 Tcrbd^−/− recipients reconstituted with 5 × 10⁵ TCR75 CD4 T cells demonstrated (left) widespread myocyte death (loss of striation), endothelial plumping and peri-vascular edema, and (right, arrows) strong endothelial complement C4d deposition. (B) Representative photomicrographs of immunofluorescence staining showing interstitial capillary staining for C4d (red, left); scale bar−50 μm and IgG deposition (green, right; scale bar−50 μm).

Figure 4

Extrafollicular and germinal center humoral responses during acute AMR. (A) Histogram of secondary (GL7^+ve) splenic follicles expressed as percentage of total follicles within spleens of BL/6 Tcrbd^−/− recipients reconstituted with 5 × 10⁵ of WT or Sh2d1a^−/− (SAP^−/−) TCR75 CD4 T cells. ^*P = 0.03, two-tailed Student's t-test. (B) Representative confocal imaging of splenic sections of help-unlimited BL/6 Tcrbd^−/− recipients; at day 10 (top panel; scale bar−500 and 250 μm, respectively), IgG-switched B cells (green) are located predominantly in the extrafollicular space, close to the marginal sinus (MadCAM-1; red), whereas at day 50 (bottom panel; scale bar–250 μm), IgG switched cells and GL7⁺ GCs (green) are located within the follicle (B220; blue). Data represents mean ± S.E.M of a minimum of 5 animals/group, with each dot representing the biological replicate in a distinct group.

Figure 5

AMR is mediated by extrafollicular alloantibody responses in help-unlimited recipients. BL/6 Tcrbd^−/− recipients were reconstituted with 5 × 10⁵ wild-type (help-unlimited WT) TCR75 CD4 T cells (n = 7) or 5 × 10⁵ Sh2d1a^−/− (help unlimited SAP^−/−) TCR75 CD4 T cells (n = 4) at challenge with a BALB/c heart allograft. (A) Compared with the WT group (copied from Figure 2B for ease of comparison), anti-H-2K^d IgG alloantibody responses in the help-unlimited SAP^−/− group were not sustained. ^*P < 0.0001 two-way ANOVA. (B) Representative immunofluorescent staining of day 50 splenic sections from help-unlimited SAP^−/− and WT recipients, confirming an absence of GL7^+ve (green) GC activity in the SAP^−/− group: scale bar−250 μm. (C) ELISPOT assay of splenic and bone-marrow (BM) anti-K^d IgG antibody secreting cells (ASCs) 50 days after transplantation. Numbers of BM ASCs in recipients reconstituted with SAP^−/− TCR75 CD4 T cells were not above background; ^*P = 0.03, ^**P = 0.004, Mann-Whitney test. (D) Splenic H-2K^d -specific B cells were identified by flow cytometric detection of binding of CD19^+ve B cells to FITC-conjugated and APC-conjugated synthetic H-2K^d tetramers in naïve unchallenged BL/6 Tcrbd^−/−, help-unlimited WT and help-unlimited SAP^−/− recipients, 6–7 weeks after challenge with a BALB/c graft. Gated cells in middle and right column of representative dot plots show percentage of enriched CD19^+ve B cells binding H-2K^d tetramer, and percentage of GC-specific (FAS^hiGL7^pos) tetramer bound CD19^+ve B cells, respectively. Whereas, transplantation provoked expansion of the H-2K^d specific B cell population in the help unlimited SAP^−/− recipients, GC B cells were not detectable. (E) BALB/c heart allografts were rejected acutely in the help-unlimited WT (n = 7, MST-9 days) and help unlimited SAP^−/− (n = 4, MST-13.5 days) recipient groups. (F) Day 10 BALB/c hearts explanted from help unlimited SAP^−/− recipients showing widespread myocyte death (loss of striation pattern), but an absence of cellular lymphocytic infiltration.

Figure 6

Alloantibody mediates endothelial activation and complement deposition. (A) Representative photomicrographs of immunofluorescence staining with no evidence of interstitial capillary staining for C4d (red, left) and IgG deposition (green, right; scale bars−50 μm) in BALB/c cardiac allografts explanted (at day 50) from Rag2^−/− recipients reconstituted with 5 × 10⁵ TCR75 CD4 T cells. (B) Anti-H-2K^d IgG antibody (mean ± S.D.) in Rag2^−/− recipients of BALB/c allografts injected with day 50 serum pooled from either help-unlimited BL/6 Tcrbd^−/− (immune serum, n = 5) or help-unlimited Rag2^−/− recipients (control serum, n = 5). (C) Transfer of immune serum led to acute graft loss, whereas transfer of control serum resulted in indefinite heart allograft survival (P < 0.001, log-rank test). (D) Histology of explanted hearts (day 7) demonstrated widespread myocyte damage and endothelial C4d deposition following transfer of immune serum (left), in comparison with normal histology and negative staining at day 30 following control serum transfer (right). Images are representative of 3 animals; scale bars−100 μm (H&E) and 150 μm (C4d).

Figure 7

Numbers of antigen-specific B cells and helper T cells determine the size of the germinal center vs. extrafollicular response. Humoral responses in BL/6 Tcrbd^−/− mice immunized with Hen Egg Lysosyme (HEL) protein and adoptively transferred with different numbers of HEL-specific SW_HEL B cells and TCR7 CD4 T cells. (A) Anti-HEL IgG (mean ± S.E.M, with each dot representing the biological replicate in a distinct animal) measured 1 and 3 weeks after immunization; ^*1P = 0.01, ^*2P = 0.04 Mann-Whitney test. (B) ELISPOT assay of splenic and bone marrow anti-HEL IgG antibody-secreting cells revealed abundant numbers after 1 week in mice adoptively transferred with large (10⁵) numbers of TCR7 CD4 T cells; ^#P = 0.06, Mann-Whitney test. (C) Representative confocal photomicrographs (scale bars-−500 μm) of immunofluorescence staining of splenic sections at 1 and 3 weeks after immunization, depicting B220 B cells (blue) and IgG positive (green) antibody secreting cells: numbers of adoptively transferred TCR7 T cells and SW_HEL B cells as indicated. IgG secreting cells are readily identified within extrafollicular foci at week 1 in mice transferred with large numbers (10⁵) of TCR7 CD4 T cells. (D) Representative confocal photomicrographs depicting HEL-specific germinal center activity (B220 B cell—Blue; GL7—green; HEL binding—red) at 3 weeks following HEL challenge in mice adoptively transferred with HEL-specific CD4 T and B cells, as indicated (left; scale bar−100 μm; right; scale bar-−20 μm). (E) Frequency of HEL-specific GL7⁺ GCs detected by immunofluorescence (expressed as percentage of total follicles within spleens of recipient BL/6 Tcrbd^−/− mice). Very few HEL-specific GCs were present at 1 week, but were clearly present by 3 weeks in the group that received proportionally greater numbers of HEL-specific B cells (and low numbers of HEL-specific CD4 T cells); ^*1P = 0.04, ^*2P = 0.03 Mann-Whitney test. (F) Flow cytometric enumeration (see Figure S3) of splenic HEL-specific B cells (left histogram) and percentage with GC (FAS^hi GL7^+ve) phenotype (right histogram), 3 weeks after HEL-challenge in mice reconstituted with HEL-specific SW_HEL B cells and TCR7 CD4 T cells, as indicated. Left histogram, ^*P = 0.05 and ^**P = 0.41; right histogram, ^*P = 0.09 and ^**P = 0.003; two-tailed Student's t-test.