Tracing Donor-MHC Class II Reactive B cells in Mouse Cardiac Transplantation: Delayed CTLA4-Ig Treatment Prevents Memory Alloreactive B-Cell Generation

Abstract

Background: The dual role of B cells as drivers and suppressors of the immune responses have underscored the need to trace the fate of B cells recognizing donor major histocompatibility complex class I and class II after allograft transplantation.

Methods: In this study, we used donor class II tetramers to trace the fate of I-E-specific B cells after immunization with BALB/c spleen cells or cardiac transplantation, in naive or sensitized C57BL/6 recipients. We combined this approach with genetic lineage tracing of memory B cells in activation-induced cytidine deaminase regulated Cre transgenic mice crossed to the ROSA26-enhanced yellow fluorescent protein reporter mice to track endogenous I-E-specific memory B cell generation.

Results: Immunization with BALB/c splenocytes or heart transplantation induced an expansion and differentiation of I-E-specific B cells into germinal center B cells, whereas BALB/c heart transplantation into sensitized recipients induced the preferential differentiation into antibody-secreting cells. A 10.8-fold increase in the frequency of I-E-specific memory B cells was observed by day 42 postimmunization. Treatment with CTLA4-Ig starting on day 0 or day 7 postimmunization abrogated I-E-specific memory B cell generation and sensitized humoral responses, but not if treatment commenced on day 14.

Conclusions: The majority of donor-specific memory B cells are generated between days 7 and 14 postimmunization, thus revealing a flexible timeframe whereby delayed CTLA4-Ig administration can inhibit sensitization and the generation of memory graft-reactive B cells.

Figure 1. Kinetics of anti-BALB/c and anti-I-E^d antibody production and endogenous B cells in sensitized mice

(A) C57BL/6 mice were sensitized with s.c. immunization with 20 million BALB/c splenocytes. Presence of anti-BALB/c antibodies was assessed by flow cytometry and anti-I-E^d antibody titers by ELISA. (B) C57BL/6 mice were injected with 20 million BALB/c or BALB.B splenocytes, and 10 days later, the total number of I-E^d-binding B cells was defined in the draining and non-draining lymph nodes (DLN, NDLN) of mice sensitized with BALB/c (I-E^d-positive) but not BALB.B (I-E^d-negative) splenocytes. (C) The percentage of I-E^d-binding B cells expressing a GC (Fas⁺GL7⁺) phenotype in the DLN, NDLN and spleen. Each dot represents an individual mouse, data are presented as mean ± SD, and statistically significant differences are indicated (*p<0.05; **p<0.01).

Figure 2. Tracking Class II-binding endogenous B cells in heart transplanted mice

On day 10 after heterotopic BALB/c heart transplant into the abdomen of C57BL/6 recipients, a significant increase in the total number (A) and percentage of I-E^d-binding B cells with a GC phenotype (B) in the draining LN (mediastinal; DLN), non-draining peripheral LNs (NDLN) and spleen. Each dot represents an individual mouse, data are presented as mean ± SD, and statistically significant differences are indicated (*p<0.05; **p<0.01).

Figure 3. A plasmablast response predominates in sensitized mice receiving BALB/c heart transplantation

(A) Experimental design of BALB/c heart transplantation into sensitized C57B/6 recipients. (B) Total number of I-E^d double positive B cells in LNs and spleen at day 6 after transplantation. (C) Only 1–7% of I-E^d double binding B cells in sensitized mice had a germinal center (Fas⁺GL7⁺) phenotype. (D) ELISPOT quantifying strong I-E^d-specific plasmablast response in sensitized HTx mice. Each dot represents an individual mouse, data are presented as mean ± SD, and statistically significant differences are indicated (*p<0.05; **p<0.01).

Figure 4. Tracking the changes in the percentage and total number of total and I-E^d memory B cells in AID-Cre-Rosa26-stop-EYFP mice

(A) Detailed gating strategy of memory B cells. (B) The percentage and total number of AID⁺ splenic B cells was not significantly different between the experimental and control naïve groups (p>0.05). (C) Mice receiving CTLA4-Ig from day 7 demonstrated reduced percentage and total number of I-E^d-specific AID⁺ memory B cells at day 42 postimmunization, compared to untreated sensitized mice. In contrast, delayed treatment with CTLA4-Ig from day 14–42 failed to show the significance when compared with no treatment sensitized mice. (C) Naïve and C57BL/6 mice at >10 weeks post-BALB/c heart transplant, the majority of AID⁺ B cells, for both bulk and I-Ad-specific, were CD73⁺CD273⁺, whereas AID⁻ B cells were CD73⁻CD273⁻. Each dot represents an individual mouse, data are presented as mean ± SD, and statistically significant differences are indicated (**p<0.01).

Figure 5. Kinetics of anti-BALB/c and anti-I-E^d antibody production during the recall response

A) Experimental design of recall response with CTLA4-Ig treatment. (B) Presence of anti-BALB/c antibodies was assessed by flow cytometry and anti-I-E^d antibody titers by ELISA. (C) On day 63, 1 week after recall, CTLA4-Ig D14+ mice showed similar I-E^d-specific antibody response compared to untreated mice, whereas CTLA4-Ig D7+ mice showed significantly reduced titer of I-E^d-specific antibodies. Each dot represents an individual mouse, data are presented as mean ± SD, and statistically significant differences are indicated (*p<0.05).