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. 2017 Jan;101(1):63-73.
doi: 10.1097/TP.0000000000001258.

Preemptive CD20+ B Cell Depletion Attenuates Cardiac Allograft Vasculopathy in CD154-Treated Monkeys

Free PMC article

Preemptive CD20+ B Cell Depletion Attenuates Cardiac Allograft Vasculopathy in CD154-Treated Monkeys

Agnes M Azimzadeh et al. Transplantation. .
Free PMC article


Background: Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive "induction" B cell depletion.

Methods: αCD154 (IDEC-131)-treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (αCD20) alone or with rabbit antihuman thymocyte globulin.

Results: Relative to previously reported reference groups, αCD20 significantly prolonged survival, delayed Ab detection, and attenuated CAV within 3 months in αCD154-treated recipients (αCD154 + αCD20 graft median survival time > 90 days, n = 7, vs 28 days for αCD154 alone (IDEC-131), n = 21; P = 0.05). Addition of rabbit antihuman thymocyte globulin to αCD154 (n = 6) or αCD154 + αCD20 (n = 10) improved graft protection from graft rejection and failure during treatment but was associated with significant morbidity in 8 of 16 recipients (6 infections, 2 drug-related complications). In αCD20-treated animals, detection of antidonor Ab and relatively severe CAV were anticipated by appearance of CD20 cells (>1% of lymphocytes) in peripheral blood and were associated with low αCD154 trough levels (below 100 μg/mL).

Conclusions: These observations support the hypothesis that efficient preemptive "induction" CD20 B cell depletion consistently modulates pathogenic alloimmunity and attenuates CAV in this translational model, extending our prior findings with calcineurin inhibitors to the context of CD154 blockade.

Conflict of interest statement

The authors declare no conflicts of interest.


Figure 1
Figure 1. Primary graft survival by treatment regimen
Cynomolgus monkey recipients of MHC- mismatched heterotopic cardiac allografts were treated with αCD154 alone (blue line) or with additional rATG (green line), or αCD20 (red line), or both antibody preparations (black line). Graft function was monitored by telemetric ECG and pressure waveforms. Historical controls that were either untreated or given αCD20 alone are not shown (see Table 1, Refs and 26). Clinical rejection was treated in some animals as indicated in Table 1. Addition of αCD20 to αCD154 prolonged rejection-free primary graft survival (P < 0.05 vs. αCD154 alone). Numbers in parentheses indicate grafts without clinical rejection by 90 days/uncensored grafts at risk.
Figure 2
Figure 2. Alloantibody production in various treatment groups
IgM (left) and IgG (right) anti-donor antibodies were measured by flow cytometry in serum collected serially (weekly for a month, bi-weekly thereafter) after cardiac allotransplantation as described in Methods. With addition of αCD20, alloantibody was detected (levels repeatedly above 20%) only when αCD154 antibody levels were low and/or B cell depletion less durable.
Figure 3
Figure 3. B cell depletion inhibits cardiac allograft vasculopathy (CAV)
A. Representative pictures from cardiac allografts explanted at graft failure (M350, M643, αCD154 therapy, original magnification ×100) or at approximately day 90 (MA008, MB301, αCD154+αCD20 therapy, original magnification ×200) and stained with H&E. Vascular intimal proliferative lesions are prominent with αCD154 monotherapy, but not with additional αCD20. Perivascular cellular infiltration and some degree of hemorrhage and endothelial activation were detected in rejected allografts, consistent with cellular and humoral immune injury. B. Median quantitative severity scores are shown for explanted grafts surviving for >21 days that were treated with αCD154 (n=11) and rATG (n=5), αCD20 (n=6), or αCD20+rATG. *p<0.05 vs αCD154 alone.
Figure 4
Figure 4. Immunochemistry analysis of graft-infiltrating B cells
Explanted allograft specimens were stained for CD20 and scored as previously. A. Representative pictures for explanted control (M364, M10670) or αCD20-treated grafts (DJ2F1, MA008). Presence of intra-graft B cells at d84 in DJ2F1 correlated with graft failure and CAV lesions (score: 1.8), detectable alloantibody and blood B cells (30% of baseline on d84) while absence of intra-graft B cells at d90 in MA008 was associated with graft protection (CAV score: 0), absence of alloantibody, and low blood B cell levels (3.6% of baseline, 39/µl). Original magnification ×100. B. CD20 intra-graft score across treatment regimens for all specimens evaluated. Each dot represents the average score for 1 cardiac allograft; horizontal bar, group median. *P<0.05 vs. untreated controls.
Figure 5
Figure 5. B cell depletion increased serum BAFF levels
Serum BAFF levels were measured at d0, 7, 14, 21, 28, 35, ~50, 64, 84 and 90 in recipients of a cardiac transplantation. BAFF levels increased after transplantation in all groups, but the increase was more modest in untreated controls (3.8±1.8 fold on d7 vs. baseline, n=5) compared to αCD20 monotherapy (11.3±4.5, n=2, p=0.95). BAFF increased to similar levels as untreated controls in graft recipients treated with αCD154 with or without ATG (3.4±0.8 fold on d28 vs. baseline, n=5), whereas levels were dramatically increased with αCD20-based regimens (12.8±7.7, n=7, p=0.03). The longitudinal profile of BAFF levels in B cell-depleted animals was very heterogeneous, and no obvious correlation was found between levels or kinetics of BAFF and alloantibody or CAV. Therefore, B cell depletion was associated with an increase in BAFF levels compared to reference groups.

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