Inhibition of spleen tyrosine kinase decreases donor specific antibody levels in a rat model of sensitization

Abstract

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.

Figure 1

Whole F344 blood transfusion to LEW rats induces an alloantibody response. Flow cytometry T lymphocyte crossmatch was performed to detect allogenic (A) IgM levels, (B) IgG levels and IgG subsets (C) in sensitized LEW rats (n = 4).

Figure 2

Fostamatinib treatment prevented production of donor specific antibody. T Lymphocyte crossmatch was performed to detect DSA levels in sensitized fostamatinib treated and vehicle treated LEW rats. (A) IgM, (B) IgG and (C–F) IgG subset production was inhibited by fostamatinib treatment at all time points measured (n = 6 rats/group). *P ≤ 0.05, **P ≤ 0.01. Blue Filled circle—vehicle, red filled triangle—fostamatinib.

Figure 3

Fostamatinib treatment had no effect on total circulating IgM and IgG. Serum was analysed by ELISA for (A) total IgM and (B) total IgG levels. There was no significant difference at any measured time point between fostamatinib treated rats and vehicle treated rats for both IgM and IgG levels (n = 6 rats/group). Blue filled circle—vehicle, red filled triangle—fostamatinib.

Figure 4

Fostamatinib treatment did not affect B cell or plasma cell populations. Flow cytometry was used to measure mature B lymphocytes populations in fostamatinib treated and vehicle treated sensitized LEW rats. Each graph shows number of cells per gram of spleen. Mature B lymphocyte numbers remained similar between treatment groups. (A) Memory cells were defined as CD45R⁺CD27⁺, (C) switched cells as CD45R⁺CD27⁺IgD⁻, (D) non-switched cells CD45R⁻CD27⁺ IgD⁺, (F) plasma cells as IgD⁻CD45R⁻IgM⁻CD138⁺. (n = 6 rats/group). Graphs show cell numbers per gram of spleen (n = 6 rats/group). Representative flow cytometry data of (B) memory B lymphocytes (E) switched and non-switched B lymphocytes (G) plasma cells. Number shown represents percentage of cells in gate. Blue filled circle—vehicle, red filled triangle—fostamatinib.

Figure 5

Fostamatinib prevents DSA production up to 5 weeks post completion of treatment. Treatment was given from day 1 to day 14 after blood transfusion. Then, the antibody levels were monitored until day 49. T lymphocyte crossmatch analysis was performed to detect DSA levels in sensitized fostamatinib and vehicle dosed LEW rats. (A) IgM and (B) IgG production was inhibited up to 5 weeks post-termination of the fostamatinib treatment regimen (n = 6 rats/group) (*P ≤ 0.05, **P ≤ 0.01). Blue filled circle—vehicle, red filled triangle—fostamatinib.

Figure 6

Delayed fostamatinib treatment reduces levels of DSA IgG in sensitised rats. T lymphocyte crossmatch analysis was performed to detect DSA levels in sensitized fostamatinib and vehicle treated LEW rats. Treatment initiation was delayed until 7 days post-transfusion. (A) IgM levels remained the same in both treatment groups. (B) Circulating allogenic IgG levels were significantly lower in fostamatinib treated rats at days 14, 17 and 21 post-transfusion. (C) From 17 days onwards IgG1 levels were significantly lower in fostamatinib treated animals, from 14 days onwards IgG2a (D) levels were significantly lower in fostamatinib treated animals, and by 21 days post-transfusion IgG2b (E) levels were significantly lower in fostamatinib treated animals. No differences were observed in IgG2c levels at any measured time point (F) (n = 6 rats/group) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Blue filled circle—vehicle, red filled triangle—fostamatinib.