In vivo effects of horse and rabbit antithymocyte globulin in patients with severe aplastic anemia

Abstract

We recently reported that rabbit antithymocyte globulin was markedly inferior to horse antithymocyte globulin as a primary treatment for severe aplastic anemia. Here we expand on our findings in this unique cohort of patients. Rabbit antithymocyte globulin was detectable in plasma for longer periods than horse antithymocyte globulin; rabbit antithymocyte globulin in plasma retained functional capacity to bind to lymphocytes for up to 1 month, horse antithymocyte globulin for only about 2 weeks. In the first week after treatment there were much lower numbers of neutrophils in patients treated with rabbit antithymocyte globulin than in patients receiving horse antithymocyte globulin. Both antithymocyte globulins induced a "cytokine storm" in the first 2 days after administration. Compared with horse antithymocyte globulin, rabbit antithymocyte globulin was associated with higher levels of chemokine (C-C motif) ligand 4 during the first 3 weeks. Besides a much lower absolute number and a lower relative frequency of CD4(+) T cells, rabbit antithymocyte globulin induced higher frequencies of CD4(+)CD38(+), CD3(+)CD4(-)CD8(-) T cells, and B cells than did horse antithymocyte globulin. Serum sickness occurred around 2 weeks after infusion of both types of antithymocyte globulin. Human anti-antithymocyte globulin antibodies, especially of the IgM subtype, correlated with serum sickness, which appeared concurrently with clearance of antithymocyte globulin in blood and with the production of cytokines. In conclusion, rabbit and horse antithymocyte globulins have very different pharmacokinetics and effects on neutrophils, lymphocyte subsets, and cytokine release. These differences may be related to their efficacy in suppressing the immune system and restoring hematopoiesis in bone marrow failure. Clinicaltrials.gov identifier: NCT00260689.

Figure 1.

Pharmacokinetics of rabbit ATG and horse ATG. (A) Concentrations of rATG (n=39) and hATG (n=43) detected by ELISA. ELISA plates were coated with rabbit anti-horse IgG (Fab’2) or chicken anti-rabbit IgG to capture hATG and rATG in the plasma, respectively. The bars represent mean ± standard error (SE). (B) Western blotting for the different types of ATG in the plasma. One microliter of plasma obtained from patients at different time points was used for western blotting. Representative data from at least three experiments with similar results are shown. (C) Binding of rATG and hATG in patients’ plasma to lymphocytes from healthy volunteers. Representative data from at least three experiments with similar results are shown. Cells in the plots were gated from lymphocytes based on forward and side scatter.

Figure 2.

Changes of absolute neutrophil count (ANC) in patients treated with different types of ATG. (A) Changes of ANC in all hATG (n = 60) and rATG (n = 60)-treated patients (including responders and non-responders). (B) Changes of ANC in responder patients treated with rATG (n = 22) or hATG (n = 41). The bars represent mean ± SE. *P<0.05; **P<0.01; ***P<0.001.

Figure 3.

Recovery of different lymphocyte populations after hATG or rATG treatment. CD38⁺ cells were gated from CD45⁺CD3⁺CD4⁺CD8⁻ T cells; double-negative T cells were gated from CD45⁺CD3⁺CD4⁻CD8⁻ T cells; B cells (CD19⁺) were gated from CD45⁺ cells. Left panel shows frequencies and right panel shows absolute numbers of different cell populations. The bars represent mean ± SE. *P<0.05; **P<0.01; ***P< 0.001.

Figure 4.

Effect of ATG on cytokine levels. (A) “Cytokine storm” on day 2 in patients treated with rATG (n = 36) or hATG (n = 41). (B) Different effects of rATG (n = 36) or hATG (n = 41) on the cytokine CCL4. (C) Different cytokine levels between responders (n = 44) and non-responders (n = 33). The bars represent median and interquartile range. *P<0.05; **P< 0.01; ***P<0.001.

Figure 5.

Pharmacokinetics of the two types of ATG in patients with serum sickness (SS) and without serum sickness (NSS). (A) Pharmacokinetics of ATG in SS (hATG n = 5, rATG n = 10) and NSS (hATG n = 49, rATG n = 43) patients detected by ELISA. The bars represent mean ± SE. *P<0.05; **P<0.01; ***P<0.001. (B) Western blotting of rATG in the plasma of SS and NSS patients.

Figure 6.

Different immune responses in ATG-treated AA patients with serum sickness (SS) or without serum sickness (NSS). (A) Human anti-ATG antibodies, including IgG, IgA, and IgM, in SS (hATG n = 5, rATG n = 10) and NSS (hATG n = 49, rATG n = 43) patients detected by ELISA. ELISA plates were coated with rATG or hATG for capture anti-rATG and anti-hATG antibodies in the plasma, respectively. (B) Comparison of platelet counts between SS and NSS patients. The bars represent mean ± SE. *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

Cytokine signature of serum sickness. Red dots represent SS (n = 15), while green dots represent NSS (n = 77). The bars represent mean ± SE. Asterisks indicate differences between SS and NSS at the same time points: *P<0.05; **P<0.01; ***P<0.001.