Abstract
PurposeThe anion channel protein band 3 is the main target of the pathogenic red blood cells (RBC) autoantibodies in New Zealand black (NZB) mice. CD4 T cells from NZB mice with autoimmune hemolytic anemia respond to band 3. Previously, we have shown that IL-10 and peptides containing a dominant T cell epitope from red cell band 3 modulate autoimmune hemolytic anemia in NZB mice. Because of the immunoregulatory role of IL-10 in autoimmune diseases, we aim to identify individual band 3 peptides that induce high IL-10 production and simultaneously suppress CD4 T cell proliferation and to investigate the effect intranasal administration of IL-10 producing band 3 peptides on autoantibody responses of NZB mice.MethodsSplenic CD4 T cells of NZB mice were isolated and stimulated by co-culture of T cells with individual band 3 peptides. IL-10 production was measured by enzyme-linked immunosorbent assay and proliferative response of CD4 T cells was estimated by incorporation of [3H] thymidine assay. NZB mice were given either PBS, or peptides 25 (241–251) and 29 (282–296) or both peptides intranasally on three occasions at 2-day intervals. The mice were bled at 6, 10 and 18 weeks after peptide inhalation, and the number of RBC auto-antibodies was measured by DELAT and hematocrit values were assessed.ResultsPeptides 25 (241–251) and 29 (282–296) induced the highest IL-10 production by CD4 T cells. These peptides also inhibited the peak T cell proliferative response. 6 and 10 weeks after peptide inhalation, the total IgG, IgG1 and IgG2a in mice treated with both peptides 241–251 and 282–296 were significantly higher than control (P < 0.05). However, no significant difference in the mean hematocrit between of the peptide-treated mice and the control group was found.ConclusionsAlthough band 3 peptides 241–251 and 282–296 induced to the highest IL-10 production by CD4 T cells in vitro but fail to reverse the RBC autoantibody response in vivo. Modifications to improve solubility these peptides might help to modulate the immune response toward a T helper-2 profile and decrease the severity of anemia.
Highlights
There is a pressing need to develop specific immunotherapies for autoimmune diseases, and considerable attention is focused on the potential beneficial effects of peptides recognized by auto-reactive CD4 T cells
Cell proliferation was measured over four days from day 3 to day 6 of culture. 100 ll samples of each CD4 T cell culture were added in triplicate to a 96-well plate and incubated for 6 h with 1 lCi per well of [3H] thymidine
In this study we mapped band 3 peptides that are able to produce IL-10 and we further investigated the effect of selected IL-10-producing peptides on CD4 T cell proliferation
Summary
There is a pressing need to develop specific immunotherapies for autoimmune diseases, and considerable attention is focused on the potential beneficial effects of peptides recognized by auto-reactive CD4 T cells. Before such treatments can become a reality for human disease, it is important to understand the mechanisms underlying the action of these peptides, in antibody-mediated conditions, where their effects are less well understood. NZB mice produce red blood cell (RBC)-bound IgG autoantibodies from as early as 6 weeks of age [1, 2] and develop signs of anemia some 5 months later [3]. The severity of anemia is reduced in NZB mice given a soluble analog of the peptide [9], showing that this disease is susceptible to peptide therapy
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