CD4⁺CD25⁺ regulatory T cells control induction of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is an autoimmune disease in which red blood cells (RBCs) are destroyed prematurely due to the production of antibodies against the patient's own red cells.¹  The most common form of AIHA is characterized by the presence of “warm”-type autoantibodies, which are immunoglobulin G (IgG) type and react optimally at 37°C, causing RBC destruction extravascularly by tissue macrophages.¹  Playfair and Marshall-Clarke described a mouse model of AIHA, in which repeated injections with rat RBCs result in the development of autoantibodies against self RBCs and a disease process similar to warm-type AIHA in humans with evidence of anemia, reticulocytosis, shortened survival of RBCs, and a direct Coombs-positive state.²  As expected, the mice develop rat-specific xenoantibodies, thus providing an inbuilt control for the autoantibody response.^3,4  Spleen cells or T cells from mice immunized with rat RBCs transferred to naive recipients suppress the subsequent induction of autoantibodies, but not the xenoantibody response, indicating a regulatory mechanism in the induction of autoantibody response.^4,5  The nature of this suppressive activity remains to be elucidated.

T-regulatory CD4⁺ cell populations characterized by coexpression of CD25 (interleukin-2 receptor alpha chain) have been shown to play an important role in the generation and maintenance of peripheral tolerance.⁶  Although studies have not directly addressed the role of CD4⁺CD25⁺ T cells in the development of AIHA per se, it was noted that about 25% of older CD25 knockout mice develop autoimmune disorders, including hemolytic anemia.⁷ 

We have used the Marshall-Clarke and Playfair model of murine AIHA to determine the role of CD4⁺CD25⁺ T-regulatory cells for induction of AIHA. Depletion studies using anti-CD25 increased the incidence of AIHA in C57/Bl6 mice from 30% to 90%. In addition, adoptive transfer of purified CD4⁺CD25⁺, but not CD4⁺CD25^- cells, from immunized mice prevented the induction of AIHA. Altogether, our data demonstrate the importance of CD4⁺CD25⁺ cells for control and induction of AIHA.

Rat RBCs were isolated from whole rat blood using histopaque (Sigma-Aldrich, St. Louis, MO) gradient and adjusted to 10⁹ cell/mL. Female C57/Bl6 mice between 8 and 10 weeks old were immunized intraperitoneally with 2 × 10⁸ rat RBCs in 200 μL RPMI on a weekly basis.

C57/BL6 mice were given 500 μg anti-CD25 (clone 7D4; BD Pharmingen, San Diego, CA) on a weekly basis for a total of 3 weeks, 8 hours prior to immunization with rat RBCs. As control, mice were treated with 500 μg isotype control rat IgG2b (BD Pharmingen) followed by immunization with rat RBCs or with 500 μg anti-CD25 alone without the rat RBC immunization regimen.

Blood samples (25 μL) by retro-orbital sinus bleeding were obtained on a weekly basis, 5 days after each immunization. Reticulocyte counts were performed using the Advia 120 Hematology System (Bayer, Tarrytown, NY). In addition, levels of IgG sensitization (autoantibodies) on the RBCs were determined by flow cytometry using fluorescein isothiocyanate (FITC)–conjugated anti–mouse IgG (Vector Laboratories, Burlingame, CA). For analysis of rat RBC-specific xenoantibodies, rat RBCs were incubated with diluted mouse plasma for 1 hour at 37°C and after several washes, were stained with FITC-conjugated anti–mouse IgG. Analysis of levels of anti–double stranded (ds) DNA was performed with plasma diluted at a final concentration of 1 in 100 using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Alpha Diagnostic, San Antonio, TX).

Mouse RBCs obtained from naive female C57/Bl6 mice were labeled with PKH-26 (Sigma)⁸  and injected by the tail-vein into control mice or those that had developed AIHA. Blood samples were obtained by retro-orbital sinus bleeding at the time points indicated after transfusion and the clearance of fluorescent RBCs was measured by flow cytometry as previously described.⁸ 

Splenocytes from animals immunized with rat RBCs on a weekly basis for 12 weeks were harvested, stained with phycoerythrin (PE)–labeled anti-CD4 (clone) and PE-Cy7–labeled anti-CD25 (clone 7D4) (both from BD Pharmingen) and purified using high-speed sorting (MoFlo; Dako Cytomation, Carpinteria, CA) to separate CD4⁺CD25^- and CD4⁺CD25^hi cells. Approximately 2 × 10⁵ cells in 0.2 mL of phosphate-buffered saline (PBS) was injected intravenously into 10-week-old female C57/Bl6 recipient mice. After one day, the mice were immunized with rat RBCs on a weekly basis.

The significance of differences between groups of mice were calculated using a single-factor analysis of variance (Anova) test, and only P values below .05 were considered significant.

Approximately 30% of female C57/Bl6 mice immunized on a weekly basis with rat RBCs develop AIHA, as evidenced by presence of red cell–specific autoantibodies on their RBCs, increased levels of reticulocytes, and increased destruction of transfused syngeneic mouse RBCs (Figure 1A-C). However, all the mice developed rat RBC-specific xenoantibodies (Figure 1D), consistent with previous data.⁴  To examine whether CD4⁺CD25⁺ cells play a role in the induction of AIHA in this experimental model, we depleted mice (n = 10) of CD25⁺ cells prior to immunization with rat RBCs on a weekly basis for 3 weeks. Administration of an anti-CD25 antibody almost completely depleted CD4⁺CD25⁺ cells in C57/Bl6 mice (Figure 2A). Immunization of rat RBCs in the depleted mice increased the frequency of AIHA to 90% (Figure 2B-C), demonstrating a critical role for T-regulatory CD25⁺ cells in development of this type of autoimmune disease. Weekly treatment with an isotype-control antibody followed by rat RBC immunization (n = 5) (Figure 2B) or treatment with anti-CD25 alone (n = 5) (Figure 2B-C) for 3 weeks did not result in increased incidence of AIHA as observed in the anti-CD25/rat RBC–immunized mice.

We also measured the levels of anti–ds DNA characteristic of systemic autoimmune disease and found significantly elevated levels in anti-CD25/rat RBC–immunized mice, as compared with control mice treated with only rat RBCs or treated with anti-CD25 alone (Figure 2D). In addition, the levels of xenoantibodies against rat RBCs in anti-CD25/rat RBC–immunized mice were elevated as compared with mice treated with rat RBCs alone (Figure 2E), consistent with a heightened immune hypersensitive state.

Splenocytes taken from mice immunized with rat RBCs at weekly intervals for 12 weeks and transferred to naive recipients suppress the subsequent induction of autoantibodies.⁴  To determine if CD4⁺CD25⁺ cells play a role in this suppressive activity in this animal model, we adoptively transferred CD4⁺CD25⁺ or CD4⁺CD25^- sorted cells from mice immunized with rat RBCs on a weekly basis for 12 weeks, into naive C57/Bl6 mice. One day after the transfer, mice were immunized with rat RBCs on a weekly basis. Analysis of the mice after 9 immunizations demonstrated that autoantibody production was completely suppressed in mice adoptively transferred with CD4⁺CD25⁺ (n = 5), but not those transferred with CD4⁺CD25^- (n = 6) cells (Figure 2F). Analysis of anti–rat RBC levels indicated that CD4⁺CD25⁺ adoptively transferred mice developed xenoantibodies as effectively as control mice (Figure 2G), demonstrating that CD4⁺CD25⁺ suppressive activity is autoantibody-response specific.

Although there may be multiple factors that contribute to the induction of AIHA, our data indicate that defective suppressive activity of CD4⁺CD25⁺ cells could play an essential role in the onset and/or maintenance of this autoimmune disease. Current therapies for AIHA include administration of immunosuppressive drugs together with transfusion or splenectomy in patients with relapsing cases, therapies that typically control, rather than cure, the disease.¹  The immunotherapeutic potential of CD4⁺CD25⁺ cells may represent a more effective strategy for treatment of AIHA.

We thank Yazan Abdullah for his technical assistance in setting up the immunization protocol and performing the initial measurements of the auto- and xenoantibodies. We are grateful to GabrielAlespeti (New York Blood Center) for cell sorting and advice on some of the FACS analysis. We wish to thank Dr Barry Coller (Rockefeller University) for allowing us to use hisAdvia 120 hematology system and are especially grateful to Thomas Hoffman for his help with the instrument.