In this study, we have demonstrated that naïve Tregs participate in the control of RBC alloimmunization. Specifically, using RBCs from mice transgenic for GPA and transfusing them together with an adjuvant into wild-type antigen negative recipients to induce RBC alloimmunization, we found blunted alloantibody responses with concomitant reduction in activated splenic B cells and macrophages in mice treated with naïve Tregs. Similarly, in a second model using xenogenic rat RBCs to induce RBC alloimmunization, antibody responses were dampened following transfer of naïve Tregs. Thus, despite using disparate blood group antigens as antigenic stimuli with or without adjuvant in our two mouse models, Treg immunotherapy was effective in suppression of RBC alloimmunization.
Immunization with huGPA RBCs required an inflammatory signal through the use of the CpG ODN adjuvant to induce robust alloantibody production (), whereas administration of rat RBCs alone was sufficient to induce anti-rat RBC production, probably reflecting the highly immunogenic nature of xenogeneic RBCs. Hendrickson et al. recently found that mouse RBC alloimmunization to hen egg lysozyme is significantly increased in the presence of poly(I:C) known to induce viral-like inflammation [8
]. Using a different inflammatory signal (CpG ODN) [23
], we also found enhanced RBC alloimmunization to human GPA blood group antigen in our mouse model. Possible mechanisms that contribute to the adjuvant activity of CpG ODN include their effect on B cells, antigen-presenting cells as well as T cells [25
]. Specifically, CpG ODN treatment results in B cell stimulation [26
]; increased antigen uptake [27
]; enhanced maturation and differentiation of antigen-presenting cells, which in turn result in activation of T cells [28
]; as well as a decrease in Treg suppressive activity, which in turn enhances T effector cell function [29
]. Interestingly, we found an increase in both the activation status of macrophages in mice treated with CpG ODN alone (although it did not reach significance) as well as B cells (see ). However, antigenic stimulation using huGPA RBCs in combination with CpG ODN resulted in significant upregulation of activation markers on both B cells and macrophages (see ), consistent with increase in alloantibody production. Treatment with Tregs prior to immunization with huGPA RBC/CpG ODN significantly reduced the macrophage and B cell activated cell frequencies and these were comparable to the frequencies in mice injected with CpG ODN alone. We have also found that that there is a reduction in the frequency of Tregs in mice that have made an alloantibody response to huGPA RBCs alone, and that the Treg numbers are further reduced when mice are challenged with huGPA RBCs in combination with CpG ODN (our unpublished data). Although preliminary, these data indicate that the suppression of Treg activity may also contribute to the mechanism of enhancement of RBC alloimmunization by inflammatory signals. Consistent with this data, we found that 5 times more naïve Tregs were needed to suppress the alloantibody responses to huGPA RBC plus CpG ODN than for suppression of anti-rat RBC antibody responses where no adjuvant was used (see Materials and Methods).
The exact mechanism of Treg-mediated suppression of RBC alloimmunization remains to be elucidated. Naturally occurring naïve Tregs inhibit the responses of T effector cells by direct cell contact in vitro [9
]. In contrast, adaptive Tregs that develop from naive CD4 cells in the periphery during the course of an immune response [30
] are thought to modulate immune responses exclusively via cytokine-mediated effects and can include IL-10- and TGF-β-producing subsets (Tr1 and Th3, respectively) [32
]. More recent data indicate that contacts with antigen-presenting cells dictate whether Tregs directly or indirectly alter the activation and differentiation of pathogenic T cells [34
]. Specifically, it appears that the type of antigen-presenting cells that the antigen is targeted to as well as the immunogenicity of antigen are among the factors thought to contribute to the cytokine secretion phenotypes of adaptive Tregs [35
]. There are also in vitro studies indicating that Tregs can suppress B cells directly without having to suppress T helper cells [36
] which appears to be dependent on activation status of Tregs as well as that of the B cells [37
] Based on the potential mechanism of Treg suppression in other experimental systems [38
], it may be that by transferring Tregs in our mouse models the ratios of naïve Tregs to target cells such as T effector cells, B cells, and/or antigen-presenting cells are increased. Alternatively, the cytokine environment may be altered by the increase in the absolute numbers of Tregs in the transferred mice, which in turn affect the outcome of the immune responses to RBC immunization [40
]. Using the rat RBC immunization model in which repeated injections with rat RBCs results in breakdown of tolerance against self RBC antigens, we recently showed that autoantibody production against mouse red cell is also under the control of Tregs, albeit a subset of Tregs that had been previously exposed to autoantigens [24
]. Interestingly, these primed Tregs had little effect on suppression of alloantibody responses to rat RBC [24
]. Conversely, adoptive transfer of naïve Tregs at concentrations that suppressed anti-rat RBC responses did not have an effect on the development of autoantibodies following rat RBC immunization (our unpublished data), indicating that at least in this mouse model naïve Tregs and primed Tregs differ in their regulation of allo- and autoantibody responses against RBC antigens. Elucidating the mechanism of suppression responsible for prevention of RBC allo- and autoimmunization by naïve and primed Tregs, respectively, remains to be defined. Similarly, defining the target cells such as activated CD4+
effector cells, B cells, and/or antigen-presenting cells that are either directly or indirectly via inhibitory cytokines under the control of naïve or primed Tregs [9
] will be critical for the future manipulation of these cells for use in cell-based therapies for prevention of red cell allo- and autoimmunization in the transfusion setting.
In conclusion, in this study we have shown that naturally occurring Tregs participate in regulation of transfusion-mediated RBC alloantibody responses, opening up the possibility that Treg immunotherapy may be applied for induction of transfusion tolerance.