The consequences of antibody binding to red blood cells in alloantibody responses Público
Mener, Amanda (Summer 2018)
Abstract
Red blood cell (RBC) transfusions provide a life-saving intervention in the setting of hematologic conditions and trauma. However, it is not without risk. Repeated RBC transfusions can lead to formation of alloantibodies against allogeneically distinct antigens on the surface of transfused RBCs. These RBC alloantibodies increase the risk of hemolytic transfusion reactions, or in the case of pregnancy, hemolytic disease of the fetus and newborn, as well as causes difficulty in finding compatible RBCs for future transfusions. The mechanism(s) of how RBC alloantigens induce alloantibodies and furthermore, the immunologic consequences of antibody binding to RBCs following transfusion of RBCs, remain poorly understood. Currently, there is only one pharmacologic agent available to actively prevent the development of RBC alloantibodies, Rh immune globulin (RhIg), which consists of pooled plasma from donors purposely exposed to RhD+ RBCs. The ability of passive administration of antibodies to prevent the de novo development of alloantibodies is termed antibody-mediated immunosuppression (AMIS). However, the mechanism of how AMIS occurs is not well-understood. Furthermore, with the decreasing availability of plasma donors and the ethical limitations in purposefully alloimmunizing patients against RBC alloantigens other than RhD, there is a critical need to better understand the mechanism(s) of AMIS in order to develop alternatives to RhIg. Given previous studies suggesting that decreases in the level of surface antigen, termed antigen modulation, may account for one mechanism of AMIS, we utilized a murine model of RBC alloimmunization, in which RBCs express a fusion protein consisting of hen egg lysozyme (HEL), ovalbumin (OVA) and human Duffy (HOD RBCs). Through exposure of mice to HOD RBCs in the presence or absence of anti-HEL monoclonal antibodies, we explore how monoclonal antibody-induced changes in the level of the target RBC antigen impacts the development of an anti-HOD alloantibody response. Additionally, through utilizing RBCs that express both the clinically-relevant HOD and KEL RBC antigens on the same RBC, we also investigate whether AMIS is antigen-specific and whether this antigen-specific AMIS occurs through antigen modulation.
Furthermore, as RhD and HOD RBCs do not induce complement fixation and complement has been shown to be important in the development of antibody responses, we next sought to determine how complement impacts the development of RBC alloantibodies utilizing a model of RBC alloimmunization that induces complement fixation, the KEL RBC antigen. Unlike previous studies describing complement as an adjuvant in the development of an antibody response, we actually observe that mice deficient in complement component 3 (C3 KO) exhibited increased alloantibody responses against the KEL antigen due to a decrease in antibody-induced antigenic changes on the RBC surface, indicating that complement negatively regulates the development of an antibody response to the KEL RBC antigen.
Finally, given the differences in IgG subclass development observed between C3-deficient and wild type mice in response to KEL RBC transfusion and the involvement of CD4+ T cells in regulating class switching, we explore the role of CD4+ T cells in the development of the alloantibody response to KEL. We find that in the presence of C3, mice develop alloantibody responses to KEL even in the pharmacologic or genetic absence of CD4+ T cells. However, C3 KO and mice deficient in complement receptors 1 and 2 are completely dependent on CD4+ T cells to develop an anti-KEL IgG response, suggesting that C3 fixation and interaction with complement receptors regulates whether the antibody response to KEL is T-independent or T-dependent.
Taken together, these findings suggest that the consequences of antibody binding to the RBC surface, acquired either through passive administration of antibody, in the case of AMIS, or following de novo antibody development, include both complement-independent and -dependent antigen modulation. This antigen modulation impacts not only the subsequent development of an alloantibody response to RBC antigens, but also the immunologic pathways involved. These findings contribute to our understanding regarding how the mechanism of alloantibody development to RBC antigens may vary by the individual antigen.
Table of Contents
Chapter 1: Introduction
A brief history of transfusion medicine
Antibody-mediated immunosuppression
Introduction to complement and historical perspective
Complement and humoral immunity
Complement’s role in T cell immunity
Figure and Figure Legend
References
1-71
2-6
6-18
18-28
28-43
43-49
50
51-71
Chapter 2: Antibody-mediated immunosuppression can result from RBC antigen loss independent of Fcg receptors in mice
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Figures and Figure Legends
72-115
73-74
75-77
78-80
81-87
88-92
93-104
105-115
Chapter 3: Antibody-Mediated Immunosuppression by Antigen Modulation is Antigen-Specific
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Figures and Figure Legends
116-155
118-119
120-122
123-125
126-132
133-135
136-144
145-155
Chapter 4: Complement component 3 Negatively Regulates Antibody Response by Modulation of RBC Antigen
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Figures and Figure Legends
156-223
157
168-160
161-167
168-177
178-187
188-205
206-223
Chapter 5: Complement serves as a switch between CD4+ T cell independent and dependent RBC antibody responses
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Figures and Figure Legends
224-284
225
226-228
229-234
235-244
245-250
251-267
268-284
Chapter 6: Discussion
Summary
Antibody-mediated immunosuppression
Complement fixation and RBC alloantibody development
Negative regulatory role of complement and implications for vaccination
Complement as a regulator of T cell independent antibody responses
General conclusions
Figures and Figure Legends
References
285-329
286-287
287-292
292-297
297-299
299-306
306-308
309-312
313-329
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