Answer by Tirumalai Kamala:
B cells make antibodies, even against RBCs (Red Blood Cells). After all anti-RBC antibodies are the original reason to type blood for transfusions. Why then are anti-RBC antibodies rare as in hemolytic anemia, an autoimmune disease?
First, autoantibody does not equal autoimmunity. Autoantibodies, antibodies against body’s own components, are normal while autoimmunity is a disease. Key difference? Autoantibodies represent controlled/regulated immunity, autoimmunity its reverse. Context is a prevailing theme in biology, especially so with immune responses. While anti-RBC antibodies may even help clear out senescent RBCs (not proven yet), when B cells make antibodies against healthy, non-senescent RBCs in numbers that overwhelm normal physiology, autoimmunity ensues.
To understand the ‘what’ and ‘how’ of anti-self RBC antibodies let’s briefly explore two phenomena, one, the general mechanism of B cell tolerance*, i.e. how autoantibody-producing B cells are deleted/constrained during B cell development, and two, specific breakdowns in immune tolerance that drive the pathologic anti-RBC antibodies associated with Autoimmune Hemolytic Anemias (AIHA).
* More than one B cell subset exists, B-1 (innate) and B-2 (adaptive). Only B-2’s covered here.
How B cells are made tolerant to body components during their development
B cells develop from hematopoietic precursors in the bone marrow. A fully formed B cell undergoes a two-fold test, one, capacity of cell surface B cell receptor (BCR) to bind antigen, and two, signals sent inside cell after binding antigen. Should the cell divide or die? B cells presenting BCRs that respond too strongly at this stage die.
Unique among body’s somatic (non-reproductive) cells, B and T cells turn on RAG (Recombination Activating Gene) and TdT (Terminal deoxynucleotidyl Transferase) to re-arrange and recombine their receptors. Re-arranged and recombined receptors, i.e. different from the germ line sequence. Makes B and T cells phenomenally powerful double-edged swords. Their vast range of receptors (repertoire) bind billions of antigens including auto-antigens. Anticipation is a hallmark of B and T cells, i.e. specific for unknown, as-yet-unmet dangers. Thus multiple checkpoints necessary to allow sufficiently anticipatory BCR diversity (repertoire) while restraining fulminant autoimmunity. A difficult balance necessitating trade-offs at every step of B cell development.
- Checkpoint one. In the bone marrow where B cells first express a fully formed BCR, the IgM. If their BCR binds too strongly to antigens, they die (deleted), the fate of some, not all, auto-reactive B cells including those that bind RBCs. Many auto-reactive B cells do escape deletion through receptor editing (light chain swap). Why? Presumably capacity to bind similar antigens from potentially dangerous pathogens outweighs low risk of autoimmunity, a trade-off built into the system. Immature B cells then leave bone marrow and differentiate into transitional and mature B cells in the spleen.
- Checkpoints two and beyond. In the ‘periphery’ where mature B cells that bind antigens but don’t get T cell help also die (deleted). Autoimmunity thus typically requires break in both B and T cell tolerance.
Another way to interpret B cell development checkpoints? Assessing how much the B cell is in charge of its fate. Arriving at bone marrow checkpoint one, the B cell’s successfully re-arranged its immunoglobulin heavy and light chains to display a BCR on its cell surface, i.e. it’s been a free agent more or less in deciding to live or die. When the B cell proceeds beyond checkpoint one, it’s subject to extrinsic controls that determine its fate, controls such as T cell help, survival factors such as BAFF. This implies two things
- Post-checkpoint one, proof of functional BCR in hand, the B cell is different in kind. Henceforth no more free agent or much less so.
- B cells are either inherently auto-reactive or retain capacity thereof, else why the need for stringent B cell-extrinsic controls once they prove they have functional BCRs?
Thus, newly formed B cells tend to be both auto-reactive (self-reactive) and poly-reactive. Poly-reactive means BCR binds more than one type of antigen. The cell-surface BCR, when secreted, is the antibody.
Anti-RBC antibodies are hallmark of Autoimmune Hemolytic Anemia (AIHA). Could this autoimmune disease explain how our B cells become tolerant to our RBCs?
In AIHA, anti-RBC antibodies deplete healthy, non-senescent RBCs. This reduces normal RBC lifespan of approx. 120 days to just a few days. Hence anemia.
- Annual incidence of approx. 1 to 3 per 100, 000, AIHA comes in many forms.
- Auto-antibody either reactive in warm or cold, the former, more common, predominantly IgG, has optimal affinity for RBC at 37 ◦C.
- In contrast, cold anti-RBC auto-antibodies (cold agglutinins), present in healthy individuals as well, are predominantly IgM and react more strongly at lower temperatures. Cold agglutinins’ target is the I/i antigen, a sialylated glycolipid (Sia-Ib), different in fetus and newborn RBCs versus adults, and a purported receptor for Mycoplasma pneumoniae.
What’s different in humans with AIHA? Human immunology lags decades behind mouse immunology. This 2008 table of unanswered AIHA questions shows even basic ones such as which cells participate remain unanswered.
No definitive mechanism yet identified but data suggest defective B and T cell tolerance.
- Defective B and T cell tolerance? AIHA is usually associated with other autoimmune disorders such as SLE (systemic lupus erythematosus), RA (rheumatoid arthritis), Sjogren’s syndrome, primary biliary cirrhosis, hypothyroidism, inflammatory bowel disease, immune thrombocytopenia, primary hypogammaglobulinemia, and other lymphoproliferative disorders such as chronic lymphocytic leukemia or lymphoma.
- Defective T cell tolerance? AIHA circulating T cells respond to the RBC-specific protein, Rh factor (5, 6).
- Defective complement regulation? AIHA patients may be deficient in CD59 (7), an important protein that regulates complement activation.
- Molecular mimicry. Do infectious agents have antigen(s) similar to those on RBCs? Immune response to such infections would drive and sustain anti-RBC T and B cell responses. e.g. Mycoplasma pneumoniae.
Caveats to human AIHA studies
- Associations with other autoimmune and lymphoproliferative diseases. When more than one thing is dysfunctional, it’s anybody’s guess which one is necessary and sufficient for driving anti-RBC antibody-based autoimmunity.
- Observational, not quantitative. For example, Rh-specific T cells in AIHA (5,6)? Requires more stringent tests such as limiting dilution assays that compare Rh-specific T cell frequencies between AIHA and healthy.
- Most studies looked at small numbers of patients. Some observations are limited to single studies (7).
- Other than Mycoplasma, association with other infections remain correlations.
Data suggesting mechanisms for B cell non-tolerance to RBC come from two transgenic mouse model studies.
Transgenic mouse model study one (10)
- They generated transgenic mice that express human anti-human RBC IgM isolated from an AIHA patient.
- IgM specific for Sia-Ii, a silaylated carbohydrate present on human RBC surface.
- Human IgM+ B cells, i.e. auto-reactive B cells, were largely deleted.
- Small number of human IgM+ B cells were found in mouse peritoneal cavity.
Transgenic mouse model study two (11)
- Same transgenic mouse.
- Intranasally infect with Mycoplasma pulmonis.
- Spontaneous RBC agglutination at room temperature in pipette used for blood collection.
- High levels of auto-antibodies?
- Interpretation? Mycoplasma pulmonis and RBC have similar antigens (molecular mimicry) —> anti- Mycoplasma pulmonis antibody cross-reacts with RBC —> breakdown in B cell tolerance.
- Something even more remarkable that escaped notice? This mouse has transgenic human IgM but its blood is still fully mouse. Yet this IgM which was originally human anti-human RBC not really reacts with Mycoplasma pulmonis but also with mouse RBC!
- Implies Mycoplasma pulmonis antigens are similar to both human and mouse RBC.
- Back full-circle to trade-off. An anticipatory system mandates considerable overlap between BCR diversity (repertoire) and auto-reactivity. Autoimmunity? An ever-present risk.
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- Wardemann, Hedda, and Michel C. Nussenzweig. “B‐Cell Self‐Tolerance in Humans.” Advances in immunology 95 (2007): 83-110.
- Berentsen, Sigbjørn, and Tatjana Sundic. “Red Blood Cell Destruction in Autoimmune Hemolytic Anemia: Role of Complement and Potential New Targets for Therapy.” BioMed Research International (2014).
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- Barker, Robert N., et al. “Identification of T-cell epitopes on the Rhesus polypeptides in autoimmune hemolytic anemia.” Blood 90.7 (1997): 2701-2715.
- Hall, Andrew M., et al. “Interleukin-10-mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen.” Blood 100.13 (2002): 4529-4536.
- Barros, Melca MO, et al. “Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal‐regulatory protein‐α, β on monocytes from patients with warm autoimmune hemolytic anemia.” Transfusion 49.1 (2009): 154-160.
- Barker, Robert N., Mark A. Vickers, and Frank J. Ward. “Controlling autoimmunity—Lessons from the study of red blood cells as model antigens.” Immunology letters 108.1 (2007): 20-26.
- Semple, John W., and John Freedman. “Mechanisms underlying autoimmunity in hematology.” Drug Discovery Today: Disease Mechanisms 3.2 (2006): 231-235.
- Havouis, Séverine, et al. “Negative regulation of autoreactive B cells in transgenic mice expressing a human pathogenic cold agglutinin.” European journal of immunology 30.8 (2000): 2290-2299.
- Havouis, Severine, et al. “Transgenic B lymphocytes expressing a human cold agglutinin escape tolerance following experimental infection of mice by Mycoplasma pulmonis.” European journal of immunology 32.4 (2002): 1147-1156.