**2. Knowledge analysis and hypotheses induction**

Basically, when an antigen is introduced into a body, the immune system (IS) does either one of two reactions: immune tolerance (IT) or immune response (IR). IT-reaction is never

absolute [2]. It is usually accompanied by a weak IR. In normal IR, one cannot identify if there is a degree of IT, because there is no defined laboratory method/test that can measure the degree of IT. Meanwhile, by logical implication, some degree of IT should exist with the normal IR. This entails that there is an equivalence relation between IT and IR.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 15

*Hypothesis III: There is an injection function (one-to-one) between circulating antibodies and* 

*Hypothesis IV: In immune response, antibodies concentration in plasma against a particular antigen in hemolysate is higher than this antigen concentration in hemolysate. Meanwhile, in immune tolerance, antibodies concentration in plasma against a particular antigen in* 

It should be remarked that Humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multi-organ autoimmune disease (autoimmune polyendocrinopathy syndrome type 1) [11]. Liston et all [12] prove that this autoimmune syndrome is caused by failure of a specialized mechanism for deleting forbidden T cell

The methodology applied will demonstrate the existence of particular self tolerogens and particular foreign antigens in RBC (Hypothesis I & II) and show that innumerable antigens exist in RBC which react with innumerable antibodies that exist in plasma. This partially proves that RBC play a role in immune reaction. To proof Hypothesis IV, it will be demonstrated that the concentration of foreign antigens in RBC varies by time in relation to

5. RBC hemolysate antigens are precipitated by plasma obtained from the same individual

Couples that have children, pregnant females, and single females were selected from relatives and friends. The purpose of the experiments was explained to them. Not all the combinations could be found, after blood grouping. The combinations presented, in Table-1, were used to conduct the experiments. Blood samples were taken on heparin. Some of the blood samples were used to prepare RBC and plasma and the rest was used to prepare

*hemolysate is lower than this antigen concentration in hemolysate.* 

clones, establishing a central role for this tolerance mechanism.

IR known behavior. The experiments done are the following:

4. RBC transport self Tissue Specific Antigens (TSAs).

3. RBC transport self HLA antigens

6. RBC transport bacterial antigens.

**3.1. Materials for experiments 1, 2, and 3** 

lymphocytes using the Ficoll hypaque technique [13].

2. RBC of pregnant females transport male spouse HLA antigens

and cross reacted with plasma from different individuals.

7. RBC antigens and plasma antibodies concentration vary with time.

1. RBC of pregnant females transport male spouse ABO blood group antigens

To proof that RBC have role in immune reactions (IR and IT), one need to proof that there is an inverse correlation between antibodies concentration in plasma and antigens

*RBC's hemolysate antigens.* 

concentration in RBC.

**3. Experiments** 

*Hypothesis I: There is no absolute immune tolerance, if and only if there is no absolute immune response* 

In central IT, immature self-reactive T lymphocytes recognize antigens in the thymus and undergo negative selection (deletion) [3]. Consequently, in normal IR against a particular antigen, measuring the concentration of this antigen in the thymus can be correlated to the degree of the accompanied IT. The transport mechanism of antigens to the thymus is a critical issue because of the remarkable capacity of IS which can recognize any antigen [4]. In [5], authors claim that Dendritic Cells (DCs) have several functions, not only, in innate and adaptive immunity, but also there is increasing evidence that DCs in situ induce antigen specific unresponsiveness or tolerance in central lymphoid organs and in the periphery. The evidence that DCs transport antigens to thymus in central tolerance is very weak while the evidence that DCs have role in peripheral tolerance is more acceptable based on the review article [6]. In conclusion RBC may be vehicles which transport self antigens to induce central IT.

The role of RBC in transporting antigens has not been investigated before. If RBC are capable of antigen transport to induce IT, this will unveil important knowledge. For instance, in hemolytic disease of fetus and newborn (HDFN), maternal anti D alloantibody and feto-maternal ABO incompatibility are the two major causes of HDFN, Meanwhile, with the implementation of Rhesus D immunoprophylaxis, hemolytic disease due to ABO incompatibility and other alloantibodies have now emerged as major causes of this condition. [7].

In pregnancy, most of delivered infants are normal when there is no anti D alloantibody which means that there is an efficient mechanism that can handle the other incompatibilities. The mechanisms explained in literature explain why ABO incompatibilities, only, do not occur [8], [9] and [10], but these mechanisms do not explain why those incompatibilities occur. The mechanism may be based on trapping those antibodies in placenta through RBC catering of ABO and other incompatible blood groups antigens. Consequently, the occurrence of HDFN may be due to depletion of those antigens' store from RBC. Also, if this RBC transport function is the mechanism a body tolerates his self antigens, this will explain how a pregnant woman is able to tolerate her fetus and placenta, assuming that they are part of self.

#### *Hypothesis II: RBC hide antigens to transport them to target organs.*

From these *hypotheses I & II*, if RBC play role in antigen transport, one can deduce that in any mammal, blood circulating antibodies against self and foreign, either antigens or tolergens, will react with hemolysate.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 15

*Hypothesis III: There is an injection function (one-to-one) between circulating antibodies and RBC's hemolysate antigens.* 

To proof that RBC have role in immune reactions (IR and IT), one need to proof that there is an inverse correlation between antibodies concentration in plasma and antigens concentration in RBC.

*Hypothesis IV: In immune response, antibodies concentration in plasma against a particular antigen in hemolysate is higher than this antigen concentration in hemolysate. Meanwhile, in immune tolerance, antibodies concentration in plasma against a particular antigen in hemolysate is lower than this antigen concentration in hemolysate.* 

It should be remarked that Humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multi-organ autoimmune disease (autoimmune polyendocrinopathy syndrome type 1) [11]. Liston et all [12] prove that this autoimmune syndrome is caused by failure of a specialized mechanism for deleting forbidden T cell clones, establishing a central role for this tolerance mechanism.
