RhD Specific Antibodies Are Not Detectable in HLA-DRB1*1501 Mice Challenged with Human RhD Positive Erythrocytes

The ability to study the immune response to the RhD antigen in the prevention of hemolytic disease of the fetus and newborn has been hampered by the lack of a mouse model of RhD immunization. However, the ability of transgenic mice expressing human HLA DRB1*1501 to respond to immunization with purified RhD has allowed this question to be revisited. In this work we aimed at inducing anti-RhD antibodies by administering human RhD+ RBCs to mice transgenic for the human HLA DRB1*1501 as well as to several standard inbred and outbred laboratory strains including C57BL/6, DBA1/J, CFW(SW), CD1(ICR), and NSA(CF-1). DRB1*1501 mice were additionally immunized with putative extracellular immunogenic RhD peptides. DRB1*1501 mice immunized with RhD+ erythrocytes developed an erythrocyte-reactive antibody response. Antibodies specific for RhD could not however be detected by flow cytometry. Despite this, DRB1*1501 mice were capable of recognizing immunogenic sequences of Rh as injection with Rh peptides induced antibodies reactive with RhD sequences, consistent with the presence of B cell repertoires capable of recognizing RhD. We conclude that while HLA DRB1*1501 transgenic mice may have the capability of responding to immunogenic sequences within RhD, an immune response to human RBC expressing RhD is not directly observed.


Introduction
The RhD antigen is a clinically important human blood group that can be a primary target in hemolytic disease of the fetus and newborn (HDFN) as well as some cases of autoimmune hemolytic anemia. Antibodies to RhD (anti-D) have been used for many years to prevent HDFN. The ability to manipulate and study the immune response to the RhD antigen in the prevention of HDFN has been hampered by the lack of a murine model to study this antigen. Although never formally published, it has been generally considered that standard laboratory mice do not make an immune response to the RhD antigen [1]. However, the more recent ability of creating transgenic mice expressing functional human HLA antigens has allowed this question to be revisited in a murine model.
The Aberdeen group has successfully induced an immune response to solubilized RhD protein in humanized mice that express the human HLA-DRB1 * 1501 allele [1]. Human HLA class II DR has been found as a major restricting element for human T-helper cells specific for RhD protein [2], and the HLA-DRB1 * 1501 allele is significantly overrepresented in RhD negative donors who have produced anti-RhD antibodies in response to RhD-positive RBCs [3]. In particular, the expression of the HLA DRB1 * 1501 transgene was found to confer on mice the ability to respond to immunization with purified RhD protein [1].
In addition to being able to stimulate an immune response, T cell epitopes derived from RhD protein sequences were also shown to induce oral tolerance to the RhD antigen in the HLA-DRB1 * 1501 murine model. While an immune response to purified RhD protein is of interest, the ability of 2 Advances in Hematology an immune response to be generated to naturally expressed RhD on the surface of red cells is needed to move forward with relevant murine models. Thus far, an immune response to RhD expressed on the surface of erythrocytes in mice expressing HLA-DRB1 * 1501 has not yet been addressed.
In this work we aimed at inducing an anti-RhD antibody response by administering human RBCs expressing RhD in mice expressing HLA DRB1 * 1501 [4]. It is important to mention that the HLA DRB1 * 1501 mouse strain used here is different from the one used by Hall and his collaborators in 2005. Specifically, the HLA DRB1 * 1501 mice used in our study lack the expression of functional murine MHC class II, forcing the restricting element for immune responses through HLA DRB1 * 1501 [4]. In addition, conventional inbred and outbred mouse strains were also challenged with human RhD-positive RBCs to formally assess if standard strains of mice can generate anti-RhD specific antibody responses.
The results showed that when HLA DRB1 * 1501 transgenic mice are challenged with RhD positive RBC under a variety of conditions, despite the development of an immune response to the red cells, no antibodies to RhD were detected by flow cytometry. However, the results of the peptide studies were consistent with the presence of a B cell repertoire capable of recognizing each of the three immunogenic sequences evaluated. We conclude that while HLA DRB1 * 1501 transgenic mice have the capability of responding to sequences from RhD, an immune response specific for human RBC expressing RhD is not directly observed.  Untreated mice were used as negative controls ( = 2). All the mice were bled for serum fifteen days after the first or seven days after the second challenge.

RhD Peptide Design and Immunization.
Peptides were designed and selected according to the human RhD sequence published in gene bank (accession number L08429). Putative linear epitopes were predicted using the antibody epitope prediction tool of the Immune Epitope Data Base (IEDB) Analysis Resource (http://tools.immuneepitope.org/tools/bcell/iedb input). Four peptides that theoretically correspond to extracellular regions of the human RhD protein and that are different in sequence from mouse RhD [5] were selected (Table 1, Figure 1). Peptides were synthesized by Peptides International and shipped lyophilized (Louisville, Kentucky, USA). Peptides 1 to 3 were successfully solubilized in 1 M ammonium bicarbonate. Unfortunately, peptide 4 was not soluble in up to 10% organic solvent and was therefore not used. Peptides 1 to 3 were successfully linked to Keyhole Limpet Haemocyanin (KLH) (Sigma-Aldrich, St Louis, MO, USA) using a cysteine added at the C-terminus and Sulfo-SMCC (4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt) (Sigma-Aldrich, St Louis, MO, USA) used as a cross-linker. The coupling efficiency of the cysteine containing peptides to KLH was determined by a cysteine assay using 5,5 -Dithiobis(2-nitrobenzoic acid) (DTNB or Ellman's reagent) which reacts with sulfhydryl groups at pH 8.0 to produce   .7% diethanolamine, pH 9.6, was added to the plates. Plates were read by an ELISA reader at 405 nm after 15 to 30 minutes. Antibody titers were defined as the highest serum dilution that showed a positive value.

Analysis of the RBC-Specific Antibody Response.
Sera from mice challenged with human RhD positive RBC were tested for antibodies using RhD positive and RhD negative RBC by flow cytometry [6]. RBCs were washed three times in PBS, pH 7.2, and 2 × 10 6 cells incubated with 20 L of serum diluted to 1/100 at 22 ∘ C for 1 hour, followed by 2.5 g/mL goat F(ab ) 2 anti-mouse IgG (FITC conjugated) before analyzing by a Guava EasyCyte Mini System cell analyzer. To selectively detect RhD specific antibodies, the sera were first adsorbed with RhD negative RBC followed by an assessment of binding to RhD positive RBC. Serum adsorptions were performed by incubating packed RhD negative RBC with sera at 22 ∘ C for 1 hour under shaking conditions. Tubes were then centrifuged and the supernatant containing the serum dilutions was collected. A second adsorption cycle was also done at 4 ∘ C.
Adsorbed serum dilutions were assessed against human RhD positive RBC as above. Human polyclonal anti-D serum (WINRHO, Cangene bioPharma Inc., MD, USA) was used as a positive control.

Response of Selected Standard Mice to Immunization with Human RhD Positive Red Blood Cells.
Although it has been considered that standard laboratory mice do not make an immune response to the RhD antigen [1], there is no published data that we are aware of regarding the immune response to the RhD antigen when mice are challenged with human RBCs. To address this, some of the most commonly used inbred and outbred (Swiss and non-Swiss origin) mouse strains were challenged with human RhD positive RBCs and the antibodies reactive with human RBC as well as RhD specific antibodies were evaluated by flow cytometry. With the exception of SFW(SW), all the mouse strains tested [C57BL/6 (H2 b ), DBA/1J (H2 q ), CD1(ICR), and NSA(CF-1)] developed high levels of IgG antibodies reactive with human RhD positive RBC (Figure 2(a)). However, reactivity against human RhD positive RBC was not detected when sera were first adsorbed with RhD negative RBCs (Figure 2(b)), indicating that significant levels of RhD specific antibodies could not be detected. The antibody response of SFW(SW) mice to human RBCs was particularly low. A potential explanation is that this strain contains a deletion in the promoter region of H2-Ea (which encodes the alpha chain of the MHC class II E heterodimer), which strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes [7].

Immunization of HLA-DRB1 * 1501 Transgenic Mice with Human RhD Positive Red Blood Cells.
Considering that the expression of the HLA DRB1 * 1501 transgene was previously found to confer on mice the ability to respond to immunization with purified RhD protein [1], we examined HLA DRB1 * 1501 transgenic mice for an immune response by challenging these mice with human RhD positive RBC. Mice were challenged with one or two doses of 10 8 human RhD positive RBC administered 21 days apart, or the same number of cells in the presence of the CpG ODN adjuvant. HLA-DRB1 * 1501 mice developed antibodies reactive with human RBC after challenge and the administration of two immunizations or the use of adjuvant increased the magnitude of the antibody response (Figure 3(a)). However, when sera produced from these mice were first adsorbed with RhD negative RBCs, antibodies specific for RhD could not be detected (Figure 3(b)).

Immunization of HLA-DRB1 * 1501 Transgenic Mice with Synthetic Peptides Corresponding to Human RhD Sequences.
As in no case was there evidence that HLA-DRB1 * 1501 mice were capable of making a humoral immune response specific for the RhD antigen on the surface of red cells, we performed experiments with peptides from putative immunogenic regions of RhD to evaluate if these mice possess an appropriate B cell repertoire reactive with human RhD. Based on the predicted structure of the RhD protein on the RBC membrane (Figure 1), we synthesized four peptides that contain putative extracellular immunogenic regions of RhD. It is important to note that peptide 1 is identical between both Rh gene loci (RHD and RHCE) while peptides 2 and 3 displayed 82% and 67% sequence identity between RHD and RHCE, respectively. HLA-DRB1 * 1501 transgenic mice were then challenged with three doses of peptide 1, 2, or 3 conjugated to KLH. The fourth peptide was insoluble in buffers compatible for KLH conjugation and was not evaluated.
Anti-peptide antibodies were successfully raised in HLA-DRB1 * 1501 mice from the sera collected 28 and 35 days after immunization; as detected by ELISA, using the immunizing peptides as antigens (Figure 4).

Discussion
Our studies provide evidence that selected inbred and outbred laboratory mice do not make an antibody response specific for naturally expressed human RhD. When we challenged standard inbred (H2 b and H2 q ) and outbred mice with human RhD positive RBC, the mice responded to the human RBC but antibodies specific for RhD protein were not observed from any of the mouse strains tested, despite using multiple RBC exposures or the addition of adjuvant to potentiate the immune response. Different incubation temperatures were also evaluated (data not shown). To the best of our knowledge, this is the first published study to show that selected conventional mice injected with human RBC do not develop antibodies specific to RhD protein detectable by flow cytometry.
An important consideration for the immunogenicity of the human RhD protein in mice is the degree of sequence homology with the mouse Rh protein. In the mouse, Rh protein is encoded by a single RH gene on chromosome 4 and exhibits only 60% sequence identity to the human proteins [8], which does not explain the lack of responsiveness to human Rh proteins. For instance, human RhD and RhCE are homologous proteins that have more than 90% sequence identity and still exposure to RhD can result in a potent immune response in a D-negative individual (RhC positive) [9][10][11].  Figure 4: Immunization of DRB1 * 1501 mice with KLH-conjugated synthetic RhD peptides induces peptide specific IgG. Mice were immunized with each peptide (P) conjugated to KLH in Freund's adjuvant as indicated in Materials and Methods (2 mice per peptide). Detection of IgG specific for KLH (a), peptide 1 (b), peptide 2 (c), or peptide 3 (d) was assessed by ELISA. The sera tested were collected at days 28 and 35 (i.e., 14 days after 2nd and 7 days after the 3rd immunization, resp.).
A likely explanation could be that standard mice do not have the proper B or T cell repertoire to respond to the RhD protein. However, it has been previously demonstrated that mice transgenic for HLA DRB1 * 1501 respond to immunization with RhD purified protein [1], demonstrating that at least these mice (capable of expressing both human HLA-DRB1 * 1501 and murine MHC class II) have B cells specific for RhD epitopes. Conversely, when we challenged mice expressing only human HLA-DRB1 * 1501 with intact human RhD positive RBCs, antibodies specific to RhD were not observed though these mice successfully developed antibodies reactive with human RBCs. It could be possible that the naturally expressed human RhD protein is not a sufficiently dominant antigen in mice to generate a response.
B or T cell lymphocyte responses are usually limited to a small proportion of the potential determinants on a protein antigen. Thus, when mice are challenged with human RBC expressing a variety of foreign proteins, the RhD protein could behave as a cryptic antigen. It is also likely that the antibody response to the RhD protein may be very low and that the flow cytometry assay is not sensitive enough to detect it. While flow cytometry has the advantage of measuring antibodies against the naturally expressed antigen on the red blood cell, it needs at least 100 molecules bound per cell to be detectable [12].
To confirm if mice expressing only HLA DRB1 * 1501 as a potential restricting element have the proper B cell repertoire to respond to human RhD sequences, these mice