Biochemical analysis of CTLA-4 immunoreactive material from human blood
© Tector et al; licensee BioMed Central Ltd. 2009
Received: 1 April 2009
Accepted: 22 September 2009
Published: 22 September 2009
CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear.
Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material.
Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins.
We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.
Alternate splicing of the CTLA-4 mRNA transcript can give rise to at least three mRNA species that encode different polypeptides . The most well characterized of these is a type I transmembrane protein (CTLA-4-TM) expressed on activated T-lymphocytes [2, 3]. CTLA-4-TM is a co-receptor for the B7.1 (CD80) and B7.2 (CD86) molecules expressed on antigen presenting cells [4, 5]. CTLA-4-TM inhibits immune activity in multiple ways. It regulates signaling through the T-cell receptor [6, 7], induces expression of immunoregulatory factors such as TGF-β and ICAM-1 [8, 9], alters the organization of the immunological synapse , increases tryptophan catabolism by antigen presenting cells [11, 12] and binds B7.1 and B7.2 preventing activation of lymphocytes through the costimulatory lymphocyte receptor CD28 . Another transcript of the CTLA-4 gene encodes a molecule lacking the transmembrane domain, thus producing a soluble CTLA-4 polypeptide referred to as sCTLA-4 [14, 15]. Like CTLA-4-TM, sCTLA-4 appears to bind B7.1 and B7.2, and may have immunomodulatory properties [15–17]. Finally, a variant transcript  has been identified in mouse (although not humans) that encodes a membrane-spanning molecule with an intact cytoplasmic tail, but lacks the extracellular domain. As such, the molecule does not bind the B7 family ligands  and has been referred to as ligand-independent CTLA-4 (liCTLA-4).
The expression of these three CTLA-4 transcripts and their polypeptide products has been associated with immunoregulatory function, and differences in their expression have been associated with immune-mediated disease. For example, CTLA-4 knockout mice express none of the possible alternate transcripts and show a profound lymphoproliferative disorder with fatal multiorgan destruction [20, 21]. Although it is commonly believed that the absence of the CTLA-4-TM transcript is solely responsible for the observed immunological disorder in CTLA-4-knockout mice, the role(s) of the other transcripts have not been studied as intensively. LiCTLA-4 may have immunoregulatory functions, as transfection of it into CTLA-4 deficient T cells partially corrects the tendency for hyperresonsiveness , and the liCTLA-4 transcript has been associated with the development of insulin dependent diabetes mellitus in the NOD mouse . Finally, a variety of reports implicate a role for sCTLA-4 in human autoimmune disease. The CT60 single nucleotide polymorphism of the CTLA-4 gene has been associated with autoimmunity and with reduced levels of the sCTLA-4 transcript . Various studies have demonstrated elevated levels of the sCTLA-4 protein in the blood of patients with a variety of immunologically mediated diseases including autoimmune thyroid disease , systemic lupus erythematosis [23, 24], cutaneous systemic sclerosis , allergic asthma [26, 27], and psoriasis vulgaris . This apparent inverse relationship between levels of sCTLA-4 mRNA and circulating levels of the sCTLA-4 protein is not understood.
Several years ago, we  and others  described immunoassays for the detection of sCTLA-4 in human plasma. Presumably, such material was the gene product of the sCTLA-4 transcript; however, this was never formally proven. In order to characterize sCTLA-4 in human blood, we performed biochemical analyses of blood-derived molecules that are recognized by multiple CTLA-4-specific antibodies. Our results suggest that the immunoreactive material in human blood is not the direct product of the sCTLA-4 alternate transcript and has several biochemical features of human immunoglobulin. In addition, CTLA-4 immunoreactive material from human plasma binds the B7.1 and B7.2 proteins, and may have immunomodulatory function.
Monoclonal Antibodies and fusion proteins
The following monoclonal antibodies against CTLA-4 (CD152) were used in these studies: BNI3 (BD Pharmingen, SanDiego, CA), AS32 and AS33 (Antibody Solutions, Palo Alto, CA), are monoclonal antibodies that recognize extracellular epitopes in the CTLA-4 molecule. The MOPC-21C antibody (Sigma-Aldrich, St. Louis, MO) was used as a negative control. ELISA assays for CTLA-4 were done as described previously . B7.1-Ig (CD-80) and B7.2-Ig (CD86) fusion proteins (R&D Systems, Minneapolis, MN) were biotinylated with the use of a commercially available kit (Pierce Immunochemical, Rockford, IL). The Muc18-Ig protein (Muc-Ig) fusion protein was produced by transfecting CHO cells with a commercially available plasmid (Novagen) encoding a Muc18-Ig fusion as described [15, 29].
Purification of blood-derived sCTLA-4
Plasma samples used in these studies were obtained from humans undergoing therapeutic plasmapheresis for myasthenia gravis. Documented informed consent was obtained for each of the subjects enrolled in this study. This study was performed under the oversight of the Institutional Review Board of Aurora Health Care (Protocol # L-04-35E). Plasma was frozen at -20°C until use. 200 mls of plasma were diluted with 20 mM Tris-HCl in water until the pH of the solution reached 6.8. Typically this required a mixture 1 volume of plasma to 9 volumes of 20 mM Tris-HCl. This mixture was then passed through a column containing 300 mls of Q sepharose (GE healthcare). Flow through was collected and solid ammonium sulfate was added to create a 45% saturated solution. After overnight incubation at 4°C, precipitate was collected by centrifugation at 1,800 g for 15 minutes. The pelleted material was resuspended in 20 mls of phosphate buffered saline (PBS, pH 7.4) and dialyzed using a 1 kDa molecular weight dialysis tubing in PBS with 2 buffer exchanges. This material was passed over a 2 ml column, linked to the MOPC-21 monoclonal antibody using the Pierce Aminolink Plus Immobilization kit, and flow through was then passed over a column containing the antibody AS32. Material was eluted from the AS32 column using elution buffer (Pierce) and collected in 1 ml fractions.
Blood-derived sCTLA-4 binding to B7.1-Ig and B7.2-Ig
96 well ELISA plates were coated with 100 ul of fractions collected from the AS32 column overnight at 4°C. After coating, plates were washed and incubated with 250 μl block solution (PBS containing 1% w/v bovine serum albumin and 5% w/v sucrose) for 1 hour at room temperature to block nonspecific protein binding sites. Blocked wells were incubated with the biotinylated proteins B7.1-Ig, B7.2-Ig, Muc18-Ig, or AS33 at room temperature for 1 hour. Next, wells were washed, incubated with streptavidin HRP (Zymed Carlsbad, CA) for 20 minutes and washed again. TMB (Pierce Immunochemicals) was added to the wells, the HRP reaction proceeded for approximately 15 minutes, was stopped by addition of 1N H2SO4, and absorbance was read during illumination with light at 450 nm.
Protein A binding of sCTLA-4
Recombinant sCTLA-4, produced as described , or plasma diluted 1:10 with 150 mM NaCl and 10 mM Tris pH 7.4 (TBS), was passed over a column containing 1 ml of protein A sepharose (Pierce). Flow through was collected in 1 ml fractions. 10 mls of TBS was passed over the column to wash unbound material and was collected in 1 ml fractions. Bound material was eluted from the column in 100 mM glycine pH 3, collected in 1 ml fractions and neutralized with 100 ul of 10xTBS. Pre-column, flow through, wash, and eluted fractions were tested in ELISA for the presence of sCTLA-4.
1.5 mls of serum was diluted with 1.5 mls of PBS containing recombinant sCTLA-4, and passed through a column (1.5 cm diameter) containing approximately 150 ml of sephacryl S200 (GE healthcare). The first 4 mls were discarded and then 2 ml fractions were collected by hand. Each fraction was tested for the presence of blood-derived sCTLA-4 or recombinant sCTLA-4 using elisa assays.
Identification of proteins from 1D-SDS PAGE was performed by Proteomic Research Services (http://www.nextgensciences.com/LCMSMS.htm. Ann Arbor, MI). Proteins that eluted from the AS32 column were precipitated in 70% ethanol and pelleted by centrifugation 14,000 rpm in a microcentrifuge for 5 minutes. The pellet was dissolved at room temperature in 5% SDS, 10 M Urea, 10 mM Tris buffer (pH 6.8), and 10% 2-mercapto ethanol, and separated using NuPage 12% bis-tris SDS PAGE gels in MES running buffer (Invitrogen, Carlsbad, CA) and visualized by staining gels with coomassie blue. Proteins were reduced, alykylated and trypsin digested in isolated gel fragments, and were identified using LC/MS/MS.
Protein identification by 2D-DIGE was performed by Applied Biomics (http://www.appliedbiomics.com/index.html. Hayward, CA). Purified proteins were labeled with either Cy3 or Cy5. The first dimension separation consisted of isoelectric focusing over pH 3-10. The second dimension separation was carried out using 8-14% gradient SDS-PAGE. Differentially expressed proteins were cut out and digested with trypsin before analysis with mass spectroscopy.
The CTLA-4-specific monoclonal antibodyAS32 isolates B7-binding proteins from blood
Identification of proteins enriched by an AS32 column
LC/MS/MS identification of proteins in the 150 kd band unique to subjects testing positive in ELISA using CTLA4 specific antibodies.
% Sequence Coverage
Chain A, Human Factor Vii C2 domain
Protein NIG51 lambda Bence-Jones
Ig heavy chain V-III region CAM
Immunoglobulin heavy chain [homo sapiens]
Unnamed protein product
Anti HBs antibody light-chain Fab fragment [homo sapiens]
Unnamed protein product [homo sapiens]
Keratin 1 [homo sapiens]
Ig A L
Anti HBs antibody light-chain Fab fragment [homo sapiens]
IGHG1 protein [homo sapiens]
IGHM protein [homo sapiens]
Keratin type I cytoskeletal 9 [homo sapiens]
Recombinant sCTLA-4 and anti-CTLA-4 affinity-purified material from human plasma differentially bind protein A and have different molecular masses
For comparison, serum from a donor lacking molecules recognized by CTLA-4 specific antibodies was spiked with recombinant sCTLA-4 and then passed over a sizing column. Recombinant sCTLA-4 clearly eluted from the column after IgG (Figure 6B). The minimal overlap of the IgG fractions with fractions containing recombinant sCTLA-4 demonstrates that recombinant sCTLA-4 is smaller than 150 kD.
2D gel electrophoresis and mass spectroscopy of proteins enriched on an anti-CTLA-4 affinity column
Mass Spectroscopy Identification of Proteins Enriched by Affinity Purification of Plasma with a CTLA4 Affinity Column and Analyzed by 2D-DIGE
Fold Increase As32:MOPC
Immunoglobulin heavy chain
Immunoglobulin heavy chain
This CDS feature is included
complement C1q subcomponent, α
complement C1q subcomponent, α
immunoglobulin lambda light chain C region
Unknown (protein for MGC:31941)
Chain B, Globular Head Of The Complement System
anti HBs antibody light-chain Fab
α-Entamoeba histolytica immunoglobulin κ light chain
anti HBs antibody light-chain Fab
Our biochemical analyses demonstrate that the molecules in human blood that are recognized by anti-CTLA-4 antibodies are not a simple product of the sCTLA-4 alternate transcript. We previously described sCTLA-4 immunoreactive material in blood plasma from patients with autoimmune thyroid disease . In that study, Western blotting of material from immunoprecipitates, using a pool of commercially available monoclonal antibodies to CTLA-4, identified proteins with a molecular mass of 23 kDa consistent with the size of recombinant sCTLA-4. We also noted the observation of larger material (> 100 kDa) in those immunopreciptates but did not characterize this species further. The CTLA-4 immunoreactive material described in this communication may be the larger molecules noted in our previous studies.
This study identifies material that reacts with multiple anti-CTLA-4 antibodies and with known ligands of CTLA-4, B7.1 (CD80) and B7.2 (CD86). Additionally, its molecular size is approximately 150 kDa, it binds protein A with high affinity, and when analyzed by mass spectroscopy, we find human antibodies. Despite the fact that the molecules studied exhibit characteristics of CTLA-4-Ig fusion proteins, none of our plasma donors received exogenous CTLA-4-Ig (Orencia) as a therapy. Therefore the isolated molecules are endogenous to our plasma donors.
Though we did not conclusively identify these molecules, we found 2 separate light chains that originated from human immunoglobulins specific for Hepatitis B surface antigen (Tables 1 and 2). One of the Hepatitis B specific light chains was found in 2 different subjects, and both light chains were isolated from a single donor. While it is possible that we have identified an idiotype network involving epitopes on the Hepatitis B surface antigen, CTLA-4, and some immunoglobulins, comparing the sequences with the bl2seq program on the NCBI website http://www.ncbi.nlm.nih.gov/ did not reveal any obvious sequence homology among these molecules. A more detailed structural analysis is warranted.
Our gel filtration and protein A binding data suggest that if sCTLA-4 exists in the blood samples we analyzed, it appears to be complexed with immunoglobulin. Another possibility is that blood contains protein(s), either immunoglobulins or a novel protein associated with immunoglobulins, that interact with the ligands of CTLA-4 and with CTLA-4 specific antibodies.
We believe the presence of CTLA-4 immunoreactivity in plasma is not likely due to heterophile antibodies that are known to interfere with two site immunoassays [30, 31]. The samples selected for analysis in these studies were screened to be negative for binding to irrelevant mouse IgG. Furthermore, the Muc-Ig negative control used in the B7 binding assays contained the identical Ig fusion partner as the B7-Ig proteins. Finally, if present, heterophile antibodies should have been removed by the MOPC column during the isolation procedure.
Our findings may reconcile the apparent discrepancy between reports of elevated levels of sCTLA-4 in plasma from patients with autoimmune disease and the report of decreased levels of the sCTLA-4 transcript among individuals with the CT60 allele of the CTLA-4 gene. CT60 is a single nucleotide polymorphism within the CTLA-4 locus  and the "G" allele is associated with both susceptibility to Type 1 diabetes and low levels of the sCTLA-4 transcript. Because sCTLA-4 in blood is probably not the direct product of the sCTLA-4 transcript, the lack of correlation between transcript and plasma protein, is not surprising. In addition, immunoassays designed to measure sCTLA-4 levels may not reliably quantify blood-derived sCTLA-4 because of its uncertain identity. In this regard, it is important to interpret reports of sCTLA-4 levels with caution, and to re-examine the possible relationship of circulating CTLA-4 levels and human disease. To this point, we have recently reported a lack of association between levels of sCTLA-4 protein in blood and several of the common polymorphisms that show population genetic associations with a variety of autoimmune disease .
Molecules from human blood that have been labeled sCTLA-4 are not simply a direct product of an alternatively spliced transcript of the CTLA-4 gene. These molecules exhibit properties of CTLA-4 and of immunoglobulins. Though some studies have found a correlation between circulating levels of the putative sCTLA-4 molecule and the presence of autoimmunity, this relationship is poorly understood. This study improves understanding of the biochemical nature of what has been called sCTLA-4 and will help subsequent analyses to elucidate the role of this molecule in autoimmune disorders.
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