Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types, but may be masked by Fab complementarity-determining region peptide in the native sera

Abstract

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti-HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA-I/-II alleles, indicating that anti-HLA IgG may be masked in normal sera – either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity-determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti-HLA IgG purified from normal sera – and serum IgG from a few donors – indeed recognized the HLA types of the corresponding donors, confirming the presence of auto-HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto-HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto-HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.

Introduction

Anti-human leucocyte antigen (HLA) immunoglobulin (Ig)G antibodies increase in patients waiting for organ transplantation 1. After transplantation, many patients have IgG antibodies reactive against HLA types of the allograft 2, i.e. donor-specific antibodies (DSA). Most of these patients also produce non-donor-specific HLA antibodies (NDSA), both DSA and NDSA correlating with allograft rejection 3. One of the therapeutic countermeasures developed is to administer intravenous immunoglobulin (IVIg), purified from plasma pooled from thousands of donors 1,2. In 2004, the US Food and Drug Administration (FDA) approved IVIg for lowering the incidence of anti-HLA antibodies in allograft recipients. High-dose IVIg is given to reduce the anti-HLA IgG in patients waiting for an allograft 1 and to those developing DSA post-transplant 2. IVIg is also used with several autoimmune diseases to suppress effector T and B cells involved in antibody production 4,5. Paradoxically, all available therapeutic IVIg preparations contain high levels of IgG that react to a wide array of HLA antigens 6 – possibly defeating the very purpose of reducing the levels of anti-HLA antibodies.

Perhaps IVIg recognizes a wide array of HLA alleles because the pooled sera or plasma of normal individuals from which it is derived contains naturally occurring anti-HLA IgG antibodies 7, or perhaps because of purification of the IgG from the pooled plasma. If the latter is the reason, we hypothesized that the IgG purified from normal sera should also display a similar wide variety of HLA reactivity. If so, it is essential to ascertain whether the natural IgG, purified or in native sera, recognizes corresponding donor HLA types, and it is also essential to identify the factors that mask the HLA reactivity of IgG in native sera. Identification of such masking factors may lead to a therapeutic benefit in that it may facilitate clinical minimization of the HLA reactivity of the sera of sensitized patients, allograft recipients and even patients with autoimmune diseases. In addition, elucidation of the masking factors may lead to an understanding of the innate regulation of anti-HLA antibody expression.

Accordingly, the objectives of this investigation were:

• to determine whether purification and isolation of normal individual sera exposes IgG antibodies that react, like IVIg, to a wide variety of HLA alleles;

• to examine whether the HLA reactivity of IgG – before or after purification from the sera – recognizes the HLA types of the corresponding donors, thus establishing the presence of auto-HLA antibodies; and

• to ascertain which serum factors, possibly derived from degraded circulating HLA or IgG, mask expression of the HLA antibodies in sera.

Materials and methods

Sources of the therapeutic preparations of IVIg

Previously, four therapeutic preparations of IVIg were examined for anti-HLA-I reactivity 6. Here, we examined the same IVIg preparations for anti-HLA-II reactivity. These were: GamaSTAN™ S/D (15–18%, Lot 26NHCVI) (Telacris Biotherapeutics, Inc., Research Triangle Park, NC, USA); Sandoglobulin® (6%, Lot 4305800026) (CSL Behring, Kankakee, IL, USA); Octagam® (6%, Lot A913A8431) (Octapharma Pharmazeutika, Switzerland/Austria); and Gamunex®-C (10%, Lots 26NKLG1 and 26NKLK1) (Telacris). All IVIg was serially diluted with phosphate-buffered saline (PBS) (pH 7·2), respectively, to 1/512, 1/1024, 1/2048 and 1/4096.

Source of normal non-alloimmunized male sera

After informed consent, 5 ml of blood was drawn from each of 20 normal, non-alloimmunized male volunteers – researchers at the Terasaki Foundation Laboratory (TFL) or their family members or visiting scientists. The sera were analysed along with LABScreen® negative control serum from Lot 9 (LSNC-9) (One Lambda, Inc. Canoga Park, CA, USA), a pool of sera from eight to 10 donors. Volunteer TFL3 had received tuberculosis (TB) vaccine, and R2 had received anthrax, meningococcal C, hepatitis B and smallpox vaccines within 10 months prior to donation; TFL5 had had a titanium implant after a leg injury 6 months earlier; TFL1 had had an IVIg infusion 3 years earlier. Fresh sera were used for purification of IgG; sera and purified IgG were frozen and stored. Both fresh and thawed sera and purified IgG were analysed simultaneously. For peptide inhibition study, frozen sera of TFL5 and TFL12 were used.

IgG repertoire purified from the sera of non-alloimmunized males

To isolate IgG from the sera, the sera were passed through protein-G columns (Thermo Fisher Scientific, Rockford, IL, USA). Each serum sample was centrifuged for 5 min at 9300 g before 132 μl was applied to the protein-G agarose resin washed in PBS, pH 7·2; the mixture was incubated for 15 min, centrifuged and collected as IgG-free flow-through. Purified IgG was eluted three times, after three washes of the column with 400 μl binding buffer (PBS, pH 7·2), by adding 400 μl acidic buffer (pH 2·8) each time and recovery by centrifugation (for 1 min) in alkaline buffer (pH 8·5; 40 μl). The protein concentrations (mg/ml) in the three eluates (E1, E2, E3) were determined using a BioPhotometer (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany), calculated with standard human IgG curve. The unpurified sera were tested at 1:10 dilution, but the protein-G eluates were tested neat because they were diluted 1/10 during protein-G elution. The IgGs in the sera and eluates were tested at similar concentrations of IgG. The mean fluorescence intensity (MFI) of E1 was always low or minimal compared with that of E2 and E3.

Luminex-based immunoassay using microbeads coated with single HLA antigen

To detect IgG reactivity to HLA-I and/or -II alleles in IVIg, human sera and purified IgG, a Luminex-based immunoassay was used 6,7. Single antigen assays were carried out using xMAP® Luminex dual-laser multiplex flow cytometry (LABScan™ 100; One Lambda) 6,7. The array of various recombinant HLA alleles on the beads is listed under ‘Antibody detection products/LABScreen Single Antigen Product: HLA-II LS2A01009 (Lot 9)’ (see http://www.onelambda.com). The single recombinant HLA-Ia alleles in LS1A04, Lots 007 and 008, were also used for screening IVIg, sera, LSNC and purified IgG, as reported earlier 6,7. HLA-I and -II microbeads have built-in control beads – positive (coated with human IgG) and negative (coated with serum albumin). MFI [mean ± standard deviation (s.d.)] for each allele was recorded as a .csv file, corrected against negative control (normalized), and archived from mid-2012. Basic statistics were calculated with Excel software.

Isolation of different molecular size fragments of peptides, polypeptides and proteins from wash fluid

For molecular sieving of the peptide/polypeptide fractions in flow-through and wash fluid (WF), Millipore concentrator columns were used (EMD Millipore, Billerica, MA, USA). First, 500 μl of WF1 was transferred to an Amicon® Ultra-0·5 100 K device (Millipore ref. UFC510024), centrifuged for 5 min at 14 000 g, then 100 K of the concentrate (30 μl) was recovered and used in the studies detailed below. The rest of the filtrate was centrifuged further in an Ultra-0·5 10 K device (ref. UFC501024) and the “10 K” concentrate recovered and used (see below). Finally, the filtrate was centrifuged in an Ultra-0·5 3 K device (ref. UFC500324), centrifuged, and the 3 K-10 K concentrate recovered and used, as was the “< 3 K” filtrate.

Inhibition of HLA reactivity of IgG with different molecular sizes of peptides from the serum wash fluid 1

For inhibition of HLA reactivity of purified IgG, the concentrates obtained after molecular sieving were admixed with serum E2 or E3 and incubated 30 min at room temperature (RT) on a shaker. WF was admixed with E2 or E3 in the ratio: E : WF or PBS, 20:10, 25:5. The final volume was 30 μl, but only 20 μl of the mixture was added in a Luminex tray to 2 μl of microbeads coated with HLA. Eluates without WF, and eluates with PBS at different ratios, were used for calculating the percentage of change at each dilution. The beads were washed three times with LABScreen wash buffer. Phycoerythrin (PE)-conjugated anti-human IgG (50 μl) was added, incubated 30 min at RT and washed three times with wash buffer. The MFI was measured by Luminex.

Inhibition of HLA reactivity of purified IgG from normal serum using HLA-I and IgG-derived peptides

Sera may contain peptides derived from fragmented or proteolyzed HLA-I or HLA-II or non-HLA molecules, or from proteolyzed Fab or Fc fragments of antibodies. HLA-I may have peptides specific for HLA alleles or commonly shared peptides that are masked by β2-microglobulin (β2m). This investigation used these shared peptides, which are common to the loci of both HLA-Ia (HLA-A, HLA-B and HLA-Cw) and -Ib (HLA-E, HLA-F and HLA-G), and identified as ¹¹⁵QFAYDGKDY¹²³ and ¹²⁶LNEDLRSWTA¹³⁵; their source, purity and characteristics were also reported earlier 8,9. In addition, the conserved complementarity-determining region (CDRII) peptide sequence of IgG was used for peptide inhibition with the recommended peptides 10–12. LifeTein® LLC (South Plainfield, NJ, USA) synthesized at 98% purity the amino acid sequence ASRNKANDYTTEYSASVKGRFIVS (Mw: 2664·2 g/mol; water-soluble) of the peptide TEPC15 (50–73), also called ‘T15H’ 10,11, and the amino acid sequence QIYPGDGDTKYNNGKFKGKATLT of the control peptide 2FIOH (50–73), which has a similar Mw and solubility.

Specific peptide inhibition was also tested with the serum E2 fraction of three different volunteers (TFL5 for HLA-I; TFL6 and TFL12 for HLA-II). Commonly shared peptide sequences of HLA and conserved CDRII peptide, with the control peptide, were used. Eluted IgG fraction (E2) (25 μl) was mixed with 5 μl of peptide at different dilutions; the final volume was 30 μl. After incubating for 30 min at RT on a shaker, 20 μl of the mixture was tested with antigen-coated microbeads. For HLA-I beads, HLA-E was used because it carries the common shared peptide sequences tested. Because loss of sensitivity is likely with multiple HLA-I allele-coated single antigen beads (n = 90), single beads of this kind were used. Sensitivity was preserved by using a mixture of HLA-II beads with the serum E2 fraction of TFL6 and TFL12, which is why observations on peptide inhibition were restricted to the most prevalent HLA-II molecules, as described in the Results. Due to the restricted availability of eluates 2 and 3 from one donor at the same time, all our observations were carried out either on the two eluates separately or from the eluates of three different sera to validate the observations.

Immunoassay using iBeads

One Lambda has generated beads with reduced amounts of β2m-free HLA (iBeads); these are regular HLA-I-coated microbeads subjected to proprietary enzymatic treatment to remove or reduce the amount of heavy chains (denatured antigens). The manufacturer provided iBeads as Felix beads for in-house use.

Murine monoclonal antibody reacting to all HLA class-II alleles

Mouse monoclonal antibody (mAb) FJ5109 (One Lambda), reactive to all HLA-II alleles, was used after being passed through a protein-G column to determine whether that caused any change in the reactivity profile.

HLA molecular typing

DNA from the donors' blood was isolated by using the QIAamp DNA Mini Kit (Qiagen Group, Valencia, CA, USA), then polymerase chain reaction (PCR)-amplified and typed using the LABType® SSO Typing Test Kit (One Lambda).

Results

Basis of the experiments

We examined the HLA reactivity of sera, and IgG purified from those sera, from 20 volunteers: 11 TFL researchers, three visiting scientists and six members of three researcher/visitor families. Serum of a 12th TFL visitor was used in a study of peptide inhibition of antibody binding to microbeads. The HLA reactivity of four IVIg preparations was also examined: GamaSTAN, Octagam, two lots of Gamunex-C and Sandoglobulin. The number of alleles examined was: 31 HLA-A, 50 HLA-B, 16 HLA-Cw, 36 HLA-DRB, 29 HLA-DQ and 26 HLA-DP.

Anti-albumin IgG: natural autoantibody in non-alloimmunized males and IVIg

IgG reactivity to HLA-I/-II alleles in IVIg and purified human IgG was measured by single antigen assay. The MFI of anti-albumin IgG in normal sera is < 150. After purification of IgG from the sera, the MFI measured several fold higher. The MFI of the purified anti-albumin IgG (at 1:10 dilution) of the donors ranged extensively; note that the seventh highest, ‘LSNC’, is not one of the 20 original donors, but the control serum: G1 (MFI, 5140), TFL3 (3477), W1 (3399), TFL4 (3237), R1 (1830), TFL5 (1549), LSNC (1394), TFL1 (1299), W2 (1186), TFL9 (1152), TFL7 (1103), T2 (910), TFL6 (808), TFL8 (778), W3 (772), TFL10 (673), R2 (663), TFL2 (633), T3 (485), TFL11 (467) and UCLA1 (213). There was no correlation between age and MFI, but the sometimes anomalous disparities will be addressed in the Discussion. The anti-albumin values were used as negative control to normalize the respective MFIs of the HLA-I/-II antibodies obtained from purified serum. Interestingly, all IVIg preparations except Sandoglobulin showed high reactivity to the albumin-coated beads, indicating the presence of differing levels of anti-albumin IgG antibodies in different preparations (data not shown). The two lots of Gamunex-C had the highest level of HLA reactivity.

Anti-HLA-II IgG: profiles before and after purification of IgG from the sera

The profile and incidence of anti-HLA-II IgG antibodies before and after purifying the IgG were examined in three cohorts of sera: the LSNC (Table 1); from different generations of non-alloimmunized, healthy males in three families (Table 2a–c); and from 11 other normal, non-alloimmunized, healthy TFL males (Table 3a–c).

Table 1.

Mean fluorescence intensity (MFI) of IgG antibodies in negative control sera [LABscreen negative serum [LSNC)] and in purified LSNC eluate 2 (E2) that reacted to human leucocyte antigen (HLA) class II alleles and albumin

HLA class II IgG†† in LSNC before and after purification of IgG and normalization of MFI
DRB	LSNC (1/10)	Normalized E2	DQA/DQB	LSNC (1/10)	Normalized E2	DPA/DPB	LSNC (1/10)	Normalized E2
Anti-albumin	20	2698	DQA1*01:01\DQB1*03:02	38	992	DPA1*01:03\DPB1*02:01	44	697
DRB1*08:01	33	1260	DQA1*01:01\DQB1*06:02	43	1580	DPA1*01:03\DPB1*11:01	38	1540
DRB1*09:01	46	556	DQA1*02:01\DQB1*03:02	31	951	DPA1*01:03\DPB1*19:01	54	2811
DRB3*01:01	29	1668	DQA1*02:01\DQB1*04:01	46	1052	DPA1*01:05\DPB1*13:01	32	1010
DRB3*03:01	167	7616	DQA1*03:01\DQB1*03:01	32	2457	DPA1*02:01\DPB1*13:02	31	951
			DQA1*03:01\DQB1*03:02	33	561	DPA1*02:01\DPB1*15:01	22	747
						DPA1*02:01\DPB1*23:01	66	6929
						DPA1*02:01\DPB1*28:01	57	1499

^†The antibody with the highest MFI value for each locus is shown in bold type.

Table 2.

Mean fluorescence intensity (MFI) of serum and purified IgG from different generations of three families that reacted to human leucocyte antigen (HLA) class II alleles and albumin

(a) HLA class II IgG antibodies in serum and in eluate 2 after purification of IgG and normalization of MFI
DR	Son		Father		Paternal grandfather		DQA/DQB	Son		Father		Paternal grandfather		DPA/DPB	Son		Father		Paternal grandfather
	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG		Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG		Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin				910		1299	DQA1*01:01\DQB1*03:02		701		778	512	1443	DPA1*01:03\DPB1*02:01		590		845
DRB1*03:02		555				649	DQA1*01:01\DQB1*06:02		509		515			DPA1*01:03\DPB1*03:01			597	530
DRB1*08:01		758		767		763	DQA1*01:02\DQB1*06:02		797		1004		859	DPA1*01:03\DPB1*04:01			572
DRB1*09:01		687		542		664	DQA1*02:01\DQB1*03:01					908	503	DPA1*01:03\DPB1*04:02			1136	712
DRB1*11:01				507			DQA1*02:01\DQB1*03:02		748		815	1264	2440	DPA1*01:03\DPB1*11:01		1014	694	1338	519	1551
DRB1*11:04			707	1026			DQA1*02:01\DQB1*04:01		645		759		670	DPA1*01:03\DPB1*19:01		1701	560	1520		1682
DRB1*13:01					606		DQA1*02:01\DQB1*04:02				539			DPA1*01:05\DPB1*03:01			572	697
DRB1*14:02				660			DQA1*03:01\DQB1*03:01		708		791	581	1148	DPA1*01:05\DPB1*11:01		683		1089	1216	1394
DRB1*15:03			673				DQA1*03:01\DQB1*03:02					1447	2125	DPA1*01:05\DPB1*13:01		962		996		790
DRB3*01:01		1038		1102		1104	DQA1*05:03\DQB1*03:01					1608	740	DPA1*01:05\DPB1*18:01			1060
DRB3*02:02				508			DQA1*05:05\DQB1*03:01					1523	765	DPA1*01:05\DPB1*28:01			642
DRB3*03:01		3436	467	3637		7939	DQA1*06:01\DQB1*03:01					1595	745	DPA1*02:01\DPB1*01:01		578	1276	1594
DRB4*01:01			786	710										DPA1*02:01\DPB1*03:01			733	528
DRB5*01:01				553		552								DPA1*02:01\DPB1*05:01				559
														DPA1*02:01\DPB1*13:01		1198		1156		1242
														DPA1*02:01\DPB1*15:01		752	646	1151		678
														DPA1*02:01\DPB1*23:01		3449		2950		5018
														DPA1*02:01\DPB1*28:01		862	523	1029		1337
Serum of the son did not show reactivity for any of the DRB/DQA\DQB/DPA\DPB alleles. † The antibody with the highest MFI value for each locus is shown in bold type.

(b)HLA class II IgG antibodies † in serum and in eluate 2 after purification of IgG and normalization of MFI
DR	Son		Father		Maternal grandfather		DQA/DQB	Son		Father		Maternal grandfather		DPA/DPB	Son		Father		Maternal grandfather
	Serum	Normalized Purified IgG	Serum	Normalized Purified IgG	Serum	Normalized Purified IgG		Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG		Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin		772		1186	1832	3399	DQA1*01:01\DQB1*03:02		910		844	579	1566	DPA1*01:03\DPB1*01:01					640
DRB1*03:02	501	1373			609		DQA1*01:01\DQB1*06:02		533			604		DPA1*01:03\DPB1*02:01		927		644	849	892
DRB1*04:01		513					DQA1*01:02\DQB1*05:02						520	DPA1*01:03\DPB1*11:01		611			1105
DRB1*04:02					602		DQA1*01:02\DQB1*06:02		1061		954	673	1914	DPA1*01:03\DPB1*19:01		2256		1351	1106	2431
DRB1*04:03		717					DQA1*01:02\DQB1*06:04		507		382	643		DPA1*01:05\DPB1*03:01					591
DRB1*04:04	519						DQA1*01:02\DQB1*06:09					546		DPA1*01:05\DPB1*11:01				647	665
DRB1*08:01		813		595	642	525	DQA1*02:01\DQB1*03:02		981		884		1686	DPA1*01:05\DPB1*13:01		1109		999	843	1402
DRB1*09:01		590			956		DQA1*02:01\DQB1*04:01		699		880	564	1060	DPA1*02:01\DPB1*01:01		633			744
DRB1*11:04					780		DQA1*02:01\DQB1*04:02					508	537	DPA1*02:01\DPB1*03:01					693
DRB1*12:01					1608	834	DQA1*03:01\DQB1*03:01		939		881		2185	DPA1*02:01\DPB1*05:01					833
DRB1*12:02		612			551		DQA1*03:01\DQB1*03:02		528		508		559	DPA1*02:01\DPB1*06:01					1030
DRB1*14:02					671		DQA1*03:01\DQB1*03:03					524		DPA1*02:01\DPB1*10:01					662
DRB1*14:54					615		DQA1*05:01\DQB1*02:01					693		DPA1*02:01\DPB1*13:01		1672		1520	902	2831
DRB1*15:01					1208									DPA1*02:01\DPB1*14:01					674
DRB1*15:02					504									DPA1*02:01\DPB1*15:01		827		625	1041	1226
DRB1*16:01					714									DPA1*02:01\DPB1*17:01					598
DRB1*16:02					519									DPA1*02:01\DPB1*23:01		3251		3021	1433	5886
DRB3*01:01		1022		825	1377	1999								DPA1*02:01\DPB1*28:01		934		673	1326	1043
DRB3*02:02					584
DRB3*03:01		3013	552	2497	2541	5617
DRB4*01:01					690
DRB5*01:01					841
Serum of the son did not show reactivity for any of the DQA\DQB/DPA\DPB alleles. † The antibody with the highest MFI value for each locus is shown in bold type.

(c)HLA class II IgG antibodies† in serum and in eluate 2 after purification of IgG and normalization of MFI
DR	Father		1st son		2nd son		DQA/DQB	Father		1st son		2nd son		DPA/DPB	Father		1st son		2nd son
	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG		Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG		Serum	Normalized purified IgG	serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin		633		1830		663	DQA1*01:01\DQB1*03:02		841		1162		815	DPA1*01:03\DPB1*02:01		741		639
DRB1*03:01					991	2211	DQA1*01:01\DQB1*06:02		696		581			DPA1*01:03\DPB1*11:01		1189		826		766
DRB1*03:02		907			1834	4199	DQA1*01:02\DQB1*05:02		525				847	DPA1*01:03\DPB1*19:01		1392		2020		1052
DRB1*08:01		834		1210		748	DQA1*01:02\DQB1*06:02		1087		1178		750	DPA1*01:05\DPB1*11:01		528		531		513
DRB1*09:01		797					DQA1*01:02\DQB1*06:04						778	DPA1*01:05\DPB1*13:01		963		1312		823
DRB1*10:01	574	1988	2864	2201			DQA1*02:01\DQB1*03:02		880		1308		757	DPA1*02:01\DPB1*05:01		519
DRB1*12:01		705					DQA1*02:01\DQB1*04:01		649		938		612	DPA1*02:01\DPB1*13:01		1126		1991		1167
DRB1*12:02		571		628			DQA1*03:01\DQB1*02:01						682	DPA1*02:01\DPB1*15:01		813		715		670
DRB1*14:02		558					DQA1*03:01\DQB1*03:01		800		1325		588	DPA1*02:01\DPB1*23:01		3891		5816		2541
DRB3*01:01		1153		1214		779	DQA1*03:01\DQB1*03:02		485				677	DPA1*02:01\DPB1*28:01		1447		1055		717
DRB3*03:01		4964		4786		3300	DQA1*03:01\DQB1*03:03		606
DRB4*01:01		743			500	925

Sera of neither father nor sons showed reactivity for any of the DQA\DQB/DPA\DPB alleles. ^†The antibody with the highest MFI value for each locus is shown in bold type.

Table 3.

Mean fluorescence intensity (MFI) of serum and purified IgG of male volunteers that reacted with human leucocyte antigen (HLA) class II alleles and albumin

(a) Anti-HLA-DRB reactivity
HLA class II IgG antibodies† in serum and in eluate 2 after purification of IgG and normalization of MFI
DRB	TFL3		TFL4		TFL5		TFL6		TFL7		TFL8		TFL9		TFL10		TFL11		UCLA1		G1
	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin		3477		3237		1549		808		1103		778		1 152		673						5 140
DRB1*01:02	526
DRB1*03:01										715				1 262		544
DRB1*03:02	671					812			648	2695			582	1 826	606	1676
DRB1*04:01	677				529	933										635
DRB1*04:02	589					506
DRB1*04:03	853				606	723									582	762
DRB1*04:04	944				892										610	575
DRB1*04:05						843										670
DRB1*08:01						863		713				706		548		1276						2 611
DRB1*09:01						939		613				662		1 612		1360
DRB1*11:01						721
DRB1*11:04					1117	1289										1019
DRB1*12:02																523
DRB1*14:02						729						732				1034
DRB1*14:54															593	1131
DRB1*15:03																690
DRB1*16:01	934															788
DRB3*01:01						1398		994		565		998		1 717		2128		630				4 394
DRB3*02:02																575
DRB3*03:01		2096		2040		5013		5385		4133		4304		10 088	510	6371		2576		987		12 401
DRB4*01:01	625		540					752						777		552
DRB5*01:01																628

Sera of TFL6/8/11, UCLA1 and G1 did not show reactivity for any of the DRB alleles. ^†The antibody with the highest MFI value is shown in bold type.

(b) Anti-HLA-DQ reactivity
HLA class II IgG antibodies† in serum and in eluate 2 after purification of IgG and normalization of MFI
DQA/DQB	TFL3		TFL4		TFL5		TFL6		TFL7		TFL8		TFL9		TFL10		TFL11		UCLA1		G1
	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin		3477		3237		1549		808		1103		778		1152		673						5140
DQA1*01:01\DQB1*03:02						1342		631				537			727	1263						3429
DQA1*01:01\DQB1*06:02						641									1604	2344						784
DQA1*01:02\DQB1*05:02						1183									2439	3252						738
DQA1*01:02\DQB1*06:02	567	538				1873		748				677		508	1502	2337		514				5181
DQA1*01:02\DQB1*06:04						935									1482	2030						536
DQA1*02:01\DQB1*02:01															1607	1752
DQA1*02:01\DQB1*02:02						514									1793	2136
DQA1*02:01\DQB1*03:01	543														1059	1208
DQA1*02:01\DQB1*03:02						1540		603				506			922	1476						3429
DQA1*02:01\DQB1*04:01						1777		596							577	945						2362
DQA1*02:01\DQB1*04:02						739									825	1169						517
DQA1*03:01\DQB1*02:01															806	1267
DQA1*03:01\DQB1*03:01	500					2170		606							620	1155						4391
DQA1*03:01\DQB1*03:02						656									758	1237						1559
DQA1*03:01\DQB1*03:03															909	1188
DQA1*03:02\DQB1*03:02																				549
DQA1*04:01\DQB1*02:01															749	828
DQA1*04:01\DQB1*04:02						575									1074	1199
DQA1*05:03\DQB1*03:01	675
DQA1*05:05\DQB1*03:01	625

(c) Anti-HLA-DP reactivity
HLA class II IgG antibodies† in serum and in eluate 2 after purification of IgG and normalization of MFI
DPA/DPB	TFL3		TFL4		TFL5		TFL6		TFL7		TFL8		TFL9		TFL10		TFL11		UCLA1		G1
	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG	Serum	Normalized purified IgG
Anti-albumin		3477		3237		1549		808		1103		778		1152		673						5140
DPA1*01:03\DPB1*01:01						588										795
DPA1*01:03\DPB1*02:01						1288						553				792						828
DPA1*01:03\DPB1*04:02	586														500
DPA1*01:03\DPB1*11:01						833		990		645		1029		2843	522	2846		627				726
DPA1*01:03\DPB1*19:01	793	1040			614	2355		1450		728		1382		1977		1964		925				5862
DPA1*01:05\DPB1*03:01																507
DPA1*01:05\DPB1*11:01						848		556							725	1266						734
DPA1*01:05\DPB1*13:01						1570		844				874		679		1344		582				2082
DPA1*01:05\DPB1*18:01	527
DPA1*01:05\DPB1*28:01															602	520
DPA1*02:01\DPB1*01:01	519				1561	1611										1034		572
DPA1*02:01\DPB1*03:01																544
DPA1*02:01\DPB1*05:01																1009
DPA1*02:01\DPB1*06:01																769
DPA1*02:01\DPB1*10:01																660
DPA1*02:01\DPB1*13:01		1037				2142		1104		632		1003		995		1604		763				7057
DPA1*02:01\DPB1*15:01	506					1423		578				581		528		1206						2815
DPA1*02:01\DPB1*23:01		2411		857		4921		4419		2477		2891		8460		4706		2317		930		13746
DPA1*02:01\DPB1*28:01						1550		808		566		912		1319	589	2168		667				2564

Only sera of TFL3, TFL5 and TFL10 showed reactivity for any of the DPA\DPB alleles. ^†The antibody with the highest MFI value is shown in bold type.

(d) Incidence of purified and normalized IgG from E2 that reacted to HLA-DRB, DQA/DQB and DPA/DPB haplotypes in all cohorts of sera examined
HLA class II	n = 20	%
Albumin negative control	17	85
DRB
DRB1*03:02	9	45
DRB1*08:01	15	75
DRB1*09:01	10	50
DRB3*01:01	17	85
DRB3*03:01	20	100
DQA/DQB
DQA1*01:01\DQB1*03:02	14	70
DQA1*01:02\DQB1*06:02	17	85
DQA1*02:01\DQB1*03:02	14	70
DQA1*02:01\DQB1*04:01	13	65
DQA1*03:01\DQB1*03:01	13	65
DPA/DPB
DPA1*01:03\DPB1*02:01	11	55
DPA1*01:03\DPB1*11:01	15	75
DPA1*01:03\DPB1*19:01	18	90
DPA1*01:05\DPB1*11:01	11	55
DPA1*01:05\DPB1*13:01	16	80
DPA1*02:01\DPB1*13:01	18	90
DPA1*02:01\DPB1*15:01	15	75
DPA1*02:01\DPB1*23:01	20	100
DPA1*02:01\DPB1*28:01	17	85

Cohort 1

With the LSNC, four DRB, five DQA/DQB and nine DPA/DPB alleles showed high MFI after normalization of the eluate (E2). Among the DRA/DRB loci, the highest MFI was found with DRB3*03:01; among the DQA/DQB loci, with DQA1*01:02\DQB1*06:02; and among the DPA/DPB loci, with DPA1*02:01\DRB1*23:01 (Table 1).

Cohort 2

Table 2a shows the antibody profiles of TFL1, his son and grandson. The grandson's serum had no detectable levels of antibodies while there were antibodies in the sera of his father (anti-DR and anti-DP) and grandfather (anti-DQ). The MFI of antibodies for all alleles was notably higher after purification of IgG. The unpurified sera showed no reactivity to several HLA-II alleles whereas purification of the IgG resulted in strong reactivity (high MFI) against the same alleles. The most prevalent purified IgG antibodies of the grandfather, father and son were the same as those observed with LSNC (Table 1 and 2a).

Table 2b compares the HLA-II antibody profiles of TFL visitor W1, his father and maternal grandfather. Their sera contained very low levels of antibodies, but upon purification the IgG fraction of all three showed much higher reactivity. Their most prevalent purified IgG antibodies were the same as those with LSNC and family 1.

Table 2c compares the antibody profiles of TFL2 and his two sons. While their sera contained no detectable antibodies against DQA/DQB or DPA/DPB haplotypes, there were antibodies in all three sera against one or two DRB alleles. Upon purification, the IgG fraction of all three sera showed much higher reactivity to HLA-II alleles. Again, the most prevalent purified IgG antibodies were the same as with LSNC and families 1 and 2.

Cohort 3

Examination of anti-HLA-II antibodies in sera of the 11 TFL researchers or visitors in this cohort, before and after purification (Table 3a–c), confirmed that the most prevalent purified IgG antibodies in normal males are, apparently, DRB3*03:01 (Table 3a), DQA1*01:02\DQB1*06:02 (Table 3b) and DPA1*02:01\DPB1*23:01 (Table 3c). Table 3d summarizes the results of the analyses of all the cohorts, which elucidates the incidence of IgG reactivity against HLA DRB, DQA/DQB and DPA/DPB alleles. The frequency/incidence was 100% for DRB3*03:01 and for DPA1*02:01\DPB1*23:01. Other major alleles and their frequency are shown in the table.

Anti-HLA-II IgG: profiles of therapeutic IVIg

The preparations of IVIg examined included, as noted, GamaSTAN, Octagam, two lots of Gamunex-C and Sandoglobulin. Table 4 compares their titrimetric MFI profile of anti-HLA-II reactivity. Among IgG antibodies reacting to DRB alleles, reactivity to DRB3*03:01 ranked first in all IVIg preparations (Table 4a), analogous to the highest incidence of the same anti-HLA-DRB IgG purified from the sera of our cohorts. (Henceforth, ‘purified IgG’ means IgG purified from normal sera.) GamaSTAN had the highest titre of antibodies; the profiles and ranks of the other anti-HLA-DRB alleles presented in Table 4a show a striking similarity to those of our cohorts' purified IgG.

Table 4.

Titrimetric mean fluorescence intensity (MFI) values of anti-human leucocyte antigen (HLA) class II reactivity of different therapeutic preparations of IVIg

(a) Anti-HLA-DR reactivity
GamaSTAN (15–18%)								Gamunex-C (10%)
Rank	Lot: 26NHCVI	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]	[1/2048]	[1/4096]	Lot: 26NKLK1	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]
1	DRB3*03:01	23 214	20 352	13 059	8575	3017	1124	708	DRB3*03:01	8665	6552	3973	1784	1466
2	DRB1*09:01	8 850	7 136	2 997	1529				DRB1*03:02	2956	1660	894
3	DRB3*01:01	6 931	4 644	1 069	1313				DRB1*09:01	1900	1064	622
4	DRB1*08:01	3 989	2 146	810	703				DRB1*08:01	2529	1034	523
5	DRB1*11:04	3 761	3 075	1 150	603				DRB3*01:01	2241	1001	520
6	DRB1*14:02	3 284	2 609	1 187	590				DRB1*11:04	1601	886
7	DRB1*03:0	2 910	1 105	553	571				DRB1*14:02	1668	861

	Octagam (6%)								Gamunex-C (10%)
	Lot: A913A8431	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]			Lot : 26NKLG1	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]
1	DRB3*03:01	9344	5024	n/a	1766	699			DRB3*03:01	4640	4183	1873	1108	904
2	DRB1*09:01	1946	662						DRB1*03:02	2801	1713	942	556
3	DRB1*03:0	1194	649						DRB1*09:01	1404	871	840
4	DRB1*11:04	1133	594						DRB1*08:01	2121	1278	611
5	DRB3*01:01	1102	574						DRB3*01:01	2137	1228	550
6	DRB1*14:02	1005	552						DRB4*01:01	1430	907	505
7	DRB1*08:01	970							DRB1*03:01	1181	643

	Sandoglobulin (6%)
	Lot: 4305800026	[1/64]	[1/128]	[1/256]	[1/512]
1	DRB3*03:01	6197	4659	2041	748

(b) Anti-HLA-DQ reactivity
Rank	GamaSTAN (15–18%)			Gamunex-C (10%)
	Lot: 26NHCVI	[1/32]	[1/64]	Lot: 26NKLK1	[1/32]	[1/64]	[1/128]
1	DQA1*01:02\DQB1*06:02	2010	1177	DQA1*01:02\DQB1*06:02	3546	2141	1274
2	DQA1*01:01\DQB1*06:02	1918	1132	DQA1*01:01\DQB1*03:02	3243	1966	1015
3	DQA1*03:01\DQB1*03:01	1176	757	DQA1*02:01\DQB1*03:02	3098	2015	1028
4	DQA1*03:01\DQB1*03:02	1095	680	DQA1*03:01\DQB1*03:01	2624	1678	966
5	DQA1*01:02\DQB1*06:04	1261	770	DQA1*03:01\DQB1*03:02	2488	1664	916
6	DQA1*02:01\DQB1*03:02	1061	692	DQA1*02:01\DQB1*04:01	2265	1678	739
7	DQA1*01:02\DQB1*05:02	1050	828	DQA1*01:01\DQB1*06:02	2250	1522	948

	Octagam (6%)			Gamunex-C (10%)
	Lot: A913A8431	[1/32]	[1/64]	Lot: 26NKLG1	[1/32]	[1/64]	[1/128]
1	DQA1*01:02\DQB1*06:02	1007	613	DQA1*01:02\DQB1*06:02	3661	1927	815
2	DQA1*03:01\DQB1*03:01	1398	899	DQA1*01:01\DQB1*03:02	4036	1755	735
3	DQA1*01:01\DQB1*03:02	926	572	DQA1*03:01\DQB1*03:01	3793	1429	646
4	DQA1*02:01\DQB1*03:02	757		DQA1*02:01\DQB1*03:02	3544	1654	679
5	DQA1*01:01\DQB1*06:02	643		DQA1*03:01\DQB1*03:02	2567	1488	702
6	DQA1*03:01\DQB1*03:02	661		DQA1*01:01\DQB1*06:02	2244	1394	661
7	DQA1*01:02\DQB1*06:04	713		DQA1*03:01\DQB1*02:01	1497	911

	Sandoglobulin (6%)
	Lot: A913A8431				[1/2]			[1/4]
1	DQA1*05:05\DQB1*03:01				1102			625
2	DQA1*03:02\DQB1*03:02				1085			565
3	DQA1*06:01\DQB1*03:01				1027			545
4	DQA1*01:02\DQB1*06:04				869			571
5	DQA1*03:01\DQB1*03:01				845			572
6	DQA1*03:01\DQB1*03:02				848			512
7	DQA1*02:01\DQB1*03:01				811			548

(c) Anti-HLA-DPA\DPB reactivity
Rank	GamaSTAN (15–18%)								Gamunex-C (10%)
	Lot: 26NHCVI	[1/32]	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]	[1/2048]	Lot: 26NKL (K1)	[1/32]	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]
1	DPA1*02:01\DPB1*23:01	18 914	17 978	13 471	7736	4161	2653	540	DPA1*02:01\DPB1*23:01	7102	5075	5315	2287	1016	806
2	DPA1*01:03\DPB1*11:01	17 213	16 775	10 470	6294	3169	2471	1062	DPA1*01:03\DPB1*19:01	4598	2991	2074	981	530
3	DPA1*02:01\DPB1*28:01	12 849	9 911	6 924	3738	1861	709		DPA1*02:01\DPB1*13:01	4290	2639	1618	607
4	DPA1*01:03\DPB1*19:01	11 616	9 098	4 312	2069	1077	534		DPA1*02:01\DPB1*15:01	3427	2340	1408	549
5	DPA1*02:01\DPB1*06:01	5 425	3 678	1 665					DPA1*01:03\DPB1*11:01	1854	2015	1663	672
6	DPA1*02:01\DPB1*15:01	3 742	2 167	1 051					DPA1*01:05\DPB1*13:01	2980	1959	1288	564
7	DPA1*02:01\DPB1*05:01	4 658	2 641	898					DPA1*02:01\DPB1*28:01	2779	1954	1283	634

	Octagam (6%)								GAMUNEX-C (10%)
Lot: A913A8431	[1/32]	[1/64]	[1/128]	[1/256]					Lot: 26NKL (G1)	[1/32]	[1/64]	[1/128]	[1/256]	[1/512]	[1/1024]
1	DPA1*02:01\DPB1*23:01	9474	6105	2483	2720				DPA1*02:01\DPB1*23:01	11183	5920	3610	1960	980	629
2	DPA1*01:03\DPB1*11:01	7316	2187	1027	927				DPA1*01:03\DPB1*19:01	5701	3370	1933	912
3	DPA1*02:01\DPB1*28:01	3870	2148	1025	524				DPA1*01:03\DPB1*11:01	5551	3174	1914	1040
4	DPA1*01:03\DPB1*19:01	4018	2288	1121	575				DPA1*02:01\DPB1*28:01	4751	2424	135	814
5	DPA1*01:03\DPB1*02:01	1563	1051	630					DPA1*02:01\DPB1*15:01	4269	2193	1192	518
6	DPA1*02:01\DPB1*15:01	1327	864						DPA1*01:05\DPB1*13:01	3011	1992	998	539
7	DPA1*02:01\DPB1*05:01	1290	944						DPA1*02:01\DPB1*13:01	4622	2190	1050

	Sandoglobulin (6%)
	Lot: 4305800026	[1/32]	[1/64]	[1/128]	[1/256]
1	DPA1*02:01\DPB1*23:01	1098	3619	1789	1082
2	DPA1*02:01\DPB1*28:01	548	971

Among IgG antibodies reacting to DQA/DQB alleles, reactivity to DQA1*01:02\DQB1*06:02 ranked first in all IVIg preparations except Sandoglobulin (Table 4b), again analogous to the highest incidence of the same anti-HLA-DQA/DQB purified IgG of our cohorts, but with Gamunex-C having the highest titre of antibodies. With DPA/DPB alleles, reactivity to DPA1*01:02\DPB1*23:01 ranked first in all IVIg preparations (Table 4c) – yet again as with our cohorts' serum-derived IgG but, again, GamaSTAN now having the highest titre of antibodies.

Figure 1a–c compares the rank of HLA-II reactivity of the IgG in the four IVIg preparations with the incidence of anti-HLA-II IgG before and after purification of IgG from the sera of the study donors. Strikingly, the IgG antibodies with a high incidence of reaction to HLA-II alleles in normal individuals have a correspondingly high incidence in IVIg preparations. The rank of anti-HLA-DR was: DRB3*03:01 > DRB1*09:01 > DRB3*01:01 > DRB1*08:01. Among anti-HLA-DQ alleles, DQA1*01:02\DQB1*06:02 ranked first, and among the anti-HLA-DP it was DPA1*0201\DPB1*23:01.

Figure 1.

Comparison of the normalized mean fluorescence intensity (MFI) of human leucocyte antigen (HLA) class II immunoglobulin (Ig)G antibodies in three groups: the sera of 15 donors and the negative control [LABScreen® negative control serum (LSNC)]; the IgG in each serum sample after protein-G purification; and five different intravenous immunoglobulin (IVIg) preparations, including two lots of Gamunex-C. MFI values for the purified IgG are the mean MFI of eluates 2 and 3. The vertical white space in the middle of the bars refers to break of the x-axis, graphically generated to accommodate the entire MFI values. For all parts of this figure (a–c) the normalized trimmed mean MFI is shown for IgG reacting to the given alleles The sera were all diluted 1/10 and the IVIg diluted 1/8.

Anti-HLA-II IgG: profiles of IVIg and purified sera IgG versus mouse anti-HLA-II IgG mAb

To ensure that the high MFI was not due to an antigen load on beads, the pattern of reactivity of a mouse mAb (FJ5109) with all HLA-DRB, DQA/DQB and DPA/DPB haplotypes was examined and compared with the HLA-II reactivity of IVIg and purified IgG. Table 5 shows the most highly reactive alleles in each of the HLA-DRB, -DQA/DQB and -DPA/DPB haplotypes. Note that, in point of reactivity, almost all the alleles recognized by the highest-ranking human antibodies for each haplotype were poorly recognized by the murine mAb; conversely, high-ranking murine anti-HLA-II antibody reactivity was restricted to alleles that rank low or lowest with human anti-HLA-II IgGs. The highest-ranking human antibodies for each of the haplotypes are shown in bold type in Table 5.

Table 5.

Ranking^† and reactivity of murine monoclonal antibody (mAb) FJ5109 (at 1/16 000) to human leucocyte antigen (HLA) class-II alleles

Rank	HLA-DR	MFI	HLA-DQ	MFI	HLA-DP	MFI
1	DRB1*13:03	3462	DQA1*05:01\DQB1*02:01	5022	DPA1*02:01\DPB1*18:01	3664
2	DRB1*15:01	3359	DQA1*03:01\DQB1*02:01	3841	DPA1*01:03\DPB1*02:01‡,§	3477
3	DRB1*01:03	3317	DQA1*03:03\DQB1*04:01	3679	DPA1*01:03\DPB1*03:01	3425
4	DRB1*03:01	3145	DQA1*03:02\DQB1*03:02	3411	DPA1*01:05 DPB1*28:01	3343
5	DRB1*11:04‡	3091	DQA1*01:03\DQB1*06:01	3305	DPA1*02:01\DPB1*09:01	3325
6	DRB1*15:02	3090	DQA1*04:01\DQB1*02:01	3274	DPA1*02:01\DPB1*28:01‡,§	3268
7	DRB1*04:02	3027	DQA1*05:05\DQB1*03:01	3265	DPA1*01:03\DPB1*04:02	3262
8	DRB1*01:01	2980	DQA1*05:03\DQB1*03:01	3227	DPA1*01:05 DPB1*03:01	3240
9	DRB1*16:02	2965	DQA1*06:01\DQB1*03:01	3223	DPA1*02:01\DPB1*14:01	3168
10	DRB5*02:02	2923	DQA1*02:01\DQB1*03:03	3212	DPA1*02:01\DPB1*17:01	3103
11	DRB1*13:01	2836	DQA1*03:02\DQB1*03:03	3119	DPA1*02:01\DPB1*06:01‡	3096
12	DRB1*15:03	2808	DQA1*01:03\DQB1*06:03	2851	DPA1*02:01\DPB1*01:01	3088
13	DRB1*16:01	2753	DQA1*02:01\DQB1*02:02	2435	DPA1*02:01 DPB1*03:01	3042
14	DRB1*04:01	2721	DQA1*01:01\DQB1*05:01	2326	DPA1*02:01\DPB1*05:01‡	3024
15	DRB1*09:02	2595	DQA1*02:01\DQB1*02:01	2144	DPA1*01:03\DPB1*04:01	2980
16	DRB1*07:01	2561	DQA1*04:01\DQB1*04:02	1880	DPA1*02:01\DPB1*10:01	2847
17	DRB4*01:03	2552	DQA1*01:02\DQB1*06:09	1867	DPA1*01:03\PB1*01:0	2811
18	DRB1*04:04	2538	DQA1*01:02\DQB1*05:02‡	1560	DPA1*01:05\DPB1*18:01	2771
19	DRB1*12:01	2514	DQA1*02:01\DQB1*04:02	1493	DPA1*01:04\DPB1*18:01	2715
20	DRB1*11:01	2502	DQA1*02:01\DQB1*04:01§	1429	DPA1*01:05\DPB1*11:01§	2503
21	DRB1*01:02	2498	DQA1*02:01\DQB1*03:01	1333	DPA1*01:03\DPB1*19:01‡,§	2418
22	DRB1*03:02‡,§	2494	DQA1*03:01\DQB1*03:01‡,§	1063	DPA1*01:03\DPB1*11:01‡,§	2307
23	DRB1*04:05	2376	DQA1*03:01\DQB1*03:03	726	DPA1*02:01\DPB1*13:01‡	2209
24	DRB3*02:02	2357	DQA1*03:01\DQB1*03:02	659	DPA1*02:01\DPB1*15:01‡,§	2119
25	DRB1*08:01‡,§	2309	DQA1*02:01\DQB1*03:02‡,§	625	DPA1*02:01\DPB1*23:01‡,§	1756
26	DRB1*04:03	2290	DQA1*01:02\DQB1*06:04	526	DPA1*01:05\DPB1*13:01‡,§	1627
27	DRB1*10:01	2238	DQA1*01:01\DQB1*03:02‡,§	472	DQA1*02:01\DQB1*03:02	625
28	DRB1*14:02‡	2214	DQA1*01:01\DQB1*06:02	438
29	DRB4*01:01	2168	DQA1*01:02\DQB1*06:02‡,§	332
30	DRB5*01:01	2046
31	DRB1*14:01	1586
32	DRB1*12:02	1281
33	DRB3*03:01‡,§	1272
34	DRB1*14:54	1138
35	DRB1*09:01‡,§	1099
36	DRB3*01:01‡,§	1077

† Rankings, in general, are the same as those observed for the HLA-II reactivity of intravenous immunoglobulin (IVIg) or the purified IgG of normal males. Those that are shown in bold type correspond to the HLA-II reactivity of IVIg^‡ or purified human sera. Alleles with superscript ^‡ are the high-ranking antibodies in IVIg. Alleles with superscript ^§ are the high-ranking antibodies in purified normal sera. MFI = mean fluorescence intensity.

Anti-HLA-I IgG: IVIg versus normal sera IgG before and after purification

Table 6a compares the rank and incidence of MFI of anti-HLA-I IgG in IVIg with that of the same purified IgG. The MFI rankings of anti-HLA-C IgG in two different preparations of IVIg were strikingly similar, and paralleled the incidence of anti-HLA-C purified IgG. With the HLA-C locus, all seven of the listed Cw specificities had an immunogenicity score of 95%: Cw*0602, Cw*0702, Cw*1802, Cw*0401, Cw*1601, Cw*1701 and Cw*0202. The rankings of anti-HLA-B IgG in Octagam IVIg paralleled the incidence of anti-HLA-B IgG in the purified IgG. However, the rank and incidence of anti-HLA-A IgG in IVIg and in purified IgG differed considerably.

Table 6.

Rank of mean fluorescence intensity (MFI) of anti-human leucocyte antigen (HLA)-I IgG in IVIg (GamaSTAN and Octagam) and in IgG purified from the sera (eluate 2) of normal, non-alloimmunized males, and incidence of the IgG in the eluate

(a)	GamaSTAN (1/8)		Octagam (1/8)			Sera (n = 20)
Rank		MFI			MFI		% Incidence (E2)
						HLA-A
1	A*34:01	3042	A*36:01		3 650	A*24:02	40
2	A*80:01	2705	A*26:01		3 071	A*34:01	40
	HLA-B
1	B*44:02	4301	B*37:01		9 974	B*37:01	65
2	B*82:01	3857	B*82:01		5 026	B*40:06	55
3	B*45:01	3667	B*40:06		4 972	B*82:01	75
4	B*15:16	3536	B*44:02		3 997	B*44:02	45
	HLA-Cw
1	CW*06:02	9171	CW*06:02		16 706	CW*06:02	95
2	CW*18:02	7981	CW*07:02		15 321	CW*07:02	95
3	CW*17:01	7856	CW*18:02		10 610	CW*18:02	95
4	CW*07:02	7620	CW*04:01		9 884	CW*04:01	95
5	CW*02:02	7183	CW*02:02		9 379	CW*16:01	95
6	CW*05:01	6968	CW*16:01		9 259	CW*17:01	95
7	CW*04:01	6287	CW*17:01		8 983	CW*02:02	95

(b) Incidence of serum and purified IgG in normal males reacting to HLA class-I and the level (MFI) of anti-albumin IgG
ID	TFL1		T2		T3		W1		W2		W3		TFL2		R1		R2		TFL3
	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified
Anti-Albumin IgG (MFI)							1547												537
Incidence of anti-HLA-A IgG		4				7	13	24	2	12	2	21		1	1	3		1	20	2
MFI range 500–999		4				7	13	6	2	9	1	19		1	2	1		1	17	2
1 000–1 999								16		3	1	3				2			3
2 000–2 999								2
Incidence of anti-HLA-B IgG	2	13		3	1	16	39	39	2	21		28	1	8		2		8	31	2
MFI range 500–999	1	9		3		15	39	12	2	20		24	1	7				7	29	2
1 000–1 999	1	2			1			22		1		4		1		2		1	2
2 000–2 999		2				1		5
3 000–3 999
Incidence of anti-HLA-Cw IgG		15		16		16	15	16		16		16		16		16		16	4	13
MFI range 500–999		2		4			14			1		1		2		1			4	7
1 000–1 999		8		9		13	1	1		7		6		12		4		5		6
2 000–2 999		4		3		3		3		6		9		2		6		6
3 000–3 999		1						4		2						2		3
4 000–7 000								8								3		2
7 000–10 000
> 10 000

ID	TFL4		TFL5		TFL6		TFL7		TFL8		TFL9		TFL10		TFL11		UCLA1		G1
	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified	serum	Normalized/purified
Anti-Albumin IgG (MFI)	943
Incidence of anti-HLA-A IgG	8		1	12		2			2				20	15		1			1	7
MFI range 500–999	5		1	12		2			2				8	4		1			1	4
1 000–1 999	3												11	8						2
2 000–2 999													1	3						1
Incidence of anti-HLA-B IgG	1		3	21		8				7	4	4	23	22		6		2		15
MFI range 500–999	1		3	17		7				6			16	10		6		2		7
1 000–1 999				4		1				1	4		6	8						5
2 000–2 999												3	1	4						3
3 000–3 999												1
Incidence of anti-HLA-Cw IgG	2		6	16		16		8	2	15		13	16	16		16		11		15
MFI range 500–999	2		6					7	2	6		7	9			9		11	1
1 000–1 999				4		10		1		9		6	6	2		7
2 000–2 999				6		5							1	4						1
3 000–3 999				3		1								7						2
4 000–7 000				3										3						5
7 000–10 000																				6
> 10 000																				1

Total incidence of anti-HLA IgG is shown in bold type. TFL = Terasaki Foundation Laboratory.

Table 6b compares HLA-I reactivity of IgG before and after purification from sera. The incidence is given for anti-HLA-A/B/Cw at different ranges of MFI. Anti-HLA-I IgG antibodies are rarely observed in sera, but upon purification of IgG they are present, indicating that most already existed in the sera, but did not react with antigens until they were subjected to purification. Sera of four individuals (TFL5, TFL10, TFL3 and W1) showed reactivity to 10–98 HLA-I alleles (1–20 HLA-A, 3–39 HLA-B and 6–16 HLA-Cw). Most importantly, the level of MFI in IgG after purification and normalization was several hundredfold higher. The purified and normalized IgG from G1 showed an MFI > 10 000 for an HLA-Cw allele. Purified and normalized IgG of several donors ranged in MFI from 4000 to 7000 for HLA-Cw alleles. All these values document the existence of anti-HLA-I IgG antibodies in sera in a masked state.

Anti-HLA-I IgG: regular beads versus iBeads, distinguishing β2m-associated and β2m-free HLA heavy chains

The purified IgG from sera differed in reactivity to intact (iBeads) and heavy-chain HLA (regular beads) (Table 7), strikingly similar to the reactivity of IVIg preparations (data in [6]). Purified IgG of all the donors showed a marked increase in MFI for HLA-A on iBeads, suggesting that the purified IgG binds to intact HLA-A. The decrease in MFI of the purified IgG for HLA-B and HLA-Cw alleles suggests that the purified IgG, like different preparations of IVIg [7], binds to the β2m-free heavy chains (open conformers). The purified IgG of TFL1, TFL9 and TFL10 reacted with iBeads (containing at least one HLA-B allele) with high MFI, indicative of reactivity to intact HLA. The purified IgG of TFL3 is unique in that it reacted with HLA-B alleles only on iBeads, not regular beads; and his purified IgG, when reacting with HLA-Cw alleles, had a higher reaction to nine Cw alleles on regular beads than on iBeads; conversely, however, it had a higher reaction to nine different Cw alleles on iBeads than on regular beads. Differences in the reactivity of IgG on albumin-coated regular beads and iBeads suggest that the proprietary enzymatic treatment used to prepare iBeads affects the expression of albumin on iBeads (Table 7).

Table 7.

The reactivity (MFI) of purified IgG (eluate 2) of the sera of non-alloimmunized males to HLA-Ia and albumin differs between regular beads (rB) and iBeads (iB)

Donor ID	A alleles (31)		B alleles (50)		Cw alleles (16)		Albumin
	rB > iB	rB < iB	rB > iB	rB < iB	rB > iB	rB < iB	rB (MFI)	iB (MFI)
TFL1	3	26	20	1	16	0	< 2 K	< 1 K
T2	0	0	1	0	13	0	< 0·5	< 0·5
TFL2	1	16	11	0	16	0	< 1 K	< 0·5
R1	5	15	17	0	16	0	< 1 K	< 0·5
R2	0	2	8	0	15	0	< 0·5	< 0·5
TFL3	0	31	0	50	9	9	< 2 K	< 0·5
TFL4	0	3	0	0	0	0	< 5 K	< 1 K
TFL5	10	7	27	0	16	0	< 0·5	< 0·5
TFL6	1	14	10	0	16	0	< 1 K	< 1 K
TFL7	0	2	0	0	14	0	< 1 K	< 0·5
TFL8	1	13	12	0	16	0	< 1 K	< 1 K
TFL9	1	3	5	1	15	0	< 1 K	< 1 K
TFL10	12	13	30	1	15	0	< 1 K	< 1 K

rB contains both β2-microglobulin-associated (heterodimer) and β2-microglobulin-free (open conformer) HLA molecules; iB are enzyme-treated beads predominantly contain heterodimer. TFL = Terasaki Foundation Laboratory.

Antibodies against their own HLA in the purified sera of normal males

Does IgG reacting to HLA before and after purification from the serum of each donor match the molecular HLA types of that donor? Various anti-HLA IgG antibodies in the native sera and those found after purification were indeed directed against that donor's own HLA types (Table 8), confirming the presence of anti-HLA-I/-II reactive IgGs, corresponding to the HLA alleles expressed by those individuals. For instance, serum IgG of four donors (the father in family 1, the grandfather in family 2, TFL3 and TFL10) had HLA antibodies against their own HLA type. In two of the four, the antibodies were against HLA-II alleles, and in the other two against HLA-I alleles. After purification of IgG, several sera showed reactivity to the donor's own HLA alleles. For example, DRB typing showed that TFL9 and TFL11 were homozygous for DRB3*01:01, and their purified IgG showed those antibodies against the same allele. Such matching can be seen for almost all the other donors, with autoantibodies to DRB345 appearing to be the most frequent (Table 8). Together, the data suggest that most normal non-alloimmunized males carry antibodies to their own HLA. Using an autoantibody frequency score to compare the loci we found that, of the 20 donors, six (30%) had IgG autoantibodies to HLA-A, eight (40%) to HLA-B and 16 (80%) had anti-HLA-Cw autoantibodies. Similarly, the frequency of IgG antibodies against the donor's own HLA-II was: anti-HLA DRB1 alleles (5%), anti-DRB345 (65%), anti-DQA (20%), anti-DQB (20%), anti-DPA (0%) and anti-DPB (15%).

Table 8.

Human leucocyte antigen (HLA) autoantibodies in normal sera: normalized mean fluorescence intensity (MFI) of antibodies in sera before and after purification of immunoglobulin (Ig)G corresponds to the donor HLA class I and II types of the non-alloimmunized males

Purified and normalized IgG positive for HLA types of the individuals
	HLA class-I			HLA class-II
	A	B	Cw	DRB1	DRB345	DQA	DQB	DPA	DPB
Family 1
Paternal grandfather (TFL1)		52:01	07:02
Father (T1)			Reactive to homozygous 07:02		Both serum and purified IgG reactive to homozygous 4*0101	01:01	03:02		02:01
Son (T2)	24:02	40:06	07:02 and 08:01			03:01	03:01		02:01
Family 2
Maternal grandfather (W1)	24:02 and 31:01	51:01 and 52:01	14:02		Serum IgG reactive to homozygous 50101
Father (W2)		52:01	01:02			03:01	03:02
Son (W3)	24:02	Reactive to homozygous 52:01
Family 3
Father (TFL2)		58:01	Reactive to homozygous 15:02		3*03:01 and 4*01:01				02:01
First son (R1)			15:02		3*03:01
Second son (R2)	24:02	58:01	15:02		3*03:01
Volunteers
TFL9				03:01 and 08:01	3*01:01 and 3*01:01
TFL8			01:02 and 03:04		3*01:01
TFL6			Reactive to homozygous 07:02		4*01:01
TFL10			Both serum and purified IgG reactive to 03:02, 03:04 and 07:02		4*01:01 and 5*01:01
TFL4					3*03:01
TFL5	11:01 and 24:02		02:02		3*01:01
TFL7					3*03:01
TFL3	Serum IgG reactive to 02:06	Serum IgG reactive to 40:01	07:02			01:02
TFL11			16:01		Reactive to homozygous 3*01:01
UCLA1			7:02
G1			03:03 and 06:02				3:02

TFL = Terasaki Foundation Laboratory.

Masking efficacy of different molecular size fragments of peptides and polypeptides from wash fluid

The peptide/polypeptide fractions (> 100 K, 100-0 K, 10-3 K and < 3 K) isolated from WF1 were tested to assess their ability to mask the HLA-E reactivity of E2 and E3 from the serum of TFL5. Both eluates contained purified IgG (E2 > E3). The MFI of E2 and E3 with and without WF fractions at two different dilutions were measured and the percentage difference was calculated. Invariably, the eluate-WF mixture showed a much lower MFI than the eluate alone, indicating that the peptides or other serum factors in the WF masked the reactivity of IgG with HLA-E. All fractions inhibited the HLA-E reactivity of the eluates. The inhibition percentage of the 3 K fraction was higher than that of the 10-3 K fraction. The inhibition percentage with E2 was greater than with E3, suggesting that the serum peptides of molecular size < 3 K are capable of masking the interaction of purified IgG with HLA. Accordingly, we then performed peptide inhibition studies with different classes of peptides of molecular size < 3 K (Table 9).

Table 9.

Efficiency of different molecular size fractions obtained from the wash fluid of TFL5's serum in masking (% inhibition) the human leucocyte antigen (HLA)-E reactivity of eluates 2 and 3 of the serum

Eluate 2 WF, after post-molecular sieving	E2 (μl)	Phosphate-buffered saline (PBS) (μl)	Normal MFI	E2 (μl)	Wash fluid (WF) (μl)	Ratio of E2/WF	Normal MFI	% inhibition
									20	10	2773	20	10	(2/1)	562	80
Original	25	5	5762	25	5	(5/1)	1406	76
	20	10	2773	20	10	(2/1)	79	97
> 100 K	25	5	5762	25	5	(5/1)	429	93
	20	10	2773	20	10	(2/1)	929	66
10–100 K	25	5	5762	25	5	(5/1)	1571	73
	20	10	3746	20	10	(2/1)	4652	−24
3–10 K	25	5	5184	25	5	(5/1)	6331	−22
	20	10	3746	20	10	(2/1)	1258	66
< 3 K	25	5	5184	25	5	(5/1)	3600	31

Eluate 3 WF, after post-molecular sieving	E3 (μl)	PBS (μl)	Ratio of E3/PBS	Normal MFI	E3 (μl)	Wash fluid (WF) (μl)	Ratio of E3/WF	Normal MFI	% inhibition
	20	10	(2/1)	2064	20	10	(2/1)	496	76
Original	25	5	(5/1)	5902	25	5	(5/1)	1510	74
	20	10	(2/1)	2064	20	10	(2/1)	132	94
> 100 K	25	5	(5/1)	5902	25	5	(5/1)	211	96
	20	10	(2/1)	2064	20	10	(2/1)	433	79
10–100 K	25	5	(5/1)	5902	25	5	(5/1)	1212	79
	20	10	(2/1)	2008	20	10	(2/1)	1479	26
3–10 K	25	5	(5/1)	4099	25	5	(5/1)	2954	28
	20	10	(2/1)	2008	20	10	(2/1)	970	52
< 3 K	25	5	(5/1)	4099	25	5	(5/1)	2988	27

Inhibition of HLA reactivity of IgG with synthetic peptides representing HLA and IgG peptides

Peptides used included those derived from HLA (commonly shared peptides) and the fragments derived from the CDRII domain. The most common shared peptides are ¹¹⁵QFAYDGKDY¹²³, ¹¹⁷AYDGKDY¹²³ and ¹²⁶LNEDLRSWTA¹³⁵ (7–9). The CDRII sequence peptide T15H (now called ‘TEPC15’) in the domain of the Fab of IgG – known to bind to naturally occurring antibodies 10–12 – is found in the sera of different strains of mice and in humans, indicating that the sequence is a conserved-idiotype determining region 10–12. Two peptides were used for the inhibition studies with the IgG fragment TEPC15: ASRNKANDYTTEYSASVKGRFIVS and the control peptide of 2FIOH; 50–73, QIYPGDGDTKYNNGKFKGKATLT.

Observations of E2 of TFL5's serum indicated that the TEPC15 peptide was the potent inhibitor of IgG reactivity to HLA-E (Fig. 2a). Neither the control peptide with the same number of amino acids nor the shared peptides of HLA-I inhibited the HLA-E binding of IgG in E2. The inhibitory potency of TEPC15 was further confirmed by the HLA-II reactivity (to DRB3*03:01 and DPA1*02:01/DPB1*23:01) of purified IgG in E2 and E3 from the serum of TFL12, and by the DRB3*03:01 reactivity of E2 from the serum of TFL6 (Fig 2b; Table 10). It appears that the peptide fragment of CDRII, TEPC15, is the potent binder of anti-HLA IgG.

Figure 2.

Inhibition of human leucocyte antigen (HLA) reactivity of immunoglobulin (Ig)G in eluates purified from the sera of two volunteers – Terasaki Foundation Laboratory (TFL)5 (a) and TFL12 (b) – by a < 3 K peptide sequence. (a) The peptide inhibition of eluate 2 of TFL5 used HLA-E, representing HLA class I, with peptides shared by all HLA class I alleles (¹¹⁵QFAYDGKDY¹²³; ¹¹⁷AYDGKDY¹²³ and ¹²⁶LNEDLRSWTA¹³⁵) and with IgG peptide TEPC15 (T15H; 50–73) (ASRNKANDYTTEYSASVKGRFIVS), with a peptide derived from the CDRII domain of IgG, 2FIOH (50–73) and with a control peptide (QIYPGDGDTKYNNGKFKGKATLT). (b) The peptide inhibition of eluates 2 and 3 of TFL12 were tested with HLA class II, as labelled in the figure. The IgG peptides were, as with (a), TEPC15 and 2FIOH (50–73).

Table 10.

Inhibition of human leucocyte antigen (HLA) DRB*3*03:01 reactivity of IgG in eluate 2, purified from the serum of TFL6 by a < 3 K peptide sequence

		Beads coated with DRB3*03:01
	μl†	MFI	% decrease
Eluate	25
PBS	5	7722
Peptide TEPC15‡
5 μg/5 μl	5	2832	63
2·5 μg/5 μl	5	2822	63
1·25 μg/5 μl	5	2463	68
0·625 μg/5 μl	5	4132	46
Peptide 2F10H
5 μg/5 μl	5	2790	64
2·5 μg/5 μl	5	6810	12

† μl = total volume of the mix was 30 μl, but only 20 μl of peptide was added to wells containing beads. ^‡TEPC15 is a peptide derived from the CDRII domain of IgG; 2F10H is the control peptide. PBS = phosphate-buffered saline.

Discussion

Four major findings emerged from this investigation. First is that the profile, incidence and MFIs of HLA-II IgG differ remarkably between native sera and IgG purified from the corresponding sera, indicative of the crypticity of the anti-HLA IgG antibodies in the native sera. The exposed and cryptic anti-HLA IgG in the sera of our study's normal donors helps to explain the preformed or pre-existing DSA and NDSA in the sera of transplant patients even before they receive their first kidney 13–16, liver 16–18 or lung 19 allograft. The pre-existing DSA notably correlated with graft rejection. If these correlations are valid, then an assessment of the HLA reactivity of purified IgG of patients before transplantation can predict the outcome of transplantation, play a significant role in the selection of donors, and therefore importantly benefit transplant patients.

Second is the presence of HLA IgG in sera capable of reacting to one's own HLA types both as exposed and cryptic antibodies. Indeed, this is the first and novel documentation of anti-HLA IgG autoantibodies in the sera of virtually all normal, healthy donors tested. Anti-HLA autoantibodies were found in IgG purified from all their sera, and also occurred unmasked in the sera of a few individuals. This pattern most probably prevails in the general population. The presence of a wide variety of cryptic allo-HLA antibodies warrants careful reassessment of DSA and NDSA in allograft recipients. Possibly both pre- and post-transplant DSA could be due to loss of crypticity or detachment of the masking factor from anti-HLA IgG because of tissue inflammation or injury. Clearly, the factors masking anti-HLA autoantibodies are relevant and critical for understanding the regulation of circulating HLA autoantibodies in healthy people and in patients with allografts, infection, inflammation, cancer or autoimmune diseases.

Third is the presence of the putative regulatory factors capable of masking those antibodies' expression. The findings, as presented in Fig. 2a, did not support the hypothesis that the peptide fragments of shed HLA might be masking the expression of anti-HLA IgG in the sera. Moreover, if concealed anti-HLA-I or -II antibodies are bound with their respective shed antigen, then the antigen should be present in the tissue of the blood donors, but that cannot be the primary cause of all HLA antibodies being masked because all alleles were not found in a single donor. For example, the incidence of anti-DRB3*03:01 IgG was 100%, but the antigen was not expressed by all donors.

A more likely hypothesis is that the expression of anti-HLA IgG in the sera may be masked by the fragment from degrading IgG in circulation that can bind, as does an anti-idiotypic antibody 10–12. The CDRII sequence peptide (T15H; 50–73) that occurs in the sera of different strains of mice and in humans as a consequence of natural degradation of IgG, and is considered a conserved-idiotype determining region 10–12, was a potent inhibitor of HLA reactivity of the purified IgG of three volunteers, TFL5, TFL6 and TFL12. Neither the control peptide, which had the same number of amino acids as the CDRII peptide, nor the shared peptides of HLA-I inhibited HLA binding of the purified IgG, strengthening the contention that the highly conserved CDRII peptide may be responsible for the crypticity of the anti-HLA IgG in human sera. This observation is strengthened further by the data obtained from the two eluates of the same sera. Similar investigation of the sera of patients with autoimmune diseases, infection, inflammation, cancer or transplantation is needed to evaluate the regulatory role of the serum factors masking the HLA antibodies. Specific identification of the factors masking the expression of autoantibodies may lead to a better understanding of how to regulate the pathogenic or detrimental antibodies, particularly in patients with autoimmune diseases and those waiting for allografts.

Autoantibodies to HLA-I were found in the purified sera with the frequency HLA-Cw > -B > -A. Comparison of all HLA types showed that the donors had auto-HLA antibodies in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DPB (43%), DRB1 (21%), A (14%) and B (7%). It is too early to speculate on the functions of auto-HLA IgG antibodies in normal sera, except to observe that they remain concealed.

The fourth major finding is that the wide array of both allo-HLA-II and auto-HLA-II IgG antibodies isolated from normal sera was mirrored in the HLA-II reactivity and profiles of IVIg, which is purified from the pooled plasma using the Cohn–Oncley protocol with acidic ethanol, a method different from the one used in our study. Most interestingly, in all IVIg preparations, the IgG reacting to HLA-II exhibited a pattern of HLA-DRB, -DQA/DQB and -DPA/DPB reactivity similar to that of the antibodies purified and isolated from our donors' sera (Fig. 1a–c). Note especially the 100% incidence and strong reactivity of IgG reacting to DRB3*03:01 and DPA1*02:01\DPB1*23:01 (Table 3d). The most frequent HLA antibodies against DRB, DPA and DQA were found in more than 70% of the donors. Why these antibodies occur in everyone may be inexplicable; yet the ubiquity this study found makes the phenomenon seem unequivocal.

Both lots of Gamunex-C had very high titres of anti-albumin antibodies compared with the other IVIgs in this study. This should not, however, be taken as a blanket observation about other preparations or their manufacturers (indeed, GamaSTAN is made by the same manufacturer as Gamunex-C). Rather, our intent here is to show that anti-HLA IgG levels after normalization against the MFI of negative control beads, which are coated with albumin, can vary considerably from lot to lot. Because there is no method by which IVIg of different lots and different manufacturers can be compared with regard to potency, titres of anti-HLA-II IgG can be used to rank the different IVIg preparations.

The presence of a wide variety of HLA-I/-II IgG in IVIg 6,20 brings into question the validity of administering IVIg to lower the incidence of HLA antibodies in patients before and after organ transplantation. In fact, the effect of IVIg in lowering the HLA antibodies in presensitized patients 21–24 has been gainsaid by three recent clinical studies, each of which points out that IVIg is incapable of reducing HLA antibodies 25–27. None of these studies corroborates previous claims on the reduction of HLA antibody incidence (determined by calculated panel reactive antigens) in sensitized patients undergoing desensitization with high-dose IVIg. Is IVIg treatment for desensitization a clinical oxymoron?

Besides the questionable immunosuppressive effect of IVIg [34,35], as used routinely in transplant patients, there is the imminent danger of HLA-reactive IVIg preparations producing transfusion-related acute lung injury (TRALI). Variations in the IVIg preparation can make it more toxic. Stoclin et al. 28 detected HLA-I/-II IgG with broad specificity (determined by panel reactive antibody screening) in two batches of IVIg (Privigen®; CSL Behring). HLA-II antibodies were identified as causal factors for the induction of TRALI 29. Five instances of TRALI, following administration of IVIg, have been reported [30–33], with one death 30. Kopko and Holland 30 reported a case after transfusion of plasma that did not contain antibodies against HLA-I or human neutrophil antigens, but did contain anti-HLA-II IgG. Kopko et al. 31 proposed that HLA-II IgG binding to monocytes in patients with TRALI may result in activation of neutrophils, which may penetrate the endothelium of lungs to cause swelling and destruction of the endothelial lining. Indeed, in a rat lung model, Sachs 29 showed that HLA-II antibodies could induce TRALI indirectly by means of monocytes.

Documentation of anti-HLA-II IgG in alloimmunized females resulted in the avoidance of using blood from females for transfusion – avoidance that has become routine as a preventive measure against TRALI in several countries 31–33. Recently, Sachs et al. 32 stated that ‘this policy did indeed significantly reduce the incidence of TRALI both in large-scale surveillance studies and haemovigilance reports’. All this suggests that further use of IVIg for patients should be preceded by the assessment of the strength (MFI and titre) of their anti-HLA IgG and their HLA types because the detrimental agent in IVIg could be the HLA-II IgG reacting to the specific type or allele of HLA-II expressed on the immune cells. The presence of anti-HLA IgG in IVIg constitutes a distinct danger of inducing TRALI. The differences in IVIg preparations underscore the need for monitoring the strength of anti-HLA IgG as a way of standardizing IVIg.

A salient question is whether the anti-HLA-I IgG in sera and in IVIg is against open conformers (β2m-free HLA-I heavy chain polypeptide) or against intact antigen (β2m-associated with HLA-I) (Fig. 3); it was the question addressed in this study by testing with regular beads and iBeads. It appears that the IgG may react to intact HLA-A alleles and to the open conformers of HLA-B and HLA-Cw. However, in some individuals the reactivity may reverse, as noted: anti-HLA-I IgG to intact HLA may produce TRALI, whereas antibodies against open conformers may effect immunosuppression 30,34.

Figure 3.

Dual expression of human leucocyte antigen (HLA) class I molecules on the cell surface of lymphocytes and other cells. The intact HLA class I occurs as HLA heavy chain (HC) polypeptides associated with β2-microglobulin (β2m). On the surface of activated lymphocytes, HLA-I occurs as open conformers (β2m-free HC). All HLA-I alleles share certain amino acid sequences (¹¹⁷AYDGKDY¹²³ and ¹²⁶LNEDLRSWTA¹³⁵). In intact HLA, they remain cryptic due to masking by β2m. In open conformers, they are exposed to interact with antibodies. Anti-HLA-I IgG may recognize regular HLA beads coated with both β2m-associated and β2m-free HCs of HLA (open conformers) but not iBeads, on which β2m-associated intact HLA predominated.

The large differences in MFIs among the sera of our study's donors can be accounted for by considering that levels of anti-albumin antibodies can be elevated in people otherwise ‘normal’ and ‘healthy’ under disease conditions, mainly involving the liver, including persistent, undiagnosed hepatitis (especially among Asians), and by such things as transient viral infections and gastrointestinal issues. Furthermore, the anti-albumin IgG and IgM involved in the removal of altered (aged) albumin molecules from the circulation in normal sera are considered physiological autoantibodies 36. It is known that the concentration of albumin increases in liver diseases, stimulating an active synthesis of anti-albumin antibodies. These could be albumin-specific or albumin antibodies cross-reacting with other proteins such as peroxidase, or the viral proteins such as those of hepatitis B. Supporting this contention, it was shown that the levels of anti-albumin IgG autoantibodies were higher than normal in both acute and chronic hepatitis patients, but that those levels decreased to normal when the patients recovered 37. It was also shown that the circulatory pool of B cells from the majority of chronic hepatitis B surface antigen carriers contained sensitized B cells that, when stimulated, secreted IgG antibodies with specificity for human serum albumin 38.

To sum up, anti-HLA IgG antibodies in the sera of normal, healthy, non-alloimmunized males were tested against beads, each of which was coated with either one protein of one of 31 HLA-A alleles or one protein of 50 HLA-B, 16 HLA-Cw, 36 HLA-DRB, 29 HLA-DQ or 26 HLA-DP alleles. A remarkable difference exists between the reactivity of IgG before and after its purification of IgG from the sera. The profile and incidence of antibodies in the purified IgG mirrored the HLA-I 6 and -II reactivity of IVIg. Failure of IgG in the sera to react with the wide variety of HLA alleles could be due to factors masking the IgG expression in sera. A <3 kDa peptide from the CDRII domain of the Fab region of IgG inhibited the binding of purified IgG to the HLA-I/-II antigens. Some of the anti-HLA-purified IgG matched the HLA types of the corresponding donors, confirming the presence of autoantibodies reacting to HLA-I/-II. Serum IgG (without purification) in a few individuals showed the presence of such auto-HLA antibodies. The presence of HLA autoantibodies and the role of antibody-derived peptide in masking the expression of antibodies indicate that there may be a built-in regulation of HLA antibody expression in circulation.

Disclosure

None of the authors have any conflicts of interest.