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The WHO Nomenclature Committee for Factors of the HLA System met following the 14th International HLA and Immunogenetics Workshop in Melbourne, Australia in December 2005 and Buzios, Brazil during the 15th International HLA and Immunogenetics Workshop in September 2008. This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports (1–18).

Lymphocytotoxic antibodies to the red cell blood groups A and B were purified from human sera by elution from synthetic immunoadsorbents (Synsorbs from Chembiomed). The lymphocytotoxic activity of the eluted antibodies was better preserved when elution was carried out at pH 11, whereas it was less stable when elution was performed at pH 3. In addition, complete removal of anti-red cell blood group activity from an anti-HLA serum was easily obtained by adsorption on the corresponding Synsorb without loss of anti-HLA activity.

T, B, and null lymphocytes isolated by sheep red blood cells (SRBC) rosetting or adherence to a nylon wool column had distinct size differences according to Coulter counter size analysis. The diameters in microns were as follows: B = 6.42, T = 6.76, null = 7.16, granulocytes = 8.42, and monocytes = 9.02. Coulter size distributions of cell preparations were compared to the proportions of cells with E, EAC, and surface immunoglobulin (SIg) markers in these preparations as well as their reactivity to HLA-DR antisera and rabbit anti-B-lymphocyte antisera. Cell preparations monitored by Coulter sizing produced the expected results in mixed lymphocyte culture (MLC) testing. That is, T cells responded but did not stimulate whereas B lymphocytes and monocytes stimulated but did not respond. NULL lympocytes both stimulated and responded. In antibody-dependent cell-mediated cytotoxicity (ADCC), null lymphocytes and monocytes functioned as effectors, whereas Tlymphocytes and granulocytes did not. B lymphocyte preparations were intermediate in activity. Coulter sizing is a rapid, simple means of assessing the purity of suspensions enriched for T, B, and null lymphocytes.

A hemolysis assay was developed to detect alloantibodies to human immunoglobulin. A total of 1035 serum samples was tested. Anti-IgM antibodies were found in 8% of 59 normal persons and in 13% of 439 multiparous women, with the highest incidence of 67% in 341 dialysis patients. Although the anti-IgM antibodies were inhibited by both IgM and IgG, it appeared that they were also inhibited by F(ab')2 but not by Fc. Anti-IgG antibodies were more strongly inhibited by Fc than F(ab')2. These results suggest that anti-IgM antibodies might be analogous to antiidiotypic antibodies directed to F(ab')2, whereas anti-IgG antibodies tend to have greater reactivity to Fc.

IgG and IgM were stripped from the surface of human B lymphocytes using heterologous anti-IgG or IgM antisera. The percent SIg (surface Ig) determined by fluorescence for SIgM was reduced from 55% to 20%, or a reduction of 64%. Upon stripping SIgM, the B lymphocytes were no longer killed by seven different cold cytotoxic antibodies obtained from kidney transplant patients. We conclude that SIgM is the target of cold cytotoxins. These cold cytotoxic antibodies are postulated to be immunoregulatory antibodies. They are less specific than anti-idiotypic antibodies.

To explore possible genetic determinants of immunity to Epstein-Barr (EB) virus infection, prevalence rates and geometric mean titers (GMT) of antibody against EB viral capsid antigen (VCA) were measured in 422 adults of known HLA and ABO histocompatibility types. Deviation from the overall prevalence (87%) of anti-VCA titers > 10 was limited to subjects with HLA-A10 (95%) and those with a "blank" HLA-A locus (78%). The latter deviation was largely accounted for by the 13 subjects of HLA-A1/blank constitution (46%). Deviations from the overall mean anti-VCA titer (GMT 131.5) were found in seropositive subjects with blood group 0 (GMT 153.3) and tissue antigens HLA-A3 (GMT 150.8), HLA-B7 (GMT 152.1), HA-Bw15 (GMT 97.0), and HLA-B27 (GMT 86.4). Separate reanalysis of the data for male and female subjects verified the association of blood group 0 with elevated and HLA-B27 with reduced mean levels of anti-VCA antibody.

The histocompatibility antigen, HL-A8, was found to be elevated in patients with Sjogren's syndrome. Among 24 patients, the frequency of HL-A8 was 58% compared to 21% among controls (P less than 0.001 after correction). With the addition of this disorder to the list of other autoimmune diseases associated with HL-A8, it now seems possible to postulate a single basis autoimmune defect gene in high linkage disequilibrium with HL-A8.

Plaque-forming cells can be generated from peripheral blood lymphocytes provided that the lymphocytes are prestimulated by in vitro sensitization in the presence of pokeweed mitogen. Of 91 blood type O persons whose lymphocytes were stimulated with red cell A antigen, 52% responded with plaque formation, whereas of 40 type O persons whose lymphocytes were stimulated with red cell B antigen, only 20% produced plaques. Similarly, reciprocal stimulation of type A and B persons showed higher plaque formation (65%) when 23 type B persons were stimulated with A than when 48 type A persons were stimulated with B red cells (19%). An optimal dilution of sensitizing cells is necessary and maximum time of sensitization was approximately 7 days.

The incidence of HL—A3 among 94 patients with a diagnosis of multiple sclerosis (MS) was significantly higher than for 871 normal persons. In addition, some increase in Te58 and decrease in HL—A2 and Te60 frequencies was noted. The well-known higher incidence of MS among Caucasians than among Orientals is paralleled by the higher incidence of HL—A3 and Te58 among Caucasians and a lower incidence of Te60.

Human lymphocytes sensitized with HL-A antibodies could not be killed by the addition of complement if the lymphocytes were incubated for periods of 1 to 5 hours at 37° C. This phenomenon was not produced by simple dissociation of the antibodies or by loss of antigens (modulation), but rather is postulated to result from an active release or pinocytosis of HL-A antigens together with the attached antibody. “Escape from sensitization” is followed closely by reformation of antigens, for the cells can readily be resensitized and killed by addition of complement. Loss of sensitization is an active process, since one-day-aged lymphocytes or lymphocytes treated with actinomycin D or puromycin were unable to express this activity. A differential escape rate for different specificities was encountered indicating that HL-A9 was produced more rapidly than several other specificities present on the same lymphocytes. A correlation was noted between the rate of HL-A antigen synthesis by lymphocytes (indicated by escape rate) and the quantity of HL-A antigen in the serum. Inhibition of lymphocyte cytotoxicity was used to detect the presence of HL-A antigens in serum. Certain specificities, such as HL-A9, were generally present in serum whereas others, such as HL-A8, could not be detected. Cross-inhibition tests with over 100 sera clearly showed an association of inhibition with specificity, although nonspecific inhibition was often observed. Sephadex fractions of sera tested at different concentrations revealed the greatest inhibitory activity in the 19S and 7S fractions.

Human peripheral blood granulocytes reacted with allogeneic or xenogeneic antisera and when stained with FITC formed polar caps very rapidly. Within 8 min, 90 % of the cells had clusters and polar caps. Cap formation generally paralleled the motility function of the cells. It occurred just as rapidly on granulocytes kept in hanging drops or in constant motion within a test tube as on glass slides. Phagocytic function of the cells was not affected by capping.

The matching criteria of counting the number of HL-A incompatibilities, the number of compatible antigens, and of ratios (net histocompatibility ratio) were evaluated. In order to avoid bias, the data were analyzed with the aid of computers, taking many different cutoff points. The probability value of statistical significance can be shown to vary considerably depending on the time selected for evaluating the clinical ranks and the clinical ranks which are considered to be “successful” or “failure.” Overall, the data indicate that because of the large number of patients who do well in spite of incompatibility, a more suitable means by which successful transplants can be predicted will be required. Since HL-A antigens are still the primary transplantation antigens (as shown by the successful HL-A-identical sibling transplants), empiric knowledge on the types of incompatibilities which can result in good graft survival must be developed.

We have defined the new allele HLA-B*4012, which had been isolated from a black individual. It was initially recognized as a serologically unique allele when typing her father for renal transplantation. The HLA class I phenotype was A*0201,*6602; B*4001,*4012; Bw6; Cw*0304,*1505. Sequencing from exon 1 through intron 3 of B*4012 was performed. B*4012 is identical to B*4001 and B*4010 in exon 3, and in the 3′ part of exon 2, but it is unique in that exon 1 and the 5′ part of exon 2 are identical to B*1503, B*1509, B*1510, B*1518, B*1523, and B*1529. The generation of this allele is best explained by a recombination event in exon 2 (break point between nucleotides 205 and 222 from the beginning of the coding region) of B*4001 or B*4010 with one of these B*15 variants as a donor allele. Its unique serological feature (B48, B60, B70, and B72 reactivity) is consistent with the sequence data of its donor alleles ( Note).

1. The Naming of New Genes Within the HLA Region The Committee agreed in 1996 to defer the naming of new genes within the HLA region. However, in view of the great interest and work being carried out on the MIC (MHC Class I chain related) genes, already named by the Human Genome Nomenclature Committee as MICA, MICB, MICC, MICD and MICE in April and September 1994 [15, 16], it was agreed that they should be included within the official HLA nomenclature for the purpose of naming alleles of these genes. In addition the change of name of the gene HLADNA to HLA-DOA, as published in the monthly updates, was confirmed, following further results showing that the gene codes for the alpha chain associated with HLA-DOB. The updated list of genes in the HLA region is given in Table 1.

We have characterized two novel HLA-B alleles, B*7802 and B*51022. The Caucasian-derived variant B*7802 most resembles the African-derived variant B*7801, from which B*7802 differs by two nucleotides. Only one of these modifications, however, is translated: a tyrosine for aspartate substitution occurs at residue 74 in B*7802, while the second nucleotide difference reflects a proximal synonymous substitution in codon 23. A second variant, B*51022, differs synonymously only at codon 23 from B*51021. Comparative analysis of the B5 CREG demonstrates that other pairs of B5 alleles differ synonymously only at codon 23 or synonymously at codon 23 and non-synonymously at a second more distal location. Contrary to the genesis of like pairs of B5 alleles via introduction of coordinate yet distant mutagenic events onto a single B5 progenitor, we postulate that synonymously different B5 progenitor molecules, B5ATT and B5ATC, are evolving in convergence to generate homologous B5 allele pairs differing silently at codon 23. Our finding that B*7802 is a single amino acid away from complete convergence with B*7801 and that B*51022 and B*51021 are in complete convergence is exemplary of such evolution.

We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step microcytotoxicity test. The test is performed by adding 1 microliter of cells to 1 microliter of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.

HLA-B15 embraces a multiplicity of antigenic specificities which vary in their distribution amongst human populations. To correlate B15 molecular structure with the serological picture we have sequenced alleles encoding the various subspecificities of the B15 antigen: B62, B63, B75, B76 and B77, and a number of "variants" of these antigens including the 8w66 split of B63. HLA-B63 (B*1517) and 8w66 (B*1516) heavy chains have sequence identity to B17 in the alpha 1 helix correlating with the antigenic crossreactivity of these molecules. HLA-B77(B*1513) and B75 (B*1502) heavy chains differ solely in segments determining the Bw4 and Bw6 public epitopes, consistent with the serological description of the B77 and B75 antigens. One allele encoding the B76 antigen (B*1512) appears to be the product of gene conversion between the HLA-A and -B loci and differs from B*1501 in codons 166 and 167. In contrast, a second allele encoding the B76 antigen (B*1514) differs from B*1501 by an unrelated substitution in codon 167 which confers similarily with B45, an antigen crossreactive with B76. A third allele encoding B76, B*1519, differs from B*1512 by a unique point substitution in exon 4. Three alleles encoding variant B15 and B62 antigens (B*1508, B*1511 and B*1515) differ from B*1501 by localized clusters of substitutions that probably result from interallelic conversion. The B15 sequences described in this paper, in combination with those previously determined, define a family of 22 alleles, including those encoding the B46 and B70 antigens. Within this family the patterns of allelic substitution are analogous to those of other HLA-A and -B families, in that pairwise differences almost always involve functional positions of the antigen recognition site and recombination is the major agent of diversification.

Binding of immunoglobulins to major histocompatibility complex (MHC) molecules was demonstrated by two different assays: the binding of IgG to B cells by flow cytometry, and purified MHC antigens with an Elisa assay. Fc fragment from immune-complex binds to the Fc receptor on B lymphocytes. Here, Fab was also shown to bind to B cells. This binding was inhibited by specific human allo anti-HLA Class I and II sera directed at the polymorphic sites. Thus, in addition to the Fc receptor, MHC can also serve as a binding site for IgG. In an Elisa assay using purified antigens, IgG was shown to bind to HLA Class I and II molecules. Other proteins such as transferrin, human serum albumin, gelatin, etc., did not bind to the MHC proteins. Immunoglobulins bound to MHC molecules by sites on the Fab fragment independent of the hypervariable region. This was demonstrated by the retention of antibody activity even after binding of antibody (anti-lactoferrin) to MHC. The relative avidity between Fab and HLA Class I and II was 4-8 x 10(5) M-1.

Direct detection of the PCR, or DD-PCR is proposed as an efficient method for performing PCR assays. Following the PCR reaction, ethidium homodimer dye is added to the reaction mixture and read by fluorescence. The dye step circumvents the necessity of running reactions on agarose gel electrophoresis, which is the current standard. This simple modification should find wide application for assays utilizing the PCR reaction. Here we show the ready detection of HLA class II polymorphism.

To test the humoral theory of allograft rejection, we investigated whether all patients who reject a kidney have HLA-antibodies. Sera from 1363 patients at four transplant centers who had rejected a kidney graft and transplant or who had rejected a graft and were waiting for a regraft were first tested by cytotoxicity AHG. Sera that were negative were tested by solid phase assays (ELISA or flow cytometry). Among the 1363 patients who had rejected a kidney, 90% were found to have HLA-antibodies. Eighty-four per cent tested positive for the CDC-AHG assay and an additional 6% of sera, when tested by solid-phase assays for IgG, IgM and IgA antibodies, was found to be positive for HLA. Among the four different centers the range of positivity was 89–91%. We conclude that 90% of patients who reject a kidney transplant have detectable HLA-antibodies.

Of 39 consecutive cadaver donors tested, DR antigens could not be detected in 17. Following 3 h incubation of the B lymphocytes at 37 degrees C, eight of the donors with no detectable DR antigens had two DR antigens and nine had one DR antigen. In addition, there were nine cases in which a potential cadaver donor had one antigen before incubation and two DR antigens following incubation. The simple technique of pre-incubating B lymphocytes before testing has helped considerably in prospectively typing cadaver donors for HLA-DR.

For the past 10 years, the International Cell Exchange has been a useful tool for testing rare and unusual HLA antigens and monitoring the progress in detection of recognized specificities. Of special interest are the antigens proposed in the 8th Workshop - 8w57, 8w58, 8w59, and 8w66. Marked improvement could be seen in the detection of 8w59 (29%) and 8w66 (31%). Possible new variants of the A9 and A10 complexes were tested in several exchanges. As shown by the increasing yearly average percentages of detection, laboratories are improving in their ability to type Aw23, Aw24, A25, A26, A29; Bw39, Bw41, Bw54, Bw55, Bw57, Bw58, Bw60 and Bw62. Over 90% of the laboratories are now able to detect Aw24; B5, B17, and B40, and A29 has attained the status of being well defined.

Highly enriched preparations of monocytes, B and T lymphocytes, and granulocytes from 18 normal donors were serotyped in parallel in a complement-dependent cytotoxicity assay using allogeneic and heterologous antisera defining three independent tissue antigen systems. HLA and B-lymphocyte tissue antigens were detected on human monocytes although granulocyte antigens were absent. By cytotoxicity testing the presence of Ia-like antigens on monocytes was significantly diminished compared to the autologous B-lymphocyte population and has important implications in B-lymphocyte serology. The study indentified a number of human antisera obtained from multitransfused subjects and pre- and post-transplant organ recipients that were non-HLA and appeared to define monocyte-associated antigens. The serological implications of surface antigen expression on human monocytes compared with other peripheral blood cells are discussed.

Spleen cells from a mouse immunized with an AML cell expressing HLA-A3 produced a hybridoma secreting an anti-HLA-A3, A11 monoclonal antibody, 26D3. By complement-dependent cytotoxicity at dilutions to 1:10(4) the ascites antibody lysed 13/13 A3, 15/15 A11, and 8 other lymphocytes from a panel of 98 donors. The 26 D3 immunoprecipitated a molecule consisting of subunits of 44,000 and 12,000 daltons. Monoclonality of the antibody was demonstrated by isoelectric focusing and protein A affinity chromatography.

The variable expression of the C locus specificities at the cell surface is a major problem in HLA typing and the phenomenon was clearly documented by the International Cell Exchange Program. In contrast with HLA-A and -B, the C locus specificities are readily lost from cells following storage at room temperature for a few days or in overnight incubation at 37 degrees C in the presence of protein synthesis inhibitors. These specificities can be made to reappear by making the cells metabolize at 37 degrees C for 8 to 20 h in nutrient medium. Sixty-nine per cent of the positive C locus reactions lost after 7-day storage at room temperature were recovered after overnight incubation at 37 degrees C. The reappearance was dependent on protein synthesis since presence of cyclohexamide during incubation completely blocked the recovery. A simple overnight incubation is very useful in enhancing C locus expression on older cells.

The frequency of HL-A2 was significantly low (21%) in patients with periodontitis when compared to controls who were free of periodontal disease (61%). The effect was most pronounced in females in whom HL-A2 was present in 12.5% (2 out of 16). This finding suggests that genes controlling susceptibility to microbial agents may be linked to the HL-A LOCUS.

The frequencies of HLA antigens were determined in 32 black sarcoidosis patients and in 89 normal black controls. Aw23 was found in 23.6% of controls as compared with a lower frequency (6.2%) in patients (P = 0.06). No other associations were found.

Using a microcytotoxicity assay, the serological reactivity of human granulocytes, namely neutrophils and eosinophils, and chronic myeloid leukemia (CML) cells and cultured CML cell lines (K562, NALM-1) were examined. Mature granulocyte forms and cord granulocytes are readily lysed by specific granulocyte cytotoxins that do not react with random T and B lymphocytes, monocytes, red blood cells, or platelets. Furthermore, certain antisera were preferentially cytotoxic for eosinophil-enriched populations. Granulocytotoxins detected antigens on one of three CML blast cell populations tested and K562, but failed to react with NALM-1. By cytotoxicity, mature granulocytes were poor targets for B2-microglobulin and the appropriate HLA antisera although both sera types are absorbed with granulocytes. Furthermore, granulocytes did not possess B-lymphocytes (la-like) or blood group A, B, and Rh (D) antigens. Except for K562, both HLA and heterologous B-lymphocyte antisera were cytotoxic for the CML blast cell populations tested.

Cold non-HLA lymphocyte cytotoxins were found to be principally reactive against B lymphocytes. These antibodies were studied in 1335 patients with a wide range of diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, Hashimoto's disease, asthma, diabetes, lymphoma, psoriasis, leukemia, multiple sclerosis, and also in healthy donors. Antibodies reactive to B lymphocytes in the cold or warm test conditions were not directed against HLA specificities. Since B lymphocytes differ from T lymphocytes principally in that they have surface immunoglobulin, it is postulated that at least one target antigen of cold lymphocyte cytotoxins is not a virus, infectious agent, or a genetically determined structural antigen, but, rather, simply immunoglobulin.

We have prospectively examined 46 consecutive unrelated children with JRA, 28 females and 18 males, for HLA-A, -B, -Dw1, and -Dw3 antigens. The data were analyzed according to mode and age of onset, as well as presence of extraarticular features. Patients with JRA were found to have an elevated frequency of HLA-B27 (26% vs. 8% of controls, P < 0.01). This elevated frequency of HLA-B27 was noted in patients with either a pauciarticular or polyarticular onset and consistent with previous observations, was more common in males than females. However, the most striking associations in JRA were apparent when the MLC-defined locus HLA-Dw3 was studied. The total patient population had a 46% frequency of HLA-Dw3 as compared with 24% in controls (P < 0.01). Moreover, when the group of 46 children was subdivided, the HLA-Dw3 frequency of patients with polyarticular disease was 64% as compared with 21% in pauciarticular disease (P < 0.01). Further, although the frequency of HLA-B8 was similar in JRA to that in the control panel, a significant degree of linkage disequilibrium was shown to exist between HLA-B8 and HLA-Dw3. No such linkage was shown to exist between HLA-B27 and either HLA-Dw1 or Dw3. Although we have not multiplied the P value by the number of HLA-A, -B and -D antigens, we suggest that the presence of the MLC-defined locus HLA-Dw3 is significantly elevated in children with polyarticular mode of onset of JRA and suggest that further definition of lymphocyte-dependent loci may aid in understanding the mechanism of pathogenesis as well as the classification and prognosis of childhood onset arthritis.

Upon evaluation of 40 subjects with progressive systemic sclerosis (PSS), a significant association of HLA antigens A9 and Aw24 (a subgroup of A9) was found with diffuse scleroderma. HLA-Aw23 (a second subgroup of A9) was also increased in the patients, however, this was not statistically significant. Diffuse scleroderma appears to be one of the few diseases that shows an association with the HLA-A locus.

The HLA types of 133 patients with cleft lip and/or cleft palate were determined. Caucasian patients with isolated cleft palate showed a possible association with HLA. Ten of 11 male patients had HLA-A2 as compared to one out of eight female patients (P < 0.005). Caucasian males with cleft lip and cleft palate had a slight increased frequency of HLA—Aw24 (P= 0.07) and Mexican-American males with cleft lip and/or cleft palate showed an increase of antigen HLA—A28 (P= 0.07), though neither were statistically significant. Females with cleft lip and/or cleft palate from either racial group had no differences from the controls. The serum from 90 mothers of patients with cleft lip and/or cleft palate were reacted against their child's lymphocytes. Of these crossmatch tests, 12% were found to be positive. These preliminary results suggest that male patients with isolated cleft palate are worthy of further studies with respect of HLA associations.

Reported associations of HLA antigens with MS have varied considerably. In this study we have examined 330 clinically definite MS patients from the United States. The frequency of DW2 (52%) was highly statistically significant in MS patients as compared to controls (p less than 0.00001). Neither A3 nor B7 was significantly increased in MS; however, the joint occurrences of A3-DW2 (20% vs 7%, p less than 0.001) and B7-DW2 (30% vs 13%, p less than 0.0002) are highly associated with MS.

Thirty-eight patients with severe chronic active liver disease (CALD) were found to have a significantly higher frequency of HLA-Dw3 (68%) than 91 healthy controls (24%) (P less than 0;0001). The association of CALD was statistically significantly stronger with the D locus than with the B locus of HLA. The response to treatment was significantly worse in patients with HLA-Dw3 than in patients who lacked this antigen.

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.

A comprehensive study to elucidate the cross-reactivity between 25 specificities of the HL-A system was carried out systematically with 65 monospecific anti-HL-A antisera and nearly 150 cell donors. Antisera were absorbed by platelets in microvolumes and the degree of absorption was measured by the inhibition of lymphocytotoxicity. The following conclusions were drawn: (1). Cross-reactivity occurred only between allelic gene products of a single HL-A segregant series and not between those of the two different series. (2). Cross-reactive groups did not form isolated clusters but were interconnected. (3). Cross-reactivity was found to be one-way (unidirectional) or two-way (bidirectional). One-way cross-reactivity was observed between 24 pairs of antigen groups and two-way cross-reactivity was observed between 8 pairs of antigen groups. (4). Cross-reactivity is exhibited by certain antisera of a given specificity and not by others of the same specificity. (5). Some cells possessing a cross-reacting antigen cross-absorbed a certain anti-HL-A antiserum, whereas other cells with the same antigen did not. These results suggest the possibility that cross-reactivity depends also upon other antigens present on the cells.

HL—A typing in 80 Asian Indians has shown differences in the frequencies of some HL—A antigens as compared with American Caucasians. These include the absence of Te54 (= W14 = Maki) and Te64, and significantly low frequencies for HL—A2 and HL—A8.

Six sets of unrelated healthy individuals, phenotypically “identical” in each set for the four antigens of the two HL—A segregant series, were tested with 153 antiplatelet antisera by microcomplement-fixation. In five of the six sets the results unequivocally show that the individuals were not identical by complement-fixation reactions. It is proposed that sera which manifest nonidentity between individuals within these sets identify either “variants” of the known HL—A antigens or antigens of a third segregant series of the HL—A system or of a non-HL—A system. Accurate and complete matching for HL—A antigens among unrelated individuals will require further definition of specificities.

Forty-nine of 127 normal sera from New Zealand white rabbits were found to contain cytotoxic antibodies against human lymphocytes. Reactions with these sera did not correlate with those of known HL–A or ABO specificities. However, four distinct specificities were found in 18 sera. These antibodies are labile upon prolonged storage at −20°C and heating at 56°C for 30 min and have low avidity. Since rabbit serum is the most efficient source of complement in the cytotoxic reaction with human lymphocytes, the quantity as well as the specificity of these natural antibodies in rabbit serum have to be considered when selecting this reagent for the lymphocytotoxic test.

Antibodies which were cytotoxic to lymphocytes were found in patients with kidney transplants, Graves' disease, Hashimoto's disease, rheumatic heart disease, and in multiparous women. These cytotoxins were 19S, optimally reactive at 15° C, were autocytotoxic to lymphocytes, and were inactive against granulocytes. The specificity of their reactivity to allogeneic lymphocytes was unrelated to HL-A specificities. In fact, though the sera varied from 1% to 99% in reactivity against a panel of random donors, much of the variation seems to be of a quantitative nature with relatively little specificity. Out of 6,105 x2 comparisons between 110 sera of the diseases mentioned above, 83% were positive and none of the negative x2 values were statistically significant. It is concluded that diverse stimuli elicit the formation of these low avidity 198 antibodies which are directed against a non-HL-A antigen richly represented on lymphocytes. Although their biologic significance may be difficult to show, as with cold hemagglutinins, they could possibly act as a “natural” antilymphocytic serum.