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A relatively simple procedure using non-denaturing techniques has been established to isolate HL-A antigens from serum. The procedure involves QAE Sephadex ion-exchange chromatography, Concanavalin A Sepharose 4B chromatography, and preparative polyacrylamide gel electrophoresis. There is approximately 17% recovery of the antigenic material in the final preparation with a 120-fold increase in specific activity over the original serum. The serum HL-A antigen is a glycoprotein containing 10 to 12% carbohydrate and having an approximate molecular weight of 130,000. The final antigen preparation migrates on polyacrylamide gels as a single band in the region of serum B1 globulins which can be entirely removed by pretreatment with specific HL-A antibodies.

Comparison was made of HL-A phenotypes in 122 patients with systemic lupus erythematosus (SLE) and 1289 ethnically matched controls. The only significant association was between the HL-A antigen HL-A5 and the presence of SLE in Black Americans. A similar trend in 40 Caucasian patients did not reach a level of statistical significance. No significant association was present between any HL-A antigen and clinical or laboratory manifestations of the disease. Lymphocytotoxic antibodies were present in 36% of the patients and correlated with the presence of low total hemolytic complement and leukopenia.

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.

Sera and plasma from normal rats and rabbits were shown to be extremely toxic in vitro to autologous epidermal cells. On the other hand, mouse sera and newborn rat sera were innocuous to autologous epidermal cells. Viability of cells was assessed by the method of eosin dye exclusion upon 2 hour incubation at 37°C. Testicular cells were also killed by autologous sera, but polymorphonuclear leukocytes, lymphocytes, and lymph node cells were not affected. Autotoxicity of sera could be destroyed by the depletion of complement components with an antigen-antibody precipitate, heat, zymosan, and NH3. Moreover, activity of sera could be absorbed out by epidermal cells, though not by lymph node cells or erythrocytes. Such absorption of toxicity was not individual-specific since homologous epidermal cells also absorbed toxicity, and in addition, were killed by fresh normal serum. Enzyme inhibitors such as soybean trypsin inhibitor and ϵ-amino-n-caproic acid did not affect the activity of fresh autologous serum. It is suggested that a natural barrier exists between the basal cells of the epidermis and the plasma which prevents the autodestructive process under normal conditions. Any injury to this barrier may than lead to necrosis and death of the epidermis as seen in various pathological conditions.

SUMMARYThe microdroplet lymphocyte cytotoxicity test was examined thoroughly in an effort to increase the reproducibility of the test. The discrepancy rate in a large series of tests was reduced from 5.16% at the start of this study to the present 0.95% by introducing certain modifications in the te

Summary A rapid, precise method is presented for the quantitative assay of cytotoxic antibody and complement levels employing lymphocytes as the target cell. The technique consists essentially of enumerating with the electronic Coulter Counter the number of “normal size cells” remaining after incubation with antibody and complement. Death of lymphocytes as appraised by eosin dye exclusion was paralleled by a loss in numbers of electronically countable cells, though simultaneous counts made in the hemocytometer indicated that the cells do not actually disappear. Thus dead cells are apparently not “seen” by the electronic counter. Plots of the reduction in normal size cells produced by allogenic mouse antisera or complement were shown to be linear by the method of probits or the von Krogh equation. The technique was highly sensitive, for cytotoxicity of some mouse antisera produced against homografts could be detected with as little as 0.00005 ml.

SummaryThe sera of rabbits which were homografted with skin are shown to be toxic to lymph node cells of the skin donor rabbit. Cytotoxicity was demonstrated upon incubation in vitro for 2 hours at 37°C. Sera of grafted rabbits became toxic after graft breakdown had commenced (11 days), and reached a peak after the rejection (20-30 days). The sera were in all instances more toxic to lymph node cells of the skin donor than to cells of other rabbits.The authors wish to acknowledge the technical assistance of C. C. Chamberlain and J. D. McClelland.

Antigenic differences between certain inbred strains of mice which could not be revealed by hemagglutination techniques were readily disclosed by lymphocyte cytotoxicity. With an improved cytotoxicity test lymphotoxic titers were as high as 1:512 with non-hemagglutinating A anti CBA antisera. In other mouse strain combinations, a close parallel of both types of antibody activity was obtained. Though both activities were absorbed from antisera proportionally by erythrocytes and lymph node cells, 100 to 1000 times as many erythrocytes as lymphocytes were necessary to produce an equivalent reduction in antibody activity. These findings suggest that erythrocytes may possess only subthreshold quantities of certain antigens which are present in readily detectable levels on lymphocytes. Lymphocyte cytotoxicity therefore may assay a wider range of allogenic antigens than hemagglutination.

Summary. Negative results were obtained in an investigation of 118 selected lymphocytotoxic antisera for activity against antigens determined by a locus or loci independent of the HL-A locus. The sera studied were those not readily identifiable in terms of either generally recognized or tentative HL-A antigenic specificities, and accordingly were considered appropriate for study to search for nonHL-A antibodies. It is concluded that antisera active in lymphocytotoxicity tests against nonHL-A antigens are quite rare. The investigation utilized a sib-pair method based on the consideration that sibs alike at the HL-A locus should react alike, except for technical error, to sera detecting only HL-A antigens and that most such alike sibs should frequently react differently to sera detecting nonHL-A antigens. Details of the method are described and its sensitivity is illustrated by showing that several red cell antigens were readily detected as determined by loci independent of the HL-A locus.

Summary Normal serum from various animals has been shown to be toxic to lymphocytes and peritoneal exudate cells of various other animals. This toxicity could best be shown with fresh serum or in some cases only with fresh serum. Serum heated at 56°C for 30 min lost its activity in most instances. The activity lost upon storage or heating could not be completely restored by the addition of fresh homologous or autologous serum. Some particularly toxic combinations of serum-lymphocytes were: human-rabbit; human-mouse; rabbit-chicken; rabbit-rat; rat-chicken; chicken-rabbit; and chicken-guinea pig.

SummaryAn agglutination method for detecting antibodies against blood lymphocytes is described. By this method non-specific clumping of polymorphonuclear leucocytes and/or monocytes usually encountered in leucoagglutination tests is avoided. The humoral response following homografting in chickens was reinvestigated using this lymphoagglutination reaction. In 82 combinations of normal sera or sera from autografted chickens, and lymphocytes, lymphoagglutination was found in only 5 instances. On the other hand, 103 cases of positive agglutination were obtained in tests with 107 combinations of immune sera and normal lymphocytes. These immune sera, including one with titer of 1:1024, were from animals previously injected with pooled homologous spleens. Sera from animals injected with splenic cells or grafted with skin from one donor, also contained lymphoagglutinins against lymphocytes of spleen or skin donor chicken. These lymphoagglutinins also reacted in some instances with lymphocytes of chickens other th...

A function of human lymphocytes was studied in 66 individuals ranging in age from newborn to 100 years. The test used was antibody-dependent lymphocyte-mediated cytolysis of HL-A antibody-coated 51Cr-labelled human lymphocytes. Lymphocytes from newborn infants were ineffective in producing cell-mediated lympholysis and lymphocytes from children 1 to 6 years of age were most effective. Lymphocytes from adult and geriatric persons demonstrated variable ability in this test system. The ability of lymphocytes to perform this cytolytic function is acquired between 3 and 12 months of age.

Summary Evidence has been found which indicates that tolerance to homografts in chickens is not completely individual specific (within one breed as well as across two breeds). A total of 71 embryonic chicks were injected with blood from embryonic chicks of a different breed and, 2 and 15 days after hatching, were grafted with skin from chicks other than the blood donor but of the same breed as the blood donor. A significant percentage of these chicks had grafts which survived longer than the grafts between control chicks not previously injected with blood. The possibility that the tolerance induced by blood of one chick to skin of another was due to similarity in antigenic makeup of the 2 donor chicks, since they were of the same breed, seems discounted by 2 experiments. First, it was shown that grafts among 2-week-old chicks of the same breed did not survive to any significant degree, thus indicating the genetic disparity among chicks of the breeds used. Secondly, when the nonspecific graft survived for a fairly long period, grafts between the blood and skin donor chicks often did not survive as long.

SUMMARYCertain deesterified lipoplysaccharides (DeLPS) from gram negative bacteria were shown to specifically inhibit the lymphocytotoxie activity of some of the 17 HL-A specificities tested. The DeLPS tested were derived from Escherichia coli of strains K-235, B-4, B-5, B-8, and from Salmonella typ

The reaction of HL-A antibodies with the HL-A determinants on the surface of lymphocytes can be blocked by preincubation of lymphocytes with diverse substances such as normal human serum, autologous plasma, fetal calf serum, newborn serum, heat-inactivated rabbit serum and guinea pig serum, serum albumins from various species, lactalbumin, dextrans, synthetic polyglucose, and polyvinylpyrrolidone. The inhibitors can be washed off readily from lymphocytes. The inhibition procedure described here differs from the conventional inhibition tests in that the inhibitor is added first to the target cells. This “masking inhibition” procedure produces substantially greater inhibition than the standard inhibition methods in which the inhibitor is added first to antiserum before the addition of the target cells. The nonspecific nature of masking inhibition indicates that numerous substances adhere to the lymphocyte surface at HL-A sites or closely adjacent sites.

SUMMARYThe role of HL-A antigen compatibility in the clinical outcome of kidney transplantation was studied in 107 sibling to sibling and 147 parent to child transplants. As could be predicted on genetic grounds, certain sharp differences between the two genetic classes of donors were found. Leukocy

Forty-eight heart transplant recipients and their donors were typed for 12 HL-A antigens. Of the patients receiving grafts from donors with two antigenic groups of incompatibilities, 16 of 21 have died. Among those with transplants having one HL-A group of incompatibility, 11 of 19 have died. Four of the 7 patients with compatible grafts have died. The difference between those with two antigenic groups of mismatch and those with one or no mismatch was of marginal statistical significance ( p 0.06). Survival curves were also correlated with compatibility for HL-A antigens, although the small numbers have restricted the analysis. Preformed antibodies were found in only 7 (4.3 per cent) of 161 prospective heart transplant recipients. Thus, in contrast to kidney transplantation, immediate failures associated with preformed antibodies can be expected to be less frequent in heart transplantation

A method was developed by which Summary kidneys could be shipped in a small box containing ordinary ice for periods of up to 30 hours with almost no detectable damage. A total of 54 dog kidneys were stored in saline at 0°C with ice slush for 16, 24, and 30 hours, using surface cooling alone or combined with initial perfusion with several solutions. To evaluate the efficacy of storage, the contralateral kidney was removed at the time of reimplantation of the stored kidney and blood levels of creatinine and urea were followed daily. The final relatively simple scheme (infusion of mannitol, phenoxybenzamine, and a special perfusate) resulted in uniformly good preservation in all 7 kidneys stored for 24 hours and all 3 kidneys stored for 30 hours (there was one technical failure). Other perfusates, such as Ringer's lactate and 10% invert-sugar solutions, gave inferior results. The new perfusate extended the time of simple ice storage from 12 hours to 30 hours.

Sera from 60 patients with pernicious anemia were studied for the presence of lymphocytotoxins (LCT), blocking and binding autoantibodies to intrinsic factor, and gastric parietal cell autoantibody. LCT were found in 21 sera. Cytotoxic activity was detected at 15°C but not at 24°C and did not appear to have HL-A specificity. Autocytotoxins were present in four of eight patients tested. Blocking antibody to intrinsic factor was found in 34 sera, binding antibody in 14 sera, and parietal cell antibody in 48 sera. Sera from 14 patients contained all three types of autoantibodies, and 12 sera were void of these autoantibodies. Of the 14 sera with three types of autoantibodies, LCT were detected in ten; none of the 12 sera without autoantibodies showed cytotoxic activity. These studies suggest that LCT may reflect the degree of autoimmune derangement in pernicious anemia; alternatively, LCT may represent naturally occurring immunosuppressants.

One hundred and fifty-six psoriatic patients had three HL-A specificities significantly altered from expected values; W17 and HL-A13 were found to be markedly increased, and HL-A12 decreas...

Crossmatch tests of the prospective kidney-transplant donor's lymphocytes with the serum of the prospective recipient in 225 transplants showed that eight of 195 with negative crossmatch f...

An 11-month-old male infant with severe combined immunodeficiency was immunologically reconstituted by bone-marrow transplantation from a female sibling whose cells were HL-A compatible on mixed leukocyte culture. The patient's lymphocytes demonstrated multiple extraneous HL-A antigens (up to 10 at one time), some of which were not present in the parents or the four siblings; this typing anomaly delayed bone-marrow donor selection. These additional antigens were nonstimulatory in mixed leukocyte culture, and since they were found in two similar patients, may represent an anomaly peculiar to combined immunodeficiency. After leukocyte transfer factor was given without benefit on two occasions, bone-marrow transplantation was performed. After a severe graft-versus-host reaction subsided, clinical improvement and chimerism ensued as evidenced by an XX (donor) karyotype of cultured peripheral blood lymphocytes and restoration of immunologic functions. Direct chromosome analysis of bone-marrow cells di...

Summary The serum of rabbits which have been homografted with skin was shown to be toxic to the lymph node cells of the skin donor. Fresh serum was toxic, and the activity of the serum declined rapidly upon standing overnight, freezing or heating at 56°C for 30 min. The loss of activity could be restored to a large extent by addition of fresh normal rabbit serum or fresh serum from the lymphocyte donor rabbit. Guinea pig complement exerted some inherent toxicity. The possibility that the detection of such cytotoxins may suggest a re-evaluation of the cellular basis of homograft immunity is discussed.

Summary Over 1000 kidney transplant patients were tested for cytotoxic antibodies before transplantation. It was found that patients with preformed antibodies had a significantly poorer outcome than those without antibodies in terms of clinical ranks and survival. This effect was over and above the instances of hyperacute failures previously shown to be associated with preformed cytotoxins. Among patients who received second transplants from cadaver donors, an extremely high failure-rate was observed in patients who had developed antibodies following the first graft, whereas if antibodies were not present, the failure-rate was comparable with that of first transplants done in patients without antibodies. By analysis of survival curves using logarithmic plots, it is postulated that pre-immunization has its greatest effect in the early 3-6 month high risk period and magnifies incompatibilities which occur with unrelated cadaver donors.

THE remarkable association of the second segregant series histocompatibility antigen (HL-A) W27 with idiopathic ankylosing spondylitis is firmly established. Ninety per cent of patients with this disorder are W27 positive as compared to 8 per cent of normal white control subjects.1 , 2 W27 has also been reported with unusual frequency in anterior uveitis of diverse causes as well as in Reiter's syndrome, raising the possibility of shared pathogenetic mechanisms in these disorders.3 4 5 It is well recognized that spondylitis and a peripheral seronegative arthritis occur in a large number of patients with chronic inflammatory bowel disease.6 , 7 Although the peripheral arthropathy has several . . .

RECENTLY, an extraordinary correlation between the second-segregant series histocompatibility antigen (HL-A), W27, and a well characterized rheumatic disease, ankylosing spondylitis was reported.1 , 2 Indeed, 90 per cent of patients with the disease are W27 positive. Because ankylosing spondylitis has a strong heredofamilial distribution,3 and since the second-segregant series of HL-A antigens in certain animals is linked to immune-response genes,4 this highly important association suggests that the pathogenesis of the disease might be directly related to some aberrant, genetically controlled, cellular immune response. Reiter's syndrome is a puzzling disease with several features in common with ankylosing spondylitis — i.e., male predominance, young . . .

The frequencies of 24 HL-A antigens were examined in 40 patients with ankylosing spondylitis, 119 with rheumatoid arthritis, and 66 with gout. No significant deviation from control frequen...

SummaryCirculating white blood cells and the upper centrifugal layer of bone marrow cells taken from normal adult chickens caused a high percentage of deaths when injected into embryonic chicks. Considerably lower percentage of deaths was produced by injection of red blood cells, lower centrifugal layer of bone marrow cells, or embryonic blood.

The HL-A2 leucocyte antigen was found to be present in a significantly higher percentage of 485 patients with chronic glomerulonephritis than in 428 normal control subjects. No correlation could be shown between any of the ABO groups and chronic glomerulonephritis. There was no statistically significant difference in the frequency of HL-A2 when the controls were compared with 280 patients with a variety of other diseases. The trend toward an excess of HL-A2 positives among patients with glomerulonephritis was also seen in a study of 108 sibships, though the difference fell short of statistical significance. It was concluded that an HL-A2 positive person was about 1·5 times as likely to develop chronic glomerulonephritis as an HL-A2 negative person.

During culturing at 22 degrees C for more than 4 days, human peripheral blood lymphocytes lose their ability to stimulate allogeneic lymphocytes in mixed lymphocyte cultures. The cells retain their ability to respond to allogeneic lymphocytes or phytohemagglutinin for up to 10 days of culturing. The findings are relevant to reports on successful transplantation of cultured skin.

SUMMARY Graft survival was analyzed in over 3,000 human kidney transplants performed between 1969 and 1972, with respect to histocompatibility. The most significant factors on graft survival were found to be HL-A haplotype differences, unresponsiveness to HL-A, and presensitization to HL-A. In cadaver donor transplants, HL-A antigen differences (HL-A matching) seemed to be of some influence, although not of statistical significance; no substantial improvement was obtained when cross reactivity and homozygosity were considered. Contrary to the reports of others, second locus antigens were not more significant than the first locus antigens. The HL-A type of the recipient and mismatches for different HL-A specificities were not found to be factors of major influence on graft survival. For cadaver kidney transplantation, immune responsiveness to HL-A as measured by antibody production to HL-A was the most consistent factor influencing survival of transplants.

Many investigators consider pernicious anemia an autoimmune disease. Cellular and humoral immunity to gastric antigens, particularly intrinsic factor, has been found in most patients with ...