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SummaryValues for the serum thymol turbidity test show a delayed rise and fall following an acute attack of infectious hepatitis. Recurrences of the disease produced a marked increase in the thymol...

Summary1. When serum with abnormally hiqh gamma globulin concentration 1s diluted with a solution containing a certain small amount of copper or zinc sulfate, a turbid precipitate forms and the opt...

Human sera were found that contained antibody activity which caused agglutination of red cells or particles sensitized with immunoglobulin G that had first been degraded by pepsin proteolysis. The agglutinating activity was specific for a determinant that was not present on the untreated, native immunoglobulin. It was found most frequently in sera from rheumatoid arthritis patients and its titre showed some correlation with disease activity.

Isolated human tonsillar and peripheral blood B lymphocytes were induced to proliferate in vitro after exposure to F(ab′)2 fragments of affinity purified antibodies to IgM, IgG, Fab, κ, and λ chain determinants. Low levels of DNA synthesis were observed in cultures containing anti-IgA antibodies, whereas DNA synthesis was not detected in cultures stimulated with anti-IgD. Divalent antibodies were essential for the generation of blastogenesis. These proliferative responses were T-cell independent and susceptible to suppression by excess numbers of monocytes. In addition, they were elicitable in cultures not containing FCS or 2-mercaptoethanol. Highly specific antibodies to IgG induced marked proliferation and this was similar in degree to that induced by anti-IgM. Subfractionation studies demonstrated that the anti-IgG responsive cells were contained to a major extent within the surface IgM+ B-cell population. None of the antibody preparations elicited B-cell differentiation to antibody producing cells. Moreover, anti-μ antibodies completely abrogated Ig synthesis by pokeweed mitogen-stimulated cultures of unseparated tonsillar mononuclear cells. Anti-IgG antibodies similarly suppressed PWM-induced antibody production, although this was most apparent on the IgG response. In contrast anti-IgD antibodies both failed to suppress Ig production and in certain instances resulted in an increased level of Ig synthesis. These functional studies suggest that IgG molecules are intimately associated with the surface membrane of some B cells and that coexpression of IgG with IgM occurs. In addition, the observations emphasize further the divergent functional roles which surface IgM and IgG vs surface IgD have in B-cell proliferation and differentiation.

Multiple lymphoid cell lines were derived from 35HLA-D homozygous donors by EB-viral transformation of B lymphocytes. The expression of Ia-like alloantigens (HLA-DR) was studied by microcytotoxicity and by absorption with alloantisera exchanged through the Seventh Histocompatibility Workshop. B-lymphoid lines expressed the same specificities as normal B lymphocytes. Workshop antisera representing DRw1, DRw2, DRw3, and DRw7 gave well-defined typing patterns with cell lines derived from donors of corresponding D-locus specificities. A more complex reaction pattern was seen for antisera representing DRw4, DRw5, and DRw6. The available reagents could not discriminate between lines from donors homozygous for Dw4, Dw10, or D-KH. All lines studied, except for those from one donor homozygous for a unique D-locus determinant (D-SPO), could be assigned one of the provisional DRw specificities. The advantage of obtaining multiple cell lines from a single donor was evident. One line could not be typed by microcytotoxicity because it was lysed in all human sera tested, and some other lines gave weak cytotoxic reactions. Absorption studies, however, did indicate similar expression of DRw antigens on these lines. The availability of multiple lines from the same donor circumvented these difficulties.

Two monoclonal antibodies were obtained that showed unique specificities for the leukemic T cells used for immunization. One antibody, S160, was totally specific for the antigen. The other antibody, S511, also reacted with a small population of normal T cells. This was made especially evident by concentrating these normal T cells with the antibody. Considerable evidence was obtained that both antibodies reacted with the same membrane molecules. In the unreduced state a major component of approximately 80 kdaltons was observed; after reduction this split into two components of approximately 43 and approximately 38 kdaltons. The reaction of the two antibodies with different antigenic sites on the same molecule, one representing a private site and the other a more cross-reactive site, strongly suggests an antibodylike molecule, but composed of polypeptide chains differing from immunoglobulins.

The human Ia-like antigens, selectively expressed on B lymphocytes, are now recognized to be closely associated with, or identical to, the gene products of the major histocompatibility complex responsible for stimulation in the mixed lymphocyte reaction. The leukemic B lymphocytes of patients with chronic lymphocytic leukemia express these antigens very well. In the present study they were readily detected by several techniques utilizing both allo- and heteroantisera. However, the leukemic B cells from most patients were found to be extremely poor stimulating cells in the mixed lymphocyte reaction. This was particularly apparent when comparisons were made on a B-cell basis with isolated normal B lymphocytes. Leukemic cell death, abnormal kinetics of leukemic cell-mediated stimulation, and serum or cellular suppressor factors do not appear to explain these findings. Studies comparing cells from a leukemic patient with those of her HLA identical sibling and results of mixed lymphocyte reactions between normal and leukemic subjects discordant for D-region-associated Ia antigens ruled out genetic explanations for the differences observed. Experiments with normal peripheral blood mononuclear cells depleted of T cells and monocytes exclude the quantitative deficiency of monocytes which is found in the peripheral blood of most leukemic patients as an explanation. The present results with chronic lymphocytic leukemia cells indicate that the mere expression of the Ia-like antigens by cell populations does not render them effective stimulators. The accumulated evidence obtained indicate that abnormalities, particularly of membrane function and metabolism, known to occur in chronic lymphocytic leukemia lymphocytes may be involved in the poor stimulatory capacity of the leukemic B cells.

Structural differences between the heavy chain of membrane-bound IgM (mu m) and the heavy chain of secreted IgM (mu s) were investigated. The primary translation products of the mu-chain, free of posttranslational modifications, were synthesized in a wheat-germ cell-free system, programmed with messenger RNA derived from human lymphoblastoid cell lines positive for both membrane-bound and secreted IgM. Encoded in this sytem were two mu-chains, which shared N-terminal signal peptides and which differed both in molecular weight and in C-terminal amino acid sequence. In vivo pulse labeling of cells confirmed that, as intermediates in the rough endoplasmic reticulum, these two forms expressed the same idiotype and maintained their difference in molecular weight and in C-terminal sequence. By correlation with pulse-chase kinetics and with immunofluorescence, one form of mu-chain represents mu m, and the other, mu s. Because the molecular weight difference between the two is manifest at the level of their primary translation products, these studies demonstrate that mu m is distinguished from mu s by a difference in primary structure, at least in part at the C-terminus.

The histochemical demonstration of nonspecific acid α-naphthyl acetate esterase (ANAE) activity was evaluated as a T lymphocyte marker primarily with the sheep erythrocyte (E) assay. A distinctive staining pattern characterized T lymphocytes which could be readily distinguished from monocyte staining. The percentage of E + and ANAE + lymphocytes was nearly always comparable in the peripheral blood and lymphoid tissue from normal and selected patients, including those with acute and chronic lymphocytic leukemia. Divergences were noted in certain other tissues including spleen and thymus. Certain mitogen-stimulated cells lost their ANAE activity while retaining their ability to form e n rosettes. Atypical and variable staining patterns were observed in established lymphoid cell lines. The histochemical demonstration of ANAE is simple and reproducible; preparations may be counterstained for cytomorphologic detail and mounted as a permanent record. Certain disadvantages are discussed. The method represents a practical alternative to E rosette assays. It is particularly well suited for certain routine laboratories.

A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.

Evidence has been obtained previously indicating that the antigens reacting with the anti-Sm and anti-RNP sera are present as a large complex, and similar protein bands are obtained with both types of sera. Inthe present study, it proved possible to break up this complex using SDS treatment before immunoprecipitation. After such treatment, different protein bands were immunoprecipitated by the two antisera; Sm determinants resided, at least partially, in a 19-kd protein. Sequential immunoprecipitation with and without prior SDS treatment provided further evidence for these specificities and suggested that two classes of particles exist in different tissues, one containing proteins immunoreactive with the Sn and RNP antisera and the other containing proteins immunoreactive only with the Sm antisera. The latter particle contained all the bands seen with the first type except for the absence of the 19-kd band. Nitrocellulose blot analyses confirmed the assignment of the 25- and 16-kd polypeptides to Sm antigenic determinants; analyses for RNP proved les informative by this technique. Some differences in the banding patterns were obtained using cells from different species: the 25-kd Sm band was usually double in human cells and single in rat and rabbit tissue. Methods of extraction also caused some differences which was especially true for the rabbit thymus extract widely used for Sm and RNP studies. Additional immunoreactive bands at 68 and 70 kd also were detected when the Sm and RNP antisera were used in nitrocellulose blot analyses. Furthermore, evidence was obtained for a number of other antibodies in lupus sera which have not as yet been detected by serological methods.

The new Ia-like antigens, which are primarily expressed on B cells, were studied in detail along with other lymphocyte markers in the peripheral blood of normal individuals and patients with immune deficiency. Rabbit antisera to purified Ia antigens proved of special value in this work although alloantisera gave similar results. The Ia antigens were found on most monocytes and all lymphocytes expressing membrane Ig and in addition on a normal population of cells lacking Ig. Both of these lymphocyte populations were completely absent in patients with infantile agammaglobulinemia with a selective B cell defect. Different findings were obtained for Fc-positive cells. In addition to T cells with Fc receptors, a non-T population was apparent that lacked both surface Ig and Ia determinants; these two cell types were not reduced in patients with infantile agammaglobulinemia. These findings suggest that all the Ia-positive lymphocytes, both those with and without clearly detectable Ig, belong to the B cell lineage. The special value of the Ia antigens as markers for distinguishing lymphocyte subpopulations was apparent in this study.

Structural differences between alpha m (ther heavy chain of membrane IgA) and alpha s (the heavy chain of secretory IgA) were investigated. Messenger RNA from the human B lymphoblastoid line 32a.1, expressing both membrane and secretory IgA, was translated in a wheat germ cell-free system, resulting in the synthesis of two primary translation products for the alpha chain, that differed in molecular weight. In vivo pulse and pulse-chase experiments demonstrated that two early biosynthetic forms of the alpha chain were subsequently modified to yield three intracellular forms. As shown by endo-beta-N-acetylglucosaminidase H (endo H) treatment, these forms represent two alpha polypeptide chains, with varying compositions of N-linked oligosaccharides. Of the two forms of the alpha chain remaining after endo H treatment, only the form with the lowest molecular weight was associated with cells after long chase periods. The possible significance of this difference from the results with mu and delta chains is discussed. These results indicate that alpha m is distinguished from an alpha s by a difference in both primary structure and intracellular processing. The functional consequences of this distinction, previously shown for the heavy chain of membrane IgM (micrometer) and heavy chain of secretory IgM (microseconds), may reflect a principle common to the secretory and membrane forms of all immunoglobulin heavy chain classes.

Human cell lines that resemble precursors in the B cell lineage have been found to synthesize J chain. In vivo pulse labeling, together with in vitro translation of total cellular RNA in a wheat germ cell-free system, detected the synthesis of J chain in immunoglobulin-secreting cell lines, in a cell line with only surface IgM, as well as in the pre-B-like cell line Josh 4 and the round cell lines Josh 7 and KLM 2. The primary translation products of J chain from all of these cell lines were found to be indistinguishable from one another by serologic criteria, by relative mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by charge as judged by alkaline-urea gel electrophoresis, and by peptide mapping. These findings suggest that the onset of J chain biosynthesis represents a relatively early event in B cell ontogeny, occurring before the development of immunoglobulin polymer-secreting cells. Its role may, consequently, be fundamental to the biosynthesis of all immunoglobulins, at different stages of B cell differentiation.

In a prospective study 26 of 29 patients with systemic lupus erythematosus had cold-reactive antilymphocyte antibodies cytotoxic for autologous lymphocytes and lymphocytes from normal subjects. The level of antilymphocyte antibodies was highly correlated, by linear regression analysis, with lymphopenia in these patients. The data suggested that both the avidity and the concentration of these antibodies were important determinants in this relationship. A clear association between increased antilymphocyte antibody activity and exacerbation of SLE was demonstrated. Apart from lymphopenia, however, neither type of clinical manifestation nor any particular serologic abnormality appeared to be related to the presence of antilymphocyte antibodies.

Human helper factors were obtained from supernates of 48 h unidirectional allogeneic and autologous mixed lymphocyte reactions. These supernates were shown to induce the production of large amounts of immunoglobulin by tonsillar and peripheral blood mononuclear cells. Abundant polyclonal activation to antibody production occurred in these cultures in the absence of antigenic challenge which was similar in degree to that produced by pokeweed mitogen. This was documented by quantitating plasma cells, specific plaque-forming cells, and secreted immunoglobulin. In addition, the supplementation of companion cultures with sheep erythrocytes resulted in a significant enhancement of the specific plaque-forming cell response without an appreciable change in plasma cell number of secreted Ig.

Using the direct migration inhibition test, response to measles antigen in patients with systemic lupus erythematosus (SLE) was found to be decreased when compared with that of normal subjects. No alteration was observed in similar experiments using parainfluenza type 1 and rubella antigens. The specific decrease in measles antigen effect showed no obvious correlation with activity of SLE or with the presence of lymphocytotoxic antibodies. Whether the specificity of the decrease in reactivity is due to some particular relationship between the measles virus or antigen and SLE, or to the possibility that measles reactivity is a more sensitive indicator of a generalized defect of cell-mediated immunity, remains unclear.

Each year it has been the privilege of the President of the Association to make a few remarks to the membership. Frequently this has been a scientific presentation, but it also has taken many other forms. Last year, when I found Baruj Benacerraf vascillating on the question as to which path he should take, I urged him to seize the opportunity and give us some of his more philosophical thoughts. We had heard enough about Ir genes. I stated that I would certainly follow his example next year. He, of course, then did this very successfully. In my own case, however, as next year drew ever closer, I had some second thoughts. It suddenly occurred to me that a scientific talk published in the Journal of Immunology would be a valuable asset. I would have to be sole author on the paper, something that hasn't occurred in many years and this would be quite impressive with my grants soon coming up for renewal.

A marked homogeneity of the light chains was observed in an analysis of 17 IgM proteins with anti-γ-globulin activity. The V region subgroups of the light chains were determined by both sequence and antigenic analysis. The latter procedure permitted large scale screening for comparisons with control proteins. The two methods showed general agreement in the determination of Kappa III proteins; all proteins positive by antigenic analysis were also positive by sequence but exceptions were noted in the opposite direction. The anti-γ-globulins showed by antigenic analysis a 92% incidence of VK III light chains as compared to an incidence of 27% among 81 control proteins without this activity. A similar selection was observed for an antigen (VK III b) which subdivided the kappa III proteins. The major Wa group of anti-γ-globulins which had been delineated previously on the basis of cross-idiotypic specificity was primarily responsible for the special light-chain selection. All the proteins of this group contain VK III light chains and all were of the VK III b type. Current evidence indicates that additional light-chain similarities were involved in the cross-idiotypic specificity of the Wa group. It thus appears that for the anti-γ-globulins various levels of selection of light chains are manifest ranging from a preponderance of kappa type, of kappa III subgroup, of kappa III b sub-subgroup and perhaps of still further subdivisions to account for the cross-idiotypic specificity.

the stimulation of lymphocytes from rheumatoid arthritis patients with pokeweed mitogen produces a large number of plasma cells that express the dominant cross-reactive idiotype previously found on monoclonal IgM anti-gamma-globulins from patients with mixed cryoglobulinemia. Similar experiments with the cells of normal individuals show a much lower percentage of these cells with a lower intensity of staining with the fluorescent reagents utilized. Efforts to demonstrate rheumatoid factor in the same cells by fluorescent staining with aggregated gammaglobulin were entirely unsuccessful. This also proved to be the case for pokeweed mitogen-stimulated cells from the mixed cryoglobulinemic patients with large amounts of rheumatoid factor in the serum, despite high percentages of cells expressing the cross-reactive idiotype and also the individual idiotype. On the other hand, native plasma cells from synovial tissue of rheumatoid arthritis patients showed some cells with both the cross-reactive idiotype and aggregate staining. The exact reason for the failure to demonstrate rheumatoid factor by aggregate staining in pokeweed mitogen-stimulated cultures remains to be determined despite considerable effort to resolve the problem. The most likely possibility is that these plasma cells are relatively immature and have not accumulated polymeric IgM in their cytoplasm to the degree seen in synovial tissue plasma cells. The monomeric forms are readily recognized by the antiidiotypic antibodies and these reagents appear to be of particular value for cellular studies of this type.

The synthesis of intracellular J chains was found to be closely associated with that of intracellular immunoglobulin, regardless of its class, during the process of B-cell differentiation. This parallelism between the synthesis of J chain and immunoglobulin was particularly evident in their coincident appearance in serial observations of pokeweed mitogen (PWM)-stimulated lymphocytes. The intensity of J-chain staining by fluorescent reagents in the stimulated cells synthesizing IgG was similar to that found in cells synthesizing IgA or IgM. Evidence was obtained that the presence of J chain in the IgG-producing cells did not reflect antecedent synthesis of IgA or IgM. T cells stimulated by phytohemagglutinin and PWM failed to show J-chain synthesis. Observations on lymphoid cell lines showed a similar parallelism between intracellular Ig and J-chain synthesis; no relation to surface Ig was found.

Supernatants from PBMC obtained from certain patients with AIDS or its prodrome were capable of depressing pokeweed mitogen-driven immunoglobulin production and the proliferative response of T cells to specific antigen. These soluble suppressor factors (SSF) were present in uniquely high concentrations, and were the product of an interaction of T lymphocytes with adherent cells. T-cell independent functions were not modified by soluble suppressor factors. Restoration of immunoglobulin synthesis in SSF-treated cultures was realized by addition of reducing agents such as 2-mercaptoethanol, suggesting inhibitory mechanisms possibly related to that of Con A-induced soluble immune response suppression, and perhaps offering clues to clinically applicable substances capable of modifying such responses. A relationship between SSF-AIDS and a human retrovirus LAV/HTLV III, linked etiologically to AIDS and its prodromes, is suggested by studies of SSF-like substances released by human T-T cell hybridomas derived from LAV+ patients.

The Second International Workshop on the Primary Immunodeficiency Diseases in Man, sponsored by the National Foundation—March of Dimes, was held in St. Petersburg, Florida, February 4–8, 1973. This short review attempts to summarize the most important findings reported at the Workshop. The complete papers and discussions will be published by the National Foundation; however, brief reports of new diagnostic criteria, recently discovered disorders of the immune system, and new information about animal models may be of value immediately to those responsible for therapy of patients with these diseases. The condensed information below is derived from notes taken by the authors; it is not an official proceedings of the workshop. Detailed inquiries regarding topics discussed here should be directed to the appropriate investigators.

An analysis was made of the immunoglobulin surface markers of the cells of patients with chronic lymphatic leukemia (CLL) in view of previous evidence of their monoclonal B-cell character. The simultaneous presence of IgM and IgD on the surface of the majority of lymphocytes was demonstrated by both immunofluorescence and hemagglutination inhibition in most cases. However, cases were observed with surface IgM without IgD as well as cases with IgD without IgM. IgG and IgA were absent. Studies of the light chains indicated only a single class in a given case. In addition to bound light chains, free light chains were readily demonstrated in most cases through the use of antisera specific for "free chain" determinants. It thus appeared that there are three major types of surface Ig on CLL lymphocytes, IgM, IgD, and free light chains.

Increasing evidence has been obtained of the special value of Ia-like B-cell alloantisera for demonstrating disease associations with histocompatibility antigens. This was particularly evident for the study of the immunogenetics of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), two conditions frequently considered related. The profiles of antigens recognized by the alloantisera in patients from each disease group was distinctive. Two types of alloantisera were obtained that illustrated the divergence between the twod iseases. One type showed a higher than normal incidence in RA but lower than normal in SLE; the other showed a higher incidence in SLE. While these sera were not totally defined, evidence was obtained that the SLE-reactive alloantiserum related to two alleles of the major histocompatibility complex DRw2 and DRw3, while the RA-reactive alloantiserum related to a common specificity shared by cells positive for either DRw4, DRw7, or DRw10. The data indicate that immunogenetic factors are relevant to the development of both RA and SLE, but that these are distinct for each disease.

A murine monoclonal antibody, designated EL-1, was raised by immunization with a human malignant T cell line. It reacted specifically with a membrane antigen expressed on T and B lymphoblastoid cell lines, a subpopulation of normal thymocytes and bone marrow lymphocytes, lymphocytes from a subset of patients with non-B, non-T cell acute lymphoblastic leukemia or T cell acute lymphoblastic leukemia and epithelial stern cells. The latter reactivity was especially striking in the skin, where only basal epidermal keratinocytes and epidermal appendages, including eccrine sweat glands, sebaceous glands and hair follicles, stained positively. A human epidermoid carcinoma cell line was also stained by EL-1. Suprabasilar keratinocytes and acellular keratin did not stain. However, in vitro proliferating fetal lung fibroblasts stained positively. Membrane immunoprecipitation analysis demonstrated that the antigen recognized by antibody EL-1 is a single protein of molecular weight 105 kilodaltoris which did not change with exhaustive chemical reduction. Metabolic iadiolabeling studies demonstrated that this protein is synthesized by the cell and not merely taken up from the culture medium. This antibody can be useful in studying keratinocyte differentiation in epidermal malignancies and normal skin.

Ribonucleoprotein particles containing Sm antigen were separated from particles containing both Sm and RNP antigens by ion-exchange chromatography to study the recognition of these antigens by autoimmune sera. By using the separated antigens, anti-Sm and/or anti-RNP antibodies were detected in approximately 60% of sera from systemic lupus erythematosus patients by both enzyme-linked immunosorbent assay and immunoprecipitation of radiolabeled antigens followed by analysis on sodium dodecyl sulfate-polyacrylamide gels. These antibodies were detected in 30% of the same sera using the standard passive hemagglutination technique. Competition experiments demonstrated that all of the sera tested that contained anti-Sm antibodies also had anti-RNP-like reactivity. This latter reactivity usually represented 80% or more of the total Sm and RNP binding activity in lupus sera. The binding to RNP-like determinants by several of the sera was uniquely resistant to treatment of the antigen with snake venom exonuclease. These studies indicate that humoral immunity against Sm and RNP antigens in systemic lupus erythematosus is directed primarily against a single type of ribonucleoprotein particle in which the two antigens are physically associated. The specific binding to a single type of ribonucleoprotein particle suggests that this particle may be especially immunogenic and that it might play an important role in induction of the humoral immune response to Sm and RNP.

Antilymphocyte antibodies in serum from patients with systemic lupus erythematosus (SLE), as detected by microcytotoxicity and indirect immunofluorescence, were predominantly cold reactive and of the IgM class. These IgM antibodies were most active at 4 degrees C. IgG antibodies were infrequent, and were only minimally lymphocytotoxic. Most sera were cytotoxic for autologous lymphocytes and were equally reactive with normal and SLE lymphocytes, as well as with B- and T-cell preparations. Separate T- and B-cell specificities, which appeared not to be related to HL-A determinants, were identified by differential absorption experiments. The functional significance of these antilymphocyte antibodies is discussed.

Murine monoclonal antibodies (MoAb) were prepared by immunizing mice with human monoclonal rheumatoid factors from patients with mixed cryoglobulinaemia. Indirect solid phase radioimmunoassay and haemagglutination inhibition were used to screen the MoAb. Reactivity patterns of MoAb were determined using (a) polyclonal and monoclonal IgM proteins, (b) monoclonal IgM proteins from patients with neuropathy, (c) monoclonal and polyclonal IgM antigammaglobulins, and (d) various isolated VkIIIb-positive immunoglobulins. Several patterns were obtained: MoAb reacting with private idiotypic determinants, with VkIIIb-related determinants, and with cross-reactive idiotypes (CRI). Two MoAb of the second type reacted with VkIIIb-positive immunoglobulins and light chains regardless of their antigenic activity. Another MoAb reacted with VkIII light chains only when in association with mu heavy chains. MoAb of the third type defined distinct CRI systems. One of these was restricted to antigammaglobulins and another also involved neuropathy-associated monoclonal IgM proteins. All MoAb clearly reacted with a minor population of normal polyclonal IgM, except for the MoAb detecting private idiotypic determinants. Studies using inhibition of binding by enzyme-linked immunosorbent assay showed that polyclonal IgM antigammaglobulins may carry a CRI determinant detected by one of the MoAb. This CRI system, defined by the MoAb Glo 86.3, was similar to but not identical with the previously described Wa CRI, which is widely prevalent among IgM antigammaglobulins of rheumatoid arthritis.

Highly purified surface IgM-bearing B cells were isolated from T cell- and monocyte-depleted mononuclear cells by their ability to rosette with purified anti-IgM antibodies covalently linked to bovine red blood cells. Surface IgM-bearing B cells from both peripheral blood and tonsil were found to be excellent stimulator cells in unidirectional MLC reactions. In the majority of cases, the surface IgM-bearing B cells had a higher stimulatory capacity in the allogeneic MLC than non-T cells lacking surface IgM. Monocytes were also demonstrated to be good stimulators in the allogeneic MLC when a low number of cells was used. At high numbers, inhibition of tritiated thymidine uptake was observed, which appeared to be partly due to the release of prostaglandins and cold thymidine. The population of non-T cells lacking surface IgM also showed stimulation that was especially strong in certain individuals. Because of the heterogeneity of this fraction, the primary cell involved could not be determined. It is of interest that this population was a more potent stimulator in the autologous MLC than was the surface IgM-bearing B cell fraction, although the latter was clearly active. Results from these studies also indicated that monocytes were relatively poor stimulators in the autologous MLC. Isolated Fab′ fragments of a rabbit anti-human Ia antiserum caused significant inhibition of both the allogeneic and autologous MLC.

The Ia antigens, usually expressed primarily on B lymphocytes, are found on a small percentage of normal peripheral blood T cells (average 2.6% by fluorescence and 10.8% by rosette assay). Elevated levels up to 40% by both assays were observed in a high proportion of patients with rheumatoid arthritis. Increases also were found in patients with systemic lupus erythematosus and various types of infections. The increases were evident with a specific heteroantiserum, a hybridoma reagent, and DR specific alloantisera. Normal levels were present in multiple sclerosis and an assortment of metabolic and other disorders. A rise in similarly positive T cells occurred in normal individuals after immunization with tetanus toxoid or PPD. The cells primarily involved in all of these instances were small lymphocytes, which stained relatively weakly with the fluorescent reagents and were readily distinguishable from T-cell blasts. They were found to be enriched in isolated T gamma fractions but were also found in other T cells. The accumulated evidence indicated that these cells represent an expansion of one or more subsets of T cells found in normal individuals, and that their level in the peripheral blood may serve as an index of immunological stimulation.

Human malignant melanoma cell lines were found to bear Ia-like cell surface determinants demonstrable by hetero- or alloantisera and by direct identification of the characteristic bimolecular glycoprotein complex. Immunoprecipitation confirmed the presence of Ia determinants on the bimolecular complex. The quantity of Ia molecules determined by these methods and by absorption experiments was relatively constant for each cell line. Among different lines, however, the amount of Ia antigens ranged from a level equal to that expressed by B-cell lines to a small fraction of this amount. This variation in level of Ia contrasted with the more uniform amount of beta2-microglobulin detected on the cell surface. The Ia alloantigen specificity DRw2 was the most frequently encountered specificity. Ia determinants were also found on the surface of an epidermoid carcinoma line, but not on various other cell lines of normal or neoplastic origin.

Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed.

The Ia alloantigens as measures of different alleles of loci in the major histocompatibility complex were determined in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). The Ia specificities of reagents used were defined by their pattern of reaction with lymphoblastoid lines derived from normal donors homozygous for HLA-D determinants. The reagent specificities included those associated with a single Dw type as well as those reacting with a single specificity shared by several Dw types. Patients with RA had a marked elevation in the frequency of alloantigens detected by reagent sera that recognize various determinants shared by cell lines from HLA-Dw4, Dw7, or Dw10 individuals (Ia 4-7-10). The frequency of mixed lymphocyte culture alleles Dw4 and Dw10 was found to be increased; however this elevation did not approach the higher frequency for the serologically determined antigens of the Ia 4-7-10 group. In contrast, patients with SLE had an increased frequency of reactions with the reagent alloantisera defined by reactions with either HLA-Dw2 or Dw3 positive cell lines. The data suggest that immunogenetic factors are relevant to both groups of patients, but that these are entirely distinct for each disease.

Summary1. Sera from patients with active lupus erythematosus fixed complement with a wide variety of nuclei from different organs and species, with calf thymus nucleoprotein, and in two instances with histone. Isolated calf thymus, salmon sperm, human leukocyte and pneumococcal DNA also fixed complement with many of these sera. Similar reactions were not encountered in a limited control series including normal individuals and other pathological states. 2. Most active L.E. sera fixed complement with both nuclei and DNA in roughly parallel titer. However, exceptions were encountered and one serum reacted strongly with nuclei but failed to react with DNA. Cross-absorption experiments with nuclei and DNA suggested the presence of 2 distinct serum factors. 3. The L.E. factor appeared to be related to the factor responsible for complement fixation with nuclei but distinct from that responsible for DNA fixation. 4. The significance of these findings with respect to antibodies against nuclear constituents is disc...

Two major antigenic fragments were obtained from various purified γ-globulin preparations after papain treatment. One, the F component, had a mobility faster than the original γ-globulin and the second, the S component, had a slower mobility. Similar F and S components were also obtained with certain homogeneous myeloma proteins which were closely related to γ-globulin immunologically. Additional minor antigenic components were detected with certain antisera. The technique of immunoelectrophoresis was particularly useful for bringing out the different antigenic constituents obtained after papain treatment. The F and S components as well as a midfraction were isolated by chromatography on DEAE-cellulose. These were immunologically homogeneous and could be utilized to absorb F and S antibodies from various antisera. The relative amount of F and S antibodies varied in different antisera from individual rabbits immunized with whole γ-globulin. Whole γ-globulin was separated by zone electrophoresis into a fast migrating and a more slowly migrating fraction. Each of these gave rise to F and S components after splitting with papain. The F components of the two γ-globulins were similar in mobility, while the S components showed marked mobility differences although antigenically they were very similar. The mobility differences of the parent γ-globulin appeared to be primarily related to the differences in the S component. Certain antisera against pathological γ-globulins were found to give double lines with a wide variety of γ-globulin preparations in agar diffusion. These were shown to be related to the F and S antigenic determinants of γ-glubulin. This relationship was evident by a number of procedures utilizing both Ouchterlony plate techniques and immunoelectrophoresis. The question of whether these findings indicate heterogeneity of γ-globulin in relation to the F and S antigenic components, or whether different antigenic groups on one molecule can give rise to separate lines in certain instances, is discussed.

No explanation has been available concerning the γ globulin defect in a unique family with one member whose serum was devoid of all the usual Gm genetic antigens. In the present study, it was found that this serum lacks ordinary γG1 and γG3 proteins and contains instead hybrid molecules of the type γG3-γG1. These were demonstrated most clearly by the precipitation of γG1 proteins with antisera specific for γG3 antigens. The analogy to the delta-beta chain hybrids, established for Lepore-type hemoglobins, was striking. An unequal homologous crossover involving mispairing of heavy chain cistrons would readily explain the deletion of genetic markers.

In the sera of a number of patients with rheumatoid arthritis an unusual, high molecular weight protein component could be detected by direct ultracentrifuge analysis of whole serum. This material sedimented more rapidly than the normal 19 S component and reached concentrations up to 340 mg. per cent. Similar components were not observed in a limited control series. The high molecular weight material was present in the γ-globulin fraction of serum and joint fluid. It had an S20,w of approximately 22 S and could be dissociated into 2 fractions, one of which had a sedimentation coefficient of approximately 19 S. Evidence for a direct relationship between the 22 S component and the γ-globulin precipitation test was obtained. The latter reaction was found to occur in the presence of altered, aggregated γ globulin. Absorption of serum with altered γ globulin removed the 22 S component. There also appeared to be a connection with the sheep cell agglutination reaction and the latex fixation test. The 22 S fraction was always observed in sera giving the most positive tests. Procedures of density gradient and repeated preparative ultracentrifugation demonstrated that each of these reactions was caused by a high molecular weight fraction. The relationship between the unusual protein complex and various 19 S γ-globulins and 19 S antibodies is discussed.

19S anti-γ-globulins were isolated in a high state of purity from the sera of two patients with rheumatoid arthritis. Following reduction with ethyl mercaptan and alkylation by iodoacetamide, fragments were produced which retained the capacity to combine with 7S γ-globulin. The fragments from one of the 19S anti-γ-globulins agglutinated red cells coated with incomplete anti-Rh antibodies. This activity was shown by density gradient ultracentrifugation to be associated with low molecular weight fractions. The agglutination of the coated red cells by the fragments was strongly inhibited by normal and myeloma 7S γ-globulins and showed a greater specificity than the parent 19S material. Analytical ultracentrifuge experiments demonstrated that the fragments from either of the 19S anti-γ-globulins formed complexes with 7S γ-globulin. Reassociation of the dissociated fragments through reformation of disulfide bonds resulted in the formation of fast sedimenting molecules having properties similar to those of the untreated 19S material in respect to precipitation with aggregated γ-globulin and agglutination of coated red cells.