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The method for purification of monocytes using adherence to gelatin coated glass surfaces described by Chien et al. was optimized by drastic shortening the incubation time and modifying the culture media. After one adherence step we obtained monocytes with a purity of 73-78% and a recovery of 53%. Thiol-protein-disulfide-oxidoreductase (TPO), a new enzyme marker of monocytes, was found to be also valid for monocytes obtained by adherence methods. Comparing the number of TPO-containing monocytes with other markers (alpha-naphthylacetate esterase, peroxidase, phagocytosis of latex particles, acridine orange fluorescence, antigens detected by the monoclonal BL-M/G antibody) almost identical values were found.

Recombinational analysis has shown that the rat MHC,RT1 is divided into two regions:RT1.A, which codes for class I (transplantation) antigens, andRT1.B, which controls the humoral immune response and proliferative response to allogeneic cells as well as the expression of class II (Ia) antigens. Serological and sequence studies suggest that there might be more than one antigen-coding locus within theRT1.A region. Results obtained by sequential immunoprecipitation reveal that both regions code for at least two gene products. By implication, theRT1 complex must therefore harbor at least four loci;RT1.A andD coding for class I glycoproteins (45,000 daltons); andRT1.B andE coding for class II (Ia) glycoproteins (35,000 and 28,000 daltons).

We investigated the possibility using Dextran M 70 instead of the common Ficoll 400, both in combination with Visotrast 370, for separation of lymphocytes from human peripheral blood by density gradient centrifugation. No significant differences were found with regard to parameters such as yield and vitality and numbers of B- (surface immunoglobulins) and T-lymphocytes (sheep red blood cells-rosette assay, and the monoclonal antibody BL-T2). From the data available as yet we can conclude--at least for the markers studied--that it is possible to use Dextran M 70 instead of Ficoll 400 for the separation of mononuclear cells of human peripheral blood.

The antigenic determinants expressed on RBC and lymphocytes and coded for by the MHC, RT1,of the MNR (RT1 ( m )) rat strain were compared to those of the BN.DA(RT1 ( a )), ALB (RT1 ( b )), and AUG (RT1 ( c )) strains by direct cytotoxicity and absorption analysis with RT1 typing sera, sera produced against MNR cells, and sera produced in MNR responders against cells carrying thea, b, andc haplotype determinants. The results indicate that MNR shares major class I (A) antigens with DA, and major class II (B) determinants with AUG, but that MNR differs from DA and AUG with respect to both classes of determinant. It appears, therefore, that the MNR haplotype does not represent a simple composite of the two other haplotypes,RT1 ( a ) andRT1 ( c ), as reported earlier.

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.

The experimental data show that absorption of ATG with liver-kidney homogenate and CLL and LCL cells stepwise removed the hemopoietic toxicity, whereas the specific activity against T lymphocytes remained. Although the mode of action of absorbed ATG could not be tested in the first clinical case, the successful experiments in rodents together with the fact that the incubation treatment was tolerated by the patient may provide a new way of preventing fatal GVH reactions in man.

Ia antigens isolated from spleen cells of rats and mice are composed of two polypeptide chains, designated alpha and beta. Mouse alloantisera specific for the I-Ak and I-Ek subregions react with two distinct groups of rat Ia antigens, designated A-like and E-like, respectively. Two-dimensional gel electrophoresis and peptide map analysis demonstrate that the A-like antigens of rat are distinct from the E-like antigens. Both rat Ia antigens react with alloantiserum produced in rats congenic for the major histocompatibility complex (MHC). These results demonstrate for the first time that two distinct Ia antigens are present in the rat. Accordingly, the rat, like the mouse, may have Ia antigens encoded by at least two subregions of the rat MHC. The existence of multiple Ia gene products in rats is revealed by chemical techniques even in the absence of formal genetic evidence of more than one I subregion in the rat.

Anti-I-region associated (Ia) antigen sera specificially supress the stimulation reaction in unidirectional mouse MLR. No effect on the response was observed with these antisera. In addition, anti-H-2 sera did not affect stimulation, however, in some cases diminished the response. The data indicate that Ia antigens and LAD might be identical. Stimulation in mixed lymphocyte culture reaction (MLR) or graft vs host reaction has been shown to be governed by genes located in the I-region (immune response region) of the H-2 complex in mice (Bach et al. 1972 and Klein and Park 1973) and it appears that the H-2K and H-2D regions play only a minor role in that reaction. Recently, antigens have been described (Hauptfeld et. al. 1973, Götze et al. 1973, David et al. 1973) which are controlled by the I-region, so-called I-region associated equals Ia antigens. If these antigens are related to determinants important for the MLR, then one might predict inhibition of the MLR. Abbasi et al (1974) reported that poly-specific anti-H-2 alloantisera inhibited the H-2 complex controlled stimulation in MLR. Using antisera produced in I-region congenic strains, we have been able to show complete and specific inhibition of the stimulation in MLR, without affection of the response, thus indicating that Ia antigens and lymphocyte activating determinants might be identical.

The data confirm an earlier finding by Klein et al. that the I region harbors a strong histocompatibility locus, which by exclusion might be assigned to the IA subregion. In addition, it appears that intercalated between the IA subregion and S region there is a minor H locus since (DBA/2 X 4R)F1 rejects skin of 2R in a chronic fashion. In another combination, HTT versus 7R, which differs for the IC, S, and G regions but may also differ for genes between the D and TL locus, a similar chronic rejection pattern was observed indicating minor H gene(s) within or closely linked to the H-2 complex. The data reported here are in concordance with very recently published results by Klein et al..

In this study, data are presented on serologic H-2 typing of 320 wild mice collected in several parts of Europe and Egypt. The sample was typed for 39 class I (K, D) and 16 class II (Ia) antigens. The phenotype frequencies of class I and class II antigens showed high variability in their distribution among different areas, ranging from absence or presence in low frequency in one area to presence in about one-half of the mice in another area. The average phenotypic frequency of class I private antigens was 6.3%; at least 60% of class I alleles (blanks) could not be identified with the available reagents. The data suggest that there might be more than 100 alleles for each class I locus, H-2K and H-2D. The average phenotypic frequencies of Ia-1 private antigens was 10.8%. About 75% of Ia-1 alleles (blanks) could not be identified. The number of Ia-1 alleles was estimated to be in the range of 20 to 50.

Homologues of Ia antigens of the mouse are identified in three rat strains by partial N-terminal amino acid sequence analysis. Ia antigens of the rat were isolated by indirect immune precipitation using specific rat alloantisera. Rat Ia antigens consist of two components, alpha and beta, which have respective mol. wts. of approximately 35 000 and 28 000. Partial N-terminal sequence analysis of each of the alpha components of the H-1a, H-11 and H-1n haplotypes yields a single, apparently homogeneous sequence which is identical among the three haplotypes and is strikingly homologous to the alpha polypeptides of the I-E subregion of mouse and to the human polypeptide, p34. Partial N-terminal sequence analysis of the beta components shows that a mixture of polypeptides is present for each haplotype. There are differences in the beta sequences among the three haplotypes and potential homologies between the rat beta sequences and the sequences of the beta polypeptides of the I-A and I-E subregions of mouse. These obserations imply that the rat has at least two distinct groups of Ia molecules. The organization of genes encoding the Ia polypeptides in the major histocompatibility complex of the rat is discussed.

By means of monoclonal antibodies against surface antigens of mononuclear cells we examined alterations of T-cell-subpopulations of peripheral blood and liver tissue in the course of acute HBsAg-negative hepatitis. Used methods for determination were the immunofluorescence for blood lymphocytes, respectively the peroxidase-anti-peroxidase-(PAP)-technique for round-cell-infiltrates of the liver based on paraffin fixed sections. The focus of attention was the relation of T4-(helper/inducer) to T8-(suppressor/cytotoxic) cells, the so-called immuno-regulatory quotient. With advancing improvement in the course of the disease we observed in peripheral blood a decrease of T4+/T8+ ratio from very high (3.6) to normal (1.9) values. By examination of the tissue round-cell-infiltrates we found a contrary behaviour to the peripheral blood with a low T4+/T8+ ratio in the liver, characteristic to more acute phases and due to an T8+-cell interchange between periphery and liver. This may be common for all virus diseases and without specificity for defined types of acute hepatitis. Beside this exists the possibility T4+/T8+ ratio has any prognostic value.

Determinants controlled by the I region of the murine H-2 complex provoked the generation of cytotoxic T lymphocytes (CTL) in both a secondary and primary mixed lymphocyte culture. The stimulating determinants appeared to be controlled by loci within the I-A subregion. The target antigens of the CTL generated were present on both lipopolysaccharide- and concanavalin-induced blast lymphocytes, but were barely detectable on phytohemagglutinin-induced blast cells. The stimulating capacity for CTL induction of a complete H-2 complex incompatibility by far exceeded the sum of H-2D/K-region and I-region incompatibility, respectively.

Different methods for determination of T-lymphocytes in human peripheral blood were compared: rosetting with sheep erythrocytes (SRBC), AET-treated SRBC, immunofluorescence using a monoclonal antibody against T cells (BL-T2), complement dependent cytolysis with polyclonal antisera against thymocytes, cytochemical demonstration of unspecific acid alpha-naphthyl-acetate esterase (ANAE), and electrophoretic mobility using a cell electrophoresis system (PARMOQUANT 2). Depending on the method, mean values between 70 and 79% T cells among separated mononuclear cells (MNC) were found. All paired observations were subjected to statistical analysis using rank correlation and U-test. From this analysis it is concluded that rosetting with SRBC, immunofluorescence using the monoclonal T-cell antibody and cytochemical reactivity for ANAE are favored methods for determining the T cell content of human MNC. However, the monoclonal antibody BL-T2 and the ANAE are not generally applicable because both markers were also found on malignant B-lymphocytes (B-CLL).

Antisera against chicken bursal and thymic lymphocytes were raised in rabbits. They were made specific for the target T or B cells by appropriat absorption and then used for immunohistochemical identification of T and B lymphocytes in tissue sections of chicken spleen, applying the unlabeled antibody enzyme method. Lymphocyte reacting with anti-chicken-thymus globulin predominated in the periarteriolar lymphatic sheaths and in the red-pulp cords. A few of these lymphocytes were localized between the periellipsoid lymphatic tissue and in germinal centers. The periellipsoid lymphocytes around the Schweigger-Seidel sheaths consisted of lymphocytes positive with anti-chicken-bursa-cell globulin. The majority of lymphocytes in the germinal centers were labeled also by anti-chicken bursa-cell globulin. Age-dependent changes in the anatomical localization of T and B lymphocytes are described between late embryonic development and an age of 2 years. The different conditions of avian and mammalian T and B cell distribution in the spleen were considered.

Difficulties in the production of specific antisera against T-lymphocytes could be overcome by a stepwise absorption and purification procedure of anti-human thymocyte serum. Specific anti-T lymphocyte globulin (sATG) reacted with thymocytes, thymus-derived lymphocytes and a lymphoblastoid cell line of T-cell type whereas no activity was found against lymphoblastoid cell lines of B-cell type. Five chronic and three acute lymphatic leukemias were characterized using sATG in the cytotoxic test, electron microscopy, complement fixation test and quantitative immunoautoradiography, and compared with lymphocyte populations of normal individuals. Three chronic lymphatic leukemias with low numbers of spontaneous rosettes and high percentages of membrane-Ig-positive lymphocytes showed only few T-cell-antigen-positive lymphocytes and were therefore classified as B-cell leukemias. The cells of two chronic lymphatic leukemias with high numbers of spontaneous rosettes carried T-cell-antigen. The T-cell-antigen concentration, however, was lower than that of normal peripheral blood T-lymphocytes. The T-cell nature of two acute lymphatic leukemias with high numbers of spontaneous rosettes was confirmed by a positive reaction of the cells with sATG. In one case of acute lymphatic leukemia most leukemic cells carried T-cell-antigen although these cells did not form spontaneous rosettes. In the first two cases the T-cell-antigen concentration on the cell surface exceeded that of normal blood-T-lymphocytes, in the latter case it was slightly below that. The advantages of the characterization of leukemias with sATG in comparison with the spontaneous rosette formation and the relevance for prognosis are discussed.

Monocytes were enumerated by three different methods, cytochemical staining for alpha-naphthyl-acetate esterase-activity, immunofluorescence test using the monoclonal antibody BL-M/G and immunochemical staining for the enzyme thiol-proteindisulfide-oxidoreductase (TPO, EC 1.8.4.2) using a polyclonal rabbit anti-rat TPO immunoglobulin. For the comparison of these methods mononuclear cells from peripheral blood of healthy volunteers, patients with different diseases and adherent cell populations were tested. Statistical analysis showed no differences between the markers within these groups (H-test) and a significant correlation between the numbers of monocytes. TPO was found to be also valid for enumeration of monocytes obtained by adherence methods.

Cellular and humoral immunological parameters were examined in mononuclear cells from peripheral blood of patients with alcoholic and nonalcoholic steatosis hepatis. The ratio of T4 and T8 positive lymphocytes and the number of monocytes of these patients were in the normal range. The percentage of NK-cells, B-lymphocytes and DR-antigen-positive cells was increased.

Antibodies produced in B10.D2 mice against soluble lymphocyte membrane antigens of B10.A (H-2(a)) mice reacted only with lymphocytes of the strains carrying the Ir(k) region, i.e., B10.A(2R), B10.K, B10.BR, B10.HTT, AQR, A.TE, C3H, and CBA; they did not react with cells of strains carrying different Ir regions, i.e., B10.A(4R), B10, B10.M, A.SW, DBA/1. It is therefore concluded that the antigen detected with these antibodies is apparently controlled by the Ir region of the H-2 complex. The antigen is present on some T lymphocytes and absent on B lymphocytes. Its presence or absence seems to correlate with MLC and GVH reactivity.