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When you receive an invitation to contribute a chapter or section of a book, allow yourself 10 minutes to feel flattered. Then, read the letter again and try to figure out exactly what the editor or senior author wants you to do. Most invitation letters are rather vague because, for understandable reasons, the inviting editor does not want to give away too much information about the project before gaining a prospective author’s agreement to participate. Before you answer the invitation, ask yourself a few specific questions and try to answer them honestly.

The antigen specificity of hybridoma antibodies with activity against H-2 antigens was analyzed by testing lymphocytes of inbred lines and their recombinants, as well as B10.W lines and wild mice in relation to the reactivity of alloantisera. Within the limited test panel of 11 inbred strains and their recombinant derivatives, the hybridoma antibodies reacted concordantly to alloantisera with public (H-2.5, H-2.11, and Ia. 13) and private (H-2.23) antigens. However, when tested on cells of B10.W lines or wild mice discordant reactions were observed, indicating that the antigenic determinants recognized by the hybridoma antibodies are included in the antigens defined by alloantisera within the inbred test panel. In wild mouse populations, these determinants do also occur independently of those which comprise the alloantisera-defined specificities. The results point to an even higher complexity of MHC antigens than envisaged so far, and it appears that a certain allele might be defined only by a certain set of antigenic determinants. Because of the discrepant reactions of alloantisera and hybridoma antibodies, it has been proposed that antigenic determinants detected by hybridoma antibodies receive a numerical designation according to their description but independent of antigens defined by alloantisera, even if they match the reactivity pattern of an alloantiserum-defined antigen specificity within the inbred panel.

Thus far, the rat major histocompatibility complex (MHC), AgB or RTI, have been divided by recombinational analysis into two regions, AgB.A (RTI.A) coding for transplantation antigens (class I gene products) and AgB.B (RTI.B) controlling the humoral immune response (Ir genes) and the proliferative response to allogeneic cells (MLR, class II genes). In this study, an attempt was made by biochemical analysis to determine whether there exists more than one AgB.A (class I) gene product, as has been found in the mouse (H-2K and H-2D) and in the human (HLA.A and HLA.B antigens). NP40-solubilized 3H-fucose-labeled rat MHC membrane products were immunoprecipitated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Firstly, with a congenic anti-erythrocyte serum, only membrane structures with an approximate m.w. of 45,000 (AgB.A) were precipitated. A congenic anti-lymphocyte serum absorbed with erythrocytes precipitated antigens with an apparent m.w. of 33,000 and 28,000 (AgB.B) whereas the unabsorbed serum precipitated in addition a 45,000-dalton component (AgB.A). Secondly, sequential treatment of a DA (AgB4, RTIa) extract with a strongly cross-reactive serum followed by an (Lewis × BN.B2)F1 anti-AgB4-specific serum demonstrated that the AgB4 haplotype codes for at least two AgB.A and at least two AgB.B gene products.

SUMMARYTwo hundred and two house mice (Mus musculusL.) from 29 populations in Europe and North Africa were typed for 16 H-2K and 17 H-2D antigens, each antigen defining a different allele. Among the 13 best characterized populations, 1 to 4 common and 3 to 20 rare antigens were observed. However, an average of 37% of the H-2K and 39% of the H-2D antigens remain to be identified. Ninety-four percent of the 50 mice tested were heterozygous for H-2K antigens and 89% for H-2D antigens. In 4 of the 8 populations tested, the most common H-2K and H-2D antigens occurred in the same individual more often than if randomly associated. Associations between common H-2K and H-2D antigens and excess heterozygosities may be the consequence of the small size and instability of populations composed primarily of related individuals. Estimates of the genetic distances between populations revealed that Danish, Egyptian, and several of the Orkney Island populations were related. These were the only populations in which metacentric chromosomes were not found. In contrast, populations which were antigenically different were also karyotypically different, regardless of taxonomic status of allozymic similarity.

Bisherige Schwierigkeiten in der Herstellung spezifischer Antiseren gegen T-Lymphocyten lassen sich durch eine stufenweise Absorptions-und Reinigungsprozedur von Anti-Human-Thymocytenserum uberwinden. Spezifisches Anti-Human-Thymocyten-Globulin (sATG) reagierte mit Thymocyten, thymus-abhangigen Lymphocyten und einer lymphoblastoiden Zell-Linie vom T-Zell-Typ, wahrend keine Aktivitat gegen lymphoblastoide Zell-Linien vom B-Zell-Type nachzuweisen war. Mit Hilfe von sATG, eingesetzt im Cytotoxizitatstest, in der Elektronenmikroskopie, dem Komplementfixationstest und der quantitativen Immunoautoradiographie, wurden funf chronische und drei akute lymphatische Leukamien charakterisiert und mit Lymphocytenpopulationen von Normalpersonen verglichen. Drei chronische lymphatische Leukamien mit niedrigen Spontanrosettenzahlen und hohen Prozentzahlen Membran-Ig-positiver Lymphocyten zeigten nur wenige T-Zell-Antigen-positive Lymphocyten und wurden daher als B-Zell-Leukamien klassifiziert. Die Zellen zweier chronischer lymphatischer Leukamien mit hohen Spontanrosettenzahlen trugen das T-Zell-Antigen, die Antigen-Konzentration jedoch lag niedriger als die von normalen peripheren Blut-T-Lymphocyten. Die T-Zell-Natur zweier akuter lymphatischer Leukamien mit hohen Spontanrosettenzahlen wurde durch eine positive Reaktion der Zellen mit sATG bestatigt. In einem Fall von akuter lymphatischer Leukamie wurden trotz fehlender Spontanrosettenbildung uberwiegend T-Zell-Antigen-positive Zellen gefunden. In den beiden ersten Fallen lag die T-Zell-Antigen-Konzentration hoher als auf normalen Blut-T-Lymphocyten, im letzteren Fall geringfugig darunter. Die Vorteile der Charakterisierung von Leukamien mit sATG im Vergleich mit der Spontanrosettenbildung und die Bedeutung fur die Prognostik werden diskutiert.

An autosomal dominant immune response gene could be demonstrated in congenic resistant strains of mice which is linked to the H-2 locus and controls the antibody response to soluble calf collagen. High responsiveness was associated with the H-2 alleles, b and f, low responsiveness with the H-2 alleles, d, k, m and r. Studies with calf procollagen, which contains an additional carrier moiety, indicated that these genetic differences might be expressed at the level of T cells.

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.

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. Lymphocytes 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.

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.

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).

Publisher Summary This chapter describes T1–T2 cell interactions, which take place during in vitro cytotoxic allograft responses. T1–T2 cell interactions may represent a reflection of the antigenic dichotomy required for the induction of a mixed lymphocyte culture-induced cytolysis (MLC–CML). In regard to the stimulating H-2 alloantigens, there exist functional differences between the phenotypical products, coded by either the peripheral regions (K, D) or the central region (I) of the H-2 complex. The central (I) region is primarily involved in lymphocyte activation, whereas the peripheral regions (K, D) code for serologically detectable (SD) transplantation antigens, which function as targets for the killer cells generated. T1 cells will not differentiate in CTL when triggered to cell proliferation by allogeneic, I region controlled surface determinants (LAD) because two T cell subsets (T1 and T2) have been defined to be involved in the genesis of cytotoxic T lymphocytes (CTL), out of which T2 cells represent the prekiller cells. T1 cells are stimulated by allogeneic cell surface molecules, differing from the target antigen for CTL generated.

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..

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.

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.

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.

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.

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.

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.

Lymphocyte alloantigens controlled by genes which are closely linked or identical to immune response genes (Ir genes) are now recognized to play a major role in the immune response regulation and might be suspected to take part in the phenomenon of disease susceptibility (1, 2). Characterization of those antigens in mice is obtained by using antibodies raised in appropriate congenic recombinant lines. Such an approach is, however, not feasible in man; antibodies are obtained by chance immunization during pregnancy or multiple blood transfusions which results in reagents with multiple specificities and low activity. The use of specific and stronger xenogeneic antibodies may lead to a better characterization of those antigens and facilitate our understanding of their role in the regulation of immune responsiveness and its pathological deviations. In addition, those reagents may prove to be useful in the diagnosis and classification of hematological malignancies.

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.

When you receive an invitation to contribute a chapter or section of a book, allow yourself 10 minutes to feel flattered. Then, read the letter again and try to figure out exactly what the editor or senior author wants you to do. Most invitation letters are rather vague because, for understandable reasons, the inviting editor does not want to give away too much information about the project before gaining a prospective author’s agreement to participate. Before you answer the invitation, ask yourself a few specific questions and try to answer them honestly.

The knowledge about the major histocompatibility system (MHS) in man has evolved in very close interrelationship with the pioneer work in the mouse model with an intensive exchange of methodology and “ideology” concerning the analysis of serologic data. As will be documented in this book there is a remarkable analogy between the histocompatibility systems of many mammalian species, most notably of mouse and man. This is expressed in a phrase coined by Jean Dausset: “The mouse has never lied” relating to the fact that most of the systems found in the mouse H-2 complex (the MHS of the mouse) have also been described for man and other species. These analogies are in marked contrast to the widely different experimental approaches used for histocompatibility research in both species. Through the pioneer work of Gorer and Snell (Gorer 1938, 1961; Snell 1948, 1953) it has become possible to use inbred strains of mice, which allows the detailed genetic characterization of a relatively restricted number of H-2 haplotypes (i.e., chromosomes carrying the H-2 region) using a limited number of recombinants. In man one is faced with an outbred population containing an almost unlimited number of “different haplotypes representing a very large number of recombinations. Therefore the laws of population genetics have to be utilized in the analysis of serologic data in an outbred species. As history has shown, both types of basic approaches have provided contributions that were essential for reaching today’s state of knowledge about the major histocompatibility complex in both species.

After the discovery of several surface markers on human lymphocyte populations many attempts have been made to classify leukemias and lymphomas on the basis of cell origin or cell characteristics. Although the biological implications of the various markers on leukemic cells are unknown, an exact characterization of these markers may lead to an early diagnosis of preleukemic stages or relapses and provide guidelines for more specific therapeutic regimens of these disorders. The panel of common surface markers was increased by recent reports on specific xenogeneic antisera identifying membrane antigens on different types of leukemia cells. The identification of the T-cell antigen by specific anti-T-cell sera on leukemias that formed spontaneous rosettes provided convincing evidence that these cells derived from the thymic pathway (3, 13, 16, 12). An antigen not present on normal mature lymphocytes was recently detected on cells of a subgroup of ALL cases by antisera which were produced against so-called O-ALL lymphoblasts lacking all the known markers (7). On the other hand, raising of diagnostic antisera against human lymphatic or leukemic tissues in other species is complicated by the fact that these sera are generally directed against xeno-specific determinants including many cells other than those used for immunization (25). Extensive absorption and purification procedures must therefore be performed to guarantee a definite specificity (22). In this report we describe the reaction pattern of an absorbed anti-T-cell-globulin (ATCG) and an anti-O-ALL-globulin (A-O-ALL-G) and their application to the classification of leukemias in several test systems.

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.

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.

Several laboratories have recently reported studies of new alloantigenic specificities preferentially expressed on lymphocytes and apparently controlled by genes in the Ir region [hereafter designated the I region (Klein et al. 1974)] of the H-2 gene complex (Hauptfeld et al. 1973, David et al. 1973, Sachs and Cone 1973, G~Jtze et al. 1973, Hammerling et al. 1973). These antigens have been designated by various laboratories as Ir-l.1 (Hauptfeld et al. 1973), Lna (David et al. 1973b) and "~" (Sachs and Cone 1973). To avoid confusion in the literature and to establish a standard nomenclature for these new antigens, representatives of the laboratories concerned met during the H-2 workshop in Bar Harbor, Maine, October 8-10, 1973, and agreed upon the following notation: 1) The system of antigens and the controlling genetic element(s) will be provisionally designated Ia (I-region-associated antigens). This symbol was chosen as a general, descriptive term having no specific connotations with regard to possible relationships of these antigens with It-1 or Lad genes or the tissue or cellular distribution of these antigens. When further information on these questions becomes available, the notation can be modified as necessary. 2) If it is found that these antigens are controlled by more than one genetic locus in the I region, the separate loci will be designated by numbers, Ia-1, Ia-2, Ia-3, etc. 3) If it is found that there are multipe Ia loci, some of which control antigens specific for B lymphocytes (bursa-equivalent lymphocytes) and some of which control antigens specific for T lymphocytes (thymusderived lymphocytes), those loci may be distinguished by the symbols Iab and Iat respectively. Multiple loci of each type would be denoted Iab-1, Iab-2, etc. and Iat-1, Iat-2, etc. 4) Alleles at the Ia loci should be designated by a superscript which