Serological identification and separation of canine lymphocyte subpopulations.

In vitro treatment of bone marrow grafts with absorbed rabbit-antidog thymocyte globulin (ATG) prevented graft-versus-host disease in a substantial number of the dogs differing by one DLA haplotype. Absorbed ATG has now been used for serological identification of canine lymphocyte populations. Specific labeling of canine T-lymphocytes by absorbed ATG could be demonstrated by (a) a distribution of ATG-positive cells in suspensions of canine lymphoid organs similar to that of T cells observed in other species and by specific staining of paracortical thymus-dependent lymph node areas in immunohistochemistry, (b) complementary labeling of nylon-wool-separated cells by ATG and antiimmunoglobulin sera, and (c) correlation of ATG surface labeling with responder activities in mixed lymphocyte cultures.

Subtypes of T-cell chronic lymphatic leukemia.

Thirteen cases of T-cell chronic lymphatic leukemia (T-CLL) (including T-cell prolymphocytic leukemia) are presented. Five subtypes were distinguished according to morphologic and functional parameters of the leukemic cells: prolymphocytic; lymphocytic, small; lymphocytic, Sézary-like; lymphocytic, abundant cytoplasm; lymphocytic, abundant cytoplasm and granules. The subtype can be recognized by light and by electron microscopic investigation. Cytochemistry (APh and ANAE) may be helpful to delimit T-CLL from B-CLL, and acid phosphatase to recognize the subtype characterized by abundant cytoplasm and granules. Membrane marker investigations support the diagnosis of T-type CLL. When functional properties of the leukemic cells were tested, cells of one patient (T-PLL) were shown to help in B-lymphocyte differentiation and Ig-secretion, whilst the cells of a second patient (lymphocytic, abundant cytoplasm and granules) were proven to act as effectors in natural killing and antibody-dependent cytotoxicity. The T-helper lymphocyte nature of some of the leukemic cells was supported by demonstration of the Fc mu-receptor in three cases. In one of these patients, monoclonal IgM was detected in the serum. Response to therapy and prognosis were rather poor in this limited number of patients when compared with B-CLL.

Independent expression of the surface markers 5'-nucleotidase and cALLA on leukemic cells.

High levels of the ectozyme 5'-nucleotidase (5'-N) and the common ALL-antigen (cALLA) are coexpressed on leukemic blast cells in common ALL, in the lymphoid blast crisis of CML and also on the lymphoblastoid cell-line Nalm-1. Clinically this coexpression can help to subclassify leukemias and may be of diagnostic and prognostic significance. In an attempt to study the mechanism underlying this simultaneous expression plasmamembrane subfractionation was undertaken on Nalm-1. When membrane-shedding from intact cells is induced by sublytic concentrations of the lysophosphatidyl-choline analogue ET-12-H, membrane subfractions are obtained which contain 30-40% of total cellular 5'-N, which is most of the enzyme carried on the cell surface, in a highly enriched form. Under these conditions only a very low release of intracellular enzymes is observed. On the other hand cALLA is not accumulated in these membrane fractions to any appreciable extent. The predominant part of this antigen is still on the intact cells remaining after the shedding procedure. It is concluded that the simultaneous expression of 5'-N and cALLA on Nalm-1 and leukemic blasts is not regulated by a physical association or a close neighborhood of these antigens on the membrane level.

Antilymphocytic antibodies and marrow transplantation. VI. Absence of immunosuppression in vivo after injection of monoclonal antibodies blocking graft-versus-host reactions and humoral antibody formation in vitro.

The in vivo and in vitro effectiveness of several monoclonal antimouse T and B cell antibodies, of anti-Th-1 and of Iak serum, as well as of ATG were compared. The parameters were prolongation of skin graft survival, prevention of graft-versus-host disease (GVHD), antibody and primary and secondary plaque formation against sheep redblood cells (RBCs), and T cell depletion of lymphoid tissues. In general, in vitro effectiveness of the monoclonal antibodies exceeded their in vivo effectiveness. Skin graft survival was prolonged by ATG, but not by monoclonal anti-T, or anti-T plus anti-B antibody. GVHD was prevented by in vitro incubation of donor bone marrow with monoclonal anti-Th-1, but in vivo treatment of marrow donors was ineffective. Treatment with ATG was successful. Anti Iak antibody blocked plaque formation by spleen cells incubated with sheep RBCs, but had no effect on secondary plaque formation when given in vivo. Neither was there any in vivo effect of anti-Iak or anti-Th-1 on antisheep RBC agglutinin formation. ATG was effective in both of these assays, although its cytotoxic and complement-fixing titer did not exceed that of anti-Th-1 or anti-Iak. Although anti-Th-1 was cleared more rapidly from the serum of mice expressing the corresponding Th-1 alloantigen, than from mice with the noncorresponding alloantigen and although anti-Th-1 was shown to bind to the T cell areas of the lymphoid tissue, it did not--unlike ATG--deplete these areas of T cells. Possible reasons for the difference in effectiveness of in vitro and in vivo application of these monoclonal antibodies are discussed.

Crossreaction of monoclonal anti-T-cell antibodies: implications for classifying B-cell leukemias.

Cell samples from 62 patients with B-cell leukemias (33 CLL, 8 IC, 6 PLL, 4 HCL, 2 ALL and 9 other NHL) were tested with a series of monoclonal antibodies (A50, T101, Lyt2, Leu 1, M203) directed against T cells and shown to crossreact with B-CLL. The results demonstrate a heterogeneity of B-cell leukemias as all typical cases of CLL were reactive whereas most other cases were negative. Using the fluorescence-activated cell sorter, a somewhat stronger reaction was seen in early or benign cases of CLL, compared to advanced cases. All B-cell leukemias tested expressed the Ia antigen.

Immunohistochemical identification of T-lymphocytes in the central nervous system of patients with multiple sclerosis and subacute sclerosing panencephalitis.

T-lymphocytes were identified in frozen brain sections derived from patients with chronic inflammatory disorders of the CNS by using a specific heteroantiserum and the unlabelled antibody enzyme method. Clusters of T-cells were found in post-mortem material of cases with multiple sclerosis (MS) and subacute sclerosing panencephalitis (SSPE). The results suggest that T-lymphocytes are involved in the pathogenesis of both MS and SSPE.

Characterization of normal human mononuclear blood cells during diffusion chamber (DC) culture with respect to proliferation and expression of T-cell antigen.

Normal human mononuclear blood cells were cultured for 13 d in diffusion chambers and the cell harvest on days 3, 6, 9 and 13 was characterized according to morphology, growth kinetics and surface antigen expression. The greatest part of the cell harvest consisted on all days of lymphoid cells. It could be shown that most of the cells carried the T-cell marker, the main growing population thus being T-cells. The mean anti-T-binding capacity per cell increased during the initial lag phase and reached its peak when the cell count was lowest. It dropped again during the following exponential growth phase to preculture values. These changes indicate an initial increase in surface antigen during the transition of cells from resting to a more activated state before the onset of DNA-synthesis, and a subsequent decrease which may indicate functional maturation.

Comparison of currently available monoclonal antibodies with conventional markers for phenotyping of one hundred acute leukemias.

One hundred acute leukemia cell samples (89 ALL, 11 AMML) derived from children's bone marrow at diagnosis were typed for the reaction with 15 monoclonal antibodies (mAbs). Subdivision in ALL-subgroups was performed according to conventional markers. Only some mAbs like VIL-Al which is directed against the cALL-antigen, can substitute polyclonal rabbit antisera. Additional subclassifications became evident by the use of mAbs for T-cell differentiated ALL forms. However, unexpected negative reactions must be taken into account as observed for all T-directed mAbs with individual T-cell ALL samples.

[Bone marrow transplantation in acute leukemias: antibody treatment for suppression of graft-versus-host disease (author's transl)]. / Knochenmarktransplantation bei akuter Leukämie: Prophylaktische Antiserumbehandlung des Knochenmarks zur Unterdrückung einer Transplantat-gegen-Wirt-Reaktion.

Anti-human-thymocyte globulin (AHTZG) was applied to prevent GvHD in clinical bone marrow transplantation. AHTZG produced by absorption with several cell preparations reacted specifically with T-lymphocyte populations and was no longer inhibitory to human CFUc and bone marrow growth in diffusion chambers. Marrow grafts of 14 patients with ALL were incubated in vitro with AHTZG and transferred to the recipients conditioned with antileukemic chemotherapy and total body irradiation of 1000 rad. Ten patients were transplanted after relapse, four patients during remission. The patients tolerated the marrow without side effects and a hemopoietic engraftment was seen in 12 cases. Three patients showed signs of GvHD on the skin, two of them showed later on also manifestations in the liver. In the other cases no GvHD could be detected. Five out of 14 patients are still alive between 144 and 964 days post transplantation in remission.

Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self-major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells.

To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus-specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC-restricted CTL. By applying the limiting-dilution technique, the CTL-precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC-restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC-restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products.

Appearance of B- or T-lymphocyte markers after diffusion chamber culture of acute lymphoblastic leukemia cells.

Blast cells derived from blood and marrow samples of a patient with acute lymphoblastic leukemia (ALL), as well as from the Reh line originally established from an ALL patient, were cultured in diffusion chambers implanted i.p. into preirradiated CBA mice. At different intervals over a period of up to 20 days, surface immunoglobulins, ALL antigen, and T-cell antigen were investigated by using direct immunofluorescence. Rosette formation was tested with sheep and mouse erythrocytes. On day 0, the cells expressed only ALL antigen at the surface, and no rosette formation was observed. During culture the patient's lymphoblasts, which originally had cytoplasmic IgM in addition to ALL antigen, expressed surface immunoglobulins as well as mouse erythrocyte receptors. The Reh line cells were ambivalent in two experiments developing T-cell antigens and sheep erythrocyte receptors as well as mouse erythrocyte receptors. Our data suggest that the differentiation arrest in leukemic lymphoblasts can be overcome, thus entailing a surface pattern similar to mature T- or B-lymphocytes.

[Bone marrow transplantation for aplastic anaemia (author's transl)]. / Knochenmarktransplantation bei aplastischer Anämie.

From March 1975 until May 1980 twelve patients with severe aplastic anemia were grafted with bone marrow from HLA-identical siblings by the Munich Cooperative Group for Bone Marrow Transplantation. Six patients are alive between 10 months and more than 5 years after grafting with normal blood values and marrow. One patient is treated as an out patient for chronic localized graft-versus-host disease (GvHD), five patients are well and without treatment. Six patients have died, one patient with a cerebral hemorrhage the day before transplantation, three patients following rejection of grafts 32, 40 and 55 days after grafting, one patient with severe GvHD 85 days after grafting and one patient, probably with interstitial pneumonia, following cerebral hemorrhage. Three of 6 patients who were conditioned with Cyclophosphamide (CY) only died following rejection of the graft. Two adults who were conditioned with CY and "total lymphoid irradiation" and three children, who wer given unirradiated leukocyte concentrates from the marrow donor after grafting, did not reject their grafts. The results of the Munich-Cooperative Group for Bone Marrow Transplantation are comparable to those of large, specialized centers for bone marrow transplantation, they indicate possibilities of cure of severe aplastic anemia by marrow grafts from HLA-identical siblings. They confirm that better results are obtained with earlier transplantation in the course of the disease.

Cytochemistry and membrane markers in acute lymphatic leukaemia (ALL).

136 patients suffering from ALL were subdivided into 5 subtypes (C-ALL, C/T-ALL, pre-T-ALL, B-ALL) according to rosetting tests and using specific antisera directed against membrane antigens. In addition, leukaemic blasts of all patients were investigated according to morphological and cytochemical criteria. In APh and ANAE, indices and the percentages of cases showing a granular staining pattern were high in pre-T- and in T-ALL, but low in C/T- and in C-ALL. PAS-staining, conversely, was more pronounced in C/T- and C-ALL. APh proved to be more discriminative for recognition of the T- and pre-T-ALL subgroups than ANAE, but ANAE-cytochemistry may be useful to detect contaminating normal T-lymphocytes in ALL. Receptors for C3 were more frequent in C- and in T-ALL than in C/T- and in pre-T-ALL, receptors for Fc were distributed equally among all subtypes. Positively of C3- and Fc-receptors was not correlated with cytochemical results. Morphological criteria were not sufficient for subclassification of ALL; the combination of APh- and PAS-staining, however, is valuable to differentiate between C-subgroups and T-subgroups.