Virus and trinitrophenol hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells.

Immune spleen cells from LCM virus-infected (CBA X C57BL/6)F1 radiation chimeras entirely repopulated with CBA-T6 lymphocytes were cytotoxic for allogeneic, LCM virus infected C57BL/6 mouse-derived target cells. Normal C57BL/6 targets were not lysed. CBA-T6 lymphocytes derived from (CBA X C57BL/6) radiation chimeras sensitized in vitro against TNP-conjugated C57BL/6 spleen cells lysed TNP-conjugated C57BL/6 targets. However normal C57BL/6 mouse-derived targets were not destroyed. The magnitude of virus-specific (or TNP-specific) cytotoxic responses against H-2 incompatible targets was lower compared to that against H-2 compatible targets. These data are considered to support and to extend the altered self concept, but are not consistent with the dual recognition concept.

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.

Autologous bone marrow grafts in dogs treated with lethal doses of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Hematological and immunosuppressive effects of various single doses of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) were evaluated in the preclinical canine model. Immune function appeared not to be impaired by treatment with CCNU, but severe myelotoxicity contributed to the death of 3 of 4 dogs given 10 mg CCNU per kg and 5 of 5 dogs given 15 mg CCNU per kg within 10 days after drug administration. Infusions of autologous bone marrow protected 6 of 6 dogs receiving 15 mg CCNU per kg and 6 of 6 dogs receiving 20 mg CCNU per kg from lethal marrow failure. Dogs given 30 mg CCNU per kg and autologous marrow died within 7 days from severe gastrointestinal toxicity. We conclude that autologous bone marrow support may allow the use of high-dose CCNU regimens and thereby increase its therapeutic efficacy in the treatment of advanced cancer.

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.

Surface markers and mitogen response of cells harvested from cutaneous infiltrates in mycosis fungoides and Sézary's syndrome.

It was the purpose of this study to characterize the proliferating cells in skin lesion of Sézary's syndrome and of mycosis fungoides by means of their surface markers and their response to Phytohemagglutinine mitogen stimulation. Viable infiltrating cells were freed from skin biopsy specimens by means of a disaggregating homogenizer and the cells yielded were tested with heterologius polyvalent anti-human Ig and with anti-human T-cell globulin, as well as for spontaneous rosette formation with sheep red blood cells (SRBC) and for their response to stimulation with Phytohemagglutinine. Most of the infiltrating cells in skin lesions of mycosis fungoides and Sézary's syndrome lack receptors for anti-human Ig but form spontaneous rosettes with SRBC and have receptors for anti-T-cell globulin, indicating the T-lymphocyte nature of the infiltrating cells; however, their response to Phytohemagglutinine is weak. The results indicate the atypical, presumably neoplastic, nature of T-lymphocytes proliferating in skin lesions of mycosis fungoides and Sézary's syndrome.

Quantitative immunoautoradiography at the cellular level. I. Design of a microphotometric method to quantitate membrane antigens on single cells using 125I-labeled antibodies.

Iodine-125 has become a commonly-used radioisotope, especially for immunoautoradiographic investigations. Microphotometry of grain density, a well-established method in autoradiography with tritium and carbon-14, was applied to nucleated cells with 125I-labeled membranes. Geometric and absorption factors of radiation were investigated in order to find suitable conditions for quantitative evaluation. A preparatory device is given and a set-up of appropriate measuring conditions is presented. With these prerequisites the reflected-light bright-field photometry of immunoautoradiographs permits to determine automatically the content of surface antigens of single cells. Measurement examples were demonstrated.

Immunohistochemical identification of T- and B-lymphocytes delineated by the unlabeled antibody enzyme method. I. Anatomical distribution of theta-positive and Ig-positive cells in lymphoid organs of mice.

The unlabeled antibody enzyme method was used to delineate the anatomical distribution of lymphocytes positive with a MBtheta and a-MIg in tissue sections of thymus, spleen, lymph nodes and Peyer's patches of mice. The known T- and B-cell areas were established. Moreover, individual B-cells are detected in T-cell regions, especially in the thymus medulla, and in the periarteriolar section of spleen white pulp. Similarly, individual T-cells occur in B-cell areas, namely in the marginal zone of spleen white pulp, in the medullar cores of lymph nodes, and in germinal centers.

Discrimination in immunofluorescence between labeled and unlabeled cells by quantitative microfluorometry and computer analysis.

An automated procedure for discrimination in immunofluorescence between antibody-labeled and unlabeled cells has been developed on the basis of microfluorimetric determination of intensity distributions. After smoothing the raw data for irregularities caused by the scoring statistics optimum fit of the negative distribution to the corresponding positive one was achieved. The procedure was tested in a model system by mixing various known proportions of immunofluorescence-negative and -positive plastic beads. In addition, variable mixtures of T-negative CLL cells and normal mononuclear peripheral blood cells were labeled with FITC-conjugated anti-T-antiserum. The expected percentage of T-positive peripheral blood cells agreed satisfactorily with the data measured and computed. Finally, the measured percentage of Ig-positive mononuclear cells from normal peripheral blood was in agreement with the values obtained by other techniques.

Antilymphocytic antibodies and marrow transplantation. V. Suppression of secondary disease by host-versus-theta-graft reaction.

An approach to block secondary disease was investigated in mice sensitized against the Th-1.1 (theta-AKR) alloantigen on the marrow donor's T cells. To avoid a concomitant sensitization against the donor's histocompatibility antigens, prospective marrow recipients were sensitized against thymocytes of a third-party strain sharing the donor's Th-1 alloantigen but not his histocompatibility antigens. Advantage was taken of the fact that rats carry a Th-1.1-like theta-antigen which induces anti-Th-1.1 antibodies in Th-1.2 mice. CBA/J and (C57BL/6 x CBA)F1 Th-1.2 mice were sensitized against rat thymocytes and tranfused with spleen and bone marrow of AKR/J Th-1.1 after irradiation with 800 to 900 R. Although unsensitized recipients died within 3 weeks of acute secondary disease, sensitized mice survived the observation period of 50 days as chimaeras. Sensitized recipients were killed by the transplantation of spleen cells from congenic AKR/Cum carrying the Th-1.2 antigen. The host-versus-theta-graft approach suppressed secondary disease following H-2-compatible and -incompatible marrow grafts. Its hemopoietic and Tcell chimaeras tolerated skin grafts of the donor strain while rejecting third-party skin grafts.

Transplantation of syngeneic bone marrow incubated with leucocyte antibodies. I. Suppression of lymphatic leukemia of syngeneic donor mice.

"Antileukemic autotransplantation" an approach to eradicate by antibodies residual leukemia in the bone marrow taken during remission and regrafted in relapse is proposed and is the basis of investigations on the elimination of leukemic cells transferred to syngeneic mice. Rabbit antiserum against mouse T cells killed over 99% of cells when incubated with 10-5 lymphocytes of a T cell AKR/J leukemia transferred subsequently to syngeneic hosts. Seventy-five per cent of the recipients of antibody-coated and complement-treated leukemic cells survived the observation period of 80 days. Recipients conditioned with 800 R and given syngeneic bone marrow mixed with 10-5 leukemic cells died within 11 days, while 40% survived the observation period of 100 days without leukemia if the leukemic bone marrow had been preincubated with anti-T cell globulin (ATCG) and complement. A detrimental effect of ATCG on normal T cells could only be demonstrated in thymectomized, irradiated recipients of syngeneic ATCG-treated marrow. These mice did not reject H-2-incompatible skin grafts in contrast to the controls without ATCG or without thymectomy. ATCG thus inhibited leukemic lymphocytes while sparing stem cells for hemopoietic reconstitution. At the same time it did not interfere with the recovery of cellular immunity after bone marrow transplantation.

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.

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.

Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphomas.

Hoping to improve the systems for identifying and classifying normal and malignant lymphoid subpopulations, frozen and paraffin sections of nonmalignant lymphoid tissue and of malignant lymphomas were immunostained for surface (S) and cytoplasmic antigens using the peroxidase-antiperoxidase method. Primary follicle cells and follicle mantle cells known to be part of the recirculating B-cell pool were found to be constantly Ia and C3 receptor (C3R) positive, mostly SIgM and SIgD positive and cytoplasmic immunoglobulin (CIg) negative. The light zone of germinal centers (GC), which is rich in centrocytes, contained a large number of T cells and showed the well-known intercellular Ig network pattern; the dark zone, containing densely packed centroblasts, was usually free of T cells, but was bordered ay a mantle-like accumulation of T cells. Usually only some of the GC cells were definitely positive for SIg and CIg of different classes. All cells reacted positively for Ia and C3R. In areas described by other authors as containing marginal zone cells, cells densely bearing SIgM and deficient in SIgD were detected. The immunoblasts of the hyperplastic plasma cell reaction usually contained CIg. Cells from chronic lymphoid leukemia sections that immunostained for SIgM and SIgD were interpreted as representing a neoplasm of recirculating B cells expressing SIgM and SIgD. The immunohistologic architecture of follicular centroblastic/centrocytic lymphoma showed a more or less close similarity to the organization of secondary follicles. Lymphomas whose cells resembled reactive centrocytes were strongly SIgM positive and SIgD negative or only weakly SIgD positive. CIg was demonstrable in nearly 90% of the lymphomas whose cells resembled centroblasts and in 70% of the lymphomas whose cells resembled immunoblasts of the plasma cell reaction. Finally, immunohistologic staining results from a T-zone lymphoma are presented, which confirm that this lymphoma was composed of a neoplastic T zone and a non-malignant B zone.

Immunofluorescence study of lymphocytic infiltration in gliomas. Identification of T-lymphocytes.

Five human brain tumours (3 glioblastomas and 2 astrocytomas) and 5 rat brain tumours induced in Sprague--Dawley animals by systemic administration of N-methyl-N-nitrosourea (3 pleomorphic gliomas and 2 mixed gliomas) were studied. The human brain tumours were surgical specimens excised from patients with no cranial surgery prior to their disease. The experimental brain tumour had been adapted to tissue culture, propagated in vitro and then transplanted to immunocompetent and immunodeficient rats of the same stock. The above-described material was selected in consideration of the mononuclear cell infiltrates occurring in these tumours. Frozen sections of human and rat gliomas, the latter both primary and transplanted, were prepared and investigated as to the presence of T-lymphocytes within the mononuclear round cell infiltrates. This was done with the indirect immunofluorescence method using rabbit antisera against man and rat T-lymphocytes. With this technique a variable percentage of T-lymphocytes was demonstrated in the cell infiltrates of human and rat gliomas alike. The tumour transplanted in thymectomized rats showed only isolated, scattered, positive-reacting cells, i.e., cells recognizable as T-lymphocytes by the above method. The results can be interpreted as circumstantial evidence for the occurrence of tumour-specific and/or tumour-associated antigens in the parenchymal cells of spontaneous and chemically-induced gliomas.

Specific absorbed antithymocyte globulin for incubation treatment in human marrow transplantation.

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.