Human autologous mixed lymphocyte reactivity is primarily specific for xenoprotein determinants adsorbed to antigen-presenting cells during rosette formation with sheep erythrocytes.

We present evidence that most T cells proliferating in response to autologous sheep erythrocyte (SRBC)-separated non-T cells (NT) cells are not specific for autoantigens but for antigens derived from xenogeneic sources. The conclusion was based on the following three observations. First, we found that NT cells isolated in the absence of xenoproteins by means of density gradient centrifugation on Percoll only weakly stimulated autologous T cells. Because this weak proliferation could not be expanded in restimulation experiments, its significance as an immune recognitive event remains questionable. NT cells isolated by the above method in the absence of xenogeneic determinants readily acquired stimulatory capacity after brief exposure to either SRBC or fetal calf serum. Second, restimulation of T memory cells generated in 1 degree autologous mixed lymphocyte reaction (AMLR) against SRBC-separated autologous NT cells was exclusively seen when NT cells exposed to or separated with xenoproteins were used for restimulation. Third, T memory cells generated against SRBC-separated autologous NT cells were specifically restimulated by autologous Percoll-separated NT cells that had been pulsed with a variety of xenogeneic mammalian sera. These xenogeneic determinants were preferentially recognized in context with autologous HLA-DR+ cells. From these findings and from our previous results that indicated an absolute requirement of HLA-DR+-adherent NT cells (8), we conclude that human AMLR primarily does not represent an autoantigen but a xenoantigen response that is genetically restricted by the HLA-DR type of the antigen-presenting cell.

Effects of human recombinant alpha 2 arg-interferon and gamma-interferon on human breast cancer cell lines: dissociation of antiproliferative activity and induction of HLA-DR antigen expression.

Human recombinant gamma-interferon (rhu-IFN-gamma) and human recombinant alpha-interferon (rhu-IFN-alpha 2 arg) with a chemical purity of over 95% were compared for their antiproliferative and HLA-DR-inducing activity in five human breast cancer cell lines (BT 20, ZR 75.1, MCF 7, 734B, Hs578T). Cytostatic effects on tumor cells were evaluated in monolayer cultures. HLA-DR antigen expression was examined by an indirect immunofluorescence technique using two different anti-HLA-DR monoclonal antibodies (anti-HLA-DR, VID-1) against framework determinants. rhu-IFN-gamma and rhu-IFN-alpha 2 arg differed in their antiproliferative efficiency in terms of both dose dependency and the spectrum of sensitive target cells. Combinations of rhu-IFN-gamma and rhu-IFN-alpha 2 always resulted in higher cytostatic effects. HLA-DR expression was exclusively inducible by rhu-IFN-gamma and did not correspond to its antiproliferative activity. Furthermore, HLA-DR expression did not depend on proliferation but did require intact RNA and protein syntheses as shown by inhibition with cycloheximide and actinomycin D. HLA-DR antigen expression in mammary cancer lines was dependent on time, dose, and the continued presence of rhu-IFN-gamma. Thus, our data suggest that in particular combinations type I and type II interferons might be useful in the treatment of breast cancer because they provide effective cytostatic and cell membrane-modulating properties.

Avian antigen binding cells: enrichment methods.

Data are presented comparing different methods for the fractionation and enrichment, respectively, of specific antigen binding lymphoid cells from immunized chickens. The bovine serum albumin (BSA) anti-BSA system was chosen as a model. To enrich avian antigen binding cells (ABC) from a mixture of chicken peripheral blood and spleen lymphocytes 3 different methods were used: (1) separation of cells forming rosettes with antigen-coated sheep red blood cells (SRBC) from non-rosetting cells by density centrifugation; (2) isolation of ABC by their specific adherence to antigen bound to immunoadsorptive surfaces (gelatin, plastics); (3) column affinity chromatography with antigen-coated agarose, cross-linked dextran for plastic beads. The most efficient method was column affinity chromatography with antigen-coated polyacrylamide beads which affords up to 12-fold enrichment of ABC. Both the other methods are also suitable for separation and enrichment of specific ABC but can only with difficulty be adapted for processing the large numbers of cells which would be necessary, e.g., for in vivo transfer studies.

Direct demonstration of binding of aggregated mouse IgG1 to the 40-kDa Fc receptor for IgG (Fc gamma RII) in both high and low responders.

Several directly fluorochrome-conjugated murine monoclonal antibodies (mAb) of the IgG1 subclass and directed against B or T cell antigens were found to bind to monocytes via the 40-kDa Fc receptor for IgG (Fc gamma RII). As expected from the established polymorphism of Fc gamma RII, strong staining was observed in about 75% of individuals. In the remaining 25% staining was clearly weaker, but could be definitely demonstrated with a mAb against the B cell-specific CD19 differentiation antigen. Specificity of binding to Fc gamma RII was confirmed by the ability to block the binding of the CD19 mAb by pre-incubation with aggregated IgG1 or with mAb against Fc gamma RII. The extent of T cell proliferation induced with a CD3 mAb of the IgG1 isotype (a-Leu 4), which is dependent on the interaction of monocyte Fc gamma RII with the Fc portion of the CD3 mAb, exactly correlated with the amount of binding to Fc gamma RII in all individuals. Proliferation was dose-dependent for both high and low responders; cells of low responders did not proliferate at concentrations below 16 ng/ml of mAb, whereas there was a small but unequivocal proliferation at higher concentrations. These results confirm that monocytes from previously characterized "non-responders" are able to bind aggregated murine IgG1 via Fc gamma RII. They also demonstrate that directly labeled mAb can cause extensive nonspecific staining which may not be excluded by the use of control antibodies of the same isotype.

Flow cytometric detection of cytoplasmic antigens in acute leukemias: implications for lineage assignment.

This study aimed at optimizing the conditions for flow cytometric detection of myeloperoxidase (MPO), cytoplasmic CD3 (cCD3), and cytoplasmic CD22 (cCD22), which seem to be more reliable lineage-associated markers in acute leukemia than surface antigens. Fixation methods employing saponin as detergent resulted in accurate detection of MPO and cCD3, whereas cCD22 was detectable only after buffered-formaldehyde-acetone fixation. MPO was detected in 16/17 AML, but only in 1/6 ALL, the MPO positive ALL being also CD13 positive. MPO was detectable in 3/4 AML with T-lymphoid features; a case of myeloid antigen-positive T-ALL, however, was MPO-negative. cCD3 was expressed only in T-ALL, and five cases of lymphoid antigen-positive AML were cCD3-negative. We suggest that these flow cytometric assays are useful for the lineage assignment of poorly differentiated leukemias and contribute to the identification of truly biphenotypic acute leukemias.

Surface immunoglobulin density in the differential diagnosis of B-cell chronic lymphocytic leukemia and leukemic immunocytoma.

Using flow cytometry peripheral blood samples of 37 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 consecutive patients with leukemic immunocytoma (IC) were studied in order to determine quantitative differences in the surface immunoglobulin (slg) density. In 8/37 (21.6%) cases of B-CLL and 1/17 (5.9%) cases of IC slg staining remained in the control level. Analysis of slg-positive cases demonstrated a close association between the amount of slg and diagnosis: per case the mean calculated fluorescence intensity for IC lymphocytes was 209.7 arbitrary linear intensity units (IU) (median: 156.4, standard error of the mean (SEM): 53.7) and for B-CLL lymphocytes 10.8 IU (median: 7.3, SEM: 1.1; p less than 0.0001). Altogether, 94.6% of all B-CLL patients and 76.5% of all IC patients were correctly classified when a cut-off point was fixed at a mean fluorescence intensity value of 20.0 IU. The percentage of leukemic cells as characterized by CD19 and HLA-DR reactivity was significantly lower in cases of IC (p less than 0.03 and p less than 0.01, respectively). In both entities disease progression occurred more frequently in advanced stages (II-IV) according to the Rai classification (p less than 0.01). In progressive disease rather than in stable disease circulating T lymphocytes were shown to express decreased amounts of surface CD3 antigen (p less than 0.02). We conclude that the quantitative assessment of surface antigens in addition to their qualitative characterization provides accurate information. In particular, the diagnostic discrimination between B-CLL and IC may be improved by determining the lymphocytes' slg amount.

Immunohistological assessment of bone marrow biopsies from patients with hairy cell leukemia: changes following treatment with alpha-2-interferon and deoxycoformycin.

Forty-six bone marrow biopsies from twelve hairy cell leukemia (HCL) patients, treated with either interferon(IFN)-alpha-2 (n = 8) or 2'deoxycoformycin(DCF) (n = 4), were examined using cryostat sections and an immunoperoxidase technique. Using this sensitive method we were able to demonstrate residual hairy cell (HC) infiltration in five cases, in which evaluation with conventional staining techniques on plastic embedded biopsies revealed complete remission. The amount of HCs in these five samples ranged from 1 to 7% (mean: 3%) of bone marrow cells. Consecutive biopsies in individual HCL patients revealed no changes of the immunological phenotype (CD19, CD22, CD25, CD10, CD11c, FMC7, HLA-DR, surface immunoglobulins) during IFN and DCF treatment. Within the infiltrated bone marrow a considerable number of "reactive" T lymphocytes was identified with prevalence of the T-helper (CD4+) subtype in untreated cases, whereas T-suppressor/cytotoxic (CD8+) cells were within the normal range. IFN treatment resulted in a reduction of CD4+ T lymphocytes (p less than 0.02). Minor alterations of CD8+ T lymphocytes and NK cells (HNK-1 + lymphoid cells) were found in bone marrow during IFN treatment. In DCF-treated patients bone marrow T lymphocytes were markedly reduced below the values of normal bone marrow. This DCF-induced T-cell depression might be related to the clinical observation of persistent cellular immune dysfunctions in HCL patients despite a DCF-induced remission.

E receptors and T antigens are different antigenic entities on human T lymphocytes.

ATS contains antibodies of different specificity directed against E receptors and T specific antigens, respectively. E-receptors are trypsin-sensitive, T-antigens are trypsin-resistant. Absorption of ATS with trypsinized thymocytes thus removes anti-T, but leaves anti-E antibodies unaffected. The rosette inhibitory potential of the absorbed antiserum (anti-E) remains unaltered. Immunization with trypsinized thymocytes on the other hand results in the production of anti T-cell sera highly specific in immunofluorescence and cytotoxicity tests without contaminating anti-E antibodies and, therefore, also lacking any rosette inhibitory capacity. E receptors and T antigens are independently mobile within the cell membrane as shown by differential capping.

Spontaneous and conA-induced suppressor lymphocytes: a comparative study.

Suppressor monocytes, Concanavalin A(ConA)-induced suppressor T cells, and short-lived suppressor lymphocytes have been describe in humans. The present study was performed to evaluate spontaneous suppression in a test system similar to that employed for the demonstration of ConA-induced suppressor cells: Lymphocytes were either stimulated by ConA (= induced suppressor cells) or immediately mitomycin-treated (= spontaneous suppressor cells). Both preparations were tested for their capacity to suppress mitogen-induced proliferation of autologous cells. Depletion of monocytes or B lymphocytes did not affect spontaneous suppression. The active cells were short-lived in vitro. Therefore the net increase in suppressor activity generated by preculture with ConA is in part related to a loss of spontaneous inhibitory activity in the control cultures kept without mitogen. Spontaneous suppressor cell activity was comparable to that of ConA-induced suppressor cells.

Normal suppressor-cell activity in systemic lupus erythematosus. A study on 26 cases.

Suppressor-cell activity of 26 SLE patients suffering from active disease was compared to that of 15 healthy controls. ConA-induced and spontaneous suppression was evaluated. The mitogen-driven proliferation of normal allogeneic cells was significantly impaired by ConA-induced as well as spontaneous suppressor cells. However, no difference in suppressor-cell activity could be demonstrated between SLE patients and controls.

Infiltration density of HNK 1 positive cells in non-Hodgkin's lymphomas depends on histologic subtype: an in situ morphometric analysis.

Numbers and distribution of HNK-1 (Leu 7) positive cells within 115 malignant Non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry. Results on the infiltration were related to histological and clinical parameters. A mean of 4.099 +/- 350 HNK 1+ cells/microliter tumor tissue was found, which was comparable to normal (reactive) lymphatic tissues (4.441 +/- 1.235) and was about a quarter of the population density of T helper/inducer (TH) and T cytotoxic/suppressor (TS) lymphocytes together. The distribution of HNK 1+ cells within the tumors was diffuse except in nodular lymphomas of follicular center cell origin (centroblastic/centrocytic = cb/cc NHL). When evaluating the different histological subgroups, the highest number of HNK 1+ cells was found within the tumor areas of cb/cc, which contained about three times as many positive cells as the other NHL. High numbers were also found in the diffuse variant of cb/cc but not in centrocytic NHL. Different degrees of HNK 1+ cell densities were observed in lymphocytic lymphomas (4.426 +/- 754), with high numbers in about 30% of the patients. Splenic tissues of 6 hairy cell leukemias displayed lower numbers of HNK 1+ cells as compared with other low grade malignant NHL. In "large cell" NHL, the lymphoblastic subtype showed only sparse HNK 1+ cells (1.345 +/- 386). The number was significantly reduced in comparison to all other NHL (p less than 0.01) and markedly lower than in the other NHL of high malignancy (immunoblastic and centroblastic NHL, p less than 0.02). The diminuation was not due to a simple dilution phenomenon in a rapidly proliferating tumor, as TH and TS infiltrates were comparable to other NHL. Correlating results with the clinical course (available in 58 patients), significantly higher numbers of HNK 1+ cells were found in NHL of low malignancy (p less than 0.02), but patients with a favourable course did not differ from those with progressive disease (p less than 0.5). Patients treated by cytotoxic drugs showed higher numbers of HNK 1+ cells than those before or without treatment (p less than 0.02). Results on TH and TS cell numbers in comparison to HNK 1+ cells showed completely different patterns of infiltration.

A micro-culture system for cloning human T lymphocytes in agar.

A simple and reproducible single-layer micro-agar culture system for cloning of human T lymphocytes has been described. The system consists of an agar layer, in which mononuclear cells from peripheral blood were suspended, and a liquid overlayer containing the mitogenic substance. The advantages of the described method are a low incubation volume (0.5 ml) and the liquid overlayer. The addition of different test substances to the liquid overlayer is simple and easily controllable. Depending on the agar concentration a different number of formed colonies can be found floating in the liquid phase. The morphological, cytochemical and immunological characteristics of the cells from those aggregates could be easily studied. The T-cell characteristics of formed clusters and colonies was confirmed by immunofluorescence and E rosette formation. The effects of agar, serum and cell concentrations, as well as the mitogenic activation caused by three lectins on the development and number of colonies were studied on day 7, 10 and 14 of incubation.

Follow-up studies on proliferative T cell responses in cadaveric kidney graft recipients under combined immunosuppressive treatment with cyclosporine and prednisone.

In 5 recipients of cadaveric renal allografts, we tested the influence of prophylactic immunosuppression with cyclosporine (Cy A) and low dose prednisone on in vitro proliferative T cell responses, T-helper/T-suppressor cell ratios and spontaneous- or lectin-induced unspecific suppressor cell activity and on serum mediated inhibition of proliferative T cell responses. Results revealed a reduction of the overall proliferative T cell responsiveness, which was particularly seen in cultures supplemented with autologous serum and was maximally expressed after approximately 30 days of treatment. This impaired proliferative capacity was neither accompanied by shifts of the T-helper/T-suppressor ratios nor by alterations of spontaneous- or lectin-induced suppressor activity. The capacity of patients' plasma to inhibit lymphocyte proliferation was also tested. Results indicated that almost every plasma of Cy A patients was capable of inhibiting mixed lymphocyte culture (MLC) responses. The inhibitory capacities of these plasma, however, were not directly correlated with their Cy A content.

Macrophage infiltration in non-Hodgkin's lymphomas: a quantitative in situ study.

The frequency and distribution pattern of macrophages within 93 non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry using stereological methods. For the identification of macrophages (M phi), several antibodies (Mono 1, Mono 2, OKM 1) reactive with surface antigens on cells of the monocyte-macrophage series and cytochemical staining for acid phosphatase were applied. The average number of macrophages within lymph node tissue of NHL was 6,299 +/- 760 cells/microliter (similar to reactive lymphatic tissue: 6,559 +/- 1,027). The highest number of infiltrating macrophages was detected in immunoblastic NHL (17,306 +/- 2,773), differing significantly from other histological subtypes and reactive lymphatic tissue (p less than 0.005). The possible impact of tumor-infiltrating macrophages on lymphoma cell proliferation and differentiation is discussed.

CD4 monoclonal antibody VIT4 in human alloimmune response in vitro and in vivo.

In the present report the immunosuppressive effects of the murine anti-human CD4 monoclonal antibody (mAb) VIT4 on human alloimmune response in vitro were analyzed. Moreover, the antibody was tested for its activity to prolong allograft survival in seven patients with steroid-refractory allograft rejection. VIT4 inhibited the proliferative response to alloantigens in the mixed lymphocyte reaction (MLR) in a dose-dependent manner. At concentrations of 1 and 10 micrograms/ml VIT4 blocked MLR by 55 +/- 11% and 77 +/- 1%, respectively. Also alloantigen-specific proliferation of in vitro- generated memory T cells was dose-dependently reduced to 23 +/- 1% at a VIT4 concentration of 100 micrograms/ml. Furthermore, at the same dose level VIT4 blocked proliferation of antigen-specific short-term alloreactive CD4+ cell lines and significantly inhibited the in vitro generation of cytotoxic T lymphocytes (CTL). In a pilot study VIT4 (5 mg/d i.v.) was administered to 7 patients with steroid-refractory allograft rejection for 14 days. In 4 of 7 patients graft function transiently improved and graft survival in all patients was prolonged to a mean of 694 days (range 128-2163) from the beginning of the VIT4 treatment. In the light of our in vitro results and the preliminary clinical data, further clinical trials using higher antibody doses are greatly warranted to assess the efficacy of anti-CD4 mAb VIT4 in the treatment of allograft rejection.

"Intravascular lymphomatosis" (angioendotheliomatosis): evidence for a T-cell origin in two cases.

Intravascular lymphomatosis (IL) is a rare and potentially fatal multifocal intravascular proliferative disorder, most often involving the skin and the central nervous system. Originally considered an endothelial disorder, IL has recently been reclassified as an angiotropic lymphoma, most often of B-cell origin. We report immunocytochemical and ultrastructural findings in two patients with IL, both representing angiotropic T-cell lymphomas. In one patient, lesional tissue was examined by Southern blot analysis and monoclonal T-cell receptor rearrangement was found. As an additional feature in one patient, a myelosuppressive serum factor was demonstrated in peripheral blood progenitor cell cultures as the cause of underlying chronic anemia and leukopenia; this factor is thought to be a cytokine product of the lymphoma cells.

An unusual case of a splenic gamma/delta T-cell lymphoma with angiocentric tendency and haemophagocytic syndrome.

We report here an unusual post-thymic T-cell neoplasia of the spleen associated with a rapidly progressive haemophagocytic syndrome. The lymphoma was classified as a medium- to large sized pleomorphic T-cell lymphoma with angiocentric tendency (CD3+, CD43+, CD45RO+ CD45+). Clonality was confirmed by PCR and revealed rearrangement of the T-cell receptor gamma chain. Serological tests excluded a recent EBV infection and in situ hybridization with the EBER probe was negative. Haemophagocytic syndrome was the initial finding in an otherwise symptomless patient and this deteriorated with progression of the T-cell malignancy. Both, the T-cell lymphoma and the haemophagocytic syndrome remained unaffected by chemotherapy. Splenic gamma/delta T-cell lymphoma associated with haemophagocytosis is an uncommon entity which has until now not been widely recognized.

Evaluation and improvement of different methods for the enrichment of antigen-binding cells of chickens.

Antigen-binding cells were enriched using bovine serum albumin immunized chickens as an experimental model. A comparison of density centrifugation, adherence to derivatized surfaces (polystyrol and gelatin) and affinity chromatography revealed that optimal enrichment was obtained using the latter procedure. Polyacrylamide was superior to agarose.

Cell profiles in serial bronchoalveolar lavage after human heart-lung transplantation.

The aim of this study was to investigate serial changes in bronchoalveolar lavage (BAL) cell profiles after human heart-lung transplantation and to assess the clinical value of BAL cytology in the differential diagnosis of complications in the transplanted lung. BAL was performed serially on 23 occasions on four patients. Elevated counts of neutrophils (4-48%) were observed in all preparations, with peak values in the early postoperative phase, in bacterial infections and in cytomegalovirus pneumonitis. In the last condition, BAL cytology also showed relative lymphocytosis (less than or equal to 50%) with high proportions (less than or equal to 50%) of HLA DR-positive T lymphocytes. No characteristic light microscopic pattern was observed in acute pulmonary rejection. However, scanning electron microscopy revealed elevated counts (greater than 5%) of "villous" macrophages in BAL obtained during or shortly after episodes of rejection. BAL cytology may be helpful in differentiating viral and bacterial infections, while scanning electron microscopy seems to be more suitable to the diagnosis of acute pulmonary rejection.

[Immunologic detection and in vitro activity of suppressor cells]. / Immunologischer Nachweis und In-vitro-Aktivität von Suppressorzellen.

Suppressor cells can be identified in vitro either by specific antibodies or by functional test assays. On investigation of the latter, a close relationship was demonstrated between spontaneously active and in vitro induced (ConA) suppressor cells. The activity of these cells, however, showed a wide day to day variation. Hence, no clinically relevant conclusions could be drawn from a comparison of patients and controls. This was shown both for SLE and myeloma. However, in multiple myeloma indirect evidence of increased activity of short-lived suppressor cells emerged from a different methodological approach. Helper and suppressor cells were evaluated using monoclonal antibodies. Patients with Hodgkin's disease in long-term remission had decreased proportions of T-lymphocytes. Helper T-cells but not suppressor T-cells were strongly diminished. The helper-suppressor ratio was changed from 2.1 in controls to 1.2 in patients. The stimulation (PHA-stimulation) index of the patients was half of the control value. The interactions of suppressor and tumour cells were investigated in non-Hodgkin's lymphoma. In general, a marked reactive infiltration of neoplastic lymph nodes was found. The pattern of suppressor cell distribution argued in favour of a functional role of these cells in tumour growth.