Workshop 13 Differentian of markers of T-lymphocyte subpopulation: Murine and Human

Workshop 13 Differentiation of Markers of T -Lymphocyte Subpopulations: Murine and Human

BONNIE MATHIESON and JEFFREY LEDBETIER

tINSERM U93, H6pital Saint-Louis, 75475 Paris, France; 2Institut Gustave-Roussy, 94805 Villejuif, France

Variants of class I molecules heavy and light chains on human thymus cells

L. BOUMSELLt, C. GELIN1, H . COPPIN1, D. PHAN2, J. KALILt, C. GOSSE2, B. RAYNAL2, and A. BERNARD

We have been able to show the complexity of class I molecules on human T cells and particularly thymus cells by using several monoclonal antibodies (Mo.Ab.) recognizing a) a human cortical thymocyte antigen : T6; b) a monomorphic determinant of HLA heavy chains: W6/32; c) at least two distinct epitopes of B2 microglobulin: M18, M28 and N26. We also obtained a Mo.Ab., anti-D47, which defines an epitope, D47, restricted to corticothymocytes within the lymphohaematopoietic system. After internal labelling of human thymus cells, we showed that anti-D47 recognized 45-49K polypeptide heavy chains, two of which (46-48K) are totally associated with a 12K light chain. We were able to show that anti-D47 and T6 do not present reciprocal fixation and thus recognize different epitopes. We also demonstrated that D47 and T6 bearing molecules move independently on the thymus cell. We observed that in reciprocal sequential immunoprecipitation, D47 and T6 epitopes appear on different molecules. Finally, we showed that 10-15 % of thymus cells have T6 bearing molecules with no detectable D47 bearing molecules. We observed that the D47 associated 12K light chain was recognized by two of the three Mo.Ab. recognizing human B2 microglobulin at 37°C and also by the third one (MI8) at 4°C. In contrast, the T6 associated 12K light chain could only be detected at 4°C. We also detected the monomorphic epitope of the HLA heavy chain (defined by W6/32) on most thymic cells. The 12K light chain, associated with the HLA heavy chain on human thymus cells and also on Sezary cells with a more mature phenotype, was recognized only by M18, both at 4°C and at 37°C. In sequential immunoprecipitation, we observed that l. on thymus cells, a currently undefined 44K heavy chains is associated with a 12K light chain recognized only by M28; 2. on Sezary cells, presently undefined 45-46K heavy chains are associated with a 12K light chain recognized by both M28 and MIS. Relationships between heavy and light chain variants will be discussed.

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Department of Medicine, National Jewish Hospital, and Research Center, Denver, Colorado 80206, USA

Functional capabilities of immature thymocytes separated using a sialic acid specific lectin

PRISCILLA A. CAMPBELL, LYNNE R. HERRON, JENNIFER VANDERWALL, and C. ABEL

The N-acetylneuraminic acid-specific lectin, lobster agglutinin 1 (LAg1), selectively agglutinates mouse medullary thymocytes, which have high amounts of surface sialic acid and does not agglutinate cortical thymocytes, which have low amounts of surface sialic acid. We have used the ability of this lectin to selectively agglutinate medullary thymocytes as a criterion for preparing immature cortical thymocytes from the mouse thymus. To do this, suspensions of thymocytes are incubated with LAgl for 1 hr at 4°C, and then are layered over calf serum gradients containing 100, 50, 25 and 10 percent calf serum. Gradients are allowed to incubate for one hour at room temperature. After this time the LAgl-negative cells are collected from the top of the gradient, and the cell populations enriched for LAg1-positive cells are collected from the bottom of the gradient. When necessary, residual LAgl-positive thymocytes can be removed from the LAgl-negative cell preparation by treatment with a rabbit anti-LAg 1 antiserum and complement. LAgl-negative cells prepared in this manner do not respond to Con A, nor can their Con A response be restored by addition of macrophages. Moreover, the inability of LAgl-negative cells to respond to Con A seems a good and reproducible method for preparing relatively pure populations of immature cortical thymocytes. These data show that immature cortical thymocytes are inherently unable to respond to either Con A or IL-2.

The Walter and Eliza Hall Institute, Melbourne, Australia

The functional ability of cortical and medullary thymus cells and their Ly defined subsets

WEI-FENG CHEN, R. SCOLLAY, and K. SHORTMAN

Murine thymocyte suspensions were separated by multiparameter fluorescence activated cell sorting into the 14-15 % medullary-type population showing low binding of peanut agglutinin (PNA -) and the cortical-type high PNA binding (PNA +) population. These were also segregated on the basis of indirect staining with a monoclonal antibody into Ly 2+ and Ly 2-subpopulations. The isolated fractions were assayed using high-cloning efficiency «universal» or non-specific limit-dilution assays (as described in an accompanying abstract) for all cells able to form proliferating T cell clones (PTL-p) and all precursors of cytolytic clones (CTL-p).

Virtually all functional T cells were confined to the medullary-type PNA - subpopulation. Both Ly 2+ and Ly Z- PNA - cells were capable of clonal proliferation, but almost all CTL-p were within the Ly 2+ PNA - subset which makes up 5 % of thymocytes. The medullary population showed 50-80 % the cloning efficiency of mature peripheral T-cells, suggesting that not all medullary cells were functional. The CTL-p and PTL-p were highly enriched in the 3-5 % of thymocytes left after treatment of the animals with cortisone, but this treatment also removed about 60-70 % of the PNA - functional cells. Thus although cortisone resistant thymocyes appear to be a typical sample of medullary-type cells, they only contain 30 % of the number of functional precursors found in a normal thymus.

No activity could be attributed to small cortical-type PNA + thymocytes, even in the presence of high levels of T-cell growth factors. We question the view that these are

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~immature» cells capable of developing into «mature» functional elements. However, a low but definite level of functional precursors, representing less than 1 % of the total function pool, was found in isolated PNA + high H-2K blast cells. These might be activated versions of medullary cells, or a true transitional cell of intermediate phenotype.

LMI, NIAID, National Institutes of Health, Bethesda, Maryland, USA

Heterogeneity in the Lyt-2-bearing murine thymocyte population detected by flow microfluorometry

B. J. FOWLKES, W. M. LEISERSON, and B. J. MATHIESON

Numerous studies on murine thymus have addressed the site and maturation sequence of functional T cells. Several reports have suggested that the «immature» cortical thymocytes give rise directly to the immunocompetent medullary cells. Peanut lectin (PNA) and the surface antigen, Lyt-2, have been used to characterize and separate functional subsets of thymocytes. By flow microfluorometry (FMF) analyses, we find that the percentage of thymocytes expressing Lyt-2 highly correlates with the percentage of PNA bright staining cells (85-90 %). Since the data of DURDA and GOTILIEB suggest that Lyt antigens possess terminal galactose residues, it is even plausible that the galactose binding lectin, PNA, may bind Lyt-2 molecules. In order to determine coincidence of these two markers, Lyt-2 and PNA, on individual thymocytes, we performed two-color FMF analysis, dual parameter analysis of fluorescence and forward light scatter, and fluorescence analysis of thymocytes separated after PNA agglutination. These studies revealed four thymocyte subpopulations: a Lyt-2+ and a Lyt-2-in the PNA dull staining population (medium size thymocytes) and a Lyt-2+ and a Lyt-2- in the PNA bright population. We therefore concluded that staining with PNA and Lyt-2 are not coincident. This conclusion was also suggested by our previous ontogeny studies in which Lyt 2+ cells began appearing on days 15-16 of fetal development, while PNA binding cells occurred prior to day 15. These findings confirm and extend the phenotypic analysis made by CEREDlG, GLASEBROOK, and MACDONALD and raise the question of the relationships among the four populations. It is of interest to determine whether these subpopulations represent various maturation states or «end of the line» cells from separate lineages. The results will be discussed with respect to functional studies obtained using isolated subpopulations of thymocytes.

Scripps Clinic and Research Foundation and University of California, San Diego, La Jolla, California, USA

A 180,000 dalton cell surface antigen found on subpopulation of normal T -cells, in autoimmune disease states

R. Fox, MYRA HUENIKEN, and I. ROYSTON

We have developed a monoclonal antibody (T305) that detects a 180,000 dalton cell surface antigen present on a subpopulation of normal peripheral blood T -cells (20 ± 7) and thymocytes (29 ± 9) as well as monocytes (> 85 %). B-cells, granulocytes, erythrocytes, platelets, muscle, liver and brain were unreactive. Based on two-color staining, the subset reactive with antibody T305 is distinct from that defined by Leu 2, Leu 3, OKM-l, or Fe receptors for IgG. Functional studies demonstrated that the T305+ subpopulation contains T-helper, T-suppres-


sor and NK activity. An increased prevalence of T305 + T -cells was found in certain disease states: infectious mononucleosis (> 90 %), graft versus host disease (55 ± 8 %) (GVHD), acquired agammaglobulinemia (45 ± 8 %). The majority of cases of acute leukemia were reactive (11/12 cases of T-ALL, 11/14 of cALL, 6/6 AML) but only rare cases of chronic leukemia (0/16 B-CLL, 1/4 T-CLL, 0/2 CML, 0/3 Hairy Cell Leukemia). After stimulation with mitogens (PHA, PWM) or Epstein-Barr-infected B-cells, T-cells could be induced to express T305 (> 70 % of blast cells were reactive). Antibody T305 detects an activation antigen present on a subset of T -cells and monocytes that is different from previously reported monoclonal antibodies; the prevalence of cells bearing this antigen is greatly increased in certain autoimmune and neoplastic disease states.

Laboratory of Immunodiagnosis, NCI; Naval Medical Research Institute, and National Naval Medical Center, Bethesda, MD, USA

Hand mirror ALL cells of early T -cell lineage

M. GRAMATZKI, D. M. STRONG, B. DUVAL-ARNaULD, G. MORSTYN, and H. R. SCHUMACHER

In two cases of acute lymphoblastic leukemias (ALL) of the hand-mirror variant, surface marker studies were performed to delineate the origin of these malignant cells. The lymphoid cells of the young patients were terminal deoxynucleotidyl transferase positive, did not react with HLA-DR antibodies and did not express the common ALL antigen. Extensive studies of the bone marrow cells with monoclonal antibodies against surface determinants revealed that the lymphoid cells were strongly reactive with 3Al, an antibody which stains the majority of normal T cells and is present on ALL cells. Furthermore, cells were stained with antibody 4F2 which is expressed on rapidly dividing cells. Other antibodies directed against thymocytes, mature T cells and lymphocyte subsets and monocytes did not react with the patients' cells or gave extremely weak staining on some cells. In one patient, after treatment with chemotherapy, a decrease of hand mirror cells and 3Al positive bone marrow cells was observed. A comparison of bone marrow, lymph node and peripheral blood lymphocytes showed a similar cell population in all three locations. These data would suggest that the hand mirror variant of ALL, at least in the two cases reported here, was of early T-cell lineage. Whether the hand mirror morphology expressed by a high number, but not all, of the strongly 3Al positive ALL cells is a characteristic morphology for pre-T-lymphocytes arrested in an early stage of differentiation or whether it is a specific reaction to antigenic stimulation, as suggested by STASS et al. (Blood 56: 661, 1980), needs further investigation.

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille cedex 9, France

Effect of anti-Lyt 1.1 monoclonal antibody in the induction of anti-self + TNP cytotoxic T lymphocytes

A. GUIMEZANES and A. M. SCHMITT-VERHULST

The role of Lyt-l + T cells in the induction of CTL anti-self-TNP was investigated by the use of monoclonal antibodies (mAb) directed against these molecules. The presence of mAb anti-Lyt-l.l during the 5 days sensitization culture of CBA T cells by TNBS-treated syngeneic

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cells reduced the level of cytolytic activity detected in such cultures. The effect was Lyt-1 allotype specific since similar cultures of B6-H-2k or B10.BR cells were unaffected. The presence of mAb anti-Lyt-1.1 either enhanced or did not affect the intensity of proliferative response against TNBS-treated syngeneic cells; the level of recovered cells was also slightly augmented as compared to the recovery of control culture, indicating that the reduction of cytotoxic efficiency of the population was not due to mortality of cells during the culture but affected specifically a T cell subpopulation involved in cytotoxic activity. The fact that IL-2 containing supernatants from rat spleen cells stimulated by concanavalin A could reverse this inhibition would indicate that the anti-Lyt-1.1 affected a T helper compartment or at least an IL-2 providing cell during the culture. This possibility is being tested directly by measuring the IL-2 production of sensitized cells in the presence or absence of mAb. Implications of these results and possible reasons for apparent discrepancies with published results in other systems have been analysed.

Department of Pathology, Chemical Industry Institute of Toxicology, Research Triangle PK, NC 27709, USA

Phytohemagglutinin-induced differentiation of rat bone marrow T lymphocytes in culture via a novel pathway

R. D. IRONS, W. S. STILLMAN, and R. W. PFEIFER

Although T precursor cells can be induced to express alloantigens in vitro by non-thymic factors, bone marrow blastogenic response to phytohemagglutinin (PHA) has traditionally been assumed to be due to emigrant thymic T cells. Nevertheless, differences exist between PHA responsive cells isolated from bone marrow and other lymphoid organs in the Fischer-344 rat. We have described a bone marrow lymphocyte population that responds optimally to PHA in culture at later time points (days 5-7) than those from spleen and thymus (days 2-3). Similarly, these cells are initially unresponsive to concanavalin A but respond to the T cell mitogen after 4-5 days in culture. W3/25 and OX8 are monoclonal antibodies derived against rat T helper and suppressor cell antigens, respectively. In contrast to spleen and thymus, freshly isolated bone marrow lymphocytes exhibit negligible W3125- or OX8- associated fluorescence. Stimulation with PHA results in the expression of these markers on cultured bone marrow cells. These markers appear to be expressed on separate populations of lymphocytes in a manner similar to that observed in freshly isolated spleen cells. OX3 and OX6 are monoclonals derived against rat cell surface antigens that correlate with mouse 1<1<] and Ia17_1s specificities, respectively. These gene products have been shown to be linked to the rat major histocompatibility complex. Coincident with the appearance of W3/25 and OX8 on lymphocytes in culture OX3 and OX6 are expressed on a population of cells distinct from T and B cells, presumably macrophages or dendritic cells. W3/13, a monoclonal derived against a differentiation antigen present on rat thymocytes, including the entire subset of W3/25-positive cells in thymus, was not expressed by bone marrow lymphocytes stimulated by PHA in culture. These findings indicate differentiation of rat bone marrow T precursor cells in culture via a pathway distinct from that previously described in the thymus.


Div. Immunopathology, University Hospital, Utrecht, The Netherlands

T cell differentiation in the human thymus and tonsil. Expression of peanut agglutinin binding capacity of T cells in different maturation stages

L. KATER, P. BREKELMANS, T. DAEMEN, and H . J. SCHUURMAN

Peanut agglutinin (PNA) binding capacity is considered to be a marker of immature T lymphocytes. We found PNA binding T cells within the thymus (53 %) and the tonsil (14 %). This prompted us to compare differentiation stages of PNA binding (PNA +) and nonbinding (PNA -) T cells in both organs. Purified PNA + fractions showed a significantly less proliferative response after stimulation with concanavalin A and phytohemagglutinin than purified PNA - fractions or unseparated thymocytes and tonsil T cells. However, the mitogen response of tonsil fractions was much higher than that of thymocyte fractions. In marker tests, PNA -fractions were enriched in cells positive for acid a-naphthyl acetate esterase. Thymocyte PNA + cells showed higher percentages of cells with OKT 6 phenotype and terminal deoxynucleotidyl transferase. The tonsil fractions were negative for these immature markers. HLAABC expression was found on all tonsil T cells, whereas this marker was present in 10% PN A + and 20 % PNA - thymocytes. Analysis for purine enzymes showed a high activity ratio of adenosine deaminase/purine nucleoside phosphorylase in thymocyte fractions and a low ratio in tonsil fractions, whereas the activity ratio ecto 5'nucleotidase deoxycytidine kinase was high in tonsil T fractions and low in thymocyte fractions. In immunohistochemistry, PNA binding T cells could be localized in the thymic cortex; in the tonsil, no specific compartment could be found apart from the staining of germinal centres (containing mainly B lymphocytes).

Conclusions - Both human thymus and tonsil PNA binding T cells are immature compared with PNA

nonbinding T cells; - PNA + tonsil T cells are in further maturation stage than PNA + or PNA - thymocytes; - In the clinical evaluation of PNA binding tests, unequivocal association with very immature

cells (compared with cortical thymocytes) is not allowed.

University Hospitals of tUtrecht and 2Nijmegen; 3The Netherlands Cancer Institute, Amsterdam, The Netherlands

Purine metabolism in intrathymic T cell differentiation: relevance for T cell function

L. KATER! , J. P. R. M. VAN LAARHOVEN2, R. BROEKHUIZENI, G. TH. SPIERENBURd, P. BREKELMANSI, c. G. FIGDOR3, C. H. M. M. DE BRUYN2, and H. J. ScHUURMAN!

The changes in purine enzyme make-up of T cells in various maturation stages have been related with differences in sensitivity to (deoxy)nucleosides in proliferative responses. We studied this variation at the intrathymic level. Thymocytes were separated on cell size by

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centrifugal elutriation in two major fractions (1 and 2, 80 % of unseparated thymocytes, mainly small thymocytes with immature marker phenotype) and four minor subsets (3-6), containing mitogen responsive cells. The adenosine deaminase/purine nucleoside phosphorylase activity ratio gradually decreased from 21 in fro 1 to 7 in fro 6 (blood T cell value 0.7). This variation was related with a similar gradual decrease in expression of immature marker phenotype and with the gradual increase in mitogen responsiveness in the fractions. The ecto 5'nucleotidase/deoxycytidine kinase activity ratio was highest in fro 3 (12) with a gradual decrease to 6 in fro 4 and 2 in fro 5-6 (values in fro 1 4, fro 2 10, and blood T cells 194). This gradual decrease in fro 3-6 was correlated with sensitivity to inhibition of the phytohemagglutinin response by (deoxy)nucleosides; correlation coefficients were 0.90 for deoxyguanosine, 0.89 for deoxyadenosine and 0.77 for adenosine (under conditions of blocked ADA activity).

Conclusions - The adenosine deaminase/purine nucleoside phosphorylase activity ratio is a useful marker

for T lymphocytes in intrathymic maturation stages; - In mitogen responsive subsets the ecto 5'nucleotidase/deoxycytidine kinase activity ratio is

related inversely with the sensitivity to intoxication by (deoxy)nucleosides (under condition of blocked ADA activity). This correlation fits with the conversion of (deoxy)nucleosides to toxic ribonucleotides as the major mechanism of cell toxicity.

LMI, NIAID, and LCBGY, NCI, National Institutes of Health, Bethesda, MD, USA

Cell surface expression of TL antigen and a determinant recognized by a monoclonal antibody (B14-2-14) on thymocytes of conventional and wild strains of mice

J. LANGHORNE, 1. PAWLITA, M. POTTER, and B. J. MATHIESON

The study of thymocyte differentiation is facilitated by the use of monoclonal antibodies specific for cell surface markers which may be expressed at different stages of maturation. In this report we have used a monoclonal antibody, B14-2-14 (IgM) which detects a Thy-1-related determinant on the surface of thymocytes of all mouse strains tested. This antibody binds predominantly to a subpopulation of thymocytes which is cortisone-sensitive (SIDMAN et al. 1982 submitted for publication). Our studies using two-colour immunofluorescence and analysis by flow microfluorometry on a Fluorescence-activated Cell Sorter (FACS) confirm that all B 14-positive cells are Thy 1 positive, and that the majority of B 14-positive cells also express the TL antigen and the acceptor for peanut agglutinin. It was previously shown that the fraction of B 14-positive thymocytes varied between different mouse strains, and SIDMAN et al. suggested that although the B14 determinants is distinct from Tla, the H-2 and Tla region may playa role in its expression. We have examined by FACS analysis the expression of the B14 determinant on different TL congenic strains. Further, using three monoclonal antibodies specific for TL determinants and a conventional antiserum, we have compared the differential level of TL expression and the B14 determinant on thymocytes of different conventional and wild strains of mice. FACS analysis demonstrated the complexity of TL antigen expression and revealed at least two new patterns of TL antigen expression in the wild mouse strains. The B 14 antibody offers an additional and useful method for the study of thymocyte differentiation.


LMI, NIAID, National Institutes of Health, Bethesda, MD, USA

Analysis of a new thymus subpopulation delineated by Ly 1 and Ly 2 surface markers

W. M. LEISERSON, TH. M. CHUSED, and BONNIE J. MATHIESON

We have used dual fluorescence flow cytometry (FC) to demonstrate four murine thymus subsets with respect to forward light scatter and the markers Ly 1 and Ly 2.

Population L y 1 L y 2 Size 1 + + + small 2 ++ + large 3 +++ medium 4 + large

Many investigators have previously reported populations 1, 2 and 3 using surface markers and size analysis on normal, cortisone-resistant and PNA-fractionated thymocytes. While the results we present here are generally consistent with previous studies, subset 4 has not previously been reported, and its characteristics have yet to be elucidated. Studies in which thymuses from newborn B6 Ly 1.1 were grafted into B6 (Ly 1.2) have been used to study the phenotypes of host cell subsets as a function of time spent in the grafted thymus. By marking the incoming host cells with monoclonal antibody specific for Lyt 1.2, we have shown that, while subset 4 comprises only a small percentage of the cells in normal thymus, it is highly enriched in the population of incoming host cells. The question arises whether this subset of cells that have most recently arrived in the thymus has any immune function. It would therefore be of interest to study the relative enrichment or depletion of this population when the thymus is treated with cortisone or when thymocytes are fractionated using PNA. Such experiments are currently in progress.

Division of Immunology, Department of Pathology, University of Cambridge, U.K.

Ia + T lymphocytes in heart transplant recipients

CAROL O'TOOLE and GRA(:A S. CARVALHO

The majority of human peripheral blood T cells express la-like antigens after stimulation with mitogens or alloantigens in vitro. Infection with EB virus also results in high levels of Ia + T cells. To assess the role of this subset in vivo, we have monitored lymphocyte subsets in heart allograft patients undergoing immunosuppression with ATG and/or cyclosporin A. Surface markers were detected by direct and mixed rosetting reactions. Monoclonal antibodies to T cells or Ia-DRw-like monomorphic determinants were coupled to papain-treated ox erythrocytes with chromic chloride. SIg+ cells were measured by reaction with sheep anti human (Fab)2 coupled ox cells. T cells were also counted by rosetting with AET treated sRBC. Double markers were estimated by rosetting with equal volumes of two indicator cells one of which was labelled with FITC. Normal age matched donors were found to :s:; 2 % Ia + T cells. A TG produced a dose-dependent and selective reduction in the proportion of detectable T cells. This was accompanied by a rise in non T, SIg + and Ia + cells. After ATG was stopped, T cell levels recovered but rarely regained normal levels. In contrast, cyclosporin A caused no measurable alteration in these subsets. Infection with cytomegalovirus resulted in a rapid rise in Ia + T cells, ;;. 20 % at least 1 week before serum antibodies increased. Rejection episodes diagnosed by the intensity of lymphoid infiltrates in heart biopsies were also associated with the presence of small numbers of Ia + T cells :s:; 10% in peripheral blood.


Division of Immunology, Children's Hospital Medical Center and Center for Blood Research, Boston, MA 02115, USA

Lymphocyte glycoprotein deficiency: a basis for human immunodeficiency

R. PARKMAN, E. REMOLD-O'DoNNELL, D. KENNEY, and F. S. ROSEN

A normal lymphocyte glycoprotein with a molecular weight of 115,000 daltons (gpL115) has been identified by surface radioiodination and SDS gel electrophoresis. Seven patients have been identified who are deficient in gpL115. All patients are immunodeficient with various T and B lymphocyte abnormalities. Five patients clinically had the Wiskott-Aldrich syndrome (WAS), and their platelets demonstrated additional abnormalities in both glycoprotein Ia and Ib by 2-dimensional gel analysis. Following partial purification, a heteroantiserum to gpL115 has been produced. Immunofluorescence with the antibody has demonstrated the presence of high concentration of gpL115 on > 90 % of all normal peripheral blood lymphocytes, while staining of WAS lymphocytes demonstrated gpL115 on only a minority (25-40 %) of lymphocytes with a reduced concentration. Four of the seven patients had lymphocytes where volume was approximately half that of normal lymphocytes. The volume of the lymphocytes from two patients normalized following splenectomy suggesting that the reduced volume was secondary to the action of the spleen on the gpL115-deficient lymphocytes. These glycoprotein abnormalities represent a new class of human immunodeficiencies and may permit insight into the relationship between lymphocyte membrane structure and function.

Department of Anatomy, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105, USA

Peripheral and intragraft development of T -lymphocyte populations in immunodeficient mice grafted with low temperature organ cultured fetal thymic epithelium

G. A. PERRY, J. D. JACKSON, and D. A. CROUSE

We examined the development of T-Iymphocyte populations in adult thymectomized, lethally irradiated and fetal liver reconstituted (ATx-FL) C57Bl/6J mice which were grafted with a low temperature organ culture (L TOC) thymus lobe. Population development was examined in both the L TOC graft and in the spleen and compared with appropriate controls. Briefly, thymus lobes from gestational day 14 fetuses were floated on Nuclepore membranes over complete media in 60 mm dishes for 7 days at 24°C (5 % CO2 in air), then at 37"C for 7 more days without media change. The LTOC lobes were transplanted under the kidney capsule in syngeneic ATx-FL recipients for various time periods prior to sacrifice for morphological, functional and marker studies. Graft and spleen cells were put into single cell suspension and stained for the presence of Thy-1, Lyt-1, Lyt-2 and SMIg using indirect methods with monoclonal antibodies and biotin-avidin fluorescent reagents. Preparations were quantitatively analyzed using an Ortho SOH flow cytometer. The frequency and intensity

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distribution of positively staining cells were obtained by setting a 3 % avidin background and subtracting this from the percentage of positively staining cells. The data demonstrate that intragraft lymphocyte populations are present at 30, ISO and 360 days post-transplantation in the same frequency as in a normal control thymus for all surface marker categories examined. At 30 days post-transplantation of the thymic epithelium, both the ATxFL-negative controls and the ATxFL-grafted mice showed spleen profiles of unreconstituted animals (i.e., -3.5 % Thy 1+, -4% Lyt 1+, - 2% Lyt2 + and -15% SMIg+ cells). By 360 days, although the grafted ATxFL animals showed reconstitution to near normal spleen cell subpopulation levels, the negative control groups also showed some return toward normal. Both groups were, however, still functionally immunodeficient in mitogen, SRBC plaque, DTH and MLR assays. We propose that the paradox of intragraft reconstitution without significant peripheralization of the cells is related to an imbalance of grafted epithelial components (ectodermal vs. endodermal) or their supporting extracellular matrix products.

Supported by NIH AI 15S19,

Dept. Immunology, Medical School Hannover, and Dept. Immunology, Munich, FRG

Alteration of specific cellular cytotoxicity by reactivity of the effector cells with monoclonal antibodies identifying suppressor/cytotoxic T-cells

W. J. PICHLER, CH, BIRKE, E. P. RIEBER, and H. H. PETER

To investigate the question whether monoclonal antibodies against T-cell subpopulations recognize functionally active membrane determinants, the monoclonal antibodies OKT4 (recognizing helper/inducer T-cells) OKTS, Leu2a and TS-ll (recognizing suppressor/ cytotoxic T-cells) were studied for their ability to alter specific cellular cytotoxicity (CML) after an allogeneic MLR, The antibodies 0 KTS, Leu2a and TS-ll appear to recognize different epitopes of the same, highly immunogenic antigen, which has a molecular size of 30,000 D. Unidirectonal MLRs were set up versus two irradiated (2,000 R) target cells. After 6 d, the effector cells were incubated with various monoclonal antibodies for 30 min at room temperature, washed once and the cytotoxicity evaluated versus 51 Cr-labeled allogeneic or autologous target cells. OKT4 and Leu2a did not alter the cytotoxicity in the effector/target cell combinations tested. OKTS had no effect in 13 out of 20 experiments. In 4 experiments, an inhibiting effect (> 50 % reduction of 51 Cr release) was seen, and in 3 experiments, an enhancing effect (3-6-fold increase 51 Cr release) was observed. Reactivity of effector cells with TS-ll reduced the cytotoxicity in 6 out of 12 experiments. The effect of OKT8 or TS-ll appears to be dependent on the individual cell combinations. For example, the peripheral blood lymphocytes of individuals A, Band C gave similar cytotoxic reactions in between, which were dose-dependent and higher versus allogeneic stimulator cells. Preculture of the effector cells with OKTS reduced the cytotoxicity in the combinations A-B and C-B, while it did not influence B~A, B-.C, C~A, A~C. The antibody TS-ll had no effect in these cell combinations but reduced the cytotoxicity in other cell combinations. The effect of the monoclonal antibodies was dose-dependent; an absent effect could not be overcome by even very high antibody concentrations (500 ng per 1 X 106 cells). The data suggest that the antigen originally recognized by OKTS is involved in specific CML. Monoclonal antibodies reacting with epitopes of this antigen can, dependent on the effector/target cell combinations, alter CML.


Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, N .Y. 10021, USA

Inhibition of specific T-cell-mediated cytotoxicity by the OKT3/antiLeu 4 and anti-Leu 2A/OKT8 monoclonal antibodies occurs at different stages of the cytolytic process

CH. D. PLATSOUCAS

We have previously shown (PNAS, 78, 544, and 78, 4500, 1981) that the OKT3 and antiLeu 2A/OKT8 monoclonal antibodies inhibit specific T-cell-mediated cytotoxicity against allogeneic targets in the absence of added complement, at the effector cell level. None of these antibodies inhibit natural killer or natural killer-like cytotoxicity. Other monoclonal antibodies recognizing antigens present on T lymphocytes and other hemopoietic cells (OKTl, anti-Leu 1, OKT4, anti-Leu 3a, OKT6, OKTI 1, OKMl, OKTlO, and anti-Ia) did not inhibit the specific cytotoxicity in the absence of complement. The cytolytic process is accomplished by the following steps: 1. effector to target cell binding (antigen recognition); 2. programming for lysis, and 3. cytotoxic cell independent lysis. Using a single cell binding assay, we investigated the steps of specific T -cell-mediated cytolysis that are inhibited by these monoclonal antibodies. OKT3 or anti-Leu 4 monoclonal antibodies in the absence of complement although significantly inhibited target cell lysis, did not affect effector to target cell binding. In contrast, anti-Leu 2a and OKT8 monoclonal antibodies significantly inhibited lysis of target cells as well as binding of effector cells to target cells. Addition of the monoclonal antibodies to the cytotoxicity assay, one hour after mixing the effector and target cells (cytotoxic cell independent lysis step) had no effect. None of the other monoclonal antibodies tested (OKTl, anti-Leu 1, OKT4, anti-Leu 3a, OKTll, OKMl) had an effect on the cytotoxicity or on the binding of the effector cells to targets. These results suggest that the OKT3 or anti-Leu 4 monoclonal antibodies block specific T -cell-mediated cytotoxicity by inhibiting the lytic process after effector cell binding to target cells, at the programming for lysis step. In contrast, the anti-Leu 2a or OKT8 monoclonal antibodies inhibit by blocking binding of effector cells to target cells (antigen recognition step). These monoclonal antibodies are useful probes for further analysis of the mechanism of specific T-cell-mediated cytotoxicity in man.

Supported by NIH grant CA 32070 and ACS grant CH 151.

The Ontario Cancer Institute, Toronto, Ontario, Canada

Sequential expression of different lytic patterns by developing cytotoxic effector cells

J. REIMANN and R. G. MILLER

The early extrathymic differentiation of cytolytic T lymphocytes (CTL) was studied in vitro. Nylon wool non-adherent bone marrow cells from (RNC X BALB/c)Ft (H_2kJd) nude mice were rigorously depleted of T cells and cultured under limiting dilution conditions (10-100 cells/culture) in 20 III Terasaki wells, with semi-purified supernatant from phorbol ester-stimulated EL-4 T-lymphoma cells. Growth of «colonies» under these conditions fitted a one-hit limiting dilution curve with a frequency of about 11120. In a fraction of these colonies (30-40%), cells expressed T markers (Thy-I, Lyt-l). Furthermore, after 8-12 days of in vitro incubation, 10-20 % of these colonies contained cytolytic effector cells active against target cells (ConA blast or tumor) derived from one or both parental strains. Few could lyse Ft (i.e., self) or allogeneic target cells. Analogous studies of syngeneic and semi-syngeneic (Ft antiparent) mixed leukocyte cultures (MLC) were made: Low numbers of nylon wool non-


adherent T cell-depleted (RNC x BALB/c)Fl (H_2k/d) responder cells from bone marrow or spleen of euthymic or athymic (nude) mice were mixed with irradiated stimulator spleen cells from syngeneic F J or parental mice in 200 ~ MLCs. The analysis of the patterns of lysis produced by individual cultures in an assay against syngeneic FJ and both parental targets, at different time points of in vitro incubation, yielded unexpected results: 1. Effector cells were produced which specifically lysed, a) the Fl targets plus either or both parental targets (i.e., expressed a lytic pattern «expected» by the established transplantation laws), or b) the Fl target exclusively, or one/both parental targets exclusively (without lysing FJ targets), i.e., were «anomalous» according to the transplantation rules. 2. The lytic pattern of individual cultures was not stable as striking changes in the «specificity" of lysis of a given culture were observed within only a few days. 3. Almost all (> 95 %) cultures shifted to an «anomalous» pattern of lysis with time of in viero incubation. 4. Almost all cultures shifted away from (<<expected» or «anomalous ,,) self-reactive, and towards self-tolerant (<< anomalous » or anti-parent) lytic patterns.

Institute of Medical Anatomy, Dept. A, University of Copenhagen, The Panum Institute, Copenhagen, Denmark

Nature of mouse T-lymphocyte colony-forming cells and of colony cells

C. R OPKE

Non-adherent Thy-1.2 + cells from the thymus and the peripheral lymphoid organs of the mouse are able to proliferate and form colonies of up to 104 cells in 0.96 % methylcellulose cultures when stimulated with PHA and spleen-conditioned medium. 1 ml cultures were set up with from 103 to 105 cells per culture after FACS sorting of Lyt-l + and Lyt-2+ cells. Whether thymic or peripheral lymphocytes were sorted, it was consistently found that only cells which are both Lyt-l + and Lyt-2 + are able to form colonies. Both small and large cellssorted by their small angle and 90° scattering - are able to form colonies. Tritium-suicide experiments showed furthermore that colony-forming cells (CFCs) are non-cycling cells. Based on experiments with cell populations from fetal, newborn, and adult mice, it is concluded that the CFC is a relatively mature T-cell, which is present both in the thymic cortec and medulla as well as in the spleen and the lymph nodes. Single colonies were investigated for surface determinants. All colony-cells were Thy- t .2 +, and the vast majority of colonies was dominated by either Lyt-1 - 2+ or Lyt- +2+ cells, consistent with a development of effector cells. Cell cycle analysis showed that the large and medium-sized colony-cells are in rapid and continued cell proliferation, while the majority of the small lymphocytes die relatively quickly. Results of this culture system may lead to a better understanding of both intra- and extrathymic T-cell maturation.

Robert-Bosch-Hospital, 7000 Stuttgart, and Immunobiology Lab., Dept. Medicine, 7400 Tiibingen, FRG

Fey-receptor heterogeneity on human T-lymphocytes of different lymphoid organs

J. G. SAAL, A. FRIDRICH, and M. R. HADAM

We have recently characterized two distinct Fc-receptors for IgG (FcyR) on human peripheral blood T-lymphocytes with differing binding capacities for monomeric and aggre-


gated IgG. The recept()r inhibitable by aggregated IgG only can be directly assessed by EArosetting and is, on peripheral blood T -cells, identical to the FcyR described by MORETI A et al. (here FcyR-F). Using controlled hypotonic treatment (HT) of purified T-lymphocytes, additional FcyR-bearing cells are revealed whose FcyR bind both monomeric and aggregated IgG (here FcyR-I). HT is shown to remove in-vivo-bound immunoglobulin. Both types of FcyR have been shown by capping studies to represent independent membrane structures. Mean values in peripheral blood are 17 ± 6 % for FcyR-F and 13 ± 9 % for FcyR-I. Lower levels were observed in tonsils (14 ± 6 % vs 6 ± 4 % , resp.). Almost no FcyR-F and FcyR-Ibearing cells were seen in thymus and lymph node. The high values reported for FcyR in splenic and cord blood T-cells could be confirmed (30 ± 1 % vs. 36 ± 6 %). However, after HT, almost no FcyR-increment could be detected on these cells. In contrast to lymphocytes from other sources FcyR on splenic and cord blood T-cells were inhibitable by monomeric IgG without previous HT down to the lower levels of FcyR-F on peripheral blood T-cells. Thus FcyR on T-Iymphocytes from spleen and cord blood exhibited binding characteristics of FcyR-I-type receptors. This was confirmed by monomer arming in vitro and subsequent HT. However, these receptors are apparently not armed under in vivo conditions as opposed to Tcells isolated from peripheral blood. This differing state of IgG-arming of FcyR-I on T-cells from various sources may correlate to a particular state of cellular activation or differentiation. This is supported by observations that in some pathological conditions non-armed, monomerinhibitable FcyR-I-bearing T-cells can be found in significant numbers. From these results it appears mandatory to apply both HT and monomer inhibition for the characterization of FcyR-bearing human T -lymphocyte subpopulations.

Supported by Robert-Bosch-Foundation.

The Walter and Eliza Hall Institute, Melbourne, Australia

The phenotype, physical properties, and functional ability of recent thymus migrants

R. SCOLLAY, WEI-FENG CHEN, and K. SHORTMAN

Mouse thymocytes were labeled in vivo by intrathymic injection of a solution of fluorescein isothiocyanate (1), and 3-4 h later the recent migrant T cells in the spleen and lymph nodes were studied using flow cytometry. As described previously (1) , recent migrants possessed Thy 1, H -2K and PNA receptor levels typical of peripheral T cells and medullary thymocytes, rather than those of cortical thymocytes, and they already showed the distribution into Ly 2-and Ly 2+ subpopulations characteristic of peripheral T cells (and medullary thymocytes). However, they were found to be larger (by low angle light scatter) and lighter (by equilibrium density gradient centrifugation) than most peripheral T cells, and by these criteria to be similar to medullary thymocytes. A new monoclonal antibody, B2A2, developed by P. BARTLETI, distinguishes stages of T cell development. Thymus migrants were shown to have an intermediate level of sensitivity to complement-mediated lysis by B2A2, different from that of peripheral T cells (insensitive) or that of cortical thymocytes (highly sensitive) but again similar to medullary thymocytes.

The recent thymus migrants were isolated to 98-99 % purity using several cycles of fluorescence sorting. They were tested for T cell function using a sensitive, non-specific, Con A and growth factor driven limit dilution culture system (described in an accompanying abstract) . The isolated migrants were as active as lymph node T cells, displayed a high cloning efficiency and produced a normal proportion of cytolytic clones. These direct experiments show that T cells emerge from the thymus functionally mature, and by most criteria phenotypically mature. We see no evidence for a «post-thymic precursor cell .. requiring


further maturation before displaying function. The migrants closely resemble medullary-type thymocytes by all criteria (phenotype, physical properties and function), and so may be derived from this subset, or have a common precursor.

1. SCOLLAY, R. 1982. J. Immuno!. 128: 1566.

Hospital for Joint Diseases Orthopaedic Institute, Mount Sinai School of Medicine, New York, N.Y., USA

Four new surface antigens on T lymphocytes

1. S. SZER, A. IRANI, G. R. BURMESTER, and R. J. WINCHESTER

Human T cells have been extensively studied with monoclonal antibodies of the T and Leu series. In order to investigate the presence of novel molecules on T lymphocytes, monoclonal antibodies arising from an immunization with blood T cells were selected on the basis of specific binding to E rosette positive cells. Two reagents, 89bl and 91d4, reacted with a majority of peripheral T cells, T cell lines and fetal thymocytes. Antibody 89b1, at nanomolar concentrations, was found to be mitogenic for normal T cells. Through indirect immunofluorescence in a fluorescence-activated cell sorter, 91d4 was also found to bind weakly to some B cell chronic lymphocyte leukemias. These data indicate similarities of 89bl to T3 and Leu4 and 91d4 to Leu 1. However, evidence in support of differences of the new reagents included much brighter staining in a different distribution profile and the lack of blocking in competition experiments using one directly labelled reagent. Two other reagents, designated 91d6 and 94bl, react with the T cell subset which bears the T4 and Leu3 antigens. Co-capping experiments utilizing direct immunofluorescence demonstrated that 94bl and Leu3 detect antigens on independent entities. In contrast, the 91d6 antigen co-caps with the Leu3 antigen, suggesting that these antigens are found on the same or physically related molecules. However, comparison of the median intensity of staining given by 91d6 revealed an increase of over 80 channels with respect to Leu3. Furthermore, these two monoclonal antibodies did not block one another in competition experiments. Additional support for the distinct nature of these antigens was provided by molecular weight analyses of internally and externally labelled molecules. These data demonstrate the existence of additional molecules on the surface of human T lymphocytes and emphasize that multiple molecules, presumably with different functions, are involved in the definition of the T cell lineage and subsets.

Department of Pathology, University of British Columbia, and the Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, B.C., Canada

Cell surface antigens associated with proliferating lymphocytes detected by monoclonal antibodies

F. TAKEI

In order to study cell surface changes associated with lymphocyte activation and transformation, rat monoclonal antibodies against mouse T cell hybrid of Concanavalin A activated spleen cells and a T cell tumor line (EL 4 BU) have been raised. Three cell surface antigens associated with proliferating or transformed lymphocytes have been defined by these antibodies. The first antigen is expressed on mitogen activated T and B lymphocytes, some bone marrow cells, and all the transformed cell lines tested, but not on thymocytes or unstimulated spleen cells. Its molecular weight (Mr) is approximately 200,000 under non-reducing conditions and 100,000 under reducing conditions. The antigen molecule binds transferrin and is


probably a membrane receptor for serum transferrin. The second antigen is expressed at low levels on most cells in spleen, thymus, and bone marrow, but not on red blood cells. Its densities on lymphocytes are elevated after activation with mitogen. It is also expressed at high levels on various transformed cell lines. The antigen is approximately 250,000 Mr which has two chains, 170,000 Mr and 90,000 Mr. The third antigen, unlike the other two, is not detectable on resting or mitogen-activated lymphocytes, but is expressed on some transformed T cells. It is approximately 100,000 Mr under non-reducing conditions. Biological functions of this molecule are unknown.

Scripps Clinic and Research Foundation, La Jolla, CA, USA

Ecto-5'-nucleotidase as a cell surface marker of human T lymphocyte subpopulations

LINDA F. THOMPSON and R. 1. Fox

Ecto-5'-nucleotidase (ecto-5'-NT) is a cell surface enzyme which is found on only a subpopulation (-25 %) of adult peripheral T cells. Virtually all patients with congenital Xlinked agammaglobulinemia (XLA) and the majority of patients with common variable immunodeficiency (CVI) have marked reductions (50-75 %) in the percentage of ecto-5'-NT+ T lymphocytes. Since patients with XLA and CVI often have abnormal T cell regulation of immunoglobulin synthesis, we have attempted to determine whether the presence or absence of ecto-5'-NT activity can be used to define functional subsets of human T lymphocytes. Purified OKT4 + and OKTS+ lymphocytes were prepared from control subjects both by negative selection using complement lysis and by positive selection with a fluorescenceactivated cell sorter, and the percentage of ecto-5'-NT+ cells was determined by histochemical stain. OKTS-enriched preparations were found to contain a higher percentage of ecto-5' -NT+ cells (35 ± S %), than OKT 4-enriched preparations (14 ± 2 %). Patients with XLA and CVI and low ecto-5'-NT activity had reduced numbers of ecto-5'-NT+ cells in both OKTSenriched (7.5 ± 3 %) and OKT4-enriched (7.5 ± 5 %) preparations. In order to determine whether the reduced number of ecto-5'-NT+ cells might correlate with abnormal T cell function, the patients' T cells were tested for the ability to support immunoglobulin synthesis by control B cells stimulated with pokeweed mitogen (PWM). The T cells from 4 patients with reduced numbers of ecto-S'-NT+ T cells were unable to provide help for PWM-driven immunoglobulin synthesis unless they were first subjected to 2000 R of irradiation. These results indicate that the patients' T cells have normal helper activity and excess suppressor activity. One patient with normal numbers of ecto-5' -NT+ T cells had both normal helper and suppressor T cell activity. Additional studies are in progress to determine whether low numbers of ecto-5'-NT+ T cells is a reliable predictor of excess suppressor T cell activity in patients with agammaglobulinemia.

Department of Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, USA

Flow microfluorometry (FMF) studies of the Thy and Lyt phenotype of T lymphocytes in experimental disseminated histoplasmosis

SUSAN R. WATSON, T. J. REDINGTON, T. B. MILLER, and W. E. BULLOCK

Previous studies have shown that C57BLl6 mice infected i.v. with 6 X 105 yeast phase Histoplasma capsula tum (Hc) develop suppressed immune responses at 1-4 wk of infection.


By 8-12 wk, these responses return to normal. In this study at increasing times after inoculation, total and differential cell counts were performed on the bone marrow (BM), peripheral blood (PB), spleen, thymus and mesenteric lymph nodes (MLN) of infected mice and compared to those of normal mice. As early as day 3, there was a marked reduction in cells recovered from the PB, BM, and thymus of infected mice. In contrast, there was an increase in the number of splenocytes recovered. Differential counts showed that lymphocytes were lost from the PB, BM and thymus. This loss was most pronounced at 7-14 days of infection; by day 28, differential counts of normal vs infected mice were similar. FMF studies compared the Thy 1.2, Lyt 1, Lyt 2 and surface immunoglobulin (SIg) phenotypes of lymphocytes from normal and infected mice. Thymocytes from infected mice displayed greater relative fluorescence intensity (RFI) of the Thy 1.2 marker as compared to normal thymocytes between days 5 and 7. On day 10 the RFI was less than that of normal thymic lymphocytes. There was no change in intensity of the Lyt 1 marker on thymocytes from infected mice vs normals. The RFI of the Lyt 2 marker was less on thymocytes between days 7 and 10. Examination of the same markers in PB, spleen and MLN revealed decreases in the RFI of both the Thy 1.2 and Lyt 2 between days 5-10 of infection. No change was observed in the Lye 1 or SIg markers. By day 28 the normal and infected mice did not differ with respect to any surface marker in all organs studied. Absolute numbers of Thy 1.2+, Lyt 1 + and Lyt 2+ cells were significantly increased in the spleen (P<O.OI) and decreased in the thymus and PB (P<O.OI) of infected mice. These studies suggest a trafficking of cells from the thymus, PB and BM to the spleens of infected mice.

Supported by NIH Grant AI 17399.

Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

Development of thymus-leukemia antigen (TL) positive cells in total lymphoid irradiated (TU) mice

1. ZAN- BAR

Thymus-leukemia antigens (TL) are transient differentiation markers of T cells, appearing only on the cells residing in the thymus. Total lymphoid irradiation (TLI) causes prolonged blockage in cell maturation processes and appearance of immature cells of B or T cell lineage in the periphery. Accordingly, the effect of TLI on appearance of TL + cells in the periphery and their relevance to leukemia development was examined. Different strains of mice received 200 rad lymphoid irradiation daily for 8-16 days. At different time intervals after TLI treatment, bone marrow (BM), lymph node (LN) and spleen cells of untreated and treated mice were stained with f1uoresceinated or rhodaminated anti TL1.2.3.5 (TL.), anti TL3, anti TL4,

anti Thy 1 and anti-mouse Ig antisera. These stained cells were analyzed by the Fluorescence Activated Cell Sorter (F ACS). Double-stained cells were analyzed with a fluorescence microscope. Our results demonstrate that 10-30 % spleen, BM or LN cells of TLI -treated mice were TL + even 2 months after TLI treatment, while no TL + cells were found in those organs in normal untreated mice. The first TL + cells to emerge are TL. +, TL4 -, Thy 1 - , Igcells in the BM (3-4 days after TLI treatment). On day 10, TL + cells can be observed in the spleen and are characterized as TL. +, TL4 +, Thy 1 -, Ig - cells. At that time, BM cells carry the same phenotypic markers. Later on, the majority of the splenic TL + cells are Thy 1 +, while still no TL +, Thy 1 + cells can be found in the BM. Thus the appearance of TL markers precedes the appearance of the TL4 antigen. The TL4 antigen appears in both irradiationshielded and unshielded areas and seems to be on all TL. + cells. This study establishes that the thymus is not required for the appearance of TL + cells in the BM and the periphery, as there is no difference in the rate or sequence of appearance of TL + cells in BM and spleen of thymectomized TLI-treated mice. In addition, there seems to be no correlation between appearance of TL + cells and the phenotypic expression of those antigens on thymus cells of different strains of mice.

Documents

Workshop 13 Differentian of markers of T-lymphocyte subpopulation: Murine and Human