stb1, a mouse lymphocyte marker found on t cell and b cell subpopulations

CELLULAR IMMUNOLOGY 126, 16-30 (1990)

STBl , A Mouse Lymphocyte Marker Found on T Cell And B Cell Subpopulations

DAVIDANDREWANDJAYJAYAKUMAR

Department of Zoology and Cell Biology, University College, Gower Street, London WClE 6BT

Received April 24, 1989; accepted September 18, 1989

A set of monoclonal antibodies has been raised to cell surface markers which are either hyperexpressed, or exclusive to activated murine lymphocytes. One antigen is present on all B cells of the Ly 1 lineage and defines a novel subpopulation of classical B ceils, most of which are activated B cells. In Western blots, this antigen appears as a single glycoprotein of 95 kDa molecular weight but of greater interest, is its expression at high levels on all mature peripheral T cells but on only 30% of thymocytes. This marker is expressed at high levels on all CD4 and CD8 thymocytes, most double-negative (CD4-CDK) thymocytes, and at low levels on double-positive (CD4+CD8+) thymocytes. It may prove useful as a developmental marker for subdividing the double-positive (CD4+CDS+) and double negative (CD4-CD8-) thymocyte populations. Taken together these results indicate that mab 3.17 recognises a novel lymphocyte antigen expressed on subsets of T and B cells, which is hyperexpressed on lymphocyte activation. o 1990 Academic

Press, Inc.

INTRODUCTION

Lymphocytes comprise a heterogeneous population, with cells at different stages of development and activation. The B cell population is split into large (activated) and small (resting) B cells, which differ in their functional properties (1). Activated murine B cells require only certain lymphokines to differentiate into plasma cells, while resting B cells need an additional activation signal, perhaps provided by direct interaction with T, cells. Mabs to markers which are hyperexpressed on activation, may thus identify receptors for these various lymphokines. Such mabs may also identify other molecules of interest, for instance those active in cell adhesion. A new marker is described here that is hyperexpressed on activated B cells and is present on all B cells of the Ly 1 lineage which, due to their unique distribution, low expression of sIgM and high level of sIgD expression, are thought to represent a separate lineage of B cells (2). This mab may have a potential use in subdividing B cells into different functional subpopulations.

Mab 3.17 may also prove useful for analysing T cell development in thymus. Thy- mocytes are split into four populations on the basis of CD4 and CD8 expression (3). A minor population ( lo- 15%) of thymocytes has the phenotype of mature peripheral T cells which express either CD4 or CD8 alone, while the major population (80%) expresses both CD4 and CD8. This double-positive population is found mainly in the cortex and has been identified as the main site of positive and negative selection (4, 5). The small CD4CDV thymocytes, which make up 80% of the CD4+CD8+

16

0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed

STBI. A MOUSE LYMPHOCYTE MARKER 17

population, have a short life span and mainly die. The large CD4+CD8+ thymocytes ( 10%) proliferate vigorously and a large proportion of them express MEL-14 (6) which is believed to mark T cells about to leave the thymus (7). A third population of thymocytes expresses neither CD4 nor CD8, and makes up only l-3% of the thymocyte population but contains all the thymic progenitors able to reconstitute an entire thymus, both in vivo and in vitro (8). The lineage relationships of these subpopulations are only partly known and new markers, which make further subdivisions, are needed. Those already available for this purpose include MEL- 14,0X-44 (9) and J 11 d ( 10) and the mab described here adds to this list. We gave our marker the name “STBl” as it is expressed on subsets of T and B lymphocytes, and we describe here its cellular distribution and some initial molecular characterisation.

MATERIALS AND METHODS

Mice. BALB/c mice were obtained from the ICRF breeding facility at 6- 12 weeks of age.

Antibodies and cell lines. MAR- 18 mab was purified from culture supematant by protein A affinity chromatography and linked to fluorescein and sepharose for use in immunofluorescence and rat mab purification. BW5 147, TA3, P388D1, 7023, WEHI- 1, FDCP-2, NSl, SP20, ABE 8.2, LS109.2, A202J and A20 lines were obtained from the ATCC in addition to hybridomas MAR- 18, M 170, RL172.4, 3.168 and H013.4.

Production of monoclonal antibody. Young adult SD rats were immunised with 2 X 1 O7 large mouse B cells ip and iv, and boosted after 3 and 6 weeks. Three days after the last injection, their spleens were removed and fused to NSl myelomas ( 11). After 2-3 weeks, culture supernatants from any resulting hybridomas were screened by RIA and Western blotting, as described below, to locate mabs to markers which were expressed on lymphocyte subpopulations of interest, or identified novel markers in Western blots.

Western blotting. Organs of interest were mashed through a sieve, spun down and lysed at 5 X 1 O7 cells/ml in hypotonic lysis buffer ( 1 mM CaCl* , 10 mM Tris, 2 mM MgC&) for 15 min at 4°C. The cell debris was then spun down and an equal volume of 2X sample buffer added to the supematant. SDS-PAGE was performed in 10% discontinuous vertical slab gels ( 12) and the protein transferred overnight onto a nitrocellulose sheet, which was then treated as described below for the Dot Blot method.

Dot blot. Samples were dotted onto nitrocellulose, left overnight at room temperature and the blot then washed twice in u-is-buffered saline (0.05 MTris, pH 7.5,O. 15 M NaCl) and blocked for 1 hr in TBS 5% BSA at 37°C. Following this, the blot was washed twice as above and incubated with hybridoma supematant for 1 hr at room temperature. After two washes in TBS 0.05% NP40 and two washes in TBS, anti-rat Ig (HRPO)’ was added (diluted in TBS 5% BSA). One hour later, the wash procedure

’ Abbreviations used: APC, antigen presenting cell; CD, complementary determinant; ConA, concanav- alin A; FCS, fetal calf serum; HEV, high endothelial venules; HRPO, horse radish peroxidase; ICAM-1, intracellular adhesion molecule; Ig, immunoglobulin; IMDM, Iscove’s modified Dulbecco’s medium; ip, intraperitoneal; ISCAM, integrin supergene family of cell adhesion molecules; LFA, lymphocyte functional antigen; LPS, lipopolysaccharide; mab, monoclonal antibody; MEM-H, modified Eagle’s medium with Hepes supplement; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; PWM, pokeweed mitogen; SDS, sodiumdodecylsulphate; TcR, T cell receptor; VLA, very late antigen.

18 ANDREW AND JAYAKUMAR

was repeated and developer added for 10 min, followed by two 5-min washes in dis- tilled water, after which the blot was photographed.

Isolation of cell populations. Mouse spleen cells were treated with 0.83% NH&l, 2.06% Tris, pH 7.5, for 5 min at 4°C to remove erythrocytes and then washed twice in MEM-H. T cells were obtained by incubating cells in MEM-H 10% FCS (GIBCO) on a lo-ml nylon wool column, prewarmed to 37°C and prerun with 10 ml of MEM- H. After 45 min, the cells were eluted with 10 ml of MEM-H 10% FCS (37”C), washed twice and resuspended in IMDM (GIBCO) 5% FCS.

For preparation of B cells, red blood cells were removed from spleen cells as described above, and treated with anti-Thy 1.2 (HO1 3.4) at 4°C for 30 min, followed by 45 min with complement at 37°C to kill any T cells. The cells were then washed twice in MEM-H and separated on discontinuous Percoll (Pharmacia) gradient 1.05/ 1.07/ 1.09, to obtain small, and large, B cells at the 1.07/l .09 and 1.05/l .07 interfaces, respectively ( 13). CD4, CD8, and CD4-CD8- thymocytes were prepared by negative selection using mabs 3.168 and R172.4, plus rabbit complement as described for preparation of B cells.

CD4’CD8+ thymocytes were prepared by positive selection, using the panning method. Petri dishes were coated with anti-CD4 or anti-CD8 (20 pg/ml in PBS) overnight at 4°C then blocked with PBS 0.2% BSA for 1 hr at room temperature. Thymo- cytes (2 X 10’ cells per plate) were then panned on a anti-CD4 petri dish to select CD4CD8’ and CD4+ thymocytes for 1 hr at 4°C. Nonadherent cells were gently washed away with PBS 0.2% BSA at 4°C and adherent cells collected by vigorously pipetting PBS 0.2% BSA over the petri dish, These isolated thymocytes were then panned on anti-CD8-coated petri dishes to remove CD4 thymocytes and select CD4CD8’ thymocytes.

Zmmunofluorescence studies. lo6 cells were incubated at 4°C with saturating amounts of mab for 30 min and then washed twice in PBS 5% FCS 0.1% azide. Fluoresceinated MAR- 18 was then added at an appropriate dilution and cells left at 4°C for another 30 min. Finally, cells were washed once in PBS, fixed for 5 min in 2% paraformaldehyde, washed once in PBS, and then analysed on a FACS analyser (Becton-Dickinson).

Modulation studies. lo6 cells were incubated in 24-well flat-bottom plates in the presence of antibody overnight in IMDM (GIBCO) 5% FCS and then washed twice before an immunofluorescence assay was carried out on the cells, as above.

Equilibrium binding studies. Single cell suspensions of Geys-treated cells were prepared and lixed with 0.25% glutataldehyde for 10 min at room temperature. Binding data was obtained by incubating 1 O6 cells with serial twofold dilutions of ’ 251 labelled mab 3.17 ( 14), diluted in MEM 5% FCS 0.1% azide for 1,3, and 6 hr. Cells were then washed twice in PBS 5% FCS and transferred to tubes for counting on a gamma counter.

Ouchterlony double d@sion. This was done with a Serotec rat monoclonal typing kit. Functional assays. Spleen cells or thymocytes were resuspended in IMDM

(GIBCO) 5% FCS at 5 X lo5 cells/ml and 100 ~1 of cells added to the wells of a flat- bottom 96-well plate. Various dilutions of mitogens in 100 ~1 of the same media were then added, and, after 3 days, 1 PCi of [3H]thymidine added to each well, followed by a 12-hr incubation, before harvesting on a Dynatech cell harvester.

RESULTS

Establishment of monoclonals. A0 rats were immunised with 2 X 10’ large B cells obtained from mouse spleen B cells by Percoll centrifugation, ip three times. Three


TABLE 1

FACS Analysis of Tumours and Several Cell Populations with mab 3.17

Name Percentage

stained Mean

intensity Description

Spleen Bone marrow Lymph node Thymus ConA cells” LPS cells” PWM cells” PHA cells” NW T cells Macrophages NSI 7023 WEHI- I P388Dl WEHl3B ABE 8.2 FDCP-2 TA3, BW5 147, A202J, A20 and

LS109.2

35 46 75 80 71 35

30-40 20 95 66 80 47 85 56 94 70 78 23 98 140 93 I28 90 80 90 86 80 95 99 157 97 90 95 80

0 0 Various T and B turnouts

T cell mitogen B cell mitogen T + B cell mitogen T cell mitogen Enriched T cells Peritoneal cells Mouse fusion partner Pre-B cell tumour Early B cell tumour Macrophage tumour Macrophage tumour Pre-B cell tumour Hemopoietic cell line

’ Spleen cells were cultured with 50 pg/ml LPS, 2 pg/ml ConA, 1 pg/ml PHA and 1 fig/ml in IMDM 5% FCS for 3 days, before staining and analysis on FACS analyser.

days after the final boost, their spleen cells were isolated and fused with NS 1 myeloma cells. In FACS analysis on spleen, lymph node, bone marrow, and thymus, one mab, 3.17, was chosen for further investigation due to its high staining of lymph node cells (70-80%) and low staining of thymus cells (30-40%) (Table 1). Examination of the FACS profiles of thymus and lymph node cells identifies a large population of thymocytes which are stained dully by mab 3.17 and a small population which are stained brightly, equivalent to that seen with lymph node cells (Fig. li). Thymocytes are therefore heterogeneous in their expression of the marker recognised by mab 3.17, which we named STB 1.

Binding of mab 3. I7 to lymphoid tissue, transformed cell lines, and tumours. As shown by immunofluorescence, mab 3.17 bound to cells in all lymphoid tissues (Ta- ble 1) and a high percentage of bone marrow cells were stained by mab 3.17, which suggests that cells of the myeloid and erythroid lineage may also express STB I. Pre- and early-B cell tumours 7023 and WEHI- 1 were recognised by mab 3.17, as is FDCP-2, a hematopoietic stem cell line, and thus this marker is expressed early in development.

The most interesting point about STB 1 is its distribution on T cells. A high percentage of lymph node cells, but only 30% of thymocytes, were stained brightly by mab 3.17 (Table 1) which indicates that STB 1 expression alters during T cell development. The immature CD4+CD8+ thymocytes make up 80% of thymocytes and may ac- count for the large population of dull STBl+ cells seen in FACS analysis with mab 3.17 on thymocytes (Fig. 1). The high levels of staining on nylon wool purified T


300 THYMUS

FL1 FL1

SPLEEN PERITONEUM LYMPH NODE

~

1 ::?

0

~~ 33. 55 I c-b ‘” 4- 2 -Q-.@jb 11 1 1 0 * 7 * I - . A 1

101 102 103 104 10' 102 103104 10' 102 103 104 FL1 FL1 FL1

FLl:FLUORESCEIN FL2 : PHYCOERYTHMN

FIG. 1. (i) Binding of mab 3.17 to thymus and lymph node lymphocytes. (ii) Binding of mab 3.17 to B, T and Ly 1 Lymphocytes. Row A: mab 3.17 (y axis) versus anti-&M (x axis); Row B: mab 3.17 versus anti- Ly 1; Row C: mab 3.17 versus anti-Thy I.

cells, Con A-activated spleen cells, and thymocytes (Table 1) show that STBl expression may also change on T cell activation.

Probing with mab 3.17 on spleen, kidney, lung, brain, and heart lysates which had been absorbed onto nitrocellulose paper showed mab 3.17 to react with spleen cells


but not with cells from nonlymphoid organs (data not shown) and therefore STB 1 is probably restricted to the lymphoid system. Double diffusion identified mab 3.17 as an IgG2a mab with a K light chain.

Binding of mab 3.17 to T cells. Using biotinylated 3.17 mab and fluorescein-conju- gated anti-Thyl.2, we further investigated the distribution of STBl on T cells (Fig. lii, Row C). Mab 3.17 bound to all T cells in spleen, peritoneum and lymph node, thus confirming the data in Table 1.

To investigate the distribution of STBl on thymocyte subpopulations we under- took purification of CD4-CDS- double-negative in addition to CD4 and CD8 single- positive thymocytes. The purity of these populations was checked by staining with anti-CD4 (linked to phycoerythrin) and anti-CD8 (linked to fluorescein) mabs and was found to be always greater than 95%. Staining ofcells with biotinylated 3.17 mab, followed by streptavidin-fluorescein and anti-CD4 linked to phycoerythrin, or anti- CD8 linked to fluorescein, allowed STBl distribution on thymocytes to be deter- mined. Surprisingly, mab 3.17 bound to a high percentage of CD4-CD8- double- negative thymocytes (85%), which represent an early stage of T cell development (Fig. 2A). All the single-positive CD4 and CD8 thymocytes were stained by mab 3.17 (Fig. 2B), which was not surprising as they are thought to represent the final stage of T cell development in the thymus and all mature T cells in the periphery react with mab 3.17 (Fig. lii, Row C). The small number of dull-negative cells observed are likely to represent the STB 1 - (CD4-CD8-) double-negative thymocytes in this population.

It proved more difficult to analyse the double-positive CD4+CD8+ population. Our approach was to isolate CD4+CD8+ thymocytes by panning thymocytes initially on anti-CD4 and then on anti-CD8-coated petri dishes (Fig. 2). Analysis of these populations was carried out on a FACS (Becton-Dickinson FACSCAN) analyser of greater sensitivity than that used in the other experiments, and therefore the FACS profile is shifted along the x axis with a clear population of dull thymocytes evident and a smaller population of brighter cells (Fig. 2C). When the nonadherent cells were analysed, the population of bright cells was greatly enriched (Fig. 2D), which we expected as this nonadherent population would comprise CD8 and CD4-CD4- thymocytes, with some contaminating CD4+CD8+ thymocytes present. Also, as expected, the isolated CD4+CD8+ population was a dull-staining population (Fig. 2E) and therefore CD4+CD8+ thymocytes make up the dull population seen in immunofluorescence studies of thymus.

Binding of mab 3.17 to B cells. Mab 3.17 binds to only 35% of spleen cells of which half is due to T cells, as double immunofluorescence studies with fluoresceinated anti-Thy 1 and biotinylated 3.17 mab showed mab 3.17 to bind all T cells in spleen. Therefore, we expected mab 3.17 to mark a subpopulation of B cells and to investigate this, double immunofluorescence was carried out with biotinylated mab 3.17 and fluoresceinated anti-IgM on spleen, peritoneal, and lymph node cells (Fig. lii, Row A). The B cell population in all three organs was indeed split by mab 3.17 with STBl+ B cells being present at very low levels in lymph node but present at high levels in the peritoneum (Fig. 1 ii, Row A). Double-immunofluorescence studies also showed mab 3.17 to divide the B220 and Lyb 7 B cell populations (date not shown), and therefore mab 3.17 would appear to define a novel subpopulation of B cells.

As the peritoneum is the organ where Ly 1 B cells occur in abundance, we also performed double-immunofluorescence with fluoresceinated anti-Ly 1 and biotinylated mab 3.17, to study STB 1 expression by Ly 1 lymphocytes. All Ly 1 lymphocytes


U FL1

400 D

4

FL1

FIG. 2. FACS Analysis of mab 3.17 binding to CD4-CD8-, CD4’CD’, CD4 and CD8 thymocytes (A, C) (---) Represents the negative control, while the (-.-) represents binding of mab 3.17 to thymus. (A) CD4CD8- thymocytes. (B) CD4 (A) and CD8 (B) thymocytes. (C) Nonseparated thymus population. (D) Nonadherent population from panning on anti-CD4 coated petri dishes. (E) Adherent CD4+CD8+ thymocytes from 2nd panning on anti-CD8- coated petri dishes.

were stained by mab 3.17 (Fig. lii, Row B) and the Ly 1 B cells must fall within this group.

Binding of mab 3. I7 to large and small B cells. To define the subpopulation of B cells stained by mab 3.17 in spleen, large and small B cells were examined. As Ly 1 B cells are rare in the spleen, most of our data must apply to the classical B cell lineage. As shown in Fig. 3D, 92% of large B cells, and 30% of small B cells were stained by mab 3.17, showing that STB 1 is hyperexpressed on activation of B cells.

Changes in the expression of STBl on activation. ConA, PHA, PWM, and LPS all increased the percentage of spleen cells stained by mab 3.17, although LPS, a B cell mitogen, was not as effective as the two T cell mitogens, ConA and PHA (Table 1). A kinetic study (Fig. 4) showed the increase to commence after approximately 24 hr


i DAY 1:42%(17)

250 B

DAY 1:41%(18)

250 E 1

FIG. 3. FACS analysis of STBl expression on lymphocyte activation. (A) Thymocytes incubated in IMDM 5% FCS with 1 rg/ml PWM for 3 days and stained each day with mab 3.17, followed by FACS analysis, to determine the percentage of cells stained and the intensity of staining (in brackets). (B) As above but with 2 pg/ml ConA replacing PWM. (C) Analysis of population from A at 2 days for size and fluorescence. (D) FACS analysis on large (1) and small (2) B cells. (E) FACS analysis on 4-day MLR cells. The percentage of cells stained by mab 3.17 is shown with the mean fluorescent intensity of these cells in parentheses.

and to plateau by 3-4 days, but the increase seen with the T cell mitogens could reflect an increased proportion of T cells in the studied population. Therefore, the effect of mitogens on STB 1 expression by thymocytes was studied. Thymocytes cultured with ConA or PWM, when stained with mab 3.17 after 3 days, showed a several- fold increase in mean fluorescent intensity from 20 to 100 (Fig. 3A). Looking at the PWM-stimulated thymocyte population after 2 days, two populations of cells are evident, i.e., a population of large bright cells and a population of small dull thymocytes (Fig. 3C). Mab 3.17 also stained activated lymphocytes from a MLR very


A ConA

+ PHA

+ PWM

- LPS

0’ I I I J 0 1 2 3 4

TIME (DAYS)

FIG. 4. Actions of mitogens on STB I expression. Spleen cells were cultured for 4 days in IMDM 5% FCS with the addition of various mitogens. Each day, aliquots of cells were removed, stained with mab 3.17, and then analysed on a FACS analyser to determine the percentage of cells stained by mab 3.17.

brightly (Fig. 3E) and therefore, on lymphocyte activation, STBl expression is increased.

Biochemical characterisation of the antigen recognised by mab 3.17. Attempts to immunoprecipitate STBl using lysates of surface iodinated spleen or [S35]methio- nine-labelled WEH 1 3B cells were unsuccessful. However, mab 3.17 did recognise a band at 90-95 kDa in Western blots of spleen and thymus (Fig. 5B). Mab Ml93 was a control mab to CD45 and Western blotted a 200-kDa band as expected, while mab 6.13 was another mab raised in the fusion that defined two glycoproteins of 40 and 45 kDa molecular weight, found on all lymphocytes. Mab 3.17 also recognised a single band at approximately 1 OO- 110 kDa in 7023, WEH 13B, and P388D 1 lysates (Fig. 5C). Culturing spleen cells in the presence of mitogens ConA or PWM, caused the band to move from 95 to 110 kDa (Fig. 5C) and in one experiment with ConA, three bands were seen (Fig. 5A). Unstimulated spleen cells (Fig. 5C, lane D) and LPS stimulated spleen cells (Fig. 5C, lane C) showed no increase, the band remaining at 97 kDa. Equal amounts of lysate were added to each well but mab 3.17 showed much weaker reactivity with spleen and LPS-stimulated spleen lysates, probably due to the hyperexpression of STBl on activation with ConA and PWM. This increase in molecular weight of STB 1 upon stimulation with T cell mitogens could result from either increased glycosylation, or changes in STBl mRNA splicing on lymphocyte activation.

The antigen recognised by mab 3.17 is not related to LFA-1 B chain. As the LFA- 1 B chain ( 15) has a molecular weight of 95 kDa and a similar tissue distribution to our marker, mab 3.17 might recognise an epitope on LFA-1. To address this question, we carried out comodulation experiments in which mab M 170 was found to modu- late LFA-1 off the cell surface of P388Dl cells, but had no effect on binding of mab 3.17 to the cells (Fig. 6) and thus STB 1 and LFA- 1 are not related.

Scatchard plot analysis with mab 3.17. Mab 3.17 was purified, labelled with I’25 by the Chloramine T method, and titrated on glutaraldehyde-fixed spleen cells, as

STBl, A MOUSE LYMPHOCYTE MARKER 25

56

46

26 A.

- -... --_.. SPLEEN 3.17 643 Mm 347 6.13 ~1%

&‘,. _- py,?J kDa

-_ i -.,,’ 200 t

3 I/

kDaA S C 4, E

2Do

43

2’ c.

A : CCNA

6: PWM c: LP6 70 HOUR SPLEEN ACTIVATION

D: - E:PHA -

F: WEH136 G: 7023

H: P366Dl

FIG. 5. Biochemical characterisation of STBI. (A) Spleen cells and ConA-stimulated spleen cells probed with mab 3.17. (B) Spleen and thymus probed with mab 3.17 and control mabs M 1993 to CD45 and mab 6.13, another mab raised in the fusion to a 40 and 45-kDa glycoprotein. (C) Various cell lines and spleen cell culture lysates probed with mab 3.17.

described under Materials and Methods. Spleen cells have 100,000 STB 1 molecules per cell and the antibody has an affinity constant of 9 X IO’M-’ (Fig. 7).

Functional studies with mab 3.17. Addition of mab 3.17 to ConA, PWM, PHA, or LPS-stimulated spleen cells did not affect their proliferation (Fig. 8) and MLR proliferation was also unaffected by mab 3.17. The monoclonal did not stimulate nylon wool purified T cells to take up Ca2+, either on its own, or with the addition of MAR- 18 to crosslink STB 1 on the lymphocyte surface (Fig. 8).

However, thymocyte populations which expressed different levels of the STBl marker had distinct functional properties. Thymocytes were separated into low- and high-expressing populations by staining thymocytes with mab 3.17 and isolating dull and bright populations with a FACS sorter. These two populations were then tested for their ability to proliferate in tissue culture on stimulation with ConA. Only the brightly stained thymocyte population was found to proliferate (Fig. 8).

DISCUSSION

The marker described in this work occurs at high levels on a subpopulation of thymocytes, which contains all the single-positive thymocytes thought to represent


3

FIG. 6. Modulation studies on STBl and LFA-1. (A. I) Ml70 staining of P388Dl cells. (A.2) M 170 staining of P388Dl cells incubated with Ml70 for 4 hr at 37°C. (B.l) 3.17 staining of P388Dl cells. (B.2) 3.17 staining of P388D I cells incubated with M 170 for 4 hr at 37°C.

the mature T cells ready to leave the thymus. A large percentage of double-negative (CD4-CD8) thymocytes also express STB 1, while double-positive CD4+CD8+ thymocytes are thought to express low levels of STB 1. Preliminary functional experiments indicate STB 1 “iGH thymocytes proliferate to ConA, while STB 1 Low thymocytes do not. Therefore, mab 3.17 may identify cells on the differentiation pathway to mature T cells, while STBl Low thymocytes may have failed some selection event and be fated to die. Other functional experiments have shown mab 3.17 to have no effect on the proliferation of lymphocytes or the Ca2’ levels in these cells and so STB 1 is unlikely to be a signal transduction molecule.

Mab 3.17 may prove useful in the study of T cell lineages. Several questions about the position of thymocyte subpopulations, defined by CD4 and CD8 in T cell development, can be addressed with this monoclonal antibody. The double-negative (CD4-CD8) thymocytes are a functionally heterogeneous population (lo), which is certainly supported by the number of markers which split this population, such as J 1 Id and Ly24 (15). Single fetal CD4-CD8- thymocytes cultured in hanging drops give rise to different thymocyte populations ( 16), and therefore different subpopulations of double-negative thymocytes may give rise to the CD4, CD8, and CD4+CD8+ thymocyte populations. In reconstitution experiments, double-negative thymocytes can reconstitute all thymocyte populations (8) but markers, such as the one described here, may be used to split the double-negative thymocyte population. The thymocyte populations arising from these subpopulations in reconstituted irradiated mice, or using in vitro culture, could then be studied. Such experiments have been carried out with the murine marker, J 11 d in which it was found that CD4-CD8-J 11 d+ thymo-


1 2 3 4 5 6

MOLES OF 3.17 BOUND X 10 t-10)

X 1 HOUR + 2 HOURS * 9 HOURS(

FIG. 7. Scatchard plot analysis with mab 3.17.

cytes can reconstitute the thymuses of irradiated mice, giving rise to all four populations of thymocytes, whereas CD4-CD8-J 1 Id- thymocytes cannot do so (10).

The heterogeneity of CD4+CD8+ thymocytes is shown by their varying size and expression of TcR (17). From panning experiments, it would appear that all CD4+CD8+ thymocytes are stained dully by mab 3.17 when compared to CD4 and CD8 thymocytes, and therefore mab 3.17 will probably not prove useful in studies on CD4+CD8+ thymocyte subpopulations. However, the high level of expression of STBl by most CD4-CD8- thymocytes and all CD4, CD8 cells, and the low level of expression by CD4+CD8+ thymocytes, is of interest. The CD4+CD8+ thymocyte population is reported to lie between the CD4-CD8- and CD8, CD4 thymocyte populations in T cell development; most CD4+CD8+ fail some selective event in the thymus and die. STBl expression must go down and then up again as CD4-CD8- develop into CD4+CD8+, a minor population of which are thought to then develop into single-positive thymocytes. The level of expression of STB 1 by this minor population of CD4+CD8+ thymocytes may be of interest.

STB 1 is hyperexpressed on activation with mitogens, and is found at higher levels on activated B cells than small B cells. Ly 1 B cells, thought to represent a separate lineage of B cells, are also all stained by mab 3.17. Other markers, such as Lyb5 (18), which is expressed on B cells that do not require MHC-restricted interactions with T cells and J 1 Id ( 19) a marker for virgin B cells, have been used to split B cells into functional subpopulations. Mab 3.17 may also mark B cells with different functional properties.

STBl has a similar molecular weight to a number of other lymphocyte surface markers. Ly24 (Pgp 1 ), LFA- 1 B chain, ICAM- 1, and MEL- 14 all have a molecular weight of approximately 95 kDa and are involved in adhesion in some way, while S7 and Ly9 have no known function as yet.

Ly24 (Pgpl) has served as a marker of memory T cells (20) and prothymocytes as well as being used to split the CD4-CD8- thymocyte population ( 15). Recently this


1 2 3 4 5 6 7 8 9 10 11

Mitogen and Cell Type

48 4t7-

20 20 -

1

1; .I I I 1 I , , , 1

2

1+ ,I’, , , , , , , , 1 I

1 26 40 68 80 108 I 20 4a 6.e 88 11?@

F-2 RED/Y ?S DNA/X P? RED/Y VS DNA/X

FIG. 8. Investigation of mab 3.17’s functional properties. Spleen cells and thymocytes were set up as described under Materials and Methods with various mitogens in the presence (5,6,7,8) and absence (1,2,3,4) of mab 3.17, as shown above. Thymocytes were also separated into STBIH’GH (10) and STB ILow populations and tested for their response to ConA.

Mab 3.17 was also tested for its ability to stimulate Ca’+ uptake by nylon wool purified T cells, on its own and with the addition of MAR- 18 to crosslink STB 1 on the cell surface. Ionomycin served as a positive control and stimulated a large influx of Ca*+ into the lymphocytes.

marker has been shown to be homologous to the Hermes series of homing receptors (2 1) and therefore Ly24 may play a role in the migration of T lymphocytes. However, Ly24 has a very different distribution to STB 1, being expressed on subsets of peripheral T cells and on a minor subpopulation of CD4CD8- thymocytes, while mab 3.17 stains all peripheral T cells and most of the CD4-CD8- thymocyte population (Fig. 2).

LFA-1 (22) belongs to the integrin supergene family (ICASM), whose members express an (Y and /3 subunit. Three different groups are distinguishable on the basis of

STBI, A MOUSE LYMPHOCYTE MARKER 29

the B chain they express and LFA- 1 is a member of the B2 family that expresses the B2 chain of molecular weight 95 kDa, which mab 3.17 may recognise. Other members of the group include the surface markers Mac- 1 and p 150.95, distinguishable from LFA- 1 by their expression of unique (Y subunits. LFA-1 is involved in numerous cell:cell interactions such as T:APC (23) Tu:B cell (24) interactions and the binding of cyto- toxic T cells to their target cells (22). LFA-1 B2 chain is unlikely to be the marker recognised by mab 3.17 as STB 1 is expressed on a much lower percentage of spleen and thymus cells (35%, 30%) than LFA-1 (97%, 97%) and STBl is absent from the thymoma BW5 147 which is LFA- 1 positive. In support of this, comodulation studies showed that removal of LFA- 1 from the cell surface did not remove STB 1.

ICAM- 1 is the ligand for LFA- 1(25) and has recently been shown to be the receptor for rhinovirus (26). As yet only mabs to human ICAM- have been raised and so mab 3.17 would prove of great use if it recognised murine ICAM- 1. However, in initial studies mab 3.17 proved unable to inhibit the aggregation of PHA+IL2 activated T cells, (data not shown), which is known to be inhibited by anti-ICAM- 1 mabs. Also, ICAM- 1 is not expressed on thymocytes or resting B and T cells, whereas mab 3.17 stains subpopulations of thymocytes and resting B cells in addition to all resting T cells (27).

MEL- 14 is a homing receptor on lymphocytes for the high endothelial venules (HEV) in lymph node (6). This marker may have an identical molecular weight to STB 1 but its distribution is different, MEL- 14 being expressed on a minor subpopulation of thymocytes about to leave the thymus (3) and absent from germinal centre B cells. In vitro or in vivo activation of lymphocytes results in loss of MEL- 14 (28) while we have shown here that in vitro activation upregulates STB 1. For example after 3 days in a MLC, the activated population is MEL-14 negative (29), while STBl is expressed at high levels (Fig. 3E).

Ly9 and S7 are markers of identical molecular weight to STBl that have as yet no assigned function, and for which there is only preliminary characterisation. It is reported that Ly9 (30) is present in greater amounts on lymph node and spleen cells than on thymocytes, which is also observed for STB 1. However, 90% of spleen cells are Ly9 positive, while only 35% of these cells express STB 1 and Ly9 is also present in nonlymphoid tissue, whereas we have been unable to detect STBI outside the lymphoid system in this report.

Mab S7 defines a marker present on all murine T cells and also on LPS-stimulated B cells (31). The marker defined by S7 is found on all thymocytes but staining is broad, with a large population of dull (verging on negatively stained) thymocytes, and also a population of brighter stained cells, so the staining patterns of mab 3.17 and S7 on thymocytes are similar. However, mab 3.17 definitely stains B cells and also stains a large proportion of activated B cells (Fig. 3) in addition to all Ly 1 B cells, whereas S7 only stains B cells when they are activated with LPS. Also, mitogen studies with mab 3.17 showed LPS to be the least effective mitogen for increasing expression of STBl on lymphocytes, whilst LPS proved an effective mitogen to stimulate S7 staining of B cells. Therefore, mab 3.17 marks S7 negative subpopulations of B cells and also a lineage of B cells not recognised by S7.

It is possible that the surface markers recognised by mabs S7 and 3.17 belong to a closely related family of proteins, as is the case for the Hermes antigens (32) a set of homing receptors based around 85-90 kDa glycoprotein. Alternatively, differential splicing of a single mRNA species may result in a set of closely related molecules


sharing many epitopes, as has been found for CD45 (33). Finally, differential glycosylation of one glycoprotein may create molecules with different epitopes.

Regardless of the function of STB 1 “rGH thymocytes, the mab described here defines a novel marker which has a distribution not found with other mabs, as discussed above. The mab 3.17 defines new thymocyte subsets for further study, and may also find use in the study of B cell subsets.

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stb1, a mouse lymphocyte marker found on t cell and b cell subpopulations

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