Part VII. Vital Staining Techniques in Cell Biology, Immunohematology,and Transplantation

IMMUNOFLUORESCENCE STUDIES OF A POSSIBLE PRETHYMIC T-CELL DIFFERENTIATION IN CONGENITALLY ATHYMIC (NUDE) MICE Francis Loor* and Georges E. Roelants

Basel Institute f o r Immunology CH-4058 Basel, Switzerland

INTRODUCTION

A hairless mouse mutant, the autosomal recessive “nude” (symbol: nu) is located in chromosome 11 (formerly linkage group VII) and has pleiotropic effects.’ From the immunologic point of view, homozygous nudes are of the highest interest, because they seem t o lack t h e T-cell compartment of the immune reactivity (for review, see Reference 2). They are unable t o reject foreign tissues or cells or t o mount normal antibody responses to T-cell-dependent antigens. There is a marked deficiency in the T cells themselves: (a) there is no thymus after birth. In the embryo, some thymic tissue appears but does not become l y m p h ~ i d ; ~ - (b) ~ the areas of the secondary lymphoid organs where T cells selectively home6 are nonexistent or contain reduced numbers of lymphocytes7 (germinal centers, although typically populated by B cells8 are not organized either9); (c) the pool of circulating lymphocytes is five t o six times smaller in nudes than in normal mice3>10 and seems t o be composed almost exclusively of B cells,’ ‘ * 1 2 whereas in normal mice, if consists of about 15% B and 85% T cells;131’4 (d) the lymphoid population almost completely lacks cells that bear detectable amounts of the Thy-1-determined antigen (0 antigen), a T-cell marker,’ both by criteria of cytolysis by (114 alloantibody plus complement and of staining by fluorochrome labelled (118a1loantibodies.l The origin of this obvious lack of T cells seems t o reside in an abnormal development of the thymus rather than in a lack o r defect at the stem cell level: indeed, it has now been demonstrated that nude mice have precursors that can repopulate a grafted neonatal thymus,’ 6 * 1 7 migrate t o secondary lymphoid organs,’ 6 , 1 which become reorganized,’ and restore some T-cell-dependent immunity.’ Though the primary location of stem cells in the adult animal is likely t o be the bone marrow, the nude mouse spleen might also contain T-cell precursors. Indeed, a variety of treatments appear t o allow or increase the detection of T-cell-characteristic membrane markers o n nude mouse spleen cells.20-24

* Chercheur qualifik from the Belgian National Funds for Scientific Research. 226

Loor & Roelants: Prethymic T-cell Differentiation

227

Moreover, cocultivation of spleen cells from thymus-deprived mice o n thymic reticuloendothelial cell monolayers has been shown t o induce an important rise in T-cell-characteristic reactivities,2 and recently it was claimed that similar results could be obtained with nude spleen cells.26 There are also reports that low percentages of 8” lymphocytes ( u p t o 1.5%) can be identified by immunofluorescence (IF)? in nude spleen or lymph node^^'?^^ (instead of about 30-80%, as in normal r n i ~ e ’ ~ ~ ’Even ~ ) . higher values (5-6%) were obtained by c y t o t ~ x i c i t y . ~Finally, ’ the capacity of nude lymph node cells to absorb the cytotoxic activity of a titrated a 4 antiserum was unequivocal.’ The existence of many more cells that express small amounts of 8 antigen, too low t o be detected by the usual techniques, has been recently suggested. When analyzed by direct immunofluorescence, as many as 40% of the spleen lymphocytes from some nude mice lack easily detectable membrane immunoglobulins (Ig),l a B-cell marker.’ When reacted with fluorescent mouse antibody directed against the 8 antigen, u p t o 20% of the nude mouse spleen lymphocytes exhibit faint fluorescence, which suggests that these cells are of T lineage b u t express only a low density of the 8 antigen.16 Though specific, this fluorescence is too marginal t o be considered really satisfactory. Like all mouse antibody, a 4 is very labile t o fluorochrome conjugation and cannot be heavily tagged. Moreover, because it is mouse Ig, it excludes the use of the far more sensitive and amplifying indirect I F technique, and it does not allow a double staining for the Ig and the 8 marker on a single-cell suspension. Both disadvantages of the a8 alloantibody could be eliminated by the use of t h e a 4 antibody present in antisera raised in the rabbit against mouse brain-associated 8 antigen,30 provided that all contaminating antimouse membrane antibody could be fully absorbed out. We review here in detail the immunofluorescent techniques presently used in our laboratory and show how they permit characterization of a new type of cell of T lineage among nude mouse lymphocytes. A brief report of some of these results has already been p~blished.~



MATERIALS AND METHODS

Animals Normal and nude BALB/c 4 t h backcross (“BALB/c-nu”), normal and nude C57BL/6 3rd backcross (“C57BL/6-nu”), normal and nude NMRI (“NMRI-nu”), and AKR mice of either sex, 8-12 weeks old, were obtained from G1. Bomholtgird, Ltd. (Ry, Denmark). Two T-reconstituted C57BL/6-nu mice were kindly provided by Dr. B. Kindred. They had been grafted with neonatal C57BL/6 thymus 8 weeks before the experiment.

t List of abbreviations: AKR IgG*BC3H, IgG from AKR serum against the 0 antigen of C3H mice; C3H IgGdiBAKR, IgG from C3H serum against the 0 antigen of AKR mice; FITC-, fluorescein isothiocyanate conjugated-; IF, immunofluorescence; NMser, normal mouse serum; NRIgG, normal rabbit immunoglobulin G, adsorbed in vivo;RIgGa-MBBr, rabbit IgG anti4 antigen, adsorbed in vivo; SIgG-or-Mlg, sheep IgG antimouse immunoglobulin (polyvalent); S1gC-a-RIg, sheep IgG antirabbit immunoglobulin (polyvalent); TL, thymus-leukemia antigens; TRITC-, tetramethylrhodamine isothiocyanate conjugated-.

228

Annals New York Academy of Sciences Anti-Ig Reagents

Sheep antisera made against electrophoretically purified mouse IgG and against ion exchanger-purified rabbit IgG were generous gifts of Dr. A. S. Kelus. The IgG fractionations (SIgG-a-MIg, S1gG-a-RIg) and their conjugation with fluorochromes were performed by our usual method,32 which is based on the Wood-Thompson-Coldstein-Cebra used in extenso, the basic principles of which are summarized hereafter with some of our slight modifications. The Ig was first precipitated two or three times by 1.6 M (NH4)*S04 (final molarity, O°C) and redissolved in 0.01 M phosphate buffer (pH 7.5). Ig fractions with restricted isoelectric point were selected from DEAE-cellulose by stepwise elutions with increasing concentrations of NaCl(0, 0.05, 0.1, 0.2 M) buffered to pH 7.5 by 0.01 M sodium phosphate salts. After concentration to 10-20 mg/ml by pressure dialysis (Diaflo membrane UM2, Amicon Corp., Redwood City, Calif.) and dialysis against 0.15 M NaCl, the individual fractions obtained from the DEAE chromatography were conjugated with either tetramethylrhodamine isothiocyanate or with fluorescein isothiocyanate (30 fig TRITC or 12.5 pg FITC/mg protein, Baltimore Biological Laboratories, Becton Dickinson and Co., Cockeyville, Md.). The conjugation was performed at O°C for 2 hr at pH 9.0-9.5 maintained by diluted NaOH (!.1-0.01 N) or carbonate-bicarbonate solutions (0.1-0.01 M) and then left at 4 C overnight, which allowed the pH to decrease spontaneously. After removal of unbound and hydrolyzed fluorochrome on Sephadex G50 equilibrated in 0.01 M phosphate buffer (pH 7.5),the conjugated Ig from each individual DEAE fraction was again fractionated on DEAE-cellulose, as above. Indeed, as fluorochrome coupling increases, the isoelectric point of the conjugated Ig molecules decreases. Thus, provided one starts with homogeneous isoelectric fractions of Ig, the second DEAE fractionation allows selection of Ig molecules having bound similar quantities of fluorochrome per molecule. It is then possible to exclude the undercoupled antibody that binds to the antigenic sites but cannot be detected and overcoupled antibody that is either inactivated or adsorbs nonspecifically to cell membranes. Conjugated immunoglobulins that had an O D ~ S O ~ , , , / O D ~ (FITC) ~ S , , ~ or ODslsnm (TRITC) between 2 and 3 were used. T o obtain efficient fluorescent antibodies, it is mandatory to use the complete procedure without omitting any step, Mouse Alloantibody Reagents (a-8,a-TL)

AKR anti4C3H and C3H antiBAKR antisera were raised by the method of Reif and Allen;35 anti-TL antisera (a-TLser) were raised in A/TL- mice against the A-strain spontaneous leukemia (ASLI).36 Details about preparation of these sera and specificity controls have already been p ~ b l i s h e d . ’ ~ The ,~~a * 4~ ~ mouse Ig was separated by agarose block electrophoresis and conjugated by the “dialysis method.” (The Wood-ThompsonGoldstein-Cebra method cannot be used for mouse Ig, which sticks to DEAE.) The mouse Ig solution at 10 mg/ml was dialyzed for 2 hr at 0°C at a pH of 8.5-8.7 against saline that contained the fluorochrome at a ratio of 30 fig (TRITC) or 12.5 pg (FITC)/ml saline/mg IgG, left overnight in the cold room, and then unbound fluorochrome removed by continuous flow dialysis.’ Unlike the method originally reported,I6 we now dialyze our samples against smaller volumes of fluorochrome-saline (only two to three times the sample volume).

Loor & Roelants: Prethymic T-cell Differentiation

229

Rabbit Immunoglobulin Antimouse Brain 0 (RIgGa-MBBr)* The preparation of rabbit antimouse 0 of brain type* was based on the findings of G 0 1 u b . ~ Each ~ of 10 rabbits received at 1-week intervals two intramuscular injections of 3 CBA/J mouse brains homogenized in complete Freund's adjuvant. They were rested f o r 5 months and then received a similar injection. Sera were collected 2-4 weeks after the boost and pooled. IgG was prepared b y repeated (NH4)2S04 precipitations (1.6 M, OOC), followed by fractionation on DEAE-cellulose (DEAE-SS, Serva, Heidelberg) equilibrated in 0.0175 M phosphate buffer (pH 6 . 3 ) . At this stage, the reagent stained all mouse cells strongly when tested by indirect IF and needed to be absorbed. In vivo absorption of this antiserum in nude mice should absorb the antimouse membrane and the a n t i 4 activities differentially, because membrane antigens should be ubiquitous and 0 rare at best. T h e in vivo absorption method was inspired by the method described by Shinego et aZ.38 BALB/c-nu mice were injected intraperitoneally with 10 mg RIgGa-MBBr in 1 ml and bled out 8-10 hr later. T h e Ig fraction was prepared b y repeated precipitation with (NH4)2S04, as above, and dialyzed against 0.0175 M phosphate buffer (pH 6.3). The precipitate due to low ionic strength was discarded, and the supernatant fractionated on DEAE equilibrated in the same buffer. Under these conditions, YO-Y5% mouse lg was retained or delayed o n the column, whereas rabbit IgG passed through. The RIgGa-M0Br was then dialyzed against phosphate-buffered saline and concentrated by pressure dialysis. A small sample was conjugated with TRITC by the dialysis method16 and used o n the cells at 500 pg/ml. The unconjugated RIgG-a-MOBr left was kept at a concentration of 10-20 mg/ml at 4°C in phosphate-buffered saline that contained 0.5% bovine serum albumin and 10 mM NaN3. At this stage, the R1gG-a-MeBr stained in the thymus, spleen, and lymph nodes the expected percentages of T lymphocytes, as could be inferred from the percentages of 0' and Ig' cells obtained with other antisera.' As a control, normal rabbit IgG (NRIgG) from a pool of preimmune sera of the same rabbits was also absorbed in vivo in BALB/c-nu mice and processed exactly in the same way and so was serum from uninjected nude mice. Most of the salt-precipitated mouse serum protein was not soluble in the buffer used, and only 5-10% of the soluble Ig passed through the DEAE column. When incubated with the cells, this fraction did not change the specificity of binding of our various a-lg and a 4 reagents.

Membrane Immunofluorescence Techniques The basic technique used by us for the detection of membrane markers by IF reagents under conditions that d o or d o not permit polar redistribution (capping) have already been described in detail.3 1,37i39

Double Labeling f o r 0 and BBr AKR and BALB/c thymocytes and lymph node cells (2.5 x l o 6 cells in 0.1 ml) were treated with TRITC-C3H-IgCa4AKR or TRITC-AKR-IgC-CxUBC3H $ Abbreviated eBr to distinguish this antigenic determinant from the 0 determinants recognized by alloantibody, which might be different in size or in nature.

Annals New York Academy of Sciences

23 0

( 5 0 pg in 0.1 ml), respectively. After 15 min, R1gC-a-MeBr was added ( 5 0 pg in 0.1 ml) and the cells incubated for 15 min more. After three washes, the cells were treated with FITC-SIgG-ar-Rlg ( 5 0 pg in 0.1 ml) for 15 min, washed three times, and examined in suspension o r fixed. This procedure was chosen to give an advantage t o the antibody of alloantisera, which are weaker because detected by direct IF, and t o avoid the strong competition for binding to B determinants by the RlgC-ar-MBBr. Double Labeling f o r Surface Ig and BBr Unless otherwise stated, in the present series of experiments, cells(2.5 x lo6 in 0.1 ml) were exposed t o R1gC-a-MBBr or NRlgG ( 5 0 pg in 0. ml) for 3 0 min, washed three times, and the pellet resuspended for 3 0 min at 0 C in 0.2 ml of a mixture of sheep IgG reagents: 0.1 ml ( 5 0 pg) antimouse Ig labeled with FITC (FITC-SIgCe-MIg) and 0.1 ml ( 5 0 pg) antirabbit Ig labeled with TRITC (TRITC-SIgG-cr-Rlg). The cells were again washed three times and then examined in suspension or fixed. Microscopy The cells were observed by fluorescence microscopy, as previously described,' ,40 with Leitz Orthoplan microscopes with Osram HBO-200 mercury lamps and Opak-Fluor vertical illuminators (E. Leitz, Wetzlar, Federal Republic of Germany). After recent modifications, the following filters and mirrors are used for observing and photographing the fluorescent cells: 693

1. Selective TRITC fluorescence is obtained by filtering the excitation light with three 4-mm BG 38 filters, one 2-mm S 3 6 0 nm, and one interference filter AL 546 nm; t h e dichroic mirror of the Ploem is TK 5 8 0 nm, and the barrier filters used for filtering the emitted light are one K 580 nm and one K 5 9 0 nm. 2. Strong FITC fluorescence (but without complete blocking of TRITC fluorescence) is obtained by filtering the excitation light with three 4-mm BG 38 filters and one 5-mm BG 12; the dichroic mirror of the Ploem is TK 495 nm, and the barrier filter for filtering the emitted light is one K 495 nm. 3. Faint but selective FITC fluorescence, free of TRITC fluorescence, is obtained with the same filters as for combination 2, but, in addition, we introduce an interference filter KP 490 n m and a barrier filter S 525 nm. Recording of the fluorescence o n KODAK TRI-X-PAN 2 7 DIN requires 10-15 sec with filter combinations 1 and 2 and 2-3 min with filter combination 3 , provided that a Leitz oil 63/1.30 objective and an H 6.3 xM eyepiece are used. Higher magnification lenses require much longer exposure times. RESULTS General Properties of RIgG-a-MeBr

When assayed in direct o r in indirect I F on thymus, spleen, and lymph node cells from normal BALB/c and AKR mice, the RIgGa-MBBr was found t o stain the expected percentages of T lymphocytes, as inferred from the percentages of

Loor & Roelants: Prethymic T-cell Differentiation

23 1

B+ and Ig+ cells obtained with other a n t i ~ e r a ; ’ ~it~ does ’ ~ not discriminate OAKR (the T h y 1.1-determined antigen) and BC3H ( t h e T h y 1.2-determined antigen). No significant binding of NRIgG was detected. The specificity of the binding of the rabbit antibody to cells of the T lineage was assessed by performing a double labeling for B and BBr, with TRITC-labeled a 8 alloantibodies, and FITC-labeled a-RIg for detection of the RlgC-cw-MBBr (see MATERIALS A N D METHODS). The expected proportions of T lymphocytes in the various cell preparations used were labeled, and the labeled cells were doubly labeled for b o t h reagents. Binding of RIgG-cw-MBBr t o normal BALB/c o r AKR lymph node o r spleen cells was blocked when the cells had been preincubated with a 8 C 3 H o r a-BAKR alloantisera (diluted 1 : 40, 30 min at O”C), which are stronger reagents than fluorochrome-labeled a 8 alloantibodies. This strongly suggests that the specific alloantibodies and the heteroantibody bind t o the same membrane determinant or t o very closely associated sites on the cell surface. The binding characteristics of RIgC-cw-MBBr t o the cell membrane are similar to those of a$ alloantibody: both bind better t o thymus lymphocytes than t o peripheral T cells; with both, the binding is homogeneous, at least at the level of resolution of optical microscopy, and gives rise t o a “ring” fluorescence when the antibody is directly conjugated or when detected by fluorescent monovalent Fab-cw-Ig; neither reagent redistributes alone as spots o r caps, which are induced only if a second layer of divalent a-Ig antibody is added. The brightness of cell membrane fluorescence in direct I F is similar with the TRITC-cw-8 alloantibodies or the TRITC-RIgG-cw-MBBr, that is, relatively weak on peripheral T cells, but is markedly increased when RIgC-cu-MBBr is used in indirect IF. Unlike a43 alloantibodies (mouse Ig), a double staining for membrane Ig and BBr can be performed, as described in the previous section, and as will be seen hereafter, most of the cells show mutual exclusion of Ig and BBr markers. Taken together, these observations show the specificity of RIgG-cu-MOBr for the 0 antigen. Cell Population Analysis in Spleen and L y m p h Nodes f r o m Normaland Nude Mice by Doublestaining f o r l g a n d BBr: Evidence f o r Cellsof TLineage in Nudes When double staining f o r Ig and BBr is performed, lymphocytes can be classified into four distinct categories: “typical” B cells,§ which have only membrane Ig (Ig+BBr-); “typical” T cells,§ which have only the B antigen (Ig-BBr+); cells that lack detectable amounts of both markers (Ig-BBr-); and cells that have both markers (Ig+BBr+). The relative proportions of these four cell types in spleen and lymph nodes of normal and nude mice are given in TABLE 1. Typical B cells (Ig+BBr- lymphocytes) are not more frequent in the nude spleen (43%) than in the normal mouse (42%).It is interesting that 22% of the nude spleen lymphocytes were definitely of T lineage (Ig-BBr+) (42% in normal spleen). The other two cell types (Ig-BBr- and Ig+BBr+) are more frequent in nudes than in normal mice, by a factor of approximately 2 (nudes: 23% Ig-BBr- and 12% Ig+OBr+; normal mice 12% Ig-BBr- and 4% Ig+BBr+). In lymph nodes, which show more variability from animal t o animal, the typical

5 According to the classic definition,’S that is, B cells = lymphocytes with easily detectable membrane Ig, T cell = lymphocytes with 8 antigen and no easily detectable membrane Ig.

8.8 6.4 9.4 14.4 8.6 18.0 18.0 11.9

20.4 23.4 26.2 19.4 25.6 24.8 23.6 23.3

1 2 3 4

1 2 2t

5

23.6 23.6 22.2 24.8 19.2 21.0 18.0 0.1 21.8

48.4 48.0 47.6 42.2 38.2 37.6 32.2 0 42.0

Ig-BBr'

Spleen Ig'BBr'

Normal Mice 38.6 4.2 43.0 2.6 42.6 3.4 38.8 4.6 47.2 6.0 40.6 3.8 44.6 5.2 1.3 42.2 4.3 Nude Mice 44.2 11.8 42.2 10.8 38.6 13.0 42.6 13.2 39.2 16.4 48.0 6.2 47.0 11.4 3.2 43.1 11.8

Ig'BBr-

4.1 5.9 11.4 9.2 3.1 16.9 15.6 9.5

3.6 3.O 4.5 6.2 6.7 9.8 5.4 5-6

Ig-BBr-

5.O

0.1

3.9 2.4 4.6 2.8 4.4 9.2 7.7

45.1 56.0 70.3 55.1 54.2 35.3 60.0 0.2 53.8

Ig-BBr'

Lymph Node

78.5 76.7 80.1 83.4 85.O 68.2 74.2 78.0

37.3

-

49.5 34.4 23.2 37.8 36.8 51.7 27.4

Ig'BBr-

2.5 0.5 7.7

5.5

14.6 15.0 3.9 4.6 7.6

1.2 6.6 2.0 0.8 2.3 2.9 7.2 0.3 3.3

Ig'BBr'

* At least 500 cells were examined per group for double fluorescence. Controls with NRIgG were not systematically counted for each individual mouse. No lymphocytes picked up NRIgG in pools of five normal or five nude BALB/c spleen or lymph node cells. However, some lymphocytes from one normal NMRI and one nude NMRI bound significant amounts of NRIgC. They were counted and their percentages are included in the Table: NMRI-2t and NMRI-nu-2.t

MEAN

Ig-BBr-

1 2 3 4 5 1 2 2t

NMr-nu

1

BALB/c-nu

MEAN

I

NMRI

1

BALB/c

Animals Cells

TABLE 1 FREQUENCY OF LYMPHOCYTES WITH SURFACE Ig AND/OR BBr MARKERS IN SPLEEN AND LYMPH NODE OF NORMAL AND NUDE MICE (%)*

2

o

-+

2

B3

2 +

3

3

3; ;

k

h)

w

h)

Loor & Roelants: Prethymic T-cell Differentiation

233

B cells are more frequent in nudes (78%) than in normal mice (37%), and far fewer lymphocytes are of T lineage (nudes 5%, normal 54%). The frequency of the two other cell types is lower than in the spleen but again higher in nudes than in normals (nudes: 9% Ig-BBr- and 8% Ig+BBr+; normal mice: 6% Ig-BBr- and 4% Ig'OBr'). As in normal mice, in nude mice, RlgC-cu-MOBr also appears t o bind t o the 0 antigen itself: indeed, when spleen cells from a pool of BALB/c-nu wzre preincubated with A K R a 4 C 3 H alloantiserum diluted 1 : 40 for 30 min a t 0 C, there was an 84% reduction in the number of cells that bound detectable amounts of RIgGa-MBBr (as compared t o preincubation with normal AKR serum). NRIgG did not bind detectably t o the cells, except in two cases: in one NMRI mouse and one NMRI-nu mouse rare cells picked up NRIgG, most of them stained with antimouse Ig reagents (see TABLE 1). These false positive cells accounted for a quarter of double positives found when the RIgC-cu-MBBr was used o n the cells from these t w o animals. It should still be pointed out that when used in direct IF, the TRITC-RIgGa-MBBr is not better than the TRITC-AKR IgGa-BC3H, because it could only be detected on occasional lymphocytes (0.5-1.5%) in spleen and lymph nodes from the nude. Thus, the -20% of Ig-BBr+ cells in t h e nude spleen are not equivalent t o the -40% of Ig-BBr+ cells in the normal spleen; the latter are also detected as 0' by direct staining, whereas the former are not. Cell Population Analysis in Spleen f r o m Normal Mice, Nudes, and T-Reconstituted Nudes f o r the Presence o f Thymus Leukemia ( T L ) Antigen4

'

Heteroantibody with a-TL activity and fluorochrome-conjugated CY-TL alloantibody are not available. The possible presence of T L determinants on some nude spleen cells was approached in an indirect way by incubating the cells first with normal mouse serum (NMser) or a-TLser, then with fluorescent a-MIg reagents. Thus, if T L is expressed on some cells that d o not have membrane Ig, one can expect an increase in the number of cells detected when a-TLser is used instead of NMser. A similar method has already shown the presence of 8' cells in nude spleen, Specificity controls consist of the same procedure performed o n spleen cells from TL- mice. Results are shown in TABLE 2. For comparison, t o show the possibilities and the limits of this methodology, the same cell suspensions have also been assayed for Bby various methods, which include the same as used for TL. Compared t o the sequential incubation with NMser + a-Mlg, the procedure a4 + a-MIg stains many more cells (20-30%) in the spleen of normal BALB/c, normal C57BL/6, and T-reconstituted C57BL/6-nudes (i.e., C57BL/6-nu that were grafted with C57BL/6 neonatal thymus 8 weeks previously' 6 , 1 9 ) ; more cells (7-1576) are also detected in the spleen of BALB/c-nu and C57BL/6-nu. Due t o the technology used, these cells can only be Ig-8'. The rabbit a4 detects more cells, because both Ig-BBr+ and Ig+BBr+ are detected, and no attempts were made here t o distinguish them. The directly conjugated mouse 01-19 does not appear t o detect all 8' cells. This may be because the direct staining is weaker and also because this particular fluorescent a4 was not very potent for detecting peripheral T cell, although it was good for detecting thymus cell, as was found afterward. Spleen cells of normal mice and T-reconstituted nudes d o n o t show any TL.

41.5 42 44 26 28 40 32.5

ND

ND

ND 0.6 0.9 18.0 20.0

:By procedure

Ig'OBr'

90.5 83 79 90.5 91.5

23.5 I2 7 24 20.5

29

15

83 92

8 13

Cells by AKRSeraOC311 + TRITCSlgGa-MIg B 93 85

0' Cells by Difference B-A

Indirect Staining$ lg' Cells + B +

C

65 82 68 60.5 69

64

82

ND 83.5

Mk

Ig+ Cells + TL+ Cells by a-TLSer + TRITC-SIgCa-

-2

4

-4

-2 11

1

14

ND 11.5

TL+ Cells by Diffcrencr C-A

and Ig-OBr' cells are included. A, all cells with niembranr It: + a l l cells with I;c receptor$ capable of picking up Ig from the NMSer arc detected: by procedure B, all cells with membrane lg + all cells coated with a 8 antibody are detected; and by procedure C , all crlls with membrane Ig + all cells with enough a-TL antibody hound to the surface are detected. SIX te\t for details. 6 T-reconstituted C57BL/6-nu, that is, grafted with a neonatal C57BL/6 thymus, 8 weeks before the e\perinient. Thymocytes were TL -.

t

66.5 71

72

67 71

63

68

A 85 72

Ig' Cclls + I'c Receptor Cells by NMSer + TRITC SIgG a - MIg

effeciency o n peripheral T cells.

ND ND

Rkt

OBI' Cells by RIG*-MOBr + TRITC-SIgCa-

TABLI 2 AND TL' CELLS IN VARIOUS MICE, AS OBTAINED BY VARIOUS TECHNIQUES (70)

ND ND

0'

* Thc fraction of 04 used here was found t o have poor

Onc BALB/c-nu Onc BALB/c-nu Pooled 2d + 29 BAl.B/c-nu Poooled 2.3 + 29 BALB/c Pooled 2.3 + 29 C57BL/6 Onc C57BL/6-nu One C57BLl6-n~ One TC57BL/6-nu$ One TC57BL/6-nu $

Mice

8' Cells by TR ITC-AKR-IgGolBC3H*

1;REQUENCY 01;

Loor & Roelants: Prethymic T-cell Differentiation

235

When there was a deviation from controls, it was a decrease in the percentage of positive cells, presumably because NMser was not ultracentrifuged nor adsorbed on cells, as was the a-TLser; thus, it could bind to cells without Ig but with Fc receptors, whereas a-TL could not. BALB/c-nu showed a definite presence of TL' spleen cells, and one C57BL/6-nu did also, whereas the other C57BL/6-nu did not. This result will be discussed below. Cocapping of BBr and Ig b y a-Ig on Ig+BBr+ Cells

Normal and nude mouse cells wzre first treated with FITC-SIgGir-MIg, washed, put in capping cond$ions ( 3 7 C, no NaN3) for 10-30 min, transferred t o noncapping conditions (0 C, 10 mM NaN3), and treated in sequence with RIgGir-MBBr and TRITC-SIgGir-RIg. As can be seen in TABLE 3, after 30 min in capping conditions, virtually all the Ig' cells show the surface Ig in caps. Quite surprisingly, on most doubly positive cells, the anti-Ig treatment induced cocapping of Ig and BBr. However, none of the Ig-BBr' cells showed the BBr in cap. After clearance of surface Ig by capping, normal and 2ude mice spleen cells were resuspended in normal mouse serum for 30 min at 37 C and then stained as usual for surface Ig. All lymphocytes remained perfectly negative for surface Ig: they were unable t o pick up any detectable amounts of mouse Ig. After similar clearance of surface Ig, macrophages pick up large amounts of cytophilic antibody when treated in a similar way.40 DISCUSSION Though they lack a thymus and thymus-processed lymphocytes, nude mice definitely have cells of T lineage. Their detection may be achieved in various ways: (a) they appear very faintly stained by direct staining with

TABLE 3 DISTRIBUTION OF SURFACE Ig AND eBr AFTER CAPPING WITH ANTI-Ig REAGENTS* Cell Type

Surface Markerst Ig e Br

Ig-OBrIg-OBr'

-

-

-

+ NC

-

+C

Ig'OBr-

+ NC +C + NC + NC +C +C

-

Ig'OBr'

-

+ NC +C + NC +C

Proportion of Cells (%)$ Balb/c§ Balb/c-nu§ 7.2 49.1 0 1.o

38.9 0 0 0.7 3.0

26.4 13.2 0 0 43.8 1.4 0 0.3 14.9

* For experimental procedures, see text. t C: redistribution in polar caps, that is, on less than one half of the cell surface; NC: no redistribution in polar caps. $ About 500 cells of each group were examined for double fluorescence. 5 Pool of three spleens.

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fluorochrome-labeled a8 reagents (either alloantibodies or heteroantibody) ; (b) clear detection can be achieved with the high amplification of indirect immunofluorescence. When a mouse a8 reagent is employed as the first step, the fluorescent a-MIg reagent used as the second step also stains the B cells through their surface Ig, and the number of 0’ cells has to be inferred from the difference between the number of stained cells whether the first step is a 8 s e r or NMser. In this way, about 10-2076 of nude mouse spleen lymphocytes appear to have the B marker and no surface Ig. However, when the in uivo adsorbed rabbit antimouse brain-associated B(RIgCa-MBBr) is used as the first step, the fluorescent sheep*-rabbit Ig reagent detects only the cells with BBr, and one can simultaneously obtain a distinct detection of the B cells through their membrane Ig by a sheep*-mouse Ig reagent labeled with another fluorochrome. Controls made with NRIgG (also adsorbed in v i m ) instead of RIgCa-MBBr showed that the methodology for double staining is fully satisfactory. With the double immunofluorescence procedure, four categories of cells were found, both in normal and in nude mice: typical B cells (Ig+BBr-) and cells that definitely pertain to the T lineage (Ig-BBr+), but also cells that lack detectable amounts of both markers (Ig-BBr-) and cells that express both (Ig+BBr+). It should be emphasized that a classification of a cell as negative for a surface marker may depend on the sensitivity of the technique used. In the present case, even the weakest cells stained with the various fluorescent reagents are quite definitely positive; the background is nul. This is even more striking when sandwich techniques are used. Thus, even if the cells classified as negative for a given marker would possess some of this marker at a level undetected by our methodology, they would nevertheless be quite different from cells classified as positive. Ig+BBr- cells in normal and nude mice appear to be “typical B lymphocyte^"^^ and do not require many comments; they are not more frequent in the spleen of nudes than in the spleen of normal mice, but in nude they constitute the large majority. lymph nodes and in their thoracic Ig-BBr+ cells definitely pertain to the T lineage. The RIgGa-MBBr binds to the same cells as a8 alloantibodies, has the same distribution-redistribution patterns, and its binding can be blocked by the preincubation of the cells with 018 alloantibodies, both in nude and normal mice. Thus, it is likely that the RIgGa-MBBr does not recognize some kind of undefined “stem cell” antigen42*43but that it binds actually to the B antigen itself o r to a determinant closely associated with it on the cell surface. However, the 20% Ig-BBr+ cells found in nude spleen are not equivalent t o the 40% Ig-BBr+ cells found in normal mouse spleen, because the latter can also be clearly detected as 0’ by direct IF with our best fluorescent a8 alloantibodies, whereas the majority of the former cannot. These cells have definitely less B antigen than thymus-processed T cells, and it is so far unknown whether they are potential precursors, that is, able to differentiate into active T cells upon thymus i n f l ~ e n c e ~or~ cells ~ ’ ~ of~ ~T ~ lineage that have followed an abortive differentiation pathway because of the lack of thymus. Both Ig-BBr- and Ig+BBr+ cells are more frequent in nudes than in normal mice. However, nudes have less lymphocytes, and we do not know how to relate the higher frequency of double-negative and double-positive cells in nudes with their absolute number per mouse, It is, of course, difficult to know if lymphocytes that lack both markers are of T or of B lineage. They have electrophoretic characteristics that are intermediate between those of most B and T cells.44 Ig-BBr- might be still

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“immature precursors” that have not yet expressed their differentiation antigens or that are not yet committed to follow a B or a T differentiation pathway. Despite their appearance of small lymphocytes, some may even pertain to the erythroid series.45 Double-negative cells have already been d e ~ c r i b e d , ~ ~ ~ ~ ~ but that they might be related t o the T-cell lineage was rejected47 on the assumption that the only cells of T lineage homing in the spleen have t o come from the thymus. If the spleen were a transit pathway of T precursors coming from the bone marrow, this might result in their accumulation in the spleen of normal thymectomized, congenitally athymic mice, and immunodeficient (thymus deficient?) strains of mice (NZB and NZW mice). However, the higher frequency in nude mice of Ig-BBr- cells does not (and even their higher absolute amounts would not) necessarily argue for their belonging to the T lineage, because the maturation of all lymphocytes in nudes, both T and B, might be retarded o r their migration pattern altered. It should be remembered that the germinal centers, which are B-cell areas of secondary lymphoid organs,8 are not organized in nudes9 and that they are under thymic influence, as 6hown by the fact that germinal centers become reorganized after thymus grafting.’ Some Ig-BBr- cells could thus simply be immature B cells, because evidence exists that B cells bear varying amounts of surface Ig.48 They might be Ig-BBrwhen leaving the bone marrow and progressively express more Ig in t h e periphery. The significance of Ig+BBr+ cells is still more difficult t o evaluate; they might be cells of T lineage with cytophilic immunoglobulins of B-cell origin, o r cells of B lineage with Fc receptors to which the RIgCa-MBBr is adsorbed (though this should be excluded as the main cause, as NRIgG is usually not picked up), or cells that actually synthesize both Ig and 8 markers, o r even cells that acquire both markers passively. Unlike macrophages, double-positive cells d o not take up cytophilic antibody after clearance of surface Ig by capping, but it is not clear whether their surface Ig is really the product of the cell itself. F, receptors might have been removed from the cell membrane by capping and not yet resynthesized during the incubation with NMIg, whereas f o r macrophages, rapid reexposure of membrane components has been shown.38 Moreover, a t least in NMRI mice, some uptake of NRIg was observed; absorption of RlgGa-MBBr was done in BALB/c-nu and antibody that reacts with some NMRI constituents might still be present. It should be stressed again that in those mice, NRlg bound t o Ig+ cells and that t h e specificity of RlgGa-MBBr in staining lg-BBr+ cells is not in doubt. Experiments of clearance and resynthesis similar t o those used t o study T-cell receptors39 still need t o be performed to solve this puzzle. In addition t o the fact that Ig+f?Br+ cells simultaneously bear what are recognized as a B and a T marker, another puzzling characteristic of these cells is the cocapping of Ig and BBr induced by anti-lg. Cocapping is found only o n those cells, and anti-lg does not affect the distribution of BBr on the Ig-BBr+ cells at all. These double-positive cells and the cocapping could not be demonstrated for purely methodologic reasons, as long as a8 alloantibodies were used. As discussed for double-negative cells, Ig+BBr+ cells may also be precursor cells with still both T and B pathways of differentiation open, o r cells of T lineage that follow an abnormal differentiation pathway, or even changing t o a B function, because of the lack of thymic influence. Whatever the significance of the various cells described, nude mice can no longer be considered as a source of pure B cells, especially when spleen lymphocytes are used. It is true that the other cell types d o not seem to present any detectable T-cell reactivity, at least as long as they are studied in o r isolated t4’

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from a “resting” nude (reviewed in Reference 2 ) , and that grafting a whole thymus epithelium seems required t o allow important recovery of nude host-derived T-cell reactivity.’ However, the presence of the other cell types cannot be ignored either, because many stimuli might turn on the T precursor ~ e l l s . ~Spleen ~ - ~cells ~ from nude mice are thus dangerous t o use as a tool to study mechanisms of B-lymphocyte triggering, and one has t o take with caution conclusions about the role of some mitogenic lectins (reviewed in Reference 49) and of T-cell replacing factors and allogeneic T lymphocytes (reviewed in Reference SO), because those agents might not act directly or only o n B cells. Conversely, the increased susceptibility of nude mouse spleen cells t o cytotoxic anti-T lymphocyte antisera after various treatments may not be due to an actual synthesis of T markers, that is, a real differentiation event, as claimed by Scheid et ~ l . but , ~rather ~ t o a redistribution of the markers already present o n the cell surface, for example, clustering, which allows more efficient binding of antibody and complement. In this context, one could expect some other T - o r thymus-specific antigens t o be expressed also. The presence of T L was investigated, but the results are less clear-cut for technical reasons. It is known that BALB/c-nu are able t o develop thymocytes that express thymus leukemia (TL) antigens4’ when a thymus structure is providedI6 (BALB/c is a TL’ strain). It seems that some T L is expressed o n some 10- 15% Ig- cells in the spleen. N o significant numbers of Ig-TL’ cells could be found in spleen from normal BALB/c, normal C57BL/6 (a TL- strain) and two individual T-reconstituted C57BL/6-nu (whose thymuses were shown to be TL-). However, one CS7BL/6-nu showed T L in the spleen; the other one did not. This might be due t o the fact that these “CS7BL/6-nu” were only backcrossed three times to C57BL/6; thus, a large segment of the genome was not yet C57BL/6, and statistically the TL- characteristic was not established for all individual mice. Because the a-TLser used was raised against TL’ leukemia, there is still a risk that the a-TLser detects some fetus-specific antigens, common to leukemic cells and t o some undifferentiated spleen cells. Our control with C57BL/6-nu should be repeated o n more individual C57BL/6-nu or on further backcrosses t o be fully satisfactory. Further characterization of all the different cell classes detected is clearly required, but it is reasonable to assume that they represent stages o n a pathway of differentiation t o mature B and T lymphocytes. In any case, since we could find antigen-binding cells among all four lymphocyte classes in the nude (as well as in normal mice),51 it is interesting t o note that the expression of T-cell receptors for antigen does not seem to be thymus dependent. We propose as the likely candidate for the function of T precursor a cell with no membrane lg detectable by IF but with receptor for antigen, with a low density of 0 detectable only b y indirect IF and low enough t o make the cell insensitive to a8 + C ‘ 5 2 and that has some T L antigen in TL’ strains. The characterization of the physical and functional properties of the various lymphocyte types described in this study as well as their organ distribution and migration patterns in nude and normal mice is currently in progress.53 SUMMARY A rabbit antimouse brain 0 reagent was made specific for cells of the T lineage b y absorption in vivo in nude mice. When used in double fluorescence together with an antimouse immunoglobulin reagent, four types of cells were

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found in spleen and lymph nodes of both normal and nude mice: Ig+BBr-, Ig-BBr+, Ig-BBr-, and Ig+BBr+. The data show that about 20% of nude mouse spleen lymphocytes are definitely of T lineage (Ig-BBr+). O n these cells, the detection of t h e “BBr” determinant, which is identical or very close t o the ‘‘0” determinant, depends o n the large amplification produced by indirect immunofluorescence, which suggests a low density of Bantigen. Similar experiments suggest the presence of cells that express some T L antigen in the spleen of nudes made congenic t o a TL+ strain (BALB/c). It is proposed that the T-cell precursor that will further differentiate in the thymus already expresses a low density of 0 and, in TL’ strains, T L antigen, ACKNOWLEDGMENTS We appreciate the outstanding technical assistance provided by Miss L.-B. Hagg, Mrs. K. S. Mayor, and Miss A. RydCn. REFERENCES 1. FLANAGAN, S. P. 1966. Nude; a new hairless gene with pleiotropic effects in the mouse. Genet. Res. (Cambridge) 8: 295-309. 2. PANTELOURIS, E. M. 1973. Athymic development in the mouse. Differentiation 1: 437 -450. 3. PANTELOURIS, E. M. 1968. Absence of thymus in a mouse mutant. Nature (London) 217: 370-371. 4. PANTELOURIS, E. M. & J. HAIR. 1970. Thymus dysgenesis in nude (nunu) mice. J. Embryol. Exp. Morphol. 24: 615-623. 5. CORDIER, A. C. 1974. Ultrastructure of the thymus in “nude” mice. J. Ultrastruct. Res. 47: 26-40. 6. PARROTT, D. W. V., M. A. B. d e SOUSA & J. EAST. 1966. Thymusdependent areas in the lymphoid organs of neonatally thymectomized mice. J . Exp. Med. 123: 191-204. 7 . De SOUSA, M. A. B., D. M. V. PARROTT & E. M. PANTELOURIS. 1969. The lymphoid tissues in mice with congenital aplasia of the thymus. Clin. Exp. Immunol. 4: 637-644. 8. GUTTMAN, G. A. & 1. L. WEISSMAN. 1972. Lymphoid tissue architecture: experimental analysis of the origin and distribution of T cells and B cells. Immunology 23: 465-479. 9. MITCHELL, J., J. PYE, M. C. HOLMES & G. J. V. NOSSAL. 1973. Antigens i n immunity; antigen localization in congenitally athymic nude mice. Austral. J. Exp. Biol. Med. Sci. 50: 637-650. 10. WORTIS, H. H., S. NEHLSON & J. J. OWEN. 1971. Abnormal development of the thymus in “nude” mice. J. Exp. Med. 134: 681-692. 11. BASTEN, A., J. F. A. P. MILLER, J. SPRENT & J. PYE. 1972. A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J. Exp. Med. 135: 610-626. 12. SPRENT, J. & J. F. A. P. MILLER. 1972. Thoracic duct lymphocytes from nude mice: migratory properties and life-span. Eur. J . Immunol. 2: 384-387. 13. MILLER, J . F. A. P., A. BASTEN, J. SPRENT & C. CHEERS. 1971. Interaction between lymphocytes in immune responses. Cell. Immunol. 2: 469-495. 14. R A W , M. C. & J. J. T. OWEN. 1971. Thymus-derived lymphocytes: their distribution and role in the development of peripheral lymphoid tissues in the mouse. Eur. J. lmmunol. 1: 27-30. 15. R A W , M. C. 1971. Surface markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6: 52-80.

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16. LOOR, F. & B. KINDRED. 1973. Differentiation of T cell precursors in nude mice demonstrated by immunofluorescence of T cell membrane markers. J. Exp. Med. 138: 1044-1055. 17. PRITCHARD, H. & H. S. MICKLEM. 1973. Haemopoietic stem cells and progenitors of functional T lymphocytes in the bone marrow of ‘‘nude’’ mice. Clin. Exp. Immunol. 14: 597-607 18. De SOUSA, M. & H. PRITCHARD. 1974. The cellular basis of immunological recovery in nude mice after thymus grafting. Immunology 26: 769-776. 19. KINDRED, B. & F. LOOR. 1974. Activity of host derived T cells which differentiate in nude mice grafted with co-isogenic or allogeneic thymuses. J. Exp. Med. 139: 1215-1227. 20. DARDENNE, M. & J. F. BACH. 1973. Studies on thymus products. I. Modification of rosette-forming cells by thymic extracts. Determination of target RFC sub-population. Immunology 25: 343- 35 2. 21. BACH, M. A. & J. F. BACH. 1973. Studies on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette-forming cells. Interaction with thymic factor. Eur. J. Immunol. 3: 778-783. 22. KOMURO, K. & E. A. BOYSE. 1973. Induction of T lymphocytes from precursor cells in vitro by a product of the thymus. J. Exp. Med. 138: 479-482. 23. SCHEID. M. P., M. K. HOFFMANN, K. KOMURO, 0. HAMMERLING, 1. ABBOTT, E. A. BOYSE, G. H. COHEN, J. A. HOOPER, R. S. SCHULOF & A. L. GOLDSTEIN. 1973. Differentiation of T cells induced by preparations from thymus and by non-thymic agents. The determined state of the precursor cell. J . Exp. Med. 138: 1027-1032. 24. BASCH, R. S. & G. GOLDSTEIN. 1974. Induction of T-cell differentiation in vitro by thymin, a purified polypeptide hormone of the thymus. Proc. Nat. Acad. Sci. USA 71: 1474-1478. 25. WEKERLE, H., I. R. COHEN & M. FELDMAN. 1973. Thymus reticulum cell cultures confer T cell properties on spleen cells from thymusdeprived animals. Eur. J. Immunol. 3: 745-748. 26. WAKSAL, S. D., I. R. COHEN, H. W. WAKSAL & R. L. St. PIERRE. 1974. In vitro differentiation of T cells: interaction of T cell precursors and thymus epithelium. Fed. Proc. 33: 736 (Abs.). 27. LAMELIN, J. P., B. LISOWSKA-BERNSTEIN, A. MATTER, J. E. RYSER & P. VASSALLI. 1972. Mouse thymus independent and thymusderived lymphoid cells. I. Immunofluorescent and functional studies. J. Exp. Med. 136: 984-1007. 28. RAFF, M. C. 1973. &Bearing lymphocytes in nude mice. Nature (London) 246: 350, 351. 29. RAFF, M. C. & H. H. WORTIS. 1970. Thymus dependence of &bearing cells in the peripheral lymphoid tissues of mice. Immunology 18: 931 -942. 30. GOLUB, E. S. 1971. Brain associated 0 antigen: reactivity of rabbit anti-mouse brain with mouse lymphoid cell. Cell. Immunol. 2: 353-361. 31. LOOR, F. & G. E. ROELANTS. 1974. High frequency of lymphocytes o f T lineage in nude mouse spleen. Nature (London) 251: 229,230. 32. LOOR, F., L. FORNI & B. PERNIS. 1972. The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur. J. Immunol. 2: 203-212. 33. WOOD, B. T., S. H. THOMPSON & G. GOLDSTEIN. 1965. Fluorescent antibody staining. 111. Preparation of fluorescein-iso thio cyanate-labelled antibodies. J. Immunol. 95: 225-229. 34. CEBRA, J. J. & G. GOLDSTEIN. 1965. Chromatographic purification of tetramethylrhodamhe-immune globulin conjugates and their use in cellular localization of rabbit yglobulin polypeptide chains. J. Immunol. 95: 230-245. 35. REIF, A. E. & J. M. V. ALLEN. 1966. Mouse thymic isoantigens. Nature (London) 209: 521-523. 36. LOOR, F., N. BLOCK & J. R. LITTLE. 1975. Dynamics of the thymus-leukemia (TL) antigen on thymus and leukemic cells. Cell. Immunol. In press.

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37, ROELANTS, G. E., L. FORNI & B. PERNIS. 1973. Blocking and redistribution (“capping”) of antigen receptors on T and B lymphocytes b y anti-immunoglobulin antibody. J . Exp. Med. 137: 1060-1077. 38. SHINEGO, N., C. ARPELS, U. HAMMERLING, E. A. BOYSE & L. J. OLD. 1968. Preparation of lymphocyte-specific antibody from anti-lymphocyte serum. Lancet II: 320-323. 39. ROELANTS, G. E., A. RYDEN, L.-B. HAGG & F. LOOR. 1974. Active synthesis of immunoglobulin receptors for antigen by T lymphocytes. Nature (London) 247: 106 - 108. 40. LOOR, F. & G. E. ROELANTS. 1974. The dynamic state of the macrophage plasma membrane. Attachment and fate of immunoglobulin, antigen and lectins. Eur. J. Immunol. 4: 649-660. 41.BOYSE, E. A., L. J. OLD & E. STOCKERT. 1972. The relation of linkage group IX to leukemogenesis in the mouse. I n RNA Viruses and Host Genome in Oncogenesis. P. Emmelot & P. Bentvelzen, Eds: 171-185. North Holland Publishing Co. Amsterdam, The Netherlands. 42. GOLUB, E. S. 1972. Brain-associated stem cell antigen: an antigen shared by brain and hemopoietic stem cells. J. Exp. Med. 136: 369-380. 43. RODT, H., S. THIERFELDER & M. EULITZ. 1974. Anti-lymphocyte antibodies and marrow transplantation. 111. Effect of heterologous anti-brain antibodies o n acute secondary disease in mice. Eur. J. Immunol. 4: 25-29. 44. von BOEHMER, H., K. SHORTMAN & G. J. V. NOSSAL. 1974. The separation of different cell classes from lymphoid organs. X. Preparative electrophoretic separation of lymphocyte subpopulations from mouse spleen and thoracic duct lymph. J. Cell. Physiol. 83: 231-242. 45. MKULLOCK, E. A. & J. E. TILL. 1971. Regulatory mechanisms acting on hemopoietic stem cells. Some clinical implications. Annu. J. Pathol. 6 5 : 601-619. 46. STOBO, J. D., N. TALAL & W. E. PAUL. 1972. Lymphocyte classes in New Zealand mice. II. Decreased frequency of immunoglobulin-bearing lymphocytes and increased frequency of lymphocytes lacking detectable 6 or immunoglobulin determinants. J . Immunol. 109: 701-710. 47. STOBO, J. D., A. S. ROSENTHAL & W. E. PAUL. 1973. Functional heterogeneity of murine lymphoid cells. V. Lymphocytes lacking detectable surface e or immunoglobulin determinants. J. Exp. Med. 138: 71-87. 48. OSMOND, D. G. & G. J. V. NOSSAL. 1974. Differentiation of lymphocytes in mouse bone marrow. 1. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues. Cell. Immunol. 13: 117-131. 49. MOLLER, G. (Ed.) 1972. Lymphocyte activation by mitogens. Transplant. Rev. 11. 50. MOLLER, G. (Ed.) 1972. Stimulation of lymphocytes by allogeneic cells. Transplant. Rev. 12. 51. LOOR, F. & G. E. ROELANTS. 1974. Receptors for antigen on “B,T, B-T and Nul” lymphocytes in normal and nude mice. hi The Immune System. E. E. Sercarz, A. R. Williamson & C. F. Fox, Eds.: 201-215. Academic Press, Inc. New York and London. 52. EL-ARINI, M. 0. & D. OSOBA. 1973. Differentiation of thymusderived cells from precursors in mouse bone marrow. J. Exp. Med. 137: 821-837. 53. ROELANTS, G. E., F. LOOR, H. von BOEHMER, J. SPRENT, L.-B. HAGG, K. S. MAYOR & A. RYDEN. 1975. Five types of lymphocytes (Ig-0-, Ig-tPWeak, Ig-B+strong, Ig+B- and Ig+B+) characterized by double immunofluorescence and electrophoretic mobility. Organ distribution in normal and nude mice. Eur. J. Immunol. In press.

Immunofluorescence studies of a possible prethymic T-cell differentiation in congenitally athymic (nude) mice.

A rabbit antimouse brain theta reagent was made specific for cells of the T lineage by absorption in vivo in nude mice. When used in double fluorescen...
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