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The null cell compartment of the mouse spleen
CELLULAR
IMMUNOLOGY
63, 106-117 (1981)
The Null Cell Compartment JEREMIAH The Houston
of the Mouse Spleen
J. TWOMEY AND NICOLA M. KOUTTAB
Veterans Administration Medical Center, and Baylor Houston, Texas 7721 I Received February
21. 1981; accepted April
College of Medicine.
29, 1981
Null lymphocytes were defined as lymphocytes without detectable T- or B-cell markers using a battery of techniques. The null cell compartment was divided into pre-T cells, pre-B cells, and other null cells based upon their acquisition of membrane markers when incubated with ubiquitin. The null ceil subpopulations were remarkably consistent in spleen cell suspensions from young adult mice of various strains. Commitment to T- or B-cell differentiation took place at the null cell stage and did not require thymic input. Pre-T cells, but not pre-B cells, were steroid sensitive. Pre-T cells accumulate with congenital thymic deficiency. This differed from senescentthymodeprivation where the outstanding finding was an accumulation of uninducible null cells. Neonatal mouse spleens were deficient in pre-T and pre-B cells but had an accumulation of uninducible cells.
INTRODUCTION Mature lymphocytes are derived from a pool of precursor cells. Some precursors may be committed to the T- or B-lymphocyte series before the onset of recognizable differentiation (1). Subpopulations of lymphocytes acquire surface markers and functions during maturation (2-5). the overall processof lymphocyte differentiation involves a complex series of events that are modulated to a greater or lesser degree by a number of inducing agents (6, 7). Lymphoid cells that lack detectable surface markers are arbitrarily termed null lymphocytes (8). These null cells include committed precursors of T or B lymphocytes and other, probably heterogeneous, cells. In the mouse, null cells comprise about 50% of bone marrow lymphocytes (9), 3-l 3% of splenic lymphocytes (8,lO) and O-5% of lymph node lymphocytes (8, 10). There has been no systematic evaluation of this null cell compartment apart from the recognition of some lymphocytes that have undergone partial B-cell differentiation; these are identified by the presence of small amounts of cytoplasmic (but not surface) P chains (11). The composition of the null cell compartment could be altered through a number of mechanisms. It could fail to sustain itself through lowered input from primitive precursors or impaired null cell replication. Alternatively, accelerated differentiation of null cells could outstrip regenerative reserves and thereby deplete the null cell pool. The null cell compartment could be expanded by increased input from primitive precursors, accelerated null cell mitosis, or reduced differentiation toward more mature cells. The latter could result from intrinsic unresponsiveness by null cells to inductive stimuli, a lack of inductive stimuli, or an unfavorable microen106 OOOS-8749/81/130106-l2802.00/0 Copyright 0 1981 by Academic F’ress. Inc. All rights of reproduction in any form rcscrvcd.
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vironment for differentiation to take place. Abnormalities could be limited to one or more subpopulations within the null cell compartment. Disorders of more mature lymphocytes may have their origins within the null cell compartment, as exemplified in man by severe combined immunodeficiency (12). This paper introduces an approach that recognized three subpopulations within the null cell compartment of the mouse spleen. These subpopulations include null cells that acquire T- or B-cell surface markers when optimally induced with ubiquitin and other null cells that are unresponsive to ubiquitin. Ubiquitin is a tissue peptide which, at the concentration employed, stimulates both T- and B-cell differentiation via P-adrenergic receptors (13, 14). We found that the composition of the null cell compartment is remarkably consistent in healthy young adult mice of different strains but is altered during the neonatal period and with advanced age. The latter cannot be ascribed to thymic involution since a different pattern was observed in congenitally athymic mice. MATERIALS
AND METHODS
Mice and cell preparations. Studies were performed on C3H/He and DBA/2 mice purchased from TIMCO Breeding Laboratories, Houston, Texas, C57BL/ 1, CBA/T6 AKR/J, and Balb/c mice purchased from Jackson Laboratories, Bar Harbor, Maine, and our colony of germ-free nude athymic mice bred on a C3H/ He background. Neonatal mice were studied 3-5 days after birth. Mice were sacrificed by cervical dislocation, their spleens removed aseptically and weighed, and cells freed by gentle teasing. Since our interest was’ in overall spleen cell composition, no attempts were made at cell fractionation. Fresh smears were prepared and stained with a-naphthyl acetate esterase (Sigma Chemical Co., St. Louis, MO.) (15) or Wright-Giemsa stains. Percentages of macrophages, hematopoietic cells, granulocytes, and lymphoid cells were determined from counts of 300 cells. Reagents. Thymopoietin, a thymic peptide of known amino acid sequence (16) and ubiquitin were purified using published techniques (13, 14, 16, 17). Antiserum to Thy 1.2 antigen was raised by immunizing AKR/J mice with C3H/He thymocytes (18). Fluorescein-labeled monoclonal IgM antibody to Thy 1.2 was purchased from New England Nuclear, Boston, Massachusetts. Adsorbed, fluoresceinconjugated antiserum to C3H/He mouse brain raised in rabbits was purchased from Litton Bionetics, Kensington, Maryland. Fluorescein-conjugated goat antisera to mouse ~1,y, or (Y chains were obtained from Meloy Laboratories, Springfield, Virginia. Fluorescein-conjugated rabbit immunoglobulin (1g)G containing antibodies to mouse K chains, unconjugated rabbit IgG containing antibodies to mouse X chains and fluourescein-conjugated goat antiserum to rabbit IgG were purchased from Litton Bionetics or procured from Dr. E. Vitetta. Affinity-purified rabbit antiserum to mouse Ig was prepared as described by Ligler et al. (19). Rhodamineconjugated goat antiserum to mouse ~1chains was obtained from Cappel Laboratories, Cochranville, Pennsylvania. Fresh frozen guinea pig serum that was absorbed with C3H/He mouse thymocytes was used as complement source. All reagents were used at optimal dilutions. Cell identification. T lymphocytes were identified using a number of different methods. In the cytotoxicity test, 2 X lo5 spleen cells in 0.05 ml medium 199 (Gibco,
108
TWOMEY
AND
KOUTTAB
Grand Island, N.Y.) enriched with 5 g% BSA (Sigma) plus 0.025 ml antiserum to Thy 1.2 antigen were incubated in duplicate with 0.025 ml complement or heatinactivated complement. Incubations were for 90 min at 37°C in a humidified atmosphere of 5% CO2 and air and for an additional 30 min after adding 0.625 mg pronase (grade B, Sigma) in 0.125 ml saline. Then 12 ml cetrimide (Eastman Kodak Co., Rochester, N.Y.) was added and cell counts performed using a Coulter counter. Injured cells were solubilized while incubated with pronase (20). This was reflected in lower cell counts in incubations containing complement compared to paired incubations that lacked complement activity. This antibody-specific cytotoxicity test was preferred to dye exclusion or membrane-bound isotope release because it is more sensitive to lesser degrees of cell injury (21). Thy 1.2 antigen was also identified using both direct and indirect immunofluorescence. For direct immunofluorescene, 1 X lo6 spleen cells and optimal concentrations of conjugated monoclonal IgM antibody to Thy 1.2 antigen were kept at 4°C for 30 min, washed, and examined for membrane flourescence using a Leitz Ploem optics uv microscope. For indirect immunofluorescence, 5 X 10’ spleen cells, suspended in BSA-enriched medium 199 were incubated with 0.025 ml unconjugated antiserum to Thy 1.2 antigen for 1 hr at 37°C. After washing twice, the cells were treated with 0.025 ml each of fluorescein-conjugated antiserum to mouse Ig heavy chains and 0.025 ml PBS at 4°C for a second hour, washed twice, and examined for membrane immunofluorescence. This procedure identified both T and B cells. Thus, to enumerate T cells, it was necessary to identify B cells concomitantly and subtract these from combined T- plus B-cell values. Cell suspensions were also reacted with fluoresceinated antiserum to mouse brain for 1 hr at 4°C washed twice, and examined directly for membrane immunofluorescence. Conjugated antisera to p, y, and a! chains were mixed at optimal dilutions to demonstrate heavy-chain determinants on B-cell membranes. Spleen cells (5 X 1OS), suspendedin 0.05 ml BSA-enriched medium 199,0.025 ml of the conjugate mixture and 0.025 ml PBS were kept at 4°C for 1 hr, washed twice, and examined directly for membrane immunofluorescence. There was minimal binding of conjugate by Fc receptors as was evidenced by there being negligible numbers of fluorescing cells after depleting B lymphocytes and macrophages by incubation with nylon wool. When testing for light-chain determinants on cell membranes, K chains were identified by direct immunofluorescence and X chains by indirect immunofluorescence using procedures outlined above. The prescence of membrane C3 receptors was demonstrated using the EAC procedure described by Mendes et al. (22). Cell sorting was done using a series 50-H cytofluorograf (Ortho, Westwood, Mass.). Excitation was done with 400 W of laser power at 488 nm. The photomultiplier tube voltage was set at 435 for direct fluorescence and at 594 for indirect fluorescence. Cells in channels 46-511 were scored positively. Gating on live cells was done using a logarithmic amplifier. The double immunofluorescence method of Raff et al. (23) was used to identify lymphoid cells with Ig limited to their cytoplasm. Cell membrane Ig was first reacted directly with mixed heavy-chain conjugates as described. The cells were then washed twice, fixed at 4°C with 5% acetic acid and 95% ethanol, stained for cytoplasmic Ig by a 1-hr incubation period at 37°C with a rhodamine conjugate to p-chain determinants, washed, and examined for immunofluorescence. Induction incubations. After allowing clumps to sediment, spleen cells were
SPLEEN NULL CELLS
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washed twice and suspended at 5 X lo6 cells/ml in medium 199 plus 5 g% BSA. Incubations were set up in duplicate in 12 X 75-mm polystyrene tubes containing 0.1 ml cell suspension and 0.1 ml medium plus 20 rig/ml ubiquitin, 1 rg/ml thymopoietin, or medium alone. Preliminary experiments indicated that these represented optimal inductive concentrations of ubiquitin and thymopoietin in this incubation system. Incubations were for 18 hr at 37°C in a humidified 5% CO1 and air atmosphere. Kinetic studies showed that most induction took place during the first 2 hr of incubation but maximum induction was achieved most consistently when incubation was extended overnight (24). At the end of 18 hr of incubation, T and B cells were identified by surface markers. Expression of data. All cell populations, except those of the null cell compartment, were identified on fresh spleen cells. First, the total number of cells freed from each spleen was determined for subsequent use in calculating absolute values. Then, percentages of granulocytes, hematopoietic cells, macrophages, and lymphocytes were determined from Wright-Giemsa- and esterase-stained smears. Tcell and B-cell percentages were detected using various procedures outlined above. Pre-T and pre-B cells were defined as null lymphocytes that acquired T- or B-cell membrane markers after overnight incubation with ubiquitin. Pre-T-cell percentages were calculated from the difference in T-cell percentages in overnight incubations with and without ubiquitin. Pre-B-cell percentages were enumerated in a similar fashion. Null lymphoid cells that could not be induced to express detectable T- or.B-cell markers were arbitrarily termed other null cells. Grouped values were expressed as means f SEMs. The statistical significance of values on the same cell preparations that were treated differently was determined using Student’s paired t test. The Wilcoxon rank sum test was used when comparing the same test results performed on different cell preparations. RESULTS The Null
Cell Compartment
in 3-Month-Old
C3H/He Mice
By morphologic and histochemical criteria, fresh spleen cell suspensionsincluded 78 + 1.4% lymphoid cells, 10 k 0.1% granulocytes, 6 f 0.3% macrophages, and 6 f 1.3% hematopoietic cells. About 25% of all spleen cells were T lymphocytes whether detected using Thy 1.2 or mouse brain antiserum (Table 1). Similar values were obtained using cytotoxicity or with direct or indirect immunofluorescence tests. Likewise, similar values were obtained using uv microscopy or a cell sorter. When enumerating B lymphocytes, 40 f 0.8% fresh spleen cells reacted with antisera to Ig heavy chains by uv microscopy and 38% using a cell sorter. When conjugates and uv microscopy were used, 43 +- 0.5% demonstrated membrane light chains. Receptors for C3 were identified on 38 + 1.1% of fresh spleen cells. After 18 hr of unstimulated incubation, 20 + 1.5% spleen cells bore Thy 1.2 antigen and 42 f 0.4% bore Ig heavy-chain determinants. The difference in T-cell percentages after overnight incubation with or without ubiquitin averaged 7% (P < 0.05) irrespective of whether Thy 1.2 or mouse brain antisera was used (Table 1). Similar values for pre-T cells were obtained using cytotoxicity, direct, or indirect immunofluorescence tests and when surface fluorescence was demonstrated by uv microscopy or using a cell sorter. The presence of ubiquitin in overnight incubations also increased the number of spleen cells with
110
TWOMEY AND KOUTTAB TABLE 1 Comparison of Different Methods Used to Identify Lymphocytes
Surface marker
Method for identification
T lymphocytes (%I
Pre-T lymphocytes (%)
Thy 1.2 antigen
Cytotoxicity Indirect IF” Direct IF Direct IF, cytofluorograf Direct IF
25 z!z 1.5 25 * 1.2 25 27 25 5 0.9
7 + 0.1 6 f 0.4 lb Tb 7 + 1.0
Direct IF Direct IF, cytofluorograf K, direct, X, indirect IF EAC rosettes
40 + 0.8 38 43 + 0.5 38 + 1.1
I + 0.9 I 8 + 0.6 6 + 1.1
Brain antigen cc,y, a chains K, X chains Cg receptors
’ IF, immunofluorescence. b Mean of two experiments.
membrane Ig heavy or light chains or receptors for C3 by 5-9% (P < 0.05). Again, similar values for pre-B cells were registered using uv microscopy or a cell sorter. Figure 1 shows quantitative differences in T and B cells and the ranges of fluorescence-registering membrane Thy 1.2 antigen or Ig heavy chains on spleen cells after incubation with and without ubiquitin. In all subsequent experiments, T cells were identified using Thy 1.2 antiserum in a complement-dependent cytotoxicity test system and B cells by the presence of Ig heavy chains using direct fluorescence microscopy. Small quantities of cytoplasmic ~1chains, without detectable membrane Ig, are thought to identify a stage in the ontogeny of B-cell differentiation (11). These comprised 4 + 0.3% of bone marrow cells from 3-month-old mice. While ubiquitin induction significantly increased the number of spleen cells bearing surface Ig, no cytoplasmic p-chain-positive and membrane Ig-negative ceils were observed in fresh spleen cell preparations or after incubation with or without ubiquitin.
h LOG
FLUOREBCENCE
INTENSITY
FIG. 1. A representative cytotluorograf analysis of T and B cells in a spleen cell suspension after overnight incubation with and without ubiquitin. T cells were identified using a monoclonal conjugate for Thy 1.2 antigen and B cells using conjugates for B, y. and a-chain determinants.
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SPLEEN NULL CELLS
FIG. 2. T- and B-cell induction of spleen cell suspensionswith ubiquitin or thymopoietin. T cells were identified by Thy 1.2 antigen in a cytotoxicity system and B cells by heavy-chain conjugates and uv microscopy.
Speci’city of Null Cell Induction Spleen cell suspensions were stimulated with 20 rig/ml ubiquitin or 1 pg/ml thymopoietin. T-Cell induction was 9 f 0.4% with ubiquitin and 6 + 0.4% with thymopoietin (P < 0.05) (Fig. 2). Ubiquitin induced 9 + 0.2% B-cell differentiation while thymopoietin did not induce significant B-cell differentiation (P < 0.001). Thus, ubiquitin is a potent inducer of both T and B cells; thymopoietin is less potent and only induces T-cell differentiation (13, 24). After a single 18-hr incubation period, thymopoietin alone induced T-cell but not B-cell differentiation while ubiquitin induced both T- and B-cell differentiation (Table 2). After two consecutive 18-hr incubation periods with thymopoietin, Tcell differentiation increased to a level that was comparable to that achieved with a single 18-hr incubation period with ubiquitin; there was still no B-cell induction. Consecutive incubations were done first with thymopoietin and then with ubiquitin. The initial stimulus with thymopoietin did not remove null cells capable of B-cell differentiation during the second incubation with ubiquitin. This representative experiment provides additional evidence that cells become committed to the T- or B-cell series during the null cell stage of differentiation. TABLE 2 Differential and Combined T- and B-Cell Induction with Thymopoietin and/or Ubiquitin
Thymopoietin, I rg/ml for 18 hr Ubiquitin, 20 rig/ml for 18 hr Thymopoietin, I pg/ml for I8 hr followed by thymopoietin, 1 pg/ml for I8 hr Thymopoietin, I pg/ml for I8 hr followed by ubiquitin, 20 rig/ml for 18 hr
Pre-T cm
Pre-B @IO)
7 9
I 9
9
I
II
IO
Note. T cells were identified by Thy 1.2 antiserum in a complement-dependent cytotoxicity test and B cells by the presence of Ig heavy chains using immunofluorescence.
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TWOMEY AND KOUTTAB
Null Cell Compartment in Different Strains of Mice Spleen cells from 3-month-old C3H/He, DBA/2, CBA/T6, Balb/c, and C57BL/ 1 mice were incubated with and without ubiquitin and induction of membrane Thy 1.2 antigen and Ig heavy chains was determined. The numbers of pre-T and preB cells were remarkably consistent among these different strains (P > 0.05) (Fig. 3). Age and the Null Cell Compartment The absolute and relative cell compositions of spleens from neonatal, 3-, 8, and 24-month-old C3H/He mice were compared. Spleens from neonatal mice weighed < 10% of spleens from young adult mice which was reflected in low absolute cell counts (Table 3). However, percentages of various cell populations differed significantly in spleens from neonatal and 3-month-old mice (P < .Ol). The neonates had elevated percentages of hematopoietic cells (49 f 6.7 vs 6 + lS%), presumably reflecting a residue from splenic hematopoiesis during intrauterine life. Percentages of both T cells (6 f 0.2 vs 24 f 1.1%) and B cells (9 + 0.4 vs 35 f 0.8%) were greatly reduced in neonatal spleens. However, no significant differences were observed in total null cell percentages. At age 24 months, absolute numbers of splenic hematopoietic cells, granulocytes, macrophages, T cells, and B cells were not significantly different from values recorded at age 3 months (Table 3). However, the total null cell content of the spleen was elevated at age 24 months (P < 0.05). The relationship of age to the composition of the null cell compartment was evaluated. Spleens from neonatal mice contained few T-cell precursors, reduced percentages of B-cell precursors, and greatly elevated percentages of ubiquitinunresponsive null cells when compared with spleens from 3-month-old mice (P < 0.01) (Fig. 4). At age 24 months, percentages of pre-T cells were reduced (P < O.Ol), pre-B cells were comparable and ubiquitin-unresponsive null cells were threefold higher than in spleens from 3-month-old mice (P < 0.01). This age-related increase in ubiquitin-unresponsive null cells appeared to commence at about 8 months of age. 12 r
m
PIE-TCELLS
0
ME-B CELLS
FIG. 3. Pre-T- and pre-B-cell percentages in spleen cell suspensionsfrom different strains of mice. A cytotoxicity system identifying Thy 1.2 antigen and uv microscopy using anti-heavy-chain determinants were used to identify T and B cells, respectively.
113
SPLEEN NULL CELLS TABLE 3 Absolute Numbers of Cell Populations in Mouse Spleens at Different Ages Cells per spleen Age of mice Hematopoietic (months) cells Neonates 3 8 24
9.8 6.4 4.9 5.7
f f + f
2.9 1.7 0.8 0.8
Granulocytes 2.7 8.0 8.3 10.6
f k k +
0.9 0.8 0.6 0.9
Macrophages 0.3 3.6 2.3 2.5
+ 2 + f
0.1 0.5 0.5 0.4
T cells 0.5 15.7 15.0 20.0
B cells
Null cells
1.9 f 0.1 0.8 + 0.1 f 0.8 22.5 + 1.7 12.4 2~ 0.9 24.1 f 1.4 15.9 f 1.8 25.1 + 2.6 28.5
f k -t +
0.2 1.3 1.7 2.2
Note. T cells were identified by membrane Thy 1.2 antigen in a cytotoxicity test and B cells by membrane heavy chains using direct immunofluorescence.
Null Cell Compartment in Athymic Mice The composition of the null cell compartment was studied at age 3 months on congenitally athymic mice, five of whom were germ free and five others were housed conventionally. These spleens lacked detectable T cells but had a normal B-cell content. Spleen cells from germ-free mice included 18 f 1.3% pre-T cells which was significantly higher than the 9 f 0.4% recorded on athymic mice that had been exposed to common environmental stresses (P < 0.01) (Fig. 5). The latter were not significantly different from age-matched C3H/He mice. The splenic content of B-cell precursors and ubiquitin-unresponsive null cells was not altered by the athymic state. Null Cell Compartment and Steroid Sensitivity We injected 5 mg of hydrocortisone acetate into the thighs of 3-month-old C3H/ He mice and studied the null cell compartment 3 days later. inducible T-cell precursors were reduced to 0.3 + 0.3% (P < 0.01) while B-cell precursors were unaltered after steroid injection.
IIEOIIATES
5 8 A&E OF IlICE INOllW
24
FIG. 4. The composition of the spleen null cell compartment at different ages. Pre-T cells were identified by acquisition of Thy 1.2 antigen in a cytotoxicity system and pre-B cells by acquisition of membrane Ig heavy chains using direct immunofluorescence.
114
TWOMEY AND KOUTTAB 20
r
T
m 0
PIE.1 CELLS ME-0 CELLS
GEMFREE COWYENTlOllAllZED CSRih WUDES NUDES
FIG. 5. The effect of congenital thymic deficiency and a germ-free environment upon the spleen null cell compartment. Pre-T cells were enumerated using antiserum to Thy 1.2 antigen and complementdependent cytotoxicity and pre-B ceils using anti-heavy-chain conjugates and direct immunofluorescence.
DISCUSSION This study indicates that the null cell compartment of the mouse spleen can be divided into three supopulations by differentiation responses to ubiquitin. Similar values were obtained with a number of cell surface markers and techniques. The number of null cells that acquired T- or B-cell membrane markers was about comparable. These inducible null cells are termed pre-T and pre-B lymphocytes. Our use of differential induction with ubiquitin and thymopoietin and an earlier study using flotation (1) suggest that commitment toward T- or B-lymphocyte differentiation takes place in the null cell compartment. A minority of normal null cells were unresponsive to ubiquitin. These could be (a) mature non-T, non-B lymphocytes (25), (b) precursors that respond to a limited number of inductive signals that do not include ubiquitin, (c) null cells whose differentiation potential does not exceed the threshold needed for identification, or (d) nonlymphoid cells such as hematopoietic precursors. There was remarkable consistency in the composition of the null cell compartment of spleens from different strains of mice. The techniques used in this study identified somewhat fewer T cells but comparable numbers of B cells to published experience in spleen cell suspensions (8, 10, 26). Some null lymphocytes bear small quantities of surface markers (27, 28); even T lymphocytes have low levels of membrane Ig (29) that are not detected using conventional techniques. Thus, cells identified in the present study as null lymphocytes may not be completely devoid of surface markers. The objective was to identify a stage of lymphocyte differentiation under standardized conditions. The null cell compartment was examined in the context of the total cell composition of the spleen. The null cell compartment comprised about 20% of crude spleen cell suspensions
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NULL
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in this study. In similar studies, Raff reported 18% (26), Roelants et al. 10% ( lo), and Stobo et al. about 7% (8) of spleen cells were null lymphocytes. Splenic null cells can differentiate in vivo as well as in vitro (30). It is not known why some null cells, although intrinsically capable of differentiation, do not do so while resident in the spleen. There may be a feedback or other mechanism that regulates the entry of precursor cells into more mature lymphocyte pools. The local splenic environment may be unfavorable for lymphocyte differentiation. Biologic initiators of lymphocyte differentiation are usually of low molecular weight (6, 14, 17) and should be distributed evenly throughout the body unless catabolism is accelerated in the spleen. The triggering of T-cell differentiation by inducing peptides takes less than 10 min ( 1) and expression of membrane markers is almost complete within 2 hr (24). Thus, it is unlikely that the presence of null cells reflects an obligatory pause in lymphocyte differentiation. It has been suggested that the appearance of p chains in the cytoplasm represents a stage in B-cell differentiation. Cells with this primitive Ig chain distribution are not found in spleens from young adult mice (11). In addition, such cells were not induced with ubiquitin in the present study. Different inducing agents induce distinct subpopulations of B cells only some of which pass through a stage where Ig chains are limited to the cytoplasm (31). Perhaps, ubiquitin only induces B-cell precursors that bypass this stage of differentiation. In that case, other B-cell precursors that respond to different inducing agents and pass through a cytoplasmic Ig stage may contribute to the subpopulation of null cells that does not respond to ubiquitin. Situations were observed where the composition of the null cell compartment was altered. It is apparent that nonthymic T-cell-inducing agents are incapable of significant T-cell differentiation in vivo with congenital thymic deficiency (32). Null cells that are committed to T-cell differentiation accumulate in spleens from unstressed congenitally athymic mice. Obviously, the commitment of null cells to T-cell differentiation is a thymic-independent event. Null cells that undergo T- or B-cell differentiation in response to ubiquitin differ in that T-cell precursors are sensitive to stress and adrenocortiocosteroids. This suggests that steroid sensitivity includes null T-cell precursors as well as relatively immature T cells (33). Conversely, the steroid resistance of our B-cell precursors suggests they may be quite primitive cells. When young adult mice are thymectomized, various T-cell functions eventually decline (34). The decline in T-cell functions with advanced age (35) is also associated with prolonged thymodeprivation due to thymic involution (36). Yet, the distribution of lymphocyte subpopulations in spleens from normal-aged or younger congenitally athymic mice differed significantly. Aged mice maintained a normal number of T lymphocytes despite a significant deficiency of ubiquitin-responsive T-cell precursors. With congenital thymic deficiency, T-cell precursors accumulated but did not sustain a detectable T-cell compartment. The B-lymphocyte series, including ubiquitin-responsive precursors, was normal with both congenital and senescent thymic deficiency. There was a gradual accumulation of ubiquitin-unresponsive null lymphocytes after age 8 months which, because of their normal numbers in spleens from 3-month-old nude mice, cannot be ascribed directly to prolonged thymodeprivation. Thus, splenic T cells of aged mice sustain themselves despite an apparent reduced input from null cell precursors. A gradual accumulation
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of ubiquitin-unresponsive null cells is a feature of aging within the null cell compartment. Their actual identity and potential to differentiate in response to other stimuli remains to be ascertained. Some studies suggest that spleen (37) or bone marrow (38) cells from aged mice are capable of normal immunologic reconstitution. The present study and the in viva study of Tyan (39) suggest that T-cell differentiation becomes defective with advanced age. Perhaps, this defect is limited to early T-cell precursors and T-cell homeostasis in aged mice is derived from replication of more differentiated cells. The potential for functional heterogeneity is likely to diminish with cell maturity. Functional restriction, due to T cells being largely derived from relatively mature precursors, could contribute to the overall waning of T-cell vigor with advanced age. The composition of lymphocyte populations was also altered in neonatal mice. T-cell, B-cell, pre-T-cell, and pre-B-cell percentages were reduced. While the total null cell compartment was not expanded, there were increased numbers of null cells that were unresponsive to ubiquitin induction. In this situation, where thymic influences are optimal, likely explanations include (a) incomplete migration of lymphoid cells to the spleen (40), (b) lymphoid precursors that are unresponsive to inductive stimuli because of immaturity, (c) immaturity of the splenic microenvironment, and (d) suppression exercising effects upon lymphocyte differentiation as well as upon lymphocyte function. This study introduces a new way to investigate lymphocyte precursors. The changes observed suggest,that this approach may help determine the approximate stage of differentiation at which lymphocyte disorders originate and, thereby, may have clinical application. Although multiple myeloma is expressed as a neoplasm of terminal B-cell differentiation, it also involves B lymphocytes (41). The accumulation of null lymphocytes in blood from patients with lymphoblastic leukemia (42) and systemic lupus erythematosus (43) suggests that abnormalities may exist within the null cell compartment with these diseases as well. These, and perhaps other disorders, would be better understood if the stage of differentiation at which abnormalities appear could be identified. ACKNOWLEDGMENTS This research was supported by NIH Grants CA 15333 and ROI NS 16325 and also by a grant from Ellem Laboratories, Milano, Italy. We are grateful to Gideon Goldstein, M. D., Ph. D., Executive Director, Immunosciences, Ortho Pharmaceutical Corporation, for providing us with ubiquitin and thymopoietin; Ellen Vitetta, Ph.D., Prof. Microbial. University of Texas Southwestern Medical School, Dallas, for reagents used for cell sorting; and David Dennison, Ph. D., Baylor College of Medicine, Houston, for his valuable assistance with use of the cytofluorograf.
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10. Roelants, G. E., Lcor, F., vonBoehmer, H., Sprent, J., Hagg, L. B., Mayor, K. S., and Ryden, A., Eur. J. Immunol. 5, 127, 1975. 1I. Raff, M. C., Magson, M., Owen, J. J. T., and Cooper, M. D., Nature (London) 247, 11, 1974. 12. Keightley, R. G., Lawton, A. R., Cooper, M. D., and Yunis, E. J., Lancer 2, 850, 1975. 13. Goldstein, G., Scheid, M., Hammerling, U., Boyse, E. A., Schlesinger, D. H., and Niall, H. D., Proc. Nat. Acad. Sci. USA 72, 11, 1975. 14. Schlesinger, D. H., Goldstein, G., and Niall,. H. D., Biochemistry 11, 2214, 1975. 15. Yam, L. T., Li, C. Y., and Crosby, W. H., Amer. J. Clin. Pathol. 55, 283, 1971. 16. Schlesinger, D. H., and Goldstein, G., Cell 5, 361, 1975. 17. Goldstein, G., Nature (London) 247, 11, 1974. 18. Reif, A. E., and Allen, J. M. V., J. Exp. Med. 120, 413, 1964. 19. Ligler, F. S., Vitetta, E. S., and Uhr, J. W., J. Immunol. 119, 1545, 1977. 20. Stewart, C. C., and Ingram, M., Blood 29, 628, 1967. 21. Twomey, J. J., Rossen, R. D., Lewis, V. M., Morgan, A. C., and McCormick K. J., Cancer 41, 1307, 1978. 22. Mendes, N. F., Tolnai, M. E. A., Silviera, P. A., Gilbertsen, R. B., and Metzgar, R. S., J. Immunol. 111,860,
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23. Raff, M. C., Magson, M., Owen, J. J. T., and Cooper, M. D., Nature (London) 259, 224, 1976. 24. Twomey, J. J., Goldstein, G., Lewis, V. M., Bealmear, P. M., and Good, R. A., Proc. Narl. Acad. Sci. USA 74, 2541, 1977. 25. Froland, S. S., and Natvig, J. B., Transplant. Rev. 16, 114, 1973. 26. Raff, M. C., Transplant Rev. 6, 52, 1971. 27. Sate, V. L., Waksal, S. D., and Herzenberg, L. A., Cell. Immunol. 24, 173, 1976. 28. Warr, G. W., Lee, J. C., and Marchalonis, J. J., J. Immunol. 121, 1767, 1978. 29. Nossal, G. J. V., Warner, N. L., Lewis, H., and Sprent, J., J. Exp. Med. 135, 405, 1972. 30. Papiernik, M., and Bach, J. F., J. Immunol. 123, 2311, 1979. 31. Muraguchi, A., Kishimoto, T., Kuritani, T., Watanabe, T., and Yamamura, Y., J. Immunol. 125, 564, 32.
33. 34. 35.
36. 37. 38. 39. 40. 41. 42. 43.
1980.
Lischner, H. W., and Huff, D. S., Birth Defecrs: Original Article Series 11, 16, 1975. Katz, D. H., “Lymphocyte Differentiation, Recognition and Regulation.” Academic Press, New York, 1977. Kappler, J. W., Hunter, P. C., Jacobs, D., and Lord, E., J. Immunol. 113, 27, 1974. Kay, M. M. B., Fed. Proc. 37, 1241, 1978. Lewis, V. M., Twomey, J. J., Bealmear, P., Goldstein, G., and Good, R. A., J. C/in. Endocrin. Metabol. 47, 145, 1978. Harrison, D. E., and Doubleday, J. W., J. Immunol. 114, 1314, 1975. Micklem, H. S., Ogden, D. A., and Payne, A. C., Ciba Found. Symp. 13, 285, 1973. Tyan, M. L., J. Immunol. 118, 846, 1977. Mosier, D. E., and Cohen, P. L., Fed. Proc. 34, 137, 1975. Abdou, N. E., and Abdou, N. L., Ann. Intern. Med. 83, 42, 1975. Pasino, M., Tonini, G. P., Rosanda, C., and Massimo, L., Lancer 1, 608, 1977. Scheinberg, M. A., Cathcart, E. S., and Goldstein, A. L., Lancer 1, 424, 1975.
IMMUNOLOGY
63, 106-117 (1981)
The Null Cell Compartment JEREMIAH The Houston
of the Mouse Spleen
J. TWOMEY AND NICOLA M. KOUTTAB
Veterans Administration Medical Center, and Baylor Houston, Texas 7721 I Received February
21. 1981; accepted April
College of Medicine.
29, 1981
Null lymphocytes were defined as lymphocytes without detectable T- or B-cell markers using a battery of techniques. The null cell compartment was divided into pre-T cells, pre-B cells, and other null cells based upon their acquisition of membrane markers when incubated with ubiquitin. The null ceil subpopulations were remarkably consistent in spleen cell suspensions from young adult mice of various strains. Commitment to T- or B-cell differentiation took place at the null cell stage and did not require thymic input. Pre-T cells, but not pre-B cells, were steroid sensitive. Pre-T cells accumulate with congenital thymic deficiency. This differed from senescentthymodeprivation where the outstanding finding was an accumulation of uninducible null cells. Neonatal mouse spleens were deficient in pre-T and pre-B cells but had an accumulation of uninducible cells.
INTRODUCTION Mature lymphocytes are derived from a pool of precursor cells. Some precursors may be committed to the T- or B-lymphocyte series before the onset of recognizable differentiation (1). Subpopulations of lymphocytes acquire surface markers and functions during maturation (2-5). the overall processof lymphocyte differentiation involves a complex series of events that are modulated to a greater or lesser degree by a number of inducing agents (6, 7). Lymphoid cells that lack detectable surface markers are arbitrarily termed null lymphocytes (8). These null cells include committed precursors of T or B lymphocytes and other, probably heterogeneous, cells. In the mouse, null cells comprise about 50% of bone marrow lymphocytes (9), 3-l 3% of splenic lymphocytes (8,lO) and O-5% of lymph node lymphocytes (8, 10). There has been no systematic evaluation of this null cell compartment apart from the recognition of some lymphocytes that have undergone partial B-cell differentiation; these are identified by the presence of small amounts of cytoplasmic (but not surface) P chains (11). The composition of the null cell compartment could be altered through a number of mechanisms. It could fail to sustain itself through lowered input from primitive precursors or impaired null cell replication. Alternatively, accelerated differentiation of null cells could outstrip regenerative reserves and thereby deplete the null cell pool. The null cell compartment could be expanded by increased input from primitive precursors, accelerated null cell mitosis, or reduced differentiation toward more mature cells. The latter could result from intrinsic unresponsiveness by null cells to inductive stimuli, a lack of inductive stimuli, or an unfavorable microen106 OOOS-8749/81/130106-l2802.00/0 Copyright 0 1981 by Academic F’ress. Inc. All rights of reproduction in any form rcscrvcd.
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vironment for differentiation to take place. Abnormalities could be limited to one or more subpopulations within the null cell compartment. Disorders of more mature lymphocytes may have their origins within the null cell compartment, as exemplified in man by severe combined immunodeficiency (12). This paper introduces an approach that recognized three subpopulations within the null cell compartment of the mouse spleen. These subpopulations include null cells that acquire T- or B-cell surface markers when optimally induced with ubiquitin and other null cells that are unresponsive to ubiquitin. Ubiquitin is a tissue peptide which, at the concentration employed, stimulates both T- and B-cell differentiation via P-adrenergic receptors (13, 14). We found that the composition of the null cell compartment is remarkably consistent in healthy young adult mice of different strains but is altered during the neonatal period and with advanced age. The latter cannot be ascribed to thymic involution since a different pattern was observed in congenitally athymic mice. MATERIALS
AND METHODS
Mice and cell preparations. Studies were performed on C3H/He and DBA/2 mice purchased from TIMCO Breeding Laboratories, Houston, Texas, C57BL/ 1, CBA/T6 AKR/J, and Balb/c mice purchased from Jackson Laboratories, Bar Harbor, Maine, and our colony of germ-free nude athymic mice bred on a C3H/ He background. Neonatal mice were studied 3-5 days after birth. Mice were sacrificed by cervical dislocation, their spleens removed aseptically and weighed, and cells freed by gentle teasing. Since our interest was’ in overall spleen cell composition, no attempts were made at cell fractionation. Fresh smears were prepared and stained with a-naphthyl acetate esterase (Sigma Chemical Co., St. Louis, MO.) (15) or Wright-Giemsa stains. Percentages of macrophages, hematopoietic cells, granulocytes, and lymphoid cells were determined from counts of 300 cells. Reagents. Thymopoietin, a thymic peptide of known amino acid sequence (16) and ubiquitin were purified using published techniques (13, 14, 16, 17). Antiserum to Thy 1.2 antigen was raised by immunizing AKR/J mice with C3H/He thymocytes (18). Fluorescein-labeled monoclonal IgM antibody to Thy 1.2 was purchased from New England Nuclear, Boston, Massachusetts. Adsorbed, fluoresceinconjugated antiserum to C3H/He mouse brain raised in rabbits was purchased from Litton Bionetics, Kensington, Maryland. Fluorescein-conjugated goat antisera to mouse ~1,y, or (Y chains were obtained from Meloy Laboratories, Springfield, Virginia. Fluorescein-conjugated rabbit immunoglobulin (1g)G containing antibodies to mouse K chains, unconjugated rabbit IgG containing antibodies to mouse X chains and fluourescein-conjugated goat antiserum to rabbit IgG were purchased from Litton Bionetics or procured from Dr. E. Vitetta. Affinity-purified rabbit antiserum to mouse Ig was prepared as described by Ligler et al. (19). Rhodamineconjugated goat antiserum to mouse ~1chains was obtained from Cappel Laboratories, Cochranville, Pennsylvania. Fresh frozen guinea pig serum that was absorbed with C3H/He mouse thymocytes was used as complement source. All reagents were used at optimal dilutions. Cell identification. T lymphocytes were identified using a number of different methods. In the cytotoxicity test, 2 X lo5 spleen cells in 0.05 ml medium 199 (Gibco,
108
TWOMEY
AND
KOUTTAB
Grand Island, N.Y.) enriched with 5 g% BSA (Sigma) plus 0.025 ml antiserum to Thy 1.2 antigen were incubated in duplicate with 0.025 ml complement or heatinactivated complement. Incubations were for 90 min at 37°C in a humidified atmosphere of 5% CO2 and air and for an additional 30 min after adding 0.625 mg pronase (grade B, Sigma) in 0.125 ml saline. Then 12 ml cetrimide (Eastman Kodak Co., Rochester, N.Y.) was added and cell counts performed using a Coulter counter. Injured cells were solubilized while incubated with pronase (20). This was reflected in lower cell counts in incubations containing complement compared to paired incubations that lacked complement activity. This antibody-specific cytotoxicity test was preferred to dye exclusion or membrane-bound isotope release because it is more sensitive to lesser degrees of cell injury (21). Thy 1.2 antigen was also identified using both direct and indirect immunofluorescence. For direct immunofluorescene, 1 X lo6 spleen cells and optimal concentrations of conjugated monoclonal IgM antibody to Thy 1.2 antigen were kept at 4°C for 30 min, washed, and examined for membrane flourescence using a Leitz Ploem optics uv microscope. For indirect immunofluorescence, 5 X 10’ spleen cells, suspended in BSA-enriched medium 199 were incubated with 0.025 ml unconjugated antiserum to Thy 1.2 antigen for 1 hr at 37°C. After washing twice, the cells were treated with 0.025 ml each of fluorescein-conjugated antiserum to mouse Ig heavy chains and 0.025 ml PBS at 4°C for a second hour, washed twice, and examined for membrane immunofluorescence. This procedure identified both T and B cells. Thus, to enumerate T cells, it was necessary to identify B cells concomitantly and subtract these from combined T- plus B-cell values. Cell suspensions were also reacted with fluoresceinated antiserum to mouse brain for 1 hr at 4°C washed twice, and examined directly for membrane immunofluorescence. Conjugated antisera to p, y, and a! chains were mixed at optimal dilutions to demonstrate heavy-chain determinants on B-cell membranes. Spleen cells (5 X 1OS), suspendedin 0.05 ml BSA-enriched medium 199,0.025 ml of the conjugate mixture and 0.025 ml PBS were kept at 4°C for 1 hr, washed twice, and examined directly for membrane immunofluorescence. There was minimal binding of conjugate by Fc receptors as was evidenced by there being negligible numbers of fluorescing cells after depleting B lymphocytes and macrophages by incubation with nylon wool. When testing for light-chain determinants on cell membranes, K chains were identified by direct immunofluorescence and X chains by indirect immunofluorescence using procedures outlined above. The prescence of membrane C3 receptors was demonstrated using the EAC procedure described by Mendes et al. (22). Cell sorting was done using a series 50-H cytofluorograf (Ortho, Westwood, Mass.). Excitation was done with 400 W of laser power at 488 nm. The photomultiplier tube voltage was set at 435 for direct fluorescence and at 594 for indirect fluorescence. Cells in channels 46-511 were scored positively. Gating on live cells was done using a logarithmic amplifier. The double immunofluorescence method of Raff et al. (23) was used to identify lymphoid cells with Ig limited to their cytoplasm. Cell membrane Ig was first reacted directly with mixed heavy-chain conjugates as described. The cells were then washed twice, fixed at 4°C with 5% acetic acid and 95% ethanol, stained for cytoplasmic Ig by a 1-hr incubation period at 37°C with a rhodamine conjugate to p-chain determinants, washed, and examined for immunofluorescence. Induction incubations. After allowing clumps to sediment, spleen cells were
SPLEEN NULL CELLS
109
washed twice and suspended at 5 X lo6 cells/ml in medium 199 plus 5 g% BSA. Incubations were set up in duplicate in 12 X 75-mm polystyrene tubes containing 0.1 ml cell suspension and 0.1 ml medium plus 20 rig/ml ubiquitin, 1 rg/ml thymopoietin, or medium alone. Preliminary experiments indicated that these represented optimal inductive concentrations of ubiquitin and thymopoietin in this incubation system. Incubations were for 18 hr at 37°C in a humidified 5% CO1 and air atmosphere. Kinetic studies showed that most induction took place during the first 2 hr of incubation but maximum induction was achieved most consistently when incubation was extended overnight (24). At the end of 18 hr of incubation, T and B cells were identified by surface markers. Expression of data. All cell populations, except those of the null cell compartment, were identified on fresh spleen cells. First, the total number of cells freed from each spleen was determined for subsequent use in calculating absolute values. Then, percentages of granulocytes, hematopoietic cells, macrophages, and lymphocytes were determined from Wright-Giemsa- and esterase-stained smears. Tcell and B-cell percentages were detected using various procedures outlined above. Pre-T and pre-B cells were defined as null lymphocytes that acquired T- or B-cell membrane markers after overnight incubation with ubiquitin. Pre-T-cell percentages were calculated from the difference in T-cell percentages in overnight incubations with and without ubiquitin. Pre-B-cell percentages were enumerated in a similar fashion. Null lymphoid cells that could not be induced to express detectable T- or.B-cell markers were arbitrarily termed other null cells. Grouped values were expressed as means f SEMs. The statistical significance of values on the same cell preparations that were treated differently was determined using Student’s paired t test. The Wilcoxon rank sum test was used when comparing the same test results performed on different cell preparations. RESULTS The Null
Cell Compartment
in 3-Month-Old
C3H/He Mice
By morphologic and histochemical criteria, fresh spleen cell suspensionsincluded 78 + 1.4% lymphoid cells, 10 k 0.1% granulocytes, 6 f 0.3% macrophages, and 6 f 1.3% hematopoietic cells. About 25% of all spleen cells were T lymphocytes whether detected using Thy 1.2 or mouse brain antiserum (Table 1). Similar values were obtained using cytotoxicity or with direct or indirect immunofluorescence tests. Likewise, similar values were obtained using uv microscopy or a cell sorter. When enumerating B lymphocytes, 40 f 0.8% fresh spleen cells reacted with antisera to Ig heavy chains by uv microscopy and 38% using a cell sorter. When conjugates and uv microscopy were used, 43 +- 0.5% demonstrated membrane light chains. Receptors for C3 were identified on 38 + 1.1% of fresh spleen cells. After 18 hr of unstimulated incubation, 20 + 1.5% spleen cells bore Thy 1.2 antigen and 42 f 0.4% bore Ig heavy-chain determinants. The difference in T-cell percentages after overnight incubation with or without ubiquitin averaged 7% (P < 0.05) irrespective of whether Thy 1.2 or mouse brain antisera was used (Table 1). Similar values for pre-T cells were obtained using cytotoxicity, direct, or indirect immunofluorescence tests and when surface fluorescence was demonstrated by uv microscopy or using a cell sorter. The presence of ubiquitin in overnight incubations also increased the number of spleen cells with
110
TWOMEY AND KOUTTAB TABLE 1 Comparison of Different Methods Used to Identify Lymphocytes
Surface marker
Method for identification
T lymphocytes (%I
Pre-T lymphocytes (%)
Thy 1.2 antigen
Cytotoxicity Indirect IF” Direct IF Direct IF, cytofluorograf Direct IF
25 z!z 1.5 25 * 1.2 25 27 25 5 0.9
7 + 0.1 6 f 0.4 lb Tb 7 + 1.0
Direct IF Direct IF, cytofluorograf K, direct, X, indirect IF EAC rosettes
40 + 0.8 38 43 + 0.5 38 + 1.1
I + 0.9 I 8 + 0.6 6 + 1.1
Brain antigen cc,y, a chains K, X chains Cg receptors
’ IF, immunofluorescence. b Mean of two experiments.
membrane Ig heavy or light chains or receptors for C3 by 5-9% (P < 0.05). Again, similar values for pre-B cells were registered using uv microscopy or a cell sorter. Figure 1 shows quantitative differences in T and B cells and the ranges of fluorescence-registering membrane Thy 1.2 antigen or Ig heavy chains on spleen cells after incubation with and without ubiquitin. In all subsequent experiments, T cells were identified using Thy 1.2 antiserum in a complement-dependent cytotoxicity test system and B cells by the presence of Ig heavy chains using direct fluorescence microscopy. Small quantities of cytoplasmic ~1chains, without detectable membrane Ig, are thought to identify a stage in the ontogeny of B-cell differentiation (11). These comprised 4 + 0.3% of bone marrow cells from 3-month-old mice. While ubiquitin induction significantly increased the number of spleen cells bearing surface Ig, no cytoplasmic p-chain-positive and membrane Ig-negative ceils were observed in fresh spleen cell preparations or after incubation with or without ubiquitin.
h LOG
FLUOREBCENCE
INTENSITY
FIG. 1. A representative cytotluorograf analysis of T and B cells in a spleen cell suspension after overnight incubation with and without ubiquitin. T cells were identified using a monoclonal conjugate for Thy 1.2 antigen and B cells using conjugates for B, y. and a-chain determinants.
111
SPLEEN NULL CELLS
FIG. 2. T- and B-cell induction of spleen cell suspensionswith ubiquitin or thymopoietin. T cells were identified by Thy 1.2 antigen in a cytotoxicity system and B cells by heavy-chain conjugates and uv microscopy.
Speci’city of Null Cell Induction Spleen cell suspensions were stimulated with 20 rig/ml ubiquitin or 1 pg/ml thymopoietin. T-Cell induction was 9 f 0.4% with ubiquitin and 6 + 0.4% with thymopoietin (P < 0.05) (Fig. 2). Ubiquitin induced 9 + 0.2% B-cell differentiation while thymopoietin did not induce significant B-cell differentiation (P < 0.001). Thus, ubiquitin is a potent inducer of both T and B cells; thymopoietin is less potent and only induces T-cell differentiation (13, 24). After a single 18-hr incubation period, thymopoietin alone induced T-cell but not B-cell differentiation while ubiquitin induced both T- and B-cell differentiation (Table 2). After two consecutive 18-hr incubation periods with thymopoietin, Tcell differentiation increased to a level that was comparable to that achieved with a single 18-hr incubation period with ubiquitin; there was still no B-cell induction. Consecutive incubations were done first with thymopoietin and then with ubiquitin. The initial stimulus with thymopoietin did not remove null cells capable of B-cell differentiation during the second incubation with ubiquitin. This representative experiment provides additional evidence that cells become committed to the T- or B-cell series during the null cell stage of differentiation. TABLE 2 Differential and Combined T- and B-Cell Induction with Thymopoietin and/or Ubiquitin
Thymopoietin, I rg/ml for 18 hr Ubiquitin, 20 rig/ml for 18 hr Thymopoietin, I pg/ml for I8 hr followed by thymopoietin, 1 pg/ml for I8 hr Thymopoietin, I pg/ml for I8 hr followed by ubiquitin, 20 rig/ml for 18 hr
Pre-T cm
Pre-B @IO)
7 9
I 9
9
I
II
IO
Note. T cells were identified by Thy 1.2 antiserum in a complement-dependent cytotoxicity test and B cells by the presence of Ig heavy chains using immunofluorescence.
112
TWOMEY AND KOUTTAB
Null Cell Compartment in Different Strains of Mice Spleen cells from 3-month-old C3H/He, DBA/2, CBA/T6, Balb/c, and C57BL/ 1 mice were incubated with and without ubiquitin and induction of membrane Thy 1.2 antigen and Ig heavy chains was determined. The numbers of pre-T and preB cells were remarkably consistent among these different strains (P > 0.05) (Fig. 3). Age and the Null Cell Compartment The absolute and relative cell compositions of spleens from neonatal, 3-, 8, and 24-month-old C3H/He mice were compared. Spleens from neonatal mice weighed < 10% of spleens from young adult mice which was reflected in low absolute cell counts (Table 3). However, percentages of various cell populations differed significantly in spleens from neonatal and 3-month-old mice (P < .Ol). The neonates had elevated percentages of hematopoietic cells (49 f 6.7 vs 6 + lS%), presumably reflecting a residue from splenic hematopoiesis during intrauterine life. Percentages of both T cells (6 f 0.2 vs 24 f 1.1%) and B cells (9 + 0.4 vs 35 f 0.8%) were greatly reduced in neonatal spleens. However, no significant differences were observed in total null cell percentages. At age 24 months, absolute numbers of splenic hematopoietic cells, granulocytes, macrophages, T cells, and B cells were not significantly different from values recorded at age 3 months (Table 3). However, the total null cell content of the spleen was elevated at age 24 months (P < 0.05). The relationship of age to the composition of the null cell compartment was evaluated. Spleens from neonatal mice contained few T-cell precursors, reduced percentages of B-cell precursors, and greatly elevated percentages of ubiquitinunresponsive null cells when compared with spleens from 3-month-old mice (P < 0.01) (Fig. 4). At age 24 months, percentages of pre-T cells were reduced (P < O.Ol), pre-B cells were comparable and ubiquitin-unresponsive null cells were threefold higher than in spleens from 3-month-old mice (P < 0.01). This age-related increase in ubiquitin-unresponsive null cells appeared to commence at about 8 months of age. 12 r
m
PIE-TCELLS
0
ME-B CELLS
FIG. 3. Pre-T- and pre-B-cell percentages in spleen cell suspensionsfrom different strains of mice. A cytotoxicity system identifying Thy 1.2 antigen and uv microscopy using anti-heavy-chain determinants were used to identify T and B cells, respectively.
113
SPLEEN NULL CELLS TABLE 3 Absolute Numbers of Cell Populations in Mouse Spleens at Different Ages Cells per spleen Age of mice Hematopoietic (months) cells Neonates 3 8 24
9.8 6.4 4.9 5.7
f f + f
2.9 1.7 0.8 0.8
Granulocytes 2.7 8.0 8.3 10.6
f k k +
0.9 0.8 0.6 0.9
Macrophages 0.3 3.6 2.3 2.5
+ 2 + f
0.1 0.5 0.5 0.4
T cells 0.5 15.7 15.0 20.0
B cells
Null cells
1.9 f 0.1 0.8 + 0.1 f 0.8 22.5 + 1.7 12.4 2~ 0.9 24.1 f 1.4 15.9 f 1.8 25.1 + 2.6 28.5
f k -t +
0.2 1.3 1.7 2.2
Note. T cells were identified by membrane Thy 1.2 antigen in a cytotoxicity test and B cells by membrane heavy chains using direct immunofluorescence.
Null Cell Compartment in Athymic Mice The composition of the null cell compartment was studied at age 3 months on congenitally athymic mice, five of whom were germ free and five others were housed conventionally. These spleens lacked detectable T cells but had a normal B-cell content. Spleen cells from germ-free mice included 18 f 1.3% pre-T cells which was significantly higher than the 9 f 0.4% recorded on athymic mice that had been exposed to common environmental stresses (P < 0.01) (Fig. 5). The latter were not significantly different from age-matched C3H/He mice. The splenic content of B-cell precursors and ubiquitin-unresponsive null cells was not altered by the athymic state. Null Cell Compartment and Steroid Sensitivity We injected 5 mg of hydrocortisone acetate into the thighs of 3-month-old C3H/ He mice and studied the null cell compartment 3 days later. inducible T-cell precursors were reduced to 0.3 + 0.3% (P < 0.01) while B-cell precursors were unaltered after steroid injection.
IIEOIIATES
5 8 A&E OF IlICE INOllW
24
FIG. 4. The composition of the spleen null cell compartment at different ages. Pre-T cells were identified by acquisition of Thy 1.2 antigen in a cytotoxicity system and pre-B cells by acquisition of membrane Ig heavy chains using direct immunofluorescence.
114
TWOMEY AND KOUTTAB 20
r
T
m 0
PIE.1 CELLS ME-0 CELLS
GEMFREE COWYENTlOllAllZED CSRih WUDES NUDES
FIG. 5. The effect of congenital thymic deficiency and a germ-free environment upon the spleen null cell compartment. Pre-T cells were enumerated using antiserum to Thy 1.2 antigen and complementdependent cytotoxicity and pre-B ceils using anti-heavy-chain conjugates and direct immunofluorescence.
DISCUSSION This study indicates that the null cell compartment of the mouse spleen can be divided into three supopulations by differentiation responses to ubiquitin. Similar values were obtained with a number of cell surface markers and techniques. The number of null cells that acquired T- or B-cell membrane markers was about comparable. These inducible null cells are termed pre-T and pre-B lymphocytes. Our use of differential induction with ubiquitin and thymopoietin and an earlier study using flotation (1) suggest that commitment toward T- or B-lymphocyte differentiation takes place in the null cell compartment. A minority of normal null cells were unresponsive to ubiquitin. These could be (a) mature non-T, non-B lymphocytes (25), (b) precursors that respond to a limited number of inductive signals that do not include ubiquitin, (c) null cells whose differentiation potential does not exceed the threshold needed for identification, or (d) nonlymphoid cells such as hematopoietic precursors. There was remarkable consistency in the composition of the null cell compartment of spleens from different strains of mice. The techniques used in this study identified somewhat fewer T cells but comparable numbers of B cells to published experience in spleen cell suspensions (8, 10, 26). Some null lymphocytes bear small quantities of surface markers (27, 28); even T lymphocytes have low levels of membrane Ig (29) that are not detected using conventional techniques. Thus, cells identified in the present study as null lymphocytes may not be completely devoid of surface markers. The objective was to identify a stage of lymphocyte differentiation under standardized conditions. The null cell compartment was examined in the context of the total cell composition of the spleen. The null cell compartment comprised about 20% of crude spleen cell suspensions
SPLEEN
NULL
CELLS
115
in this study. In similar studies, Raff reported 18% (26), Roelants et al. 10% ( lo), and Stobo et al. about 7% (8) of spleen cells were null lymphocytes. Splenic null cells can differentiate in vivo as well as in vitro (30). It is not known why some null cells, although intrinsically capable of differentiation, do not do so while resident in the spleen. There may be a feedback or other mechanism that regulates the entry of precursor cells into more mature lymphocyte pools. The local splenic environment may be unfavorable for lymphocyte differentiation. Biologic initiators of lymphocyte differentiation are usually of low molecular weight (6, 14, 17) and should be distributed evenly throughout the body unless catabolism is accelerated in the spleen. The triggering of T-cell differentiation by inducing peptides takes less than 10 min ( 1) and expression of membrane markers is almost complete within 2 hr (24). Thus, it is unlikely that the presence of null cells reflects an obligatory pause in lymphocyte differentiation. It has been suggested that the appearance of p chains in the cytoplasm represents a stage in B-cell differentiation. Cells with this primitive Ig chain distribution are not found in spleens from young adult mice (11). In addition, such cells were not induced with ubiquitin in the present study. Different inducing agents induce distinct subpopulations of B cells only some of which pass through a stage where Ig chains are limited to the cytoplasm (31). Perhaps, ubiquitin only induces B-cell precursors that bypass this stage of differentiation. In that case, other B-cell precursors that respond to different inducing agents and pass through a cytoplasmic Ig stage may contribute to the subpopulation of null cells that does not respond to ubiquitin. Situations were observed where the composition of the null cell compartment was altered. It is apparent that nonthymic T-cell-inducing agents are incapable of significant T-cell differentiation in vivo with congenital thymic deficiency (32). Null cells that are committed to T-cell differentiation accumulate in spleens from unstressed congenitally athymic mice. Obviously, the commitment of null cells to T-cell differentiation is a thymic-independent event. Null cells that undergo T- or B-cell differentiation in response to ubiquitin differ in that T-cell precursors are sensitive to stress and adrenocortiocosteroids. This suggests that steroid sensitivity includes null T-cell precursors as well as relatively immature T cells (33). Conversely, the steroid resistance of our B-cell precursors suggests they may be quite primitive cells. When young adult mice are thymectomized, various T-cell functions eventually decline (34). The decline in T-cell functions with advanced age (35) is also associated with prolonged thymodeprivation due to thymic involution (36). Yet, the distribution of lymphocyte subpopulations in spleens from normal-aged or younger congenitally athymic mice differed significantly. Aged mice maintained a normal number of T lymphocytes despite a significant deficiency of ubiquitin-responsive T-cell precursors. With congenital thymic deficiency, T-cell precursors accumulated but did not sustain a detectable T-cell compartment. The B-lymphocyte series, including ubiquitin-responsive precursors, was normal with both congenital and senescent thymic deficiency. There was a gradual accumulation of ubiquitin-unresponsive null lymphocytes after age 8 months which, because of their normal numbers in spleens from 3-month-old nude mice, cannot be ascribed directly to prolonged thymodeprivation. Thus, splenic T cells of aged mice sustain themselves despite an apparent reduced input from null cell precursors. A gradual accumulation
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of ubiquitin-unresponsive null cells is a feature of aging within the null cell compartment. Their actual identity and potential to differentiate in response to other stimuli remains to be ascertained. Some studies suggest that spleen (37) or bone marrow (38) cells from aged mice are capable of normal immunologic reconstitution. The present study and the in viva study of Tyan (39) suggest that T-cell differentiation becomes defective with advanced age. Perhaps, this defect is limited to early T-cell precursors and T-cell homeostasis in aged mice is derived from replication of more differentiated cells. The potential for functional heterogeneity is likely to diminish with cell maturity. Functional restriction, due to T cells being largely derived from relatively mature precursors, could contribute to the overall waning of T-cell vigor with advanced age. The composition of lymphocyte populations was also altered in neonatal mice. T-cell, B-cell, pre-T-cell, and pre-B-cell percentages were reduced. While the total null cell compartment was not expanded, there were increased numbers of null cells that were unresponsive to ubiquitin induction. In this situation, where thymic influences are optimal, likely explanations include (a) incomplete migration of lymphoid cells to the spleen (40), (b) lymphoid precursors that are unresponsive to inductive stimuli because of immaturity, (c) immaturity of the splenic microenvironment, and (d) suppression exercising effects upon lymphocyte differentiation as well as upon lymphocyte function. This study introduces a new way to investigate lymphocyte precursors. The changes observed suggest,that this approach may help determine the approximate stage of differentiation at which lymphocyte disorders originate and, thereby, may have clinical application. Although multiple myeloma is expressed as a neoplasm of terminal B-cell differentiation, it also involves B lymphocytes (41). The accumulation of null lymphocytes in blood from patients with lymphoblastic leukemia (42) and systemic lupus erythematosus (43) suggests that abnormalities may exist within the null cell compartment with these diseases as well. These, and perhaps other disorders, would be better understood if the stage of differentiation at which abnormalities appear could be identified. ACKNOWLEDGMENTS This research was supported by NIH Grants CA 15333 and ROI NS 16325 and also by a grant from Ellem Laboratories, Milano, Italy. We are grateful to Gideon Goldstein, M. D., Ph. D., Executive Director, Immunosciences, Ortho Pharmaceutical Corporation, for providing us with ubiquitin and thymopoietin; Ellen Vitetta, Ph.D., Prof. Microbial. University of Texas Southwestern Medical School, Dallas, for reagents used for cell sorting; and David Dennison, Ph. D., Baylor College of Medicine, Houston, for his valuable assistance with use of the cytofluorograf.
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