- Email: [email protected]
W mice
CELLULAR IMMUNOLOGY
15, 129-142 (1975)
Properties of Fractionated GEORGE
M. GARNER,
RI.
ERIC
Spleen Cells from NZB/W GERSHWIN,
AND
Arthritis and Rltc?f~rratism Branch, Natiorlal Insitzttr Ili,qrsti;v Disrascs, National Ilrstitutcs of Hmlth, Recekvd
ALFRED
Mice
D. STEINBERG
Arthritis, Metabolism, ajrd Gethrsda, Maryland 20014
of
Alay 16, 1974
The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/Z, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice. A number of functional studies performed with the separated cells proved more of cells which sediinformative than the settling patterns themselves. Fractions mented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in e-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZBjW mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.
INTRODUCTION New Zealand mice manifest autoimmune phenomena including multiple autoantibodies and a number of immunological abnormalities ( 1). Recirculating lymphocytes (2), lymph node-seeking cells (3)) and helper cells participating in graft versus host (GVH)’ disease (4) and skin allograft rejection (5) are all lost as New Zealand mice age, perhaps as a result of the spontaneous production of naturally occurring thymocytotoxic antibdoy (NTA) (3, 6). The antibody response of New Zealand mice to sheep red blood cells (SRBC) develops early in life in comparison with other strains (7, 8). Later in life there is a premature loss of responsiveness to SRBC at a time when the response to type III pneumococcal polysaccharide (SSSIII), an antigen not requiring thymic helper cells, is intact (9). All of the evidence points to a loss of helper T cells in older New Zealand mice. In view of the interest in subpopulations of lymphoid cells, we have fractionated spleen mice using a unit gravity separation techcells from NZB x NZW F1 (NZB/W) 1 Abbreviations used in this paper : GVH, graft versus host disease ; NTA, naturally occurring thymocytotoxic antibody ; SSSIII, type III pneumococcal polysaccharide ; SRBC, sheep red blood cells ; NZB/W, NZB X NZW F, mice ; MLR, mixed lymphocyte reaction; FCS, fetal calf serum ; MEM, minimal essential medium; PHA, phytohemagglutinin A ; Con A, concanavalin A. 129 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
130
GARNER,
GERSHWIN
AND
STEINBERG
nique (10, 11). The fractions were characterized with respect to responsiveness to allogencic cells and other mitogens, surface markers, and ability to restore the response of old NZB/W mice to SKBC. MATERIALS
AND
METHODS
Animals. BALB/c, NZB x NZW F1 (NZB/W), C3H/He, DBA/2, and C57BL/6 mice were obtained from colonies maintained at the National Institutes of Health (NIH), Bethesda, MD. AKR mice were obtained from Jackson Laboratories, Bar Harbor, ME. Females were used throughout for study except as donors of target cells in the mixed lymphocyte reaction (MLR). All media were obtained from the NIH media center. Spleen cell fractionation. Spleen cells from NZB/W, C3H/He, DBA/2, and BALB/c mice were fractionated in this study. Groups of 1.5 mice were sacrificed by cervical dislocation and their spleens asceptically removed. Single cell suspensions were prepared and washed in 50 ml Hanks’ Balanced Salt Solution (HBSS) buffered with 19 mlM N-2 hydroxyethylpeperazine N’-Zethanesulfonic acid (HEPES) . The cell pellet was resuspended in 80 ml of Eagle’s minimal essential medium supplemented with Earle’s salts, and buffered to pH 7.2 with Tris buffer. The cells were separated at unit gravity over a 530% fetal calf serum (FCS) linear gradient for 2 hr in the settling chamber of Mage et al. (IO) according to the method of Miller and Phillips (11)) except that Eagle’s minimal essential medium (MEM) was used as a supporting medium instead of phosphate-buffered saline (PBS). After 2 hr the gradient was harvested in I-mm/hr fractions and the settling patterns were determined. For further study the gradient was arbitrarily separated into six fractions as follows : Fraction 1 2 3 4 5 6
Settling rate 3-5.5 mm/hr 5.5-7.0 mm/‘hr 7.0-8.5 mm/hr 8.5-10.0 mm/hr 10.0-11.5 mm/hr 12.O16.0 mm/hr
Cell counts. Leukocytes were counted in a Coulter Counter Model ZBI. The cell counts were confirmed periodically in a hemocytometer. Viability was checked by trypan blue exclusion. Tissue culture: MLR and response to mitogens. Fractionated spleen cells and unfractionated control spleen cells were aliquoted and washed once in HBSS and once in RPMI-1640 with 2% FCS (Industrial Biological Laboratories, Rockville, MD) and 2 mM glutamine. Target cells were flushed from the spleens of C57BL/6 males into HBSS using a syringe and 25-gauge needle. They were then washed twice as above and irradiated with 750 R in a Westinghouse 250-Kv machine with a half-value layer of 0.9 mm copper, focal distance 54 cm, and rate bf irradiation 139 rads/min. The effector cells were centrifuged at 3658 for 10 min in 50-ml conical centrifuge tubes. The cells were 90-99s viable at the beginning of culture. Cells were resuspended in RPMI-1640 containing 2% FCS and glutamine (total volume 0.2 ml) and cultured in flat-bottomed Microtest II plates (Falcon plastics number, 3040, in a humified atmosphere of 5% COz, 95% air at 37°C for 4 days.
FRACTIONATED
NZB/W
SPLEEN
131
CELLS
Cultures were performed in sextuplicate. Four hours before harvest 0.4 &i of [3H]thymidine (New England Nuclear, Boston, MA) was added. The cultured cells were collected using an automated multiple cell harvester, washed, and the trichloroacetic acid insoluble radioactivity measured in a Nuclear-Chicago liquid scintillation counter. In the MLR, fractionated or unfractionated spleen cells from mice of different ages and strains were cultured at a concentration of 4 X 105 cells per well with 6 X lo5 irradiated C57BL/5 spleen cells as targets. Controls were 4 X lo5 effector cells alone or 6 x lo5 target cells alone, Separated or unseparated spleen cells from young or old mice of different strains were cultured at a concentration of 2 X lo5 cells per well with the previously determined optimal concentration, 1 pg/ml, of phytohemagglutinin (PHA) or concanavalin A (Con A) (Miles Laboratories, Kankakee, IL). Controls were cultured without PHA or Con A. MLR and mitogen cultures were set up simultaneously from the same pools. Surface markers. The percentage of mlfractionated spleen cells or cells from the different fractions bearing either 0 or kappa determinants was determined by a cytotoxic assay. Anti-0 C3H serum was prepared by immunization of AKR/J mice with C3H thymocytes as described by Reif and Allen (1). The anti-8 serum was used at a dilution of l/64 which was found in previous experiments to kill all splenic T cells. The anti-kappa serum described previously (13) was used at a dilution of 4 which was found in previous experiments to kill all kappa-bearing spleen cells. Cytotoxicity, performed by trypan blue exclusion, was carried out in triplicate for each fraction. The results were in agreement with those obtained by us using a 51Cr-release assay. Restoration of antibody response to SRBC. Unfractionated spleen cells or spleen cells from the different fractions obtained by unit gravity sedimentation of 1-mo NZB/W spleen cells were injected intraperitoneally into 7-mo-old NZB/W mice. The next day the recipients were immunized ip with 2 X lo8 SRBC. Four days
(
k-Fr
‘+2+3+4+-5-t--+---+ 9 II 13 7 SETTLING RATE (mm/hr)
15
17
FIG. 1. Cells from l-mo-old female mice of different strains were fractionated by unit .gravity sedimentation. The settling rate is plotted against the number of cells obtained in each I-mm/hr interval. The settling patterns of NZB X NZW (B/W F1) mice is similar to that of other strains. The fractions used in subsequent studies are indicated above the settling rate as Fr 1 through 6.
132
GARNER.
IO3
GERSHWIN
AND
STEINBERG
C--------a
B/W
F&k3 M Old
b-4
B/W
F,96
*----A
C3tVHa98
-
&lb/c
M Old M Old
$’ 6 M Old
ll,l,l,r,r,r,L, 3
5
7 9 II 13 SETTLING RATE (mm/hr)
15
17
FIG. 2. Cells from 8-mo-old BALBJc, C3H/He, and NZB X NZW (B/W R) mice and 6-mo-old B/W FI mice were fractionated by unit gravity sedimentation and the settling rate plotted against the number of cells obtained in each 1-mmjhr interval, The patterns were similar to those obtained with I-mo-old cells except that after 10 mm/hr there was a slower fall off in cells per fraction of B/W F1 and C3H/He cells.
later they were killed by cervical dislocation and the antibody-producing plaqueforming cells (PFC) of individual mice were enumerated by a modification of the method of Jerne (14). Recipient mice received 5 X 106, 12.5 X lOs, 25 X 106, or TABLE
1
MIXED LYMPHOCYTE REACTION BETWEEN BALB/c SPLEEN CELLS AND IRRADIATED C57BL/6 TARGET CELLS Fraction
thymidine & SEM
33,971c f 438df
2,812 65
1
6,96Sc f 520df
2
9,112< f 603df
Difference”
Ratio b
33,388 (P < 0.001)
77.3 (P < 0.001)
647 49
6,300 (I’ < 0.00s)
13.1 (P < 0.05)
842 63
8,364 (I’ < 0.005)
14.9 (P < 0.01)
3
282,223c f 25,613 329 3,190d It
278,888 (P < 0.005)
88.4 (P < 0.001)
4
54,96Sc f 4,654 276 2,141d z!z
52,678 (P < 0.001)
25.6 (P < 0.001)
Unfractionated
C57BL/6
Mean tritiated incorporation
5
5,900c f 2,OOSd f
535 25s
3,750 (P < o.oSj
2.9 (P > 0.05)
6
1,357c f 54Sdf
157 52
667 (P > 0.05)
2.2 (P > 0.05)
145 *
21
target cells
a (cpm stimulated cultures-cpm C57BL/6 target cells) -unstimulated cultures. b (cpm stimulated cultures-cpm C57BL/6 target cells)/unstimulated cultures. c Stimulated cultures (4 X 106 BALB/c effector cells + 6 X lo5 C57BL/6 target cells). d Unstimulated cultures (4 X lo5 BALB/c cells alone).
FRACTIONATED
NZB/W
TABLE MIXED
LYMPHOCYTE
NZB/W Fraction
REACTION
BETWEEN
MICE AND IKRADIATED
hlean tritiated incorporation
thymidine i SEM
SPLEEN
133
CELLS
2 SPLEEN
C57BL/6
CELLS FROM ~-MO-OLD TARGETS
Differencea
Ratiob -__
Unfractionated
31,050 (P < 0.001)
60.3 (I’ < 0.001)
1,726 (P > 0.05)
3.9 (P > 0.05)
1
2,520c f 594d f
288 99
2
28,73gc f 570d f
3,655 57
27,968 (I’ < 0.005)
50.1 (P < 0.001)
178,44gc =I= 22,093 389 2,81id f
175,437 (P < 0.001)
63.4 (2’ < 0.001)
1,200 137
9,854 (P < 0.05)
8.9 (P < 0.05)
3 4
C57BL/6
31,773c zk 2,589 523d f 87
11,306c f 1,251d *
5
5,576c z!z 1,662d f
764 227
3,714 (P > 0.05)
3.2 (P > 0.05)
6
3,676c f 1,408d f
436 151
2,068 (P > 0.05)
2.5 (P > 0.05)
target cells
200
3~
16
G (cpm stimulated cultures-cpm C57BL/6 b (cpm stimulated cultures-cpm C57BL/6 c Stimulated cultures (4 X lo5 NZB/W F, d Unstimulated cultures (4 X lo5 NZB/W
target target effector F1 cells
cells) --unstimulated cultures. cells)/unstimulated cultures. cells +6 X 105 C57BL/6 target cells). alone).
50 x IO6 cells as indicated. Insufficient cells were available to give the larger cell doses from fractions IV, V, and VI. The frequency of 8- and kappa-bearing cells was determined on the different fractions used in these restoration experiments. RESULTS Settling patterns. Initially we examined settling patterns of spleen cells from BALB/c, DBA/2, and C3H/He control and NZB/W mice. The settling patterns of young and old mice are shown in Figs. 1 and 2. Although there were small differences in settling between spleen cells from young and old mice and between strains they all seem to follow the same general pattern. There were no large differences in any one particular fraction between control and NZB/W mice. Although old NZB/W mice had about four times as many cells per spleen as young NZB/W mice. their settling patterns were not markedly different. Krsponstx to nlloyncic c-ells ond mito~gens, In the niIx, spleen cells from I
134
GARNER,
GERSHMYN
AND
STEINBERG
An interesting phenomenon was the change in incorporation of the unstimulated cultures (background). The background incorporation of the first two fractions of BALB/c spleen cells was near that of unfractionated controls (Table 1). while fraction 3, the region of peak response, had nearly eight times higher background. Fractions 4 and 5 had background incorporation five times that of the unfractionated cultures (Table 1). A similar phenomenon was seen with the young NZB/W cells (Table 2). The high background incorporation of several of the fractions shown in Tables 1 and 2 was observed repeatedly and caused uncertainty with regard to the most informative method of presenting the data. \I’e, therefore, have made comparisons using both ratio of stimulated response to background response and differences between these responses. For example, the high background in fraction 3 distorts the incorporation pattern using ratios, whereas the difference in cpm yields an incorporation almost seven times larger in fraction 3 than in fraction 2. The response of fraction 2 from the young NZB/\V mice is larger than fraction 2 of the BALB/c’s using both means methods of calculation : differences and ratios (Tables 1 and 2). The unfractionated cells of old NZB/W’s had a background incorporation similar to that of young NZB/W’s; however, the stimulated incorporation was less than &th as great (Tables 2 and 3). In contrast, fraction 2 of old SZB/\I’ mice had one-half of the stimulated incorporation of young NZB/\\“s but had an increase of about 14 times in background incorporation. The net result was a decrease in fraction 2 of more than 11-fold using the difference calculation and a decrease in the ratio of almost 50-fold. Fraction 3 of the old NZB/W spleen cells had a total
unfracf~onoted
4 2 3 FRACTION WMBER
5
6
FIG. 3. The response of fractionated spleen cells in the mr-\vay Illised-l?nll)huc~te reaction (MLR) against CS7BL/6 target cells is represented: G.\LB/c (O---O ). young NZB X (AA), and old NZB X NZW (B/) (O-- ---0). The response SZW (B/W) uf unfractionated cells is indicated by horizontal lines : dashed liws (B:\I.B/c), solid lines (young B/W), long and short dashes (old B/W).
FRACTIONATED
NZB/W
TXBLE
SPLEEN
135
CELLS
3
MIXED LYMPHOCYTE REACTION BETWEEN SPLEEN CELLS FROM ~-MO-OLD NZB/b. MICE AND C57BL/6 TARGET CELLS Difference”
Ratiob
224 37
1,679 (P < 0.05)
5.0 (P < 0.05)
1
1,637” f 150 490d + 46
864 (P > 0.05)
2.8 (P > 0.05)
2
10,551” f 929 8,014d zk 716
2,534 (P > 0.05)
1.3 (P > 0.05)
3
8,771c zk 769 4,291d zk 433
4,198 (P > 0.05)
2.0 (P > 0.05)
4
6,9W f 2,323d f
727 226
4,382 (P > 0.05)
2.9 (P > 0.05)
5
4,356” f 1,074d f
450 90
2,999 (P < 0.05)
3.8 (P > 0.05)
6
3,200c f 418 1,018d + 101
1,899 (P > 0.05)
2.9 (P > 0.05)
Fraction
Unfractionated
C57BL/6
target cells
-Mean tritiated incorporation
thymidine f SEM
2,379c f 417df
283
f
28
D (cpm stimulated cultures-cpm C57BL/6 * (cpm stimulated cultures-cpm C57BL/6 c Stimulated cultures (4 X 105 NZB/W F1 d Unstimulated cultures (4 X lo5 NZB/W
target target effector F1 cells
cells) --unstimulated cultures. cells)/unstimulated cultures. cells +6 X lo5 C57BL/6 target cells). alone).
mean incorporation of l/ZOth that of young NZB/W and a background two times that of young NZB/W cells. Fraction 4 had a similar pattern but was much less striking ; fractions 5 and 6 were similar to young NZB/W’s. Response to mitogens. The fractions of BALB/c spleen cells all show a good response to both PHA and Con A except fractions 1 and 6 (Table 4). The PHAto-Con A ratio which was 0.69 in unfractionated cells dropped to 0.1 in fraction 1 and then steadily increased to 0.95 and 1.0 in fractions 3 and 4, respectively. It then dropped again to 0.1 in fraction 6 (Fig. 4). Good PHA and Con A responses were observed in unfractionated cells from young NZB/W mice as well as in fractions 2, 3, and 4 (Table 5). The total incorporation did, however, tend to decrease with increasing fraction number. Fraction 5 gave a poor PHA response and fractions 1 and 6 responded poorly to both PHA and Con A. Unfractionated cells from young NZB/W mice had a PHA-to-Con A ratio of 0.68. This was approximated only in fraction 2 after which the PHA/Con A ratio fell (Table 5). When graphically presented, the differences in PHA/Con A ratios between fractions from young BALB/c and young NZB/W mice become more obvious (Fig. 4). Cells from old NZB/W mice generally had much reduced thymidine incorporation for both PHA and Con A when compared with young NZB/W cells (Table 6 and Fig. 4). The Con A response in fractions 2, 3, and 4 by the difference calculation gave incorporation of up to 10 times greater than did unfractionated cells. However, this degree of incorporation was only l/10 the response of the cor-
136
GARNER,
GERSHWIN
AND
STEINBERG
responding fractions from young NZB/W’s. PHA responses of the different fractions from old NZB/W spleen cells never increased much above those of unfractionated cells, and were l/100 those of young mice. Unfractionated cells from old NZB/W mice had a low PHA/Con A ratio of 0.34 and none of the fractions had a PHA/Con A ratio above 0.2 (Table 6 and Fig. 4). Ability of diferent fractions to restore the antibody response to SRBC. Previous study demonstrated an impaired primary response to SRBC in female NZB/W mice older than 6 mo of age, thought to be secondary to reduced “helper” T cells. Accordingly, spleen cells from l-mo-old NZB/W mice and their six fractions were injected separately into 7-mo-old NZB/W females 1 day prior to immunization and the direct PFC response to 2 X lo* SRBC was assessed 4 days later. Unfractionated spleen cells were ineffective at a cell dose of 5 X lost but did lead to a good PFC response if 50 X lo6 were given (Table 7). Only fractions 3 and 4 restored the SRBC response at 5 x lo6 cells (Table 7 and Fig. 5). Percent 0 and kappa-bearing cells in the different fractions. The percentage of cells in each fraction that could be killed by either anti-6 or anti-kappa and complement was determined for the cells used in the SRBC experiment above. We
TAKE
4
RESPONSE OF DIFFERENT FRACTIONS OF BALB/c SPLEEN CELLS (2 X 105)~o STIMULATION WITH PHA (1 pg/d)ORCON A (1 rg/d) Fraction
Mitogen
Unfractionated
PHA Con A None
Mean tritiated thymidine incorporation ItSEM 75,670 f 108,932 f 300 f
112 542 23
75,370 (P < 0.001) 108,632 (P < 0.001) 246 (P > 0.05) 1,851 (P > 0.05)
Ratio*
252 363
(P < 0.001) (P < 0.001)
1.9 (P > 0.05) 7.6 (P < 0.05)
PHA/ Con A
0.69
0.13
1
PHA Con A None
2
PHA Con A None
198,363 f 8,562 301,698 & 11,850 655 -f 79
190,708 (P < 0.001) 301,043 (P < 0.001)
303 461
(P < 0.001) (P < 0.001)
0.66
3
PHA Con A None
190,118 f 199,260 k 891 -f
6,284 5,151 143
189,229 (I’ < 0.001) 198,369 (P < 0.001)
213 244
(P < 0.001) (P < 0.001)
0.95
4
PHII Con A None
169,219 + 164,397 f 768 f
8,875 9,050 110
168,451 (P < 0.001) 163,629 (I’ < 0.001)
220 214
(I’ < 0.001) (P < 0.001)
1.00
5
1’1Ii\ Con A None
60,840 f 117,264 + 691 f
061 3,144 106
60,14Y (I’ < 0.001) 116,573 (I’ < 0.001)
101 170
(I’ < 0.001) (I < 0.001)
0.5’)
6
PHA Con A None
608 f 2,433 zk 332 zk
44 192 23
G (cpm stimulated * (cpm stimulated
527 z!z 2,132 z!z 281 I!=
3,793 8,043 52
Difference0
276 (P > 0.05) 2,101 (P < 0.05)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
1.8 (I’ > 0.05) 7.3 (P < 0.05)
0.13
FRACTIONATED
unfroctlonafed
NZB/W
SPLEEN
CELLS
137
FRACTION NUMBER
4. The PHA/Con A ratio of fractionated spleen cells from FIG. (Ayoung NZB X NZW (B/W) n), and old NZB X NZW is represented. The PHA/Con A ratio of unfractionated spleen cells lines : BALB/c (dashed lines), young B/W (solid lines), old B/W
BALB/c (O---O ), (B/W) (O-----O) is indicated by horizontal (long and short dashes).
found that fractions 3 and 4 were greatly enriched in o-bearing cells (Table 8). Fraction 1 was similar to unfractionated cells with regard to frequency of c?- and kappa-bearing cells and probably represents considerable lack of fractionation. Fraction 2 contained very few e-bearing cells, the great majority bore kappa-bearing cells. Fractions 5 and 6 were largely enriched in cells which were not killed by either anti-8 or anti-kappa serum and complement. These non-o, non-kappa-bearing cells have been termed “null” cells. DISCUSSION In this study we employed the technique of unit gravity sedimentation to separate subpopulations of spleen cells. The settling patterns of young and old mice of several strains were compared, and were found to be similar. We had expected to observe the loss of a subpopulation of cells in old NZB/W mice, corresponding to their functional loss of helper cells. However, if anything, old NZB/W mice had an increase in numbers of cells in fractions 2 and 3, fractions which had high MLR activity and PHA and Con A responsiveness in young mice. We concluded that little useful information could be obtained from the settling patterns by themselves and proceeded, therefore, to detailed functional studies of each fraction. The unit gravity sedimented cells were arbitrarily divided into six fractions which were individually studied. In BALB/c mice separation of spleen cells resulted in fractions with high and low MLR activity. Almost all the MLR activity was found in fraction 3 with a lesser amount in fraction 4. Both of these fractions had PHA/ Con A ratios of about 1.0; this is consistent with the findings of Stobo et al. (15)
138
GARNER,
GERSHWIN
AND
STEINBERG
that cells with high PHA/Con A ratios tend to be active in the MLR and represent recirculating cells with high 8 content. They also confirm the findings of Gorczynski and Rittenberg (16) who found, using unit gravity sedimentation, that cells responsive to PHA sedimented at the same rate as cells responding in an MLR. This population was found by Osoba (17) to be a fraction rich in T cells. Using a separation technique based upon centrifugation on discontinuous BSA gradients, it was observed that a subpopulation of thymocytes were especially responsive to the mitogenic effects of PHA ( 18). The separation of spleen cells from 1-mo-old NZB/W mice led to a peak MLR response which, as in BALB/c mice, was found in fraction 3. Some MLR activity also was observed in fraction 2. When the MLR activity was expressed as ratios, both BALB/c and young NZB/W peak fractions corresponded closely to the response of the unfractionated cells. It is not clear from this study or the literature whether ratio or difference is a better method for expression of this type of data. When the differences in thymidine incorporation were compared, the MLR activity was S-fold higher in BALB/c fraction 3, and $fold higher in NZB/W fraction 3 than in the respective unfractionated populations. These fractions are clearly enTABLE
5
RESPONSE OF DIFFERENT SPLEEN CELL FRACTIONS FROM ~-MO-OLD NZB/W MICE (2 X lo5 CELLS) TO PHA (1 #g/ml) AND CON A (1 pg/ml) Differencea
PHA/ Con A
Mitogen
1Jnfractionated
PHA Con A None
78,018 f 114,358 f 452 f
1,927 27,765 42
1
PHA Con A None
1,040 f 2,240 f 386 f
139 273 38
2
PHA Con A None
155,069 f 268,535 f 438 f
16,058 26,351 4.5
154,631 (P < 0.00s) 268,097 (P < 0.005)
354 613
(P < 0.001) (P < 0.001)
0.58
3
PHA Con A
47,364 f 255,862 f 596 f
4,070 22,890 62
46,768 (P < 0.001) 255,266 (I’ < 0.005)
79 429
(P < 0.001) (P < 0.001)
0.18
4
PH.4 con A
398 3,015 103
3,471 (P < 0.01) 29,661 (P < 0.005)
5.5 (P < 0.05) 39 (P < 0.001)
0.12
NOIN2
4,242 f 30,432 f 771 f
5
I’llA Con A None
1,172 f 23,644 f 843 f
101 1,967 71
329 (P > 0.05) 22,801 (P < 0.001)
1.4 (P > 0.05) 28 (I’ < 0.001)
0.01
6
PHA Con A None
226 f 414 f 173 f
34 43 27
1.3 (P > 0.005) 2.4 (I’ > 0.05)
0.22
a (cpm stimulated * (cpm stimulated
Mean tritiated thymidine incorporation GEM
Ratio*
Fraction
77,566 (P < 0.001) 113,906 (P < 0.001) 654 (P > 0.05) 1,854 (P < 0.05)
53 (P > 0.05) 241 (P > 0.05)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
173 253
(P < 0.001) (P < 0.001)
2.7 (P > 0.05) 5.8 (P < 0.05)
0.68
0.35
FRACTIONATED
NZB/W
TABLE l<~spoNSE
01: ~~IFFEKI~NT
SI'LREN
(2 X lo5 CELL) Fraction
Mitogen
SPLEEN
6
CISLL i'KAC1.IWS
TO PHA
Mean tritiated thymidine incorporation ItSElI
139
CELLS
(1 ag/ml)
lcKOM 8-MW~1.1~
N%I$/\\'
~IICli
AND CON A (1 rg/ml)
Differencea
Rntiob
PHA/ Cnn A
PHA Con A None
1,565 + 3,882 f 372 f
134 360 39
1,193 (P < 0.05) 3,510 (P < 0.05)
4.2 (P < 0.05) 10 (P < 0.05)
0.34
1
PHA Con A None
443 f 1,154 f 284f
36 112 24
159 (P > 0.05) 870 (P < 0.01)
1.6 (P > 0.05) 5.1 (P < 0.05)
0.14
2
PHA Con A None
4,494 f 459 27,364 k 2,230 360 f 36
4,134 (P < 0.05) 27,004 (P < 0.005)
12 76
3
PHA Con A None
1,685 f 138 25,545 f 2,202 548~11 48
1,137 (P < 0.05) 24,997 (1' < 0.005)
3.1 (I-’ > 0.05) 16 (I-' < 0.001)
0.05
4
PHA Con A None
1,285 f 24,327 f 536h
749 (P > 0.05) 23,791 (P < 0.00.5)
2.4 (P > 0.05) 45 (P < 0.001)
0.03
2,180 46
5
PHA Con A None
604 f 3,009 f 488f
57 271 46
116 (P > 0.05) 2,521 (P < 0.05)
1.2 (P > 0.05) 6.2 (P < 0.05)
0.05
6
PHA Con A None
466f 1,502 f 455 f
40 132 36
11 (P > 0.05) 1,047 (P < 0.05)
1.0 (P > 0.05) 3.3 (P > 0.05)
0.01
Unfrnctionated
a (cpm stimulated * (cpm stimulated
110
(P
< 0.01)
0.15
(P < 0.001)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
riched in responsive cells. However, the total response per cell of all the fractions is greater than that of unfractionated cells. These findings suggested that cells in other fractions might be inhibiting high-responding cells in the unfractionated populations. Since both background incorporation and stimulated incorporation rose proportionally, such an inhibitory population would act nonspecifically upon DNA synthesis. The most impressive findings in the old NZB/W fractions were the lack of responsiveness in MLR, and the lo- to 30-fold increase in background incorporation in fractions 2, 3, and 4. These same fractions had a 7-fold increase in Con A responsiveness but little increase in PHA responsiveness as compared with mfractionated cells. In contrast to BALB/c and young NZB/W mice, there was no increased responsiveness in individual fractions of old NZB/W mice; such an increase might have pointed to inhibition by macrophages or other cells. The low PHA/Con A ratios in old NZB/W mice are in agreement with previous observations (5) and the data collectively suggest the loss of a subpopulation of T cells from old NZB/W spleens. This is consistent with the finding that spleens of old NZB/W mice lack a subpopulation of T cells which participate in skin allograft rejection (5, 13).
140
GARNER,
GERSH\VIN
AND
‘I‘:\BI,E
STEINBERG
7
FOUR-DAY RESPONSE TO IMMUNIZATION WITH 2 X lo* SRBC IN T-MO-OLD NZB/W FEMALES GIVEN DIFFERENT FRACTIONS OF ~-MO-OLD NZB/W’ SPLEEN CELLS 1 DAY PRIOR TO IMMDNIZATION
Fraction
Number of cells added
ITnfractionated
PFC/spleen”
PFC/106
X106 x 10”
2,240 43,500
187
5
X 106
X 10’
1,770 12,150
10 46
2
50
8 17
0
79 260
71 104
5 50
1
2 3 4
5 6 None*
PFC/106 Control b 2
10
5
x 106
50
x 106
1,363 2,710
5 50
x 106 Y 106
98,500
5
x 10”
18,500
112
12.5 x 10”
30,000 12,500
167 236
2,790 3,750
16 21
8
12.5 x 106 5 X106 12.5 x 106 -
1,815 2,340 1,850
11
3
20 8
25
x 106
5
x 106
*
16,400
a Average of two to three determinations. b Control response is that of unrestored 7-mo NZB/\V cated by “None” in this table.
-
mice immunized with SRBC and indi-
The ability of the different fractions to restore helper activity in the SRBC system correlated with the mitogen and MLR studies. Fractions 3 and 4 had a profound helper effect at 5 x lo6 cells whereas the other fractions did not. These
:,
60
Y 2 ii
40
6 m 20 3 No Cells
UNFX Splesn Cells
FX I
FX 2
:rj: FX 3
FX 4
FX 5
FX 6
FIG. 5. Seven-month-old NZB/W female mice were immunized with 2 X 10’ SRBC 1 day after transfer of S X 10’ cells from 1-mo-old NZB/W mice fractionated by unit gravity sedimentation. The number of direct antibody-producing plaque-forming cells is indicated for mice receiving no cells, unfractionated spleen cells (UNFX), or cells from fractions (FX) 1 through 6. The height of each bar represents the geometric mean and the vertical bars delimit the range. Only cells from fractions 3 and 4 restored the SRBC response to the ‘I-mo-old NZB/W recipients.
FRACTIONATED
NZB/W
TABLE
SPLEEN
141
CELLS
8
PERCENT 0 AND KAPPA-BEARING CELLS IN THE DIFFERENT FRACTIONS” AFTER SEDIMENTATION OF SPLEEN CELLS FROM ~-MO-OLD NZB/\\. nlrcE
UNIT GRAVITY
Fraction
%S
% Kappa
5; Null cellsb
IJnfractionnted 1 2 3 4 5 6
29.7 31.0 9.7 73.7 78.3 16.7 9.0
47.7 46.0 70.3 7.0 5.0 5.3 5.7
19.3 26.7 19.7 19.3 16.7 78.0 88.7
o Means of three determinations. b Cells bearing neither 6 nor kappa determinants.
active populations contained the greatest percentage of &positive cells. Fraction 2, which contained ahnost no e-positive cells, was ineffective even at 50 X lo6 cells. These results imply that the subpopulation of cells lacking in old NZB/W mice which is required for the response to SRBC is a helper T cell. Earlier studies demonstrated that spleen cells were separable using equilibrium centrifugation in density gradients of Ficoll into two populations functionally distinct in the PFC response to SRBC (19). In particular, one population appeared to be helper T cells which cooperated with bone marrow cells when transferred to lethally irradiated recipients. In the present study, old NZB/W mice which lack helper T cells were restored with regard to the PFC response to SRBC by subpopulations of young spleen cells which contain a high percentage of o-bearing cells. These fractions presumably contained splenic helper T cells required for the PFC response to SRBC analogous to those separated by Gorczynski and co-workers (19). The ability to restore the SRBC response izith small numbers of cells from a fraction of young NZB/W spleen cells suggests that restoration of immunologic competence in old NZB/W mice may be achieved in a manner not previously possible. Five million spleen cells from fractions 3 and 4 increased the numbers of PFC/106 cells 40-60 times more than did 5 million unfractionated cells. Previous study has suggested some therapeutic efficacy of large numbers of yomig NZB/LV’ lymphoid cells given to syngeneic mice as they aged (20). However, the benefits of such therapy appeared to be limited by negative aspects of the therapy thought to be secondary to the large number of cells given. If much smaller numbers of cells could be given by separating and administering only the relevant subpopulations of cells, some of the disadvantages of such therapy might be avoided. It was of interest to find that bye could enrich in null cells as indicated by the small percentage of & or kappa-bearing cells in fractions 5 and 6. The null cells were found by Stobo et al. (21) to be present in increased frequency in unfractionated spleen cell suspensions from New Zealand mice, an observation which we have herein confirmed. The function of these cells is uncertain ; however, the present study suggests that unit gravity sedimentation may be helpful in enriching in these cells. In conjunction with other techniques almost pure populations of null cells can probably be obtained and their functions studied. The present study suggests that unit gravity sedimentation may be of considerable value in separating subpopulations of lymphoid cells from NZB/W mice.
142
GARNER,
GERSHWIN
AND
STEINBERG
It has been observed that NZB/W mice demonstrate marked changes in lymphoid cell subpopulations with age. Early in life they appear to lose regulatory or suppressor cells (9, 22) and later in life they lose helper cells (1, 4, 5). Use of separation techniques should be a valuable aid in further studying these processes. It would be desirable to separately enrich subpopulations of helper and suppressor cells so that their function can be independently studied. In addition, the maturation of different cell types can be studied as has already been demonstrated for B cells in other inbred mouse strains (23). ACKNOWLEDGMENTS The authors are pleased to acknowledge the excellent technical assistance of Ms. Susan Kysela. We are indebted to Drs. T. M. Chused, P. H. Plotz, M. C. Gelfand, and L. M. Parker for advice and gifts of reagents. The careful assistance of Ms. Nancy Milloy in the preparation of the manuscript is gratefully acknowledged.
REFERENCES 1. Talal, N., and Steinberg, A. D. In “Current Topics in Microbiology and Immunology,” Springer-Verlag New York, 1974. 2. Denman, A. M., and Denman, E. J., C/in. Exp. Imnzwaol. 6, 457, 1970. 3. Gelfand, M. C., Parker, L. M., and Steinberg, A. D., J. Immunol. 1974. 4. Cantor, H., Asofsky, R., and Talal, N., J. Exp. Med. 131, 223, 1970. 5. Gelfand, M. C., and Steinberg, A. D., J. Immunol. 110, 1652, 1973. 6. Shirai, T., and Mellors, R. C., Proc. Nat. Acad. Sci. USA 68, 1412, 1971. 7. Evans, M. M., Williamson, W. G., and Irvine, W. J., Clin. Exp. Iwmorol. 3, 375, 1968. 8. Playfair, J. H. L., Zmmzrnology 15, 35, 1968. 9. Barthold, D. R., Kysela, S., and Steinberg, A. D., J. Iinnzuaol. 112, 9, 1974. 10. Mage, M. G., Evans, W. H., and Peterson, E. A., Proc. Sot. Exp. Biol. Med. 127, 478, 1968. 11. Miller, R. G., and Phillips, R. A., J. Cell. Physiol. 73, 191, 1969.’ 12. Reif, A. E., and Allen, J. M. V., Nature (Londolz) 209, 521, 1966. 13. Gelfand, M. C., and Steinberg, A. D., Cell. l+n~mno/. 11, 221, 1974. 14. Jacobs, M. E., Steinberg, A. D., Gorden, J. K., and Talal, N., Arthritis Rhcum. 15, 201, 1972. 15. Stobo, J. D., and Paul, W. E., J. Immunol. 110, 362, 1973. 16. Gorczynski, R. M., and Rittenberg, M. B., 1. Imnttlnol. 112, 47, 1974. 17. Osoba, D., J. Exp. Med. 132,368, 1970. 18. Levey, R. H., and Burleson, R., Cell. Imlrnrnol. 4, 316, 1972. 19. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., 1mmzlnolog.v 20, 693, 1971. 20. Kysela, S., and Steinberg, A. D., Clin. Inzmunol. Immunopathol. 2, 113, 1973. 21. Stobo, J. D., Talal, N., and Paul, W. E., 1. Immulzol. 109, 701, 1972. 22. Steinberg, A. D., Arthritis Rhncm. 17, 11, 1974. 23. Lafleur, L., Miller, R. G., and Phillips, R. A., J. Exp. Med. 135, 1363, 1972.
15, 129-142 (1975)
Properties of Fractionated GEORGE
M. GARNER,
RI.
ERIC
Spleen Cells from NZB/W GERSHWIN,
AND
Arthritis and Rltc?f~rratism Branch, Natiorlal Insitzttr Ili,qrsti;v Disrascs, National Ilrstitutcs of Hmlth, Recekvd
ALFRED
Mice
D. STEINBERG
Arthritis, Metabolism, ajrd Gethrsda, Maryland 20014
of
Alay 16, 1974
The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/Z, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice. A number of functional studies performed with the separated cells proved more of cells which sediinformative than the settling patterns themselves. Fractions mented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in e-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZBjW mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.
INTRODUCTION New Zealand mice manifest autoimmune phenomena including multiple autoantibodies and a number of immunological abnormalities ( 1). Recirculating lymphocytes (2), lymph node-seeking cells (3)) and helper cells participating in graft versus host (GVH)’ disease (4) and skin allograft rejection (5) are all lost as New Zealand mice age, perhaps as a result of the spontaneous production of naturally occurring thymocytotoxic antibdoy (NTA) (3, 6). The antibody response of New Zealand mice to sheep red blood cells (SRBC) develops early in life in comparison with other strains (7, 8). Later in life there is a premature loss of responsiveness to SRBC at a time when the response to type III pneumococcal polysaccharide (SSSIII), an antigen not requiring thymic helper cells, is intact (9). All of the evidence points to a loss of helper T cells in older New Zealand mice. In view of the interest in subpopulations of lymphoid cells, we have fractionated spleen mice using a unit gravity separation techcells from NZB x NZW F1 (NZB/W) 1 Abbreviations used in this paper : GVH, graft versus host disease ; NTA, naturally occurring thymocytotoxic antibody ; SSSIII, type III pneumococcal polysaccharide ; SRBC, sheep red blood cells ; NZB/W, NZB X NZW F, mice ; MLR, mixed lymphocyte reaction; FCS, fetal calf serum ; MEM, minimal essential medium; PHA, phytohemagglutinin A ; Con A, concanavalin A. 129 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
130
GARNER,
GERSHWIN
AND
STEINBERG
nique (10, 11). The fractions were characterized with respect to responsiveness to allogencic cells and other mitogens, surface markers, and ability to restore the response of old NZB/W mice to SKBC. MATERIALS
AND
METHODS
Animals. BALB/c, NZB x NZW F1 (NZB/W), C3H/He, DBA/2, and C57BL/6 mice were obtained from colonies maintained at the National Institutes of Health (NIH), Bethesda, MD. AKR mice were obtained from Jackson Laboratories, Bar Harbor, ME. Females were used throughout for study except as donors of target cells in the mixed lymphocyte reaction (MLR). All media were obtained from the NIH media center. Spleen cell fractionation. Spleen cells from NZB/W, C3H/He, DBA/2, and BALB/c mice were fractionated in this study. Groups of 1.5 mice were sacrificed by cervical dislocation and their spleens asceptically removed. Single cell suspensions were prepared and washed in 50 ml Hanks’ Balanced Salt Solution (HBSS) buffered with 19 mlM N-2 hydroxyethylpeperazine N’-Zethanesulfonic acid (HEPES) . The cell pellet was resuspended in 80 ml of Eagle’s minimal essential medium supplemented with Earle’s salts, and buffered to pH 7.2 with Tris buffer. The cells were separated at unit gravity over a 530% fetal calf serum (FCS) linear gradient for 2 hr in the settling chamber of Mage et al. (IO) according to the method of Miller and Phillips (11)) except that Eagle’s minimal essential medium (MEM) was used as a supporting medium instead of phosphate-buffered saline (PBS). After 2 hr the gradient was harvested in I-mm/hr fractions and the settling patterns were determined. For further study the gradient was arbitrarily separated into six fractions as follows : Fraction 1 2 3 4 5 6
Settling rate 3-5.5 mm/hr 5.5-7.0 mm/‘hr 7.0-8.5 mm/hr 8.5-10.0 mm/hr 10.0-11.5 mm/hr 12.O16.0 mm/hr
Cell counts. Leukocytes were counted in a Coulter Counter Model ZBI. The cell counts were confirmed periodically in a hemocytometer. Viability was checked by trypan blue exclusion. Tissue culture: MLR and response to mitogens. Fractionated spleen cells and unfractionated control spleen cells were aliquoted and washed once in HBSS and once in RPMI-1640 with 2% FCS (Industrial Biological Laboratories, Rockville, MD) and 2 mM glutamine. Target cells were flushed from the spleens of C57BL/6 males into HBSS using a syringe and 25-gauge needle. They were then washed twice as above and irradiated with 750 R in a Westinghouse 250-Kv machine with a half-value layer of 0.9 mm copper, focal distance 54 cm, and rate bf irradiation 139 rads/min. The effector cells were centrifuged at 3658 for 10 min in 50-ml conical centrifuge tubes. The cells were 90-99s viable at the beginning of culture. Cells were resuspended in RPMI-1640 containing 2% FCS and glutamine (total volume 0.2 ml) and cultured in flat-bottomed Microtest II plates (Falcon plastics number, 3040, in a humified atmosphere of 5% COz, 95% air at 37°C for 4 days.
FRACTIONATED
NZB/W
SPLEEN
131
CELLS
Cultures were performed in sextuplicate. Four hours before harvest 0.4 &i of [3H]thymidine (New England Nuclear, Boston, MA) was added. The cultured cells were collected using an automated multiple cell harvester, washed, and the trichloroacetic acid insoluble radioactivity measured in a Nuclear-Chicago liquid scintillation counter. In the MLR, fractionated or unfractionated spleen cells from mice of different ages and strains were cultured at a concentration of 4 X 105 cells per well with 6 X lo5 irradiated C57BL/5 spleen cells as targets. Controls were 4 X lo5 effector cells alone or 6 x lo5 target cells alone, Separated or unseparated spleen cells from young or old mice of different strains were cultured at a concentration of 2 X lo5 cells per well with the previously determined optimal concentration, 1 pg/ml, of phytohemagglutinin (PHA) or concanavalin A (Con A) (Miles Laboratories, Kankakee, IL). Controls were cultured without PHA or Con A. MLR and mitogen cultures were set up simultaneously from the same pools. Surface markers. The percentage of mlfractionated spleen cells or cells from the different fractions bearing either 0 or kappa determinants was determined by a cytotoxic assay. Anti-0 C3H serum was prepared by immunization of AKR/J mice with C3H thymocytes as described by Reif and Allen (1). The anti-8 serum was used at a dilution of l/64 which was found in previous experiments to kill all splenic T cells. The anti-kappa serum described previously (13) was used at a dilution of 4 which was found in previous experiments to kill all kappa-bearing spleen cells. Cytotoxicity, performed by trypan blue exclusion, was carried out in triplicate for each fraction. The results were in agreement with those obtained by us using a 51Cr-release assay. Restoration of antibody response to SRBC. Unfractionated spleen cells or spleen cells from the different fractions obtained by unit gravity sedimentation of 1-mo NZB/W spleen cells were injected intraperitoneally into 7-mo-old NZB/W mice. The next day the recipients were immunized ip with 2 X lo8 SRBC. Four days
(
k-Fr
‘+2+3+4+-5-t--+---+ 9 II 13 7 SETTLING RATE (mm/hr)
15
17
FIG. 1. Cells from l-mo-old female mice of different strains were fractionated by unit .gravity sedimentation. The settling rate is plotted against the number of cells obtained in each I-mm/hr interval. The settling patterns of NZB X NZW (B/W F1) mice is similar to that of other strains. The fractions used in subsequent studies are indicated above the settling rate as Fr 1 through 6.
132
GARNER.
IO3
GERSHWIN
AND
STEINBERG
C--------a
B/W
F&k3 M Old
b-4
B/W
F,96
*----A
C3tVHa98
-
&lb/c
M Old M Old
$’ 6 M Old
ll,l,l,r,r,r,L, 3
5
7 9 II 13 SETTLING RATE (mm/hr)
15
17
FIG. 2. Cells from 8-mo-old BALBJc, C3H/He, and NZB X NZW (B/W R) mice and 6-mo-old B/W FI mice were fractionated by unit gravity sedimentation and the settling rate plotted against the number of cells obtained in each 1-mmjhr interval, The patterns were similar to those obtained with I-mo-old cells except that after 10 mm/hr there was a slower fall off in cells per fraction of B/W F1 and C3H/He cells.
later they were killed by cervical dislocation and the antibody-producing plaqueforming cells (PFC) of individual mice were enumerated by a modification of the method of Jerne (14). Recipient mice received 5 X 106, 12.5 X lOs, 25 X 106, or TABLE
1
MIXED LYMPHOCYTE REACTION BETWEEN BALB/c SPLEEN CELLS AND IRRADIATED C57BL/6 TARGET CELLS Fraction
thymidine & SEM
33,971c f 438df
2,812 65
1
6,96Sc f 520df
2
9,112< f 603df
Difference”
Ratio b
33,388 (P < 0.001)
77.3 (P < 0.001)
647 49
6,300 (I’ < 0.00s)
13.1 (P < 0.05)
842 63
8,364 (I’ < 0.005)
14.9 (P < 0.01)
3
282,223c f 25,613 329 3,190d It
278,888 (P < 0.005)
88.4 (P < 0.001)
4
54,96Sc f 4,654 276 2,141d z!z
52,678 (P < 0.001)
25.6 (P < 0.001)
Unfractionated
C57BL/6
Mean tritiated incorporation
5
5,900c f 2,OOSd f
535 25s
3,750 (P < o.oSj
2.9 (P > 0.05)
6
1,357c f 54Sdf
157 52
667 (P > 0.05)
2.2 (P > 0.05)
145 *
21
target cells
a (cpm stimulated cultures-cpm C57BL/6 target cells) -unstimulated cultures. b (cpm stimulated cultures-cpm C57BL/6 target cells)/unstimulated cultures. c Stimulated cultures (4 X 106 BALB/c effector cells + 6 X lo5 C57BL/6 target cells). d Unstimulated cultures (4 X lo5 BALB/c cells alone).
FRACTIONATED
NZB/W
TABLE MIXED
LYMPHOCYTE
NZB/W Fraction
REACTION
BETWEEN
MICE AND IKRADIATED
hlean tritiated incorporation
thymidine i SEM
SPLEEN
133
CELLS
2 SPLEEN
C57BL/6
CELLS FROM ~-MO-OLD TARGETS
Differencea
Ratiob -__
Unfractionated
31,050 (P < 0.001)
60.3 (I’ < 0.001)
1,726 (P > 0.05)
3.9 (P > 0.05)
1
2,520c f 594d f
288 99
2
28,73gc f 570d f
3,655 57
27,968 (I’ < 0.005)
50.1 (P < 0.001)
178,44gc =I= 22,093 389 2,81id f
175,437 (P < 0.001)
63.4 (2’ < 0.001)
1,200 137
9,854 (P < 0.05)
8.9 (P < 0.05)
3 4
C57BL/6
31,773c zk 2,589 523d f 87
11,306c f 1,251d *
5
5,576c z!z 1,662d f
764 227
3,714 (P > 0.05)
3.2 (P > 0.05)
6
3,676c f 1,408d f
436 151
2,068 (P > 0.05)
2.5 (P > 0.05)
target cells
200
3~
16
G (cpm stimulated cultures-cpm C57BL/6 b (cpm stimulated cultures-cpm C57BL/6 c Stimulated cultures (4 X lo5 NZB/W F, d Unstimulated cultures (4 X lo5 NZB/W
target target effector F1 cells
cells) --unstimulated cultures. cells)/unstimulated cultures. cells +6 X 105 C57BL/6 target cells). alone).
50 x IO6 cells as indicated. Insufficient cells were available to give the larger cell doses from fractions IV, V, and VI. The frequency of 8- and kappa-bearing cells was determined on the different fractions used in these restoration experiments. RESULTS Settling patterns. Initially we examined settling patterns of spleen cells from BALB/c, DBA/2, and C3H/He control and NZB/W mice. The settling patterns of young and old mice are shown in Figs. 1 and 2. Although there were small differences in settling between spleen cells from young and old mice and between strains they all seem to follow the same general pattern. There were no large differences in any one particular fraction between control and NZB/W mice. Although old NZB/W mice had about four times as many cells per spleen as young NZB/W mice. their settling patterns were not markedly different. Krsponstx to nlloyncic c-ells ond mito~gens, In the niIx, spleen cells from I
134
GARNER,
GERSHMYN
AND
STEINBERG
An interesting phenomenon was the change in incorporation of the unstimulated cultures (background). The background incorporation of the first two fractions of BALB/c spleen cells was near that of unfractionated controls (Table 1). while fraction 3, the region of peak response, had nearly eight times higher background. Fractions 4 and 5 had background incorporation five times that of the unfractionated cultures (Table 1). A similar phenomenon was seen with the young NZB/W cells (Table 2). The high background incorporation of several of the fractions shown in Tables 1 and 2 was observed repeatedly and caused uncertainty with regard to the most informative method of presenting the data. \I’e, therefore, have made comparisons using both ratio of stimulated response to background response and differences between these responses. For example, the high background in fraction 3 distorts the incorporation pattern using ratios, whereas the difference in cpm yields an incorporation almost seven times larger in fraction 3 than in fraction 2. The response of fraction 2 from the young NZB/\V mice is larger than fraction 2 of the BALB/c’s using both means methods of calculation : differences and ratios (Tables 1 and 2). The unfractionated cells of old NZB/W’s had a background incorporation similar to that of young NZB/W’s; however, the stimulated incorporation was less than &th as great (Tables 2 and 3). In contrast, fraction 2 of old SZB/\I’ mice had one-half of the stimulated incorporation of young NZB/\\“s but had an increase of about 14 times in background incorporation. The net result was a decrease in fraction 2 of more than 11-fold using the difference calculation and a decrease in the ratio of almost 50-fold. Fraction 3 of the old NZB/W spleen cells had a total
unfracf~onoted
4 2 3 FRACTION WMBER
5
6
FIG. 3. The response of fractionated spleen cells in the mr-\vay Illised-l?nll)huc~te reaction (MLR) against CS7BL/6 target cells is represented: G.\LB/c (O---O ). young NZB X (AA), and old NZB X NZW (B/) (O-- ---0). The response SZW (B/W) uf unfractionated cells is indicated by horizontal lines : dashed liws (B:\I.B/c), solid lines (young B/W), long and short dashes (old B/W).
FRACTIONATED
NZB/W
TXBLE
SPLEEN
135
CELLS
3
MIXED LYMPHOCYTE REACTION BETWEEN SPLEEN CELLS FROM ~-MO-OLD NZB/b. MICE AND C57BL/6 TARGET CELLS Difference”
Ratiob
224 37
1,679 (P < 0.05)
5.0 (P < 0.05)
1
1,637” f 150 490d + 46
864 (P > 0.05)
2.8 (P > 0.05)
2
10,551” f 929 8,014d zk 716
2,534 (P > 0.05)
1.3 (P > 0.05)
3
8,771c zk 769 4,291d zk 433
4,198 (P > 0.05)
2.0 (P > 0.05)
4
6,9W f 2,323d f
727 226
4,382 (P > 0.05)
2.9 (P > 0.05)
5
4,356” f 1,074d f
450 90
2,999 (P < 0.05)
3.8 (P > 0.05)
6
3,200c f 418 1,018d + 101
1,899 (P > 0.05)
2.9 (P > 0.05)
Fraction
Unfractionated
C57BL/6
target cells
-Mean tritiated incorporation
thymidine f SEM
2,379c f 417df
283
f
28
D (cpm stimulated cultures-cpm C57BL/6 * (cpm stimulated cultures-cpm C57BL/6 c Stimulated cultures (4 X 105 NZB/W F1 d Unstimulated cultures (4 X lo5 NZB/W
target target effector F1 cells
cells) --unstimulated cultures. cells)/unstimulated cultures. cells +6 X lo5 C57BL/6 target cells). alone).
mean incorporation of l/ZOth that of young NZB/W and a background two times that of young NZB/W cells. Fraction 4 had a similar pattern but was much less striking ; fractions 5 and 6 were similar to young NZB/W’s. Response to mitogens. The fractions of BALB/c spleen cells all show a good response to both PHA and Con A except fractions 1 and 6 (Table 4). The PHAto-Con A ratio which was 0.69 in unfractionated cells dropped to 0.1 in fraction 1 and then steadily increased to 0.95 and 1.0 in fractions 3 and 4, respectively. It then dropped again to 0.1 in fraction 6 (Fig. 4). Good PHA and Con A responses were observed in unfractionated cells from young NZB/W mice as well as in fractions 2, 3, and 4 (Table 5). The total incorporation did, however, tend to decrease with increasing fraction number. Fraction 5 gave a poor PHA response and fractions 1 and 6 responded poorly to both PHA and Con A. Unfractionated cells from young NZB/W mice had a PHA-to-Con A ratio of 0.68. This was approximated only in fraction 2 after which the PHA/Con A ratio fell (Table 5). When graphically presented, the differences in PHA/Con A ratios between fractions from young BALB/c and young NZB/W mice become more obvious (Fig. 4). Cells from old NZB/W mice generally had much reduced thymidine incorporation for both PHA and Con A when compared with young NZB/W cells (Table 6 and Fig. 4). The Con A response in fractions 2, 3, and 4 by the difference calculation gave incorporation of up to 10 times greater than did unfractionated cells. However, this degree of incorporation was only l/10 the response of the cor-
136
GARNER,
GERSHWIN
AND
STEINBERG
responding fractions from young NZB/W’s. PHA responses of the different fractions from old NZB/W spleen cells never increased much above those of unfractionated cells, and were l/100 those of young mice. Unfractionated cells from old NZB/W mice had a low PHA/Con A ratio of 0.34 and none of the fractions had a PHA/Con A ratio above 0.2 (Table 6 and Fig. 4). Ability of diferent fractions to restore the antibody response to SRBC. Previous study demonstrated an impaired primary response to SRBC in female NZB/W mice older than 6 mo of age, thought to be secondary to reduced “helper” T cells. Accordingly, spleen cells from l-mo-old NZB/W mice and their six fractions were injected separately into 7-mo-old NZB/W females 1 day prior to immunization and the direct PFC response to 2 X lo* SRBC was assessed 4 days later. Unfractionated spleen cells were ineffective at a cell dose of 5 X lost but did lead to a good PFC response if 50 X lo6 were given (Table 7). Only fractions 3 and 4 restored the SRBC response at 5 x lo6 cells (Table 7 and Fig. 5). Percent 0 and kappa-bearing cells in the different fractions. The percentage of cells in each fraction that could be killed by either anti-6 or anti-kappa and complement was determined for the cells used in the SRBC experiment above. We
TAKE
4
RESPONSE OF DIFFERENT FRACTIONS OF BALB/c SPLEEN CELLS (2 X 105)~o STIMULATION WITH PHA (1 pg/d)ORCON A (1 rg/d) Fraction
Mitogen
Unfractionated
PHA Con A None
Mean tritiated thymidine incorporation ItSEM 75,670 f 108,932 f 300 f
112 542 23
75,370 (P < 0.001) 108,632 (P < 0.001) 246 (P > 0.05) 1,851 (P > 0.05)
Ratio*
252 363
(P < 0.001) (P < 0.001)
1.9 (P > 0.05) 7.6 (P < 0.05)
PHA/ Con A
0.69
0.13
1
PHA Con A None
2
PHA Con A None
198,363 f 8,562 301,698 & 11,850 655 -f 79
190,708 (P < 0.001) 301,043 (P < 0.001)
303 461
(P < 0.001) (P < 0.001)
0.66
3
PHA Con A None
190,118 f 199,260 k 891 -f
6,284 5,151 143
189,229 (I’ < 0.001) 198,369 (P < 0.001)
213 244
(P < 0.001) (P < 0.001)
0.95
4
PHII Con A None
169,219 + 164,397 f 768 f
8,875 9,050 110
168,451 (P < 0.001) 163,629 (I’ < 0.001)
220 214
(I’ < 0.001) (P < 0.001)
1.00
5
1’1Ii\ Con A None
60,840 f 117,264 + 691 f
061 3,144 106
60,14Y (I’ < 0.001) 116,573 (I’ < 0.001)
101 170
(I’ < 0.001) (I < 0.001)
0.5’)
6
PHA Con A None
608 f 2,433 zk 332 zk
44 192 23
G (cpm stimulated * (cpm stimulated
527 z!z 2,132 z!z 281 I!=
3,793 8,043 52
Difference0
276 (P > 0.05) 2,101 (P < 0.05)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
1.8 (I’ > 0.05) 7.3 (P < 0.05)
0.13
FRACTIONATED
unfroctlonafed
NZB/W
SPLEEN
CELLS
137
FRACTION NUMBER
4. The PHA/Con A ratio of fractionated spleen cells from FIG. (Ayoung NZB X NZW (B/W) n), and old NZB X NZW is represented. The PHA/Con A ratio of unfractionated spleen cells lines : BALB/c (dashed lines), young B/W (solid lines), old B/W
BALB/c (O---O ), (B/W) (O-----O) is indicated by horizontal (long and short dashes).
found that fractions 3 and 4 were greatly enriched in o-bearing cells (Table 8). Fraction 1 was similar to unfractionated cells with regard to frequency of c?- and kappa-bearing cells and probably represents considerable lack of fractionation. Fraction 2 contained very few e-bearing cells, the great majority bore kappa-bearing cells. Fractions 5 and 6 were largely enriched in cells which were not killed by either anti-8 or anti-kappa serum and complement. These non-o, non-kappa-bearing cells have been termed “null” cells. DISCUSSION In this study we employed the technique of unit gravity sedimentation to separate subpopulations of spleen cells. The settling patterns of young and old mice of several strains were compared, and were found to be similar. We had expected to observe the loss of a subpopulation of cells in old NZB/W mice, corresponding to their functional loss of helper cells. However, if anything, old NZB/W mice had an increase in numbers of cells in fractions 2 and 3, fractions which had high MLR activity and PHA and Con A responsiveness in young mice. We concluded that little useful information could be obtained from the settling patterns by themselves and proceeded, therefore, to detailed functional studies of each fraction. The unit gravity sedimented cells were arbitrarily divided into six fractions which were individually studied. In BALB/c mice separation of spleen cells resulted in fractions with high and low MLR activity. Almost all the MLR activity was found in fraction 3 with a lesser amount in fraction 4. Both of these fractions had PHA/ Con A ratios of about 1.0; this is consistent with the findings of Stobo et al. (15)
138
GARNER,
GERSHWIN
AND
STEINBERG
that cells with high PHA/Con A ratios tend to be active in the MLR and represent recirculating cells with high 8 content. They also confirm the findings of Gorczynski and Rittenberg (16) who found, using unit gravity sedimentation, that cells responsive to PHA sedimented at the same rate as cells responding in an MLR. This population was found by Osoba (17) to be a fraction rich in T cells. Using a separation technique based upon centrifugation on discontinuous BSA gradients, it was observed that a subpopulation of thymocytes were especially responsive to the mitogenic effects of PHA ( 18). The separation of spleen cells from 1-mo-old NZB/W mice led to a peak MLR response which, as in BALB/c mice, was found in fraction 3. Some MLR activity also was observed in fraction 2. When the MLR activity was expressed as ratios, both BALB/c and young NZB/W peak fractions corresponded closely to the response of the unfractionated cells. It is not clear from this study or the literature whether ratio or difference is a better method for expression of this type of data. When the differences in thymidine incorporation were compared, the MLR activity was S-fold higher in BALB/c fraction 3, and $fold higher in NZB/W fraction 3 than in the respective unfractionated populations. These fractions are clearly enTABLE
5
RESPONSE OF DIFFERENT SPLEEN CELL FRACTIONS FROM ~-MO-OLD NZB/W MICE (2 X lo5 CELLS) TO PHA (1 #g/ml) AND CON A (1 pg/ml) Differencea
PHA/ Con A
Mitogen
1Jnfractionated
PHA Con A None
78,018 f 114,358 f 452 f
1,927 27,765 42
1
PHA Con A None
1,040 f 2,240 f 386 f
139 273 38
2
PHA Con A None
155,069 f 268,535 f 438 f
16,058 26,351 4.5
154,631 (P < 0.00s) 268,097 (P < 0.005)
354 613
(P < 0.001) (P < 0.001)
0.58
3
PHA Con A
47,364 f 255,862 f 596 f
4,070 22,890 62
46,768 (P < 0.001) 255,266 (I’ < 0.005)
79 429
(P < 0.001) (P < 0.001)
0.18
4
PH.4 con A
398 3,015 103
3,471 (P < 0.01) 29,661 (P < 0.005)
5.5 (P < 0.05) 39 (P < 0.001)
0.12
NOIN2
4,242 f 30,432 f 771 f
5
I’llA Con A None
1,172 f 23,644 f 843 f
101 1,967 71
329 (P > 0.05) 22,801 (P < 0.001)
1.4 (P > 0.05) 28 (I’ < 0.001)
0.01
6
PHA Con A None
226 f 414 f 173 f
34 43 27
1.3 (P > 0.005) 2.4 (I’ > 0.05)
0.22
a (cpm stimulated * (cpm stimulated
Mean tritiated thymidine incorporation GEM
Ratio*
Fraction
77,566 (P < 0.001) 113,906 (P < 0.001) 654 (P > 0.05) 1,854 (P < 0.05)
53 (P > 0.05) 241 (P > 0.05)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
173 253
(P < 0.001) (P < 0.001)
2.7 (P > 0.05) 5.8 (P < 0.05)
0.68
0.35
FRACTIONATED
NZB/W
TABLE l<~spoNSE
01: ~~IFFEKI~NT
SI'LREN
(2 X lo5 CELL) Fraction
Mitogen
SPLEEN
6
CISLL i'KAC1.IWS
TO PHA
Mean tritiated thymidine incorporation ItSElI
139
CELLS
(1 ag/ml)
lcKOM 8-MW~1.1~
N%I$/\\'
~IICli
AND CON A (1 rg/ml)
Differencea
Rntiob
PHA/ Cnn A
PHA Con A None
1,565 + 3,882 f 372 f
134 360 39
1,193 (P < 0.05) 3,510 (P < 0.05)
4.2 (P < 0.05) 10 (P < 0.05)
0.34
1
PHA Con A None
443 f 1,154 f 284f
36 112 24
159 (P > 0.05) 870 (P < 0.01)
1.6 (P > 0.05) 5.1 (P < 0.05)
0.14
2
PHA Con A None
4,494 f 459 27,364 k 2,230 360 f 36
4,134 (P < 0.05) 27,004 (P < 0.005)
12 76
3
PHA Con A None
1,685 f 138 25,545 f 2,202 548~11 48
1,137 (P < 0.05) 24,997 (1' < 0.005)
3.1 (I-’ > 0.05) 16 (I-' < 0.001)
0.05
4
PHA Con A None
1,285 f 24,327 f 536h
749 (P > 0.05) 23,791 (P < 0.00.5)
2.4 (P > 0.05) 45 (P < 0.001)
0.03
2,180 46
5
PHA Con A None
604 f 3,009 f 488f
57 271 46
116 (P > 0.05) 2,521 (P < 0.05)
1.2 (P > 0.05) 6.2 (P < 0.05)
0.05
6
PHA Con A None
466f 1,502 f 455 f
40 132 36
11 (P > 0.05) 1,047 (P < 0.05)
1.0 (P > 0.05) 3.3 (P > 0.05)
0.01
Unfrnctionated
a (cpm stimulated * (cpm stimulated
110
(P
< 0.01)
0.15
(P < 0.001)
cultures) - (cpm unstimulated cultures). cultures)/(cpm unstimulated cultures).
riched in responsive cells. However, the total response per cell of all the fractions is greater than that of unfractionated cells. These findings suggested that cells in other fractions might be inhibiting high-responding cells in the unfractionated populations. Since both background incorporation and stimulated incorporation rose proportionally, such an inhibitory population would act nonspecifically upon DNA synthesis. The most impressive findings in the old NZB/W fractions were the lack of responsiveness in MLR, and the lo- to 30-fold increase in background incorporation in fractions 2, 3, and 4. These same fractions had a 7-fold increase in Con A responsiveness but little increase in PHA responsiveness as compared with mfractionated cells. In contrast to BALB/c and young NZB/W mice, there was no increased responsiveness in individual fractions of old NZB/W mice; such an increase might have pointed to inhibition by macrophages or other cells. The low PHA/Con A ratios in old NZB/W mice are in agreement with previous observations (5) and the data collectively suggest the loss of a subpopulation of T cells from old NZB/W spleens. This is consistent with the finding that spleens of old NZB/W mice lack a subpopulation of T cells which participate in skin allograft rejection (5, 13).
140
GARNER,
GERSH\VIN
AND
‘I‘:\BI,E
STEINBERG
7
FOUR-DAY RESPONSE TO IMMUNIZATION WITH 2 X lo* SRBC IN T-MO-OLD NZB/W FEMALES GIVEN DIFFERENT FRACTIONS OF ~-MO-OLD NZB/W’ SPLEEN CELLS 1 DAY PRIOR TO IMMDNIZATION
Fraction
Number of cells added
ITnfractionated
PFC/spleen”
PFC/106
X106 x 10”
2,240 43,500
187
5
X 106
X 10’
1,770 12,150
10 46
2
50
8 17
0
79 260
71 104
5 50
1
2 3 4
5 6 None*
PFC/106 Control b 2
10
5
x 106
50
x 106
1,363 2,710
5 50
x 106 Y 106
98,500
5
x 10”
18,500
112
12.5 x 10”
30,000 12,500
167 236
2,790 3,750
16 21
8
12.5 x 106 5 X106 12.5 x 106 -
1,815 2,340 1,850
11
3
20 8
25
x 106
5
x 106
*
16,400
a Average of two to three determinations. b Control response is that of unrestored 7-mo NZB/\V cated by “None” in this table.
-
mice immunized with SRBC and indi-
The ability of the different fractions to restore helper activity in the SRBC system correlated with the mitogen and MLR studies. Fractions 3 and 4 had a profound helper effect at 5 x lo6 cells whereas the other fractions did not. These
:,
60
Y 2 ii
40
6 m 20 3 No Cells
UNFX Splesn Cells
FX I
FX 2
:rj: FX 3
FX 4
FX 5
FX 6
FIG. 5. Seven-month-old NZB/W female mice were immunized with 2 X 10’ SRBC 1 day after transfer of S X 10’ cells from 1-mo-old NZB/W mice fractionated by unit gravity sedimentation. The number of direct antibody-producing plaque-forming cells is indicated for mice receiving no cells, unfractionated spleen cells (UNFX), or cells from fractions (FX) 1 through 6. The height of each bar represents the geometric mean and the vertical bars delimit the range. Only cells from fractions 3 and 4 restored the SRBC response to the ‘I-mo-old NZB/W recipients.
FRACTIONATED
NZB/W
TABLE
SPLEEN
141
CELLS
8
PERCENT 0 AND KAPPA-BEARING CELLS IN THE DIFFERENT FRACTIONS” AFTER SEDIMENTATION OF SPLEEN CELLS FROM ~-MO-OLD NZB/\\. nlrcE
UNIT GRAVITY
Fraction
%S
% Kappa
5; Null cellsb
IJnfractionnted 1 2 3 4 5 6
29.7 31.0 9.7 73.7 78.3 16.7 9.0
47.7 46.0 70.3 7.0 5.0 5.3 5.7
19.3 26.7 19.7 19.3 16.7 78.0 88.7
o Means of three determinations. b Cells bearing neither 6 nor kappa determinants.
active populations contained the greatest percentage of &positive cells. Fraction 2, which contained ahnost no e-positive cells, was ineffective even at 50 X lo6 cells. These results imply that the subpopulation of cells lacking in old NZB/W mice which is required for the response to SRBC is a helper T cell. Earlier studies demonstrated that spleen cells were separable using equilibrium centrifugation in density gradients of Ficoll into two populations functionally distinct in the PFC response to SRBC (19). In particular, one population appeared to be helper T cells which cooperated with bone marrow cells when transferred to lethally irradiated recipients. In the present study, old NZB/W mice which lack helper T cells were restored with regard to the PFC response to SRBC by subpopulations of young spleen cells which contain a high percentage of o-bearing cells. These fractions presumably contained splenic helper T cells required for the PFC response to SRBC analogous to those separated by Gorczynski and co-workers (19). The ability to restore the SRBC response izith small numbers of cells from a fraction of young NZB/W spleen cells suggests that restoration of immunologic competence in old NZB/W mice may be achieved in a manner not previously possible. Five million spleen cells from fractions 3 and 4 increased the numbers of PFC/106 cells 40-60 times more than did 5 million unfractionated cells. Previous study has suggested some therapeutic efficacy of large numbers of yomig NZB/LV’ lymphoid cells given to syngeneic mice as they aged (20). However, the benefits of such therapy appeared to be limited by negative aspects of the therapy thought to be secondary to the large number of cells given. If much smaller numbers of cells could be given by separating and administering only the relevant subpopulations of cells, some of the disadvantages of such therapy might be avoided. It was of interest to find that bye could enrich in null cells as indicated by the small percentage of & or kappa-bearing cells in fractions 5 and 6. The null cells were found by Stobo et al. (21) to be present in increased frequency in unfractionated spleen cell suspensions from New Zealand mice, an observation which we have herein confirmed. The function of these cells is uncertain ; however, the present study suggests that unit gravity sedimentation may be helpful in enriching in these cells. In conjunction with other techniques almost pure populations of null cells can probably be obtained and their functions studied. The present study suggests that unit gravity sedimentation may be of considerable value in separating subpopulations of lymphoid cells from NZB/W mice.
142
GARNER,
GERSHWIN
AND
STEINBERG
It has been observed that NZB/W mice demonstrate marked changes in lymphoid cell subpopulations with age. Early in life they appear to lose regulatory or suppressor cells (9, 22) and later in life they lose helper cells (1, 4, 5). Use of separation techniques should be a valuable aid in further studying these processes. It would be desirable to separately enrich subpopulations of helper and suppressor cells so that their function can be independently studied. In addition, the maturation of different cell types can be studied as has already been demonstrated for B cells in other inbred mouse strains (23). ACKNOWLEDGMENTS The authors are pleased to acknowledge the excellent technical assistance of Ms. Susan Kysela. We are indebted to Drs. T. M. Chused, P. H. Plotz, M. C. Gelfand, and L. M. Parker for advice and gifts of reagents. The careful assistance of Ms. Nancy Milloy in the preparation of the manuscript is gratefully acknowledged.
REFERENCES 1. Talal, N., and Steinberg, A. D. In “Current Topics in Microbiology and Immunology,” Springer-Verlag New York, 1974. 2. Denman, A. M., and Denman, E. J., C/in. Exp. Imnzwaol. 6, 457, 1970. 3. Gelfand, M. C., Parker, L. M., and Steinberg, A. D., J. Immunol. 1974. 4. Cantor, H., Asofsky, R., and Talal, N., J. Exp. Med. 131, 223, 1970. 5. Gelfand, M. C., and Steinberg, A. D., J. Immunol. 110, 1652, 1973. 6. Shirai, T., and Mellors, R. C., Proc. Nat. Acad. Sci. USA 68, 1412, 1971. 7. Evans, M. M., Williamson, W. G., and Irvine, W. J., Clin. Exp. Iwmorol. 3, 375, 1968. 8. Playfair, J. H. L., Zmmzrnology 15, 35, 1968. 9. Barthold, D. R., Kysela, S., and Steinberg, A. D., J. Iinnzuaol. 112, 9, 1974. 10. Mage, M. G., Evans, W. H., and Peterson, E. A., Proc. Sot. Exp. Biol. Med. 127, 478, 1968. 11. Miller, R. G., and Phillips, R. A., J. Cell. Physiol. 73, 191, 1969.’ 12. Reif, A. E., and Allen, J. M. V., Nature (Londolz) 209, 521, 1966. 13. Gelfand, M. C., and Steinberg, A. D., Cell. l+n~mno/. 11, 221, 1974. 14. Jacobs, M. E., Steinberg, A. D., Gorden, J. K., and Talal, N., Arthritis Rhcum. 15, 201, 1972. 15. Stobo, J. D., and Paul, W. E., J. Immunol. 110, 362, 1973. 16. Gorczynski, R. M., and Rittenberg, M. B., 1. Imnttlnol. 112, 47, 1974. 17. Osoba, D., J. Exp. Med. 132,368, 1970. 18. Levey, R. H., and Burleson, R., Cell. Imlrnrnol. 4, 316, 1972. 19. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., 1mmzlnolog.v 20, 693, 1971. 20. Kysela, S., and Steinberg, A. D., Clin. Inzmunol. Immunopathol. 2, 113, 1973. 21. Stobo, J. D., Talal, N., and Paul, W. E., 1. Immulzol. 109, 701, 1972. 22. Steinberg, A. D., Arthritis Rhncm. 17, 11, 1974. 23. Lafleur, L., Miller, R. G., and Phillips, R. A., J. Exp. Med. 135, 1363, 1972.