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Ontogeny of cellular immunity in the human fetus
CELLUI.AK
IMMUNOLOGY
11,
Ontogeny Development
257-271
of Cellular
of Responses
DANIEL I)c/uwtrwnts
P.
(1974)
STITES,~
Immunity
in the
to Phytohemagglutinin MARTIN
C. CARR,
of Medicine and Obstetrics U&vcr.rity of Cnlifornia, San Rcceked
July
AND
Human
and
H.
Fetus
to Allogeneic
HUGH
Cells l
FUDENBERG
and Gynecology, School of Francisco, Culifornia 94143
Medicin?,
23, 1973
Human fetal lymphoid cells from thymus, spleen, blood, liver, and bone marrow of 22 fetuses (5-19 weeks of fetal age) were studied for their ability to respond to phytohemagglutinin and to adult allogeneic lymphocytes by the mixed lymphocyte reaction. The earliest detectable response was that by hepatic cells in the mixed 1)-mphocyte reaction at 7.5 weeks of fetal age. Phytohelnagglutinili reactivity was initially seen in the thymus at 10 weeks, in blood at 14.5 weeks, and in spleen at 13 weeks. Mixed lymphocyte reaction reactivity was first detected in the thymus at 12.5 weeks; it appeared somewhat later in peripheral organs. Few significant responses to phytohemagglutinin were d’etected in bone marrow or hepatic cells, and virtually no response to allogeneic cell,s was found in bone marrow. -411 lymphoid cells studied showed stimutatory ability in the mixed lymphocyte reaction. However, spleen, blood, and marrow cells produced higher stimulation of allogeneic cells than did thymic or hepatic cells.
INTRODUCTION Differentiation of lymphoid cells during embryogenesis leads to the development mature cells which eventually partake in either humoral or cellular immune reactions. Previous investigations of the ontogeny of immunity in man have been restricted almost entirely to elucidation of the development of immunoglobulin-producing plasma cells and surface immunoglobulin-bearing bone marrow derived cells (B cells) (l-3). Immunoglobulin M-producing cells have been found in the spleen by 10.5 wk of gestation and IgG-producing cells by 12 wk (2). J~J, 20 wk synthetic capacities for both IgG and IgM approach that of the normal newborn (1) . Sparse numbers of IgM-staining cells have been found as early as 9.5 wk in the fetal liver (3). By 11.5 wk. hoth IgG-staining and IgM-staining cells were present in the spleen and, by 12 wk, in peripheral blood. At 14.5 wk the percentage of either IgM- or TgG-bearing B lymphocytes in the spleen cqualled of functionally
that present
in normal
neonatal
(cord)
and children’s
blood
(3).
The response of lymphocytes to phytohemagglutinin (I’HA) has been used b> various investigators to map the functional development of thymus-derived cells (1‘ cells) in the human fetus. In general, thymocytes have been found to respond 1 Thiz work was supported in part by US Public Health Service Grants HI)-0.19.19 ant1 HII-05894 from the National Institute of Child Health and Human Development. 2 Supported as a postdoctoral trainee by US Public Health Service Grant HT,-05677.
Copyright 0 1974 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
258
STITES,
CARR,
AND
FUDENBERG
to PHA at about 12-14 wk of gestation; a few weeks later, positive responses can be seen in blood and splenic lymphocytes (4-7). However, these studies employed only one #dose of mitogen and generally ‘did not evaluate maximal response to PHA at various intervals of time or at various concentrations. The pitfalls of this approach and the need for systematic time- and dose-response curves has been pointed out (8). Pegrum (9) examined the response of lymphocytes to allogeneic cells in the mixed lymphocyte reaction (MLR) and, in a number of fetuses from 16 to 24 wk of gestation, found responsive cells in the thymus and variable responses in the spleen and liver. Previous work from our laboratory has emphasized the marked dichotomy in proliferative and cytotoxic capacities of human fetal lymphoid cells (10) and has suggested that different populations are responding to PHA and in the MLR (11). The present study was designed to elucidate in as quantitative a manner as possible the temporal development of the responses of human fetal lymphoid cells from thymus, spleen, blood, liver, and bone marrow to PHA and to allogeneic lymphocytes. Our results clearly demonstrate the functional heterogeneity of thymus-dependent cells during ontogeny and imply that cells in the fetal liver play a crucial role in the developing response to allogeneic cells. These studies also provide important conceptual clues to understanding the pathogenesis of human immunologic deficiency states. MATERIALS
AND
METHODS
Human fetal cells. Twenty-two human fetuses from 5 to 19 wk of fetal age were obtained by abdominal hysterotomy or hysterectomy which was performed for therapeutic abortion. The age of the fetus, counted as beginning with the formation of the zygote, was determined by measurement of crown-rump length (12). If there was evidence of fetal or maternal disease which might have influenced immunologic reactivity, the fetus was excluded from the study. Lymphocyte culture assays were initiated within 3-4 hr of delivery and, in nearly all instances, were performed simultaneously on the various lymphoid organs. Lymphoid cells were obtained from fetal thymus, spleen, (cord) blood, liver, and bone marrow. Blood lymphocytes were collected in phenol-free heparin (LipoHepin, Riker Laboratories, Northridge, CA) and purified by density-gradient centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ, and Winthrop Laboratories, New York, NY) according to the method of Biiyum (13). Thymus, spleen, and liver were finely minced in Petri dishes containing sterile phosphatebuffered saline, pH 7.4 (PBS), and gently homogenized in a glass tissue grinder to disperse clumps. The resulting suspension was rapidly passed through a glass fiber filter in a syringe to obtain a single cell suspension for each organ. Bone marrow cells were obtained by flushing the marrow cavities of the long bones with syringes containing PBS. All cells were washed three times, then centrifuged at 400g in PBS before incubation was begun. Concentration of lymphoid cells was determined in a hemocytometer. In suspensions of marrow cells, only small lymphocytes were counted; in liver suspensions, hepatic parenchymal cells were differentiated from lymphoid cells on the basis of size and nuclear/cytoplasmic ratios.
CELLULAR
IMMUNITY
IN
THE
HUNAN
FETUS
25(1
Phytoheunagglutinin-induced DNA synthesis. Phytohemagglutinin-M (PHA-M, Difco I,aboratories, Detroit, MI) from one lot (control No. 546514) was weighed, dissolved in PBS, sterilized by filtration through a 0.45-pm Millipore filter, and stored at -20°C until use. Lymphocytes were cultured by a modification of the microculture method of Hartzman and coworkers (14). 2 X lo5 lymphoid cells were placed in 12 x 75 mm plastic tubes (No. 2054, Falcon Plastics, Oxnard, CA) containing 1.0 ml of medium 199 with i2i-2-hydroxyethylpiperazine-N’-ethane sulfonic acid (pH 7.2)) penicillin (100 U/ml), streptomycin (100 pg/ml), 20 mM fresh L-glutamine (Grand Island Biological Co., Berkeley, Ca), and 20% pooled human nB plasma. The plasma was obtained from five adult donors who had never been pregnant or received blood transfusions and were not taking any medication at the time of bleeding. Phytohemagglutinit~-M was added at the initiation of cultures to give final concentrations ranging from 0 to 1000 pg/ml. Triplicates lor each observation were incubated for 66 hr at 37°C in 5% CO2 in air. T\vo Schwarz/Mann, Orangeburg, microcuries of tritiated thymidine (16 Ci/mmole; NY) were added, and the incubation 1~3s colltinuetl for 6 hr. Jn some exprimellts the time of incubation with I’HA was varied to cover the time period from 1 to 7 days of culture, but the labeling period was always 6 hr. Kesponse to allogelrcic cells in wzidirc~ctional nlixrd Iyw,blzoc~~te reactiokz. Sormal adult peripheral blood lymphocytes were separated from whole heparinizetl blood l)y density-gradient centrifugation on Ficoll-Hypaque (13 j Resulting cell suspensions contained 95% mononuclear cells, virtually all of which excluded trypan bhle dye. The lymphocytes were washed by centrifugation at 4009 in PBS. Mitotnycin-C (Calbiochem, Los Angeles, CA) was added at a final concentration of 35 pg/ml to the stimulating cell population for 30 min at 37°C in medium 199 with -7O’,$ AB plasma. The cells were then thoroughly washed, centrifuged in the same nit:tlium, counted in a hemocytometer, and adjusted to various concet1tr:ttic~tl.s for use as stimulator cells. Fetal cells to be used as stimulator cells were prepared in a similar fashion. Cultures were prepared in 1.0 ml of the same medium described for I’H,l stimulation. Usually, stitnulating and responding cells were added at a final concentration of 2 X 10” each; in some experiments the ratio of stimulator to responding cells was 1:2! l:l, 2:1, or 4: 1. Control cultures contained mitomycin-treated stimulator cells and nonmitomycin-treated respondin, m cells from the same fetal organ or frotn the same adult peripheral blood donor. Triplicate experimental and control cultures were incubated for 120 hr at 37°C in 5% COZ in air; 3 p.Ci of tritiated thymidine were added during the last IS hr of culture. Scintillation cowtijzg after PHA stimulation or MLR. At the end of the labeling period, cells were transferred into 16 X 100 mm glass tubes with three washes of iced PBS. The cells were washed twice with 3 ml of iced trichloroacetic acid and once with absolute methanol; they were centrifuged at 700g for 20 min after each wash. The precipitates were air dried, dissolved in 0.2 ml of 0.2 .Y NnOH bJ heating at 60°C for 45 min, and acidified with 0.2 ml of 10% acetic acid. The soluble material was transferred to scintillation vials with 10 ml of a mixture CWtaining 4 g of 2.5diphenyloxazole, 50 mg of 1.4~his-2.5phenyloxazolyl. an(I 100 ml of Bio-Solv (BBS-3, Beckman Instruments. Inc., Fullerton, CA) per liter of toluene. Counts per minute (cpm) were determined in a liquid scintillation spec-
260
STITES,
CARR,
AND
FUDENBERG
trorneter (Packard Instruments, Downer’s Grove, IL), employing an automatic external standard for quench correction. Karyotype analysis. Karyotype analysis was performed in selected instances to determine the sex of the cells responding to PHA stimulation or in the MLR. Chromosomes were prepared from metaphase-arrested lymphoid cells cultured for 72 (PHA) or 120 hr (MLR) . During the last 4-6 hr of culture, colchicine (final concn 0.2 pg/ml) was added, and cells were allowed to incubate at room temperature. A hypotonic solution of 0.2% sodium citrate and KC1 was added for 15 min, the cells were fixed with three successive washes in methanol-acetic acid, and smears were prepared and air dried. Cells were stained with Giemsa, complete mitotic figures were examined microscopically and photographed, and sex was determined. RESULTS Dose-response and time-response relationships for PHA stimulation. In instanceswhere adequate numbers of cells were obtained from the various lymphoid organs, dose+responsecurves for PHA at final concentrations of 0, 3.3, 10, 33, 100, 333, and 1000 a/ml were determined at 72 hr of culture. A response was considered significant if the mean of triplicate stimulated cultures was significantly greater than the mean of triplicate control cultures (P S 0.05 by t test). Significant responseswere rarely obtained in hepatic or marrow cells, and these were considered nonresponsive organs. Maximal reactivity in the three responsive organs, thymus, spleen, and blood, occurred over a fairly narrow dose range between 33 and 333 pg/ml (Fig. 1). Time-response curves for thymus and liver were determined at 100 pg of PHA (Fig. 2). The time of maximal incorporation for thymocytes occurred at 72 hr of culture. Time-response studies in the liver did not reveal a significant response at any time during the entire stimulation period of l-7 days. Acquisition of PHA response by various lymphoid organs. The youngest thymus that responded significantly was from a lo-wk fetus (Table 1). Only one thymocyte preparation, that from an 11-wk fetus, failed to respond. A significant response was seen in splenic lymphocytes at 13 wk. The first strongly positive response in the spleen (SI of 4.1) occurred at 1.5 wk of fetal age. Blood lymphocytes were available from fetuses 12.5 wk of fetal age and older. At 14.5 wk, a positive re. 3331
II
.
‘FIG. 1. Maximal responses of fetal lymphoid cells to various doses of phytohemagglutinin after 72 hr of culture. Numbers in parentheses are total numbers of specimens that had significant responses in this study.
CELLULAR
IMMUNITY
IN
THZ
HUMAN
DAYS
IN CULTURE
FETUS
161
CPM 1500 t
500
t
FJG. 2. Time-response of fetal thymic and hepatic cells after stimulation by phytohcmagglutinin (100 pg/ml). 0, Thymus; l , thymus + PHA; D, liver; A, liver + PHA; fetal ape. = 15 wks.
sponseof blood lymphocytes to PHA did occur. With few exceptions there were nap significant positive responses in either liver or marrow cultures. regardless of fetal age. Quantitative aspects of the acquisition of PHA response. Since the stimulation index is the ratio of cpm in stimulated cultures to cpm in control cultures, a high spontaneous incorporation of isotope into control cultures could mask a significant response. A quantitative representation of cpm and stimulation index for the fi\-c fetal organs is detailed in Table 1. Consistently high spontaneous incorporatiotl of [3H]thymidine was seen in blood and bone marrow. In spite of this high background, significant responsesoccurred in most blood cultures; however. with one exception the marrow was nonresponsive. In all responding organs, a tendency for increased responsiveness with incrensing fetal age was noted. This relationship was most striking in spleen, where there was a nearly linear relationship between maximal stimulation index and increasing age. The increase in stimulation index with time was a result of an increased incorporation of isotope into stimulated cultures, not of a coiicornitaiit fall iri the cpm of controls (Table 1). Dose and time relationships foY response to alloge~leic cells (MLR) Selcctetl experiments were performed in thymic and hepatic lymphocytes with dnses of stimulator cells ranging from lo5 to 8 X lo5 and an incubation period of 138 hr. Clear responses were observed over this entire dose range with both thymic ;111(1 hepatic cells (Fig. 3). Employing a ratio of stimulating to responding cells of 1: 1, cultures of thymic and hepatic lymphocytes responding to adult blood stimulator cells were harvested at 4, 6, and 8 days. For thymus and control adult lymphocyte cultures the masimal response occurred on the sixth day (Fig. 4). A response in hepatic cells was also present during the sixth day, but a further increase was detected by the eighth day. Using these dose-time relationships as a guide, all subsequent experiments were performed using 2 x 105 stimulator and 2 x 1V responding cells, nhich were cultured for 138 hr (isotope added for the last 18 hr). Acquisitiom of resfome to nllogerleic cells by fetal lyw@hoid orgatls. The rt’sponsesof lymphoid cells from 23 fetuses from 5 to 18 wk of fetal age were tested in the RILR. A positive response was assigned to those cultures with P 5 0.05 when comparing means of syngeneic to allogeneic pairs. An analysis of positive
a CR, b Not
7.5 10 10 11 11.5 12 12.5 13 13 14 14.5 15 1.5 15.5 15.5 16 16.5 18 18 19
age (wk)
Fetal
crown-rump significantly
34 58 62 70 77 85 93 98 100 110 115 120 125 130 130 140 145 160 160 178
CR* (mm)
length; different
263 459 1081 170 431
SI, stimulation from control
1995 393 530 4177 9200 2462 7459 2007 11,756 9122 12,091 7127 14,734 138,368 3408 13,921 1928 13,224
1195 342 93 273 197 197 337 235 189 276
181
-
PHA kpm>
-
Control (w-d
Thymus
at
P 5 0.05.
index.
917 940 755 1243 430 1396 1695 415 87 1066 686 1767
-
279
-
-
1.7 1.16 5.7 15.3 46.7 12.5 22.1 8.5 62.2 33.1 66.8 27.1 32.1 128.0 20.0 32.3 4.0 58.0
Control (cpm)
OF RESPONSE
SI
ACQUEITION
TABLE
282
1879 1560 1525 5133 2606 8822 9153 12,201 119 9807 32,359 33,926
-
-
--
PHA (wm)
Spleen
l.Ob 2.0 1.7 2.0b 4.1 6.1 6.3 5.4 29.4 1.4b 9.2 47.2 19.2
-
-
SI
1773 34,270 3478 1703 3611
-
27,201 4031 11,783 10,549
-
16,560
-
52,357 62,496 12,520 44,107 109,774
-
88,675 42,325 39,119 76,085
-
35,273
-
PHA (cpm)
Blood
(PHA)
km4
1
Control
TO PHYTOHEMAGGLLJTININ
29.5 1.8 3.6 25.9 30.4
-
3.3" 10.5 3.3b 7.2
2.1b
-
-
SI
OF FETAL
-
2201 621 243 820 908 390
-
1002 1752 1746 511 303 375 1349 615 124 299 254 1690
Control Gw-4
LYMPHOID
242 1 1242 364 1312 1544 546 -
1042 2821 3841 562 485 1050 1753 861 136 508 864 1605 -
PHA (cpm)
Liver
ORGANS
l.lb 2.0* 1.5 1.6 1.7* 1.4b -
1 .O* 1.6* 2.2 1.16 1.6 2.8 1.36 1.4 l.lb 1.7b 3.4 0.9b -
SI
30,414 17,218 30,521
25,345 12,656 14,534
-
-
21,110 -
-
16,888
-
16,397
14,907
-
17,503 49,324
-
-
14,698
-
PHA kpm)
marrow
14,586 32,883
-
-
16,152
-
Control kpm)
Bone
-
-
1.2” 1.46 2.lb
-
-
1.2b
l.lb
1.2” 1.56
0.96
-
SI
CELLULAR
IMMUNITY
IN
THE
HUMAN
FETUS
FIG. 3. MLR response of fetal thymic and hepatic cells to mitomycin-treated (indicated subscript M) stimulator cells. n , Fetal hepatic cells stimulated by adult blood cells; 0, l , fetal thymic cells stimulated hepatic cells stimulated by autologous fetal hepatic cells; adult blood cells; 0, fetal thymic cells stimulated by autologous fetal thymic cells.
263
by fetal by
responses in the MLR is shown in Table 2. Clearly, the first human fetal cells to respond were from the liver at 7.5 wk, and this response was the earliest cellular immune reaction detectable in these experiments. Responding cells were initially detected in the thymus at 12.5 wk of fetal age. Splenic and blood lymphocytes responded at 14.5 wk, these being the youngest specimens available for study. Four marrow specimens were studied, and only one responded. Quantitatizle aspects of crcquisitiow of respone to allogeneic cells. A detailed tabulation of cpm and stimulation index for control and stimulated cultures is shown in Table 2. A strictly quantitative analysis of response in fetal lymphoid organs is not possible because various adult blood donors were employed and responding cells possessed varying histocompatibility antigens with regard to these stimulator cells. Within these limitations, the following observations are made. In liver, 21 of 25 cultures responded (P S 0.05), b u t no consistent age-dependent trend was dis-cerned in hepatic MLR. A moderate response was first noted in fetal thymocytes
FIG. 4. Time response in MLR stimulator cells (Ax). A, Hepatic of fetus, 90 mm (12.5 wk).
of fetal lymphoid cells; 0, thymic
cells to mitomycin-treated adult cells; 0, blood cells; crown-rump
blood length
-
-
-
34
51-
52 58
75
77 85 85 90
7.5
9
9 10 11
11 12 12 12.5
577 355 510 262
-
-
-
-
-
17 30
5 7.5
TTDI~ (wm)
CRO (mm)
Fetal age (wk)
1438 670 760 1027
-
_
--
-
-
-
TAM kpm)
Thymus
RESPONSE
2.5” 1.9” 1.5” 3.9
-
-
--
-
-
-
SI
OF FETAL
-
-
-
--
-
-
-
SSy (cpm)
LYUPHOID
-
-
_
--
-
-
-
SAM bm)
Spleen
CELLS
TABLE
-
-
-
--
-
-
SI
2
-
_
--
-
-
-
_
-
-
-
-
BAM bm)
BBM
Blood
LYMPHOCYTES
kpm)
TO ALLOGENEIC
-
-
-
--
-
-
SI
HHM
1133 280 534 8879
49.5 156 156 1465 39.52
5099 49.5
749 7838
15,620 749
(wm)
6216 794 3738 25,315
2389 807.5 10,389 20,747 14,245
8368 2158
7064 9086
34,220 13,162
(wm)
HAM
Liver
IN THE MIXED-LYMPHOCYTE
5.5 2.8 7.3 2.9
5.2 51.8 66.6 14.2 3.6
1.6” 4.4
9.4 1.2~
2.2~ 17.6
SI
REACTION
MMM
-
-
-
-
(wm)
Bone
-
-
-
-
(cpm)
MAM
marrow
-
-
-
-
SI
3 ; z zi
5
“i
c
2 z “!
216 1112 4X)
135 140 160
6016 35,625 1878
1124 1046 1203 1555 11,144 2124 15,816 7676 6334
262 208 584 172 151 227 7074 332 345
9<1 98 110 115 120 12.5 1.10 130
12.5 13 14 14.5 15 15 15.5 15.5 16 16.5 18
TAM (cpm)
Thyus
TTM” kpm)
CP (mm)
Fetal age (wk)
.__
27.9 32.0 4.4
4.3 5.0 2.1~ 9.0 73.8 9.4 2.2 23.1 18.1
31
360 1446 249
2885 2085 2606 4104 --
SSM (CP)
14,531 43,532 1011
-
22,355 2803 10,779 10,699 40.4 30.1 4.1
7.7 1.3c 4.1 2.6
SI
M&f (CP)
Spleen
11,832
19,066 11,966 29,175
-
BBM (cp$
127,475 -.
80,218 24,186 58,059
-
(cv)
BAM
Blood
10.1
4.2 2.0 2.0
SI
4574 1855 l4.=78
8.9 6.3 4.2
2.7
22,975
8597 511 292 347
2.2 16.8 9.7 3.2 15.9 1.6: -.
__-
SI
19,344 7426 14,597 760 22,562 5646
JiIIY (wm)
8879 443 1512 235 1417 3518
__ HHM (cd
I.iver
ll,l.iS
502.1
‘4,784
11,392
Bone
15,1’7
lO,.ZO.i
40,289
11,690
_
marrow
..-
1.1<
1.7 .-
1.6’
1.P -~
CR” (mm)
17 30
34 34 51
52
58 75 77 85 85 90
Fetal age (wk)
5 7.5
7.5 7.5 9
9
10 11 11 12 12 12.5
197 189 228 674 2782 1382 1041 429 462 1696 4872 212 433 827 390
(cd
Adult control AAM~
-
4.6 0.8” 2.5 l.OC
975 359 2078 396
-
-
-
-
-
ATM (cm)
SI
LYMPHOID
Thymus
FETAL
CELLS
TABLE
-
-
SI
-
AS&f (cpm)
Spleen
AS STIMULATORS
3
-
-
ABm (wm)
Blood
-
-
SI
IN THE MIXED-LY~MPHOCYTE
2469 285 432 650 4253 7459 1129 686 679 13,013 11,682 642 389 1241 357
REACTION Liver
13.5 1.5” 1.9c l.Oc 1.5 6.9 1.70 1.6” 1.2” 7.7 2.4 3.oc 0.9 1.5C 0.9c
SI
-
Bone
marrow
-
SI
3 g CY
s
3
.F +
F
2 z E
93 98 110 115 120 125 130 135 140 160
12.5 13 14 15 15 15.5 16 16.5 16.5 18 3396 5464 7823 3543 954 35,811 12,083
294 137 1238 867 468 600 1578 377 241 1887 539
1.2c 1.5c 3.4 0.7c l.lC 6.9 2.3 6.7 0.7< 3.6 3.1
kpm)
346 199 4156 596 501 1188 3561 2538 172 6778 1686
;\SM (wm)
.%dult control AA&f*
” CR, crowwrump length. * Control culture consisted of adult hlootl lymphorytcs stimulatvd lymphocytes from the same adult stimulated by InitoInycin-tre~ltcd donors. SI, stimulation index; .A, adult cells (see also ‘Table 2). c Sot significantly differrnt from control at P S 0.05.
CR5 (mm)
Fetal age (wk)
7.3 9.1 5.0 9.1” 3.9 19.0 22.4
SI
3 (continued)
by nlitc,mycitl-treated fetal cells. \\‘hrn two
Spleen
T.1BLE
22.8 12.9
13,050 6946
1.3c l.OC 2.4c 0.9c 2.2 3.3 4.2 0.e 5.6 2.5
5210 1583 198 10,482 1347
SI
396 133 2981 819 1043
AHM (wm)
Liver
4885
9.1<
17.2
2936
7.9
-
7.7
marrow
12,176
3738 -
Bone
autologous adult blood Iyml~hocytcs; the experimental culture had test5 were done on cells from the same fetus, different adults served as
4.8 17.1
SI
2240 10,782
.\BM (cpm)
Blood
268
STITES,
CARR,
AND
FUDENBERG
at approximately 12.5 wk of fetal age, and it became stronger at 14.5 weeks. Three of the four younger spleen cultures responded, each moderately, while all three older specimens responded, two showing marked responses. Although all four blood lymphocyte preparations responded, no definite conclusions regarding trend of response with age is possible. In the three youngest available fetal thymuses, MLR reactivity was minimal or absent. However, hepatic cells from these same fetuses had clear-cut responses in the MLR. It was not until approximately 14 wk that MLR reactivity in thymocytes exceeded that seen in hepatic cells. Comfarison of onset of PHA and MLR reactivity. In the thymus, PHA reactivity preceded onset of MLR by 2.5 wk (10 vs 12.5 wk of fetal age). In the spleen and blood (based ‘on limited specimens available), both properties were acquired at approximately the same time of fetal gestation (14.5 wk). Identification of responsive hepatic cells. In order to determine if the hepatic cells that responded in MLR were of fetal, and not maternal, origin, karyotype analyses were performed. Thymocytes from a 12.5wk fetus cultured with PI3.A (100 pg for 72 hr) clearly demonstrated a male (XY) chromosome pattern. Hepatic lymphoid cells from the same fetus cultured in the presence of allogeneic female mitomycin-treated cells were all of male karyotypes. Stiwmlatory ability of fetal lymphoid cells irt MLR. Concurrent with experiments testing response of fetal cells to allogeneic lymphocytes, the ability of fetal cells to stimulate adult lymphocytes was examined in the MLR (Table 3). All fetal lymphoid organs were capable of stimulating DNA synthesis in adult allogeneic lymphocytes. Eight of fourteen thymic preparations and 11 of 21 hepatic cultures were capable of stimulation. All but one spleen (6 of 7), all blood (4 of 4), and all but one marrow (3 of) 4) ‘specimen produced significant stimulation in the MLR. In each case, cells from the youngest available organ were capable of stimulating adult lymphocytes to incorporate DNA. In every case studied, spleen, blood, and marrow cells produced higher stimulation of adult allogeneic lymphocytes than did hepatic and thymic cells. DISCUSSION Human lymphoid tissues from fetuses of various ages exhibit considerable cellular immunocompetence as measured by ,their ‘responses to PHA and to allogeneic cells in vitro. The earliest detectable response in these experiments was to allogeneic cells, and it occurred in hepatic cells from a 7.5-wk fetus (crown-rump length, 30 mm). For the remainder of the observation period, i.e., to 19 wk, hepatic cells responded almost exclusively in the MLR. Reactivity to PHA preceded that to allogeneic cells in fetal thymocytes, occurring at 10 vs 12.5 wk, respectively. Reactivity in both assays was first detected in the spleen and blood at about 14.5 wk. Bone marrow cells generally failed to respond to either stimulus during the entire developmental period under study. Previous studies have shown that the reactivity of fetal thymocytes to PHA begins at approximately 12 wk of gestation (4-7). At this developmental stage a well-demarcated thymic cortex and medulla are present (6). A tendency for DNA synthesis to increase with age in cultures of fetal thymocytes, splenocytes, and blood lymphocytes was noted up to 19 wk. Papiernik (7) has made the same observation and has also demonstrated a progressive diminution of responsiveness
CELLULAR
IRIMUi’iITY
IN
TlIE
HI’MAiX
FliTI-S
‘69
from the 20th week to birth. This age-dependent responsiveness correlated with the appearance of mature lymphocytes in the thymic cortex. Although the precise significance of PHA responsiveness in human lymphoid cells is unclear, studies in patients with congenital absence of the thymus (DiGeorge cells, strongly implies that these syndrome) ( 15)) who have no PHA-responsive cells are thymus-dependent or thymus-derived, Similar unresponsiveness also exists in patients with thymic dysplasia (16) and with hyporesponsiveness in conditions associated with depressed cellular immunity ( 17). Our finding that PHA responsiveness in spleen and peripheral blood follows temporally that in the thymus is also consistent with a thymic origin for these cells. We have recently demonstrated that cells forming rosettes with sheep erythrocytes follow a similar pattern of localization in the thymus and peripheral lymphoid organs (IS), and considerable evidence has been presented that the rosette is a T-cell marker in man (19, 20). Of great interest was the finding that the ability to respond to allogeneic lymphocytes in the MLR was first detectable at a very primitive stage (7.5 wk) in hepatic cells. Evidence that the cells responding in the liver were of fetal and not maternal origin was obtained by use of sex chromosome markers. Because no studies were done trf other functional properties of this population, a positive identification of the responding cells as lymphoid cannot be made. In fact, it is possible that responding cells were hematopoietic stem cells. Lafferty et al. (21) have recently reviem:ed evidence that allogenic cell interactions comprise a unique class of cellular immune reaction distinct from antigenic stimulation. In his studies of graft-vs-host reactivity in chicken embryos, proliferation of thJmic? bursal, or bone marrow cells corresponded temporally with the appearance of stem cells in these organs. If hepatic stem cells were responding in the 311-R. this \vould provide direct evidence exatninatio:l in man for a similar unique type of immune reaction. Norphologic of fetal liver at about 5-7 wk of gestation shows ;t panoply of cell types, some of which do, in fact, resemble mature small lymphocytes. One could conclude that this small population of immunocompetent, mature lymphocytes was actually responsible for allogeneic responsiveness in the early fetal liver. Further studies on the “bone marrow” colony-forming capacity of human fetal hepatic cells in vitro sholtld help clarify this point. 11 number of studies in rodents have shown that cells responding in hll,I< and to PHA can be separated on gradients, suggesting that they are separate functional populations (22). Furthermore, neonatal mouse splenocytes were relativel! insensitive to cytotosic activity (anti-theta), \\-hereas I’HA-responsive cells wer
270
STITES,
CARR,
AND
FUDENBERG
fetal hepatic cells. Tyan (27) demonstrated by means of a two-stage transfer system in viva that the mouse fetal liver gives rise to immunocompetent cells before the appearance of the thymic rudiment. In other experiments (28) he demonstrated the need for a thymic influence for the eventual development of full immunocompetence of transferred fetal hepatic cells in the final host. Furthermore, chromosomally marked (TeT,) fetal hepatic cells (29) can repopulate the thymus and probably eventually migrate to the periphery (30). However, the ,detailed experiments of Stutman et al. (31, 32) clearly demonstrated the need for thymic influence to facilitate the thymic reconstitution of neonatally thymectomized mice given hematopoietic stem cells from fetal liver. Recently, a radiation chimera model was used to demonstrate that allogeneic fetal hepatic cells and a few thymocytes could reconstitute thymectomized, lethally irradiated mice (33). The remarkable absence of graft-vs-host disease suggests that fetal hepatic and thymic transplants, under appropriate conditions, could be used to reconstitute patients with immunodeficiency diseases or bone marrow aplasia. The findings in the present study of hepatic cells responsive in MLR and of a divergence between MLR- and PHA-responsive populations has interesting parallels in certain human immunodeficiency states. In one patient with a variant of thymic aplasia (DiGeorge syndrome), peripheral blood cells were unresponsive to PHA but reacted in one-way MLR (34). T wo additional patients with congenital thymic dysplasia (lymphopenic hypogammaglobulinemia) (16) manifested a similar dichotomy of PHA-unresponsive, MLR-responsive cells. The present report suggests that such individuals are specifically lacking thymic-derived PHA-responsive cells but have liver-derived stem cells which respond in the MLR. In fact, these patients and our ontogenic data taken together add some support to the notion that the fetal liver may be a controlling lymphoid organ which gives rise to a subpopulation of cells that can respond to allogeneic cells. The anatomic proximity of the liver to blood returning from placenta places immunocompetent hepatic cells teleologically in excellent position to ward off infection from foreign cells which might gain access to the developing fetus. From a developmental standpoint, these studies represent the first systematic investigation of the ontogeny of the MLR in man. In the neonatal mouse, Howe an(d Manziello (35) have demonstrated that maximal reactivity in thymic MLR precedes reactivity to PHA. The converse pattern was detected in splenocytes. Knight and Thorbecke (36) have shown that response to allogeneic cells precedes that to xenogeneic cells during immune ontogenesis in the rat. Our study demonstrates that PHA reactivity precedes MLR reactivity in the human fetal thymus. The finding of MLR-reactive cells in fetal liver at a developmental stage before the onset of lymphopoiesis in the thymus raises further questions regarding the nature of the cells that respond to allogeneic cells, and regarding the possible role of the liver in influencing development of this cell population. ACKNOWLEDGMENTS We Rush.
gratefully Dr. Felix
acknowledge the technical assistance of Ms. Janice Perlman Conte, Department of Pediatrics, kindly performed karyotype
and Ms. analyses.
REFERENCES 1. van ‘Furth, R., Schuit, H. R. E., and Hijmans, W., J. Exp. 2. Gitlin, D., and Biasucci, A., J. Cl& Invest. 48, 1433, 1969.
Med. 122,
1173,
1965.
Joanne
CELLULAR
IMMUNITY
IN
THE
HUMAN
FETI:S
271
3. Lawton, A. R., Self, K. S., Royal, S. rl., and Cooper, M. D., Cliu. ~~I~v~I/JI~~~. I~/II~/uH~~patkol. 1, 84, 1972. 4. August, C. S., Berkel, A. I., Driscoil, S., and Merler, E., Prditrtv. 1Zv.v. 5, 5N. 1’171. 5. Kay, H. E. M., Doe, J., and Hockley, A., 1~w~z~~~olug~ 18, 393, 1970. 6. Kyriazis, A. A., and Esterly, J. R., Arclz. P&al. 90, 338, 1970. 7. Papiernik, M., Blood 36, 470, 1970. 8. Carr, M. C., Stites, D. P., and Fudenberg, H. H., Ct./l. Irr~nz~~ol. 5. 21. 1972. 9. Pegrum, G. D., I~w~wtoZog~ 21, 159, 1971. 10. Stites, I>. I’., Carr, M. C., and Fudenherg, H. H., Z’rvc. ilic/f. .1i(ld. s‘c.i. I,-.\‘:1 69. l-l-1(1. 1972. 11. Carr, M. C., Stites, D. P., and Fudcnbcrg, H. H., AVntzfrc .VCIL~ Biol. 241. 279, 1973. 12. Hertig, A. T., “Human Trophoblast,” p. 137. Charles C Thomas, Springfield, 1968. 13. Rijyum, A., Scarad. J. Clin. Lab. Invest. Suppl. 97. I, 1968. 14. Hartzman, R. J., Segall, M., Bach, M. I,., and Bach, F. H., Tmrtsplantation 11. 268, 1971. 15. Kretschmer, R., Say, B., Brown, D., and Rosen, F. S.. A7. Eir{/i. J. dl&. 279. 129.5, 1968. 16. Meuwissen, H. J., Bach, F. H., Hong, R., and Good. Ii. A., J. Peditrtr-. 72. 177, 1968. 17. Gatti, R. A., Garrioch, D. B., and Good, R. A., 1~ “Proceedings of tire Fifth Leukocyte Culture Conference,” p. 339. Academic Press, Xc\<- York, 1970. 18. \Vyhran, J., Carr, M. C., and Fudenberg, H. H., J. c‘lirl. I?zvcsf. 51, 2537, 1972. 1’). Jondal, M., Helm, G., and W?gzelt, H., J. Ext. Jrrd. 136, 207, 1972. 20. Ross, G. I)., Rabellino, E. ?II., Polley, M. J., and Grey, 11. XI.. J. C/in. Iu;v.ct. 52, 377, 1373. 21. Lafferty, K. J., Walker, K. Z., Scollay, R. G., and Killhy, \‘. :1. A., Tj.tm.~bltr~rf Kf,o. 12, 198, 1972. 22. Colley, D. G., Shih Wu, A. Y., and Waksman, B. H., J. S.rp. .l/cd. 132, 1107, 1970. 23. Howe, hf. L., Zlt “Proceedings of the Sixth Leukocyte Culture Conference,” 1’. 475. :\c:idemic Press, New York, 1972. 24. Wagner, II., J. ZVIUZZMOI. 109, 630j 1972. 25. Johnston, J. AI., and W’ilson, D. B., Cell. Iwwtz~~zol. 1, 4.10, 1970. 20. Folch, H., and Waksman, B. E-I., J. Imnzunol. 109, 1046, 1972. 27. Tyan, M. J>.. /. IUZIIULIZO~. 100, 53 j, 1968. 28. Tyan, M. I,., Sricrzcc 145, 934, 1963. 29. Taylor, Ii. B., nl-i/. J. E.r$. I’clllioi. 46, 376, 1964. 30. W~eissman. I. J.., J. E.rP. jllcd. 126, 291, 1967. 31. Stutman, O., Yunis, E. J., and Good, R. A., /. Exp. dlcd. 132, 583, 1970. 32. Stutman, O., Yunis, E. J., and Good, R. A., J. Exp. .V&. 132, 601, 1970. 33. Giller, R. H., Bortin, M. M., Rimm, A. A., Glasspiegel, J. S., and Saltzstein. E. C., [‘,-ni-. Sac. ErP. Bid. Med. 139, 1022, 1972. 34. Gatti, R. A., Gershanhik, J. J., Levkoff, -4. H., Wertelerki, WI:., and (;o(H~, R. A., /. p,,ditr(r. 81, 920, 1972. 3.5. Howe, hf. I,., and blanziello, B., J. I~zrrzm~ol. 109, 534, 1972. 36. Knight, S. C., and Thorbccke, G. J,. Cell. Imm~~nol. 2. 91, 1971.
IMMUNOLOGY
11,
Ontogeny Development
257-271
of Cellular
of Responses
DANIEL I)c/uwtrwnts
P.
(1974)
STITES,~
Immunity
in the
to Phytohemagglutinin MARTIN
C. CARR,
of Medicine and Obstetrics U&vcr.rity of Cnlifornia, San Rcceked
July
AND
Human
and
H.
Fetus
to Allogeneic
HUGH
Cells l
FUDENBERG
and Gynecology, School of Francisco, Culifornia 94143
Medicin?,
23, 1973
Human fetal lymphoid cells from thymus, spleen, blood, liver, and bone marrow of 22 fetuses (5-19 weeks of fetal age) were studied for their ability to respond to phytohemagglutinin and to adult allogeneic lymphocytes by the mixed lymphocyte reaction. The earliest detectable response was that by hepatic cells in the mixed 1)-mphocyte reaction at 7.5 weeks of fetal age. Phytohelnagglutinili reactivity was initially seen in the thymus at 10 weeks, in blood at 14.5 weeks, and in spleen at 13 weeks. Mixed lymphocyte reaction reactivity was first detected in the thymus at 12.5 weeks; it appeared somewhat later in peripheral organs. Few significant responses to phytohemagglutinin were d’etected in bone marrow or hepatic cells, and virtually no response to allogeneic cell,s was found in bone marrow. -411 lymphoid cells studied showed stimutatory ability in the mixed lymphocyte reaction. However, spleen, blood, and marrow cells produced higher stimulation of allogeneic cells than did thymic or hepatic cells.
INTRODUCTION Differentiation of lymphoid cells during embryogenesis leads to the development mature cells which eventually partake in either humoral or cellular immune reactions. Previous investigations of the ontogeny of immunity in man have been restricted almost entirely to elucidation of the development of immunoglobulin-producing plasma cells and surface immunoglobulin-bearing bone marrow derived cells (B cells) (l-3). Immunoglobulin M-producing cells have been found in the spleen by 10.5 wk of gestation and IgG-producing cells by 12 wk (2). J~J, 20 wk synthetic capacities for both IgG and IgM approach that of the normal newborn (1) . Sparse numbers of IgM-staining cells have been found as early as 9.5 wk in the fetal liver (3). By 11.5 wk. hoth IgG-staining and IgM-staining cells were present in the spleen and, by 12 wk, in peripheral blood. At 14.5 wk the percentage of either IgM- or TgG-bearing B lymphocytes in the spleen cqualled of functionally
that present
in normal
neonatal
(cord)
and children’s
blood
(3).
The response of lymphocytes to phytohemagglutinin (I’HA) has been used b> various investigators to map the functional development of thymus-derived cells (1‘ cells) in the human fetus. In general, thymocytes have been found to respond 1 Thiz work was supported in part by US Public Health Service Grants HI)-0.19.19 ant1 HII-05894 from the National Institute of Child Health and Human Development. 2 Supported as a postdoctoral trainee by US Public Health Service Grant HT,-05677.
Copyright 0 1974 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
258
STITES,
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to PHA at about 12-14 wk of gestation; a few weeks later, positive responses can be seen in blood and splenic lymphocytes (4-7). However, these studies employed only one #dose of mitogen and generally ‘did not evaluate maximal response to PHA at various intervals of time or at various concentrations. The pitfalls of this approach and the need for systematic time- and dose-response curves has been pointed out (8). Pegrum (9) examined the response of lymphocytes to allogeneic cells in the mixed lymphocyte reaction (MLR) and, in a number of fetuses from 16 to 24 wk of gestation, found responsive cells in the thymus and variable responses in the spleen and liver. Previous work from our laboratory has emphasized the marked dichotomy in proliferative and cytotoxic capacities of human fetal lymphoid cells (10) and has suggested that different populations are responding to PHA and in the MLR (11). The present study was designed to elucidate in as quantitative a manner as possible the temporal development of the responses of human fetal lymphoid cells from thymus, spleen, blood, liver, and bone marrow to PHA and to allogeneic lymphocytes. Our results clearly demonstrate the functional heterogeneity of thymus-dependent cells during ontogeny and imply that cells in the fetal liver play a crucial role in the developing response to allogeneic cells. These studies also provide important conceptual clues to understanding the pathogenesis of human immunologic deficiency states. MATERIALS
AND
METHODS
Human fetal cells. Twenty-two human fetuses from 5 to 19 wk of fetal age were obtained by abdominal hysterotomy or hysterectomy which was performed for therapeutic abortion. The age of the fetus, counted as beginning with the formation of the zygote, was determined by measurement of crown-rump length (12). If there was evidence of fetal or maternal disease which might have influenced immunologic reactivity, the fetus was excluded from the study. Lymphocyte culture assays were initiated within 3-4 hr of delivery and, in nearly all instances, were performed simultaneously on the various lymphoid organs. Lymphoid cells were obtained from fetal thymus, spleen, (cord) blood, liver, and bone marrow. Blood lymphocytes were collected in phenol-free heparin (LipoHepin, Riker Laboratories, Northridge, CA) and purified by density-gradient centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ, and Winthrop Laboratories, New York, NY) according to the method of Biiyum (13). Thymus, spleen, and liver were finely minced in Petri dishes containing sterile phosphatebuffered saline, pH 7.4 (PBS), and gently homogenized in a glass tissue grinder to disperse clumps. The resulting suspension was rapidly passed through a glass fiber filter in a syringe to obtain a single cell suspension for each organ. Bone marrow cells were obtained by flushing the marrow cavities of the long bones with syringes containing PBS. All cells were washed three times, then centrifuged at 400g in PBS before incubation was begun. Concentration of lymphoid cells was determined in a hemocytometer. In suspensions of marrow cells, only small lymphocytes were counted; in liver suspensions, hepatic parenchymal cells were differentiated from lymphoid cells on the basis of size and nuclear/cytoplasmic ratios.
CELLULAR
IMMUNITY
IN
THE
HUNAN
FETUS
25(1
Phytoheunagglutinin-induced DNA synthesis. Phytohemagglutinin-M (PHA-M, Difco I,aboratories, Detroit, MI) from one lot (control No. 546514) was weighed, dissolved in PBS, sterilized by filtration through a 0.45-pm Millipore filter, and stored at -20°C until use. Lymphocytes were cultured by a modification of the microculture method of Hartzman and coworkers (14). 2 X lo5 lymphoid cells were placed in 12 x 75 mm plastic tubes (No. 2054, Falcon Plastics, Oxnard, CA) containing 1.0 ml of medium 199 with i2i-2-hydroxyethylpiperazine-N’-ethane sulfonic acid (pH 7.2)) penicillin (100 U/ml), streptomycin (100 pg/ml), 20 mM fresh L-glutamine (Grand Island Biological Co., Berkeley, Ca), and 20% pooled human nB plasma. The plasma was obtained from five adult donors who had never been pregnant or received blood transfusions and were not taking any medication at the time of bleeding. Phytohemagglutinit~-M was added at the initiation of cultures to give final concentrations ranging from 0 to 1000 pg/ml. Triplicates lor each observation were incubated for 66 hr at 37°C in 5% CO2 in air. T\vo Schwarz/Mann, Orangeburg, microcuries of tritiated thymidine (16 Ci/mmole; NY) were added, and the incubation 1~3s colltinuetl for 6 hr. Jn some exprimellts the time of incubation with I’HA was varied to cover the time period from 1 to 7 days of culture, but the labeling period was always 6 hr. Kesponse to allogelrcic cells in wzidirc~ctional nlixrd Iyw,blzoc~~te reactiokz. Sormal adult peripheral blood lymphocytes were separated from whole heparinizetl blood l)y density-gradient centrifugation on Ficoll-Hypaque (13 j Resulting cell suspensions contained 95% mononuclear cells, virtually all of which excluded trypan bhle dye. The lymphocytes were washed by centrifugation at 4009 in PBS. Mitotnycin-C (Calbiochem, Los Angeles, CA) was added at a final concentration of 35 pg/ml to the stimulating cell population for 30 min at 37°C in medium 199 with -7O’,$ AB plasma. The cells were then thoroughly washed, centrifuged in the same nit:tlium, counted in a hemocytometer, and adjusted to various concet1tr:ttic~tl.s for use as stimulator cells. Fetal cells to be used as stimulator cells were prepared in a similar fashion. Cultures were prepared in 1.0 ml of the same medium described for I’H,l stimulation. Usually, stitnulating and responding cells were added at a final concentration of 2 X 10” each; in some experiments the ratio of stimulator to responding cells was 1:2! l:l, 2:1, or 4: 1. Control cultures contained mitomycin-treated stimulator cells and nonmitomycin-treated respondin, m cells from the same fetal organ or frotn the same adult peripheral blood donor. Triplicate experimental and control cultures were incubated for 120 hr at 37°C in 5% COZ in air; 3 p.Ci of tritiated thymidine were added during the last IS hr of culture. Scintillation cowtijzg after PHA stimulation or MLR. At the end of the labeling period, cells were transferred into 16 X 100 mm glass tubes with three washes of iced PBS. The cells were washed twice with 3 ml of iced trichloroacetic acid and once with absolute methanol; they were centrifuged at 700g for 20 min after each wash. The precipitates were air dried, dissolved in 0.2 ml of 0.2 .Y NnOH bJ heating at 60°C for 45 min, and acidified with 0.2 ml of 10% acetic acid. The soluble material was transferred to scintillation vials with 10 ml of a mixture CWtaining 4 g of 2.5diphenyloxazole, 50 mg of 1.4~his-2.5phenyloxazolyl. an(I 100 ml of Bio-Solv (BBS-3, Beckman Instruments. Inc., Fullerton, CA) per liter of toluene. Counts per minute (cpm) were determined in a liquid scintillation spec-
260
STITES,
CARR,
AND
FUDENBERG
trorneter (Packard Instruments, Downer’s Grove, IL), employing an automatic external standard for quench correction. Karyotype analysis. Karyotype analysis was performed in selected instances to determine the sex of the cells responding to PHA stimulation or in the MLR. Chromosomes were prepared from metaphase-arrested lymphoid cells cultured for 72 (PHA) or 120 hr (MLR) . During the last 4-6 hr of culture, colchicine (final concn 0.2 pg/ml) was added, and cells were allowed to incubate at room temperature. A hypotonic solution of 0.2% sodium citrate and KC1 was added for 15 min, the cells were fixed with three successive washes in methanol-acetic acid, and smears were prepared and air dried. Cells were stained with Giemsa, complete mitotic figures were examined microscopically and photographed, and sex was determined. RESULTS Dose-response and time-response relationships for PHA stimulation. In instanceswhere adequate numbers of cells were obtained from the various lymphoid organs, dose+responsecurves for PHA at final concentrations of 0, 3.3, 10, 33, 100, 333, and 1000 a/ml were determined at 72 hr of culture. A response was considered significant if the mean of triplicate stimulated cultures was significantly greater than the mean of triplicate control cultures (P S 0.05 by t test). Significant responseswere rarely obtained in hepatic or marrow cells, and these were considered nonresponsive organs. Maximal reactivity in the three responsive organs, thymus, spleen, and blood, occurred over a fairly narrow dose range between 33 and 333 pg/ml (Fig. 1). Time-response curves for thymus and liver were determined at 100 pg of PHA (Fig. 2). The time of maximal incorporation for thymocytes occurred at 72 hr of culture. Time-response studies in the liver did not reveal a significant response at any time during the entire stimulation period of l-7 days. Acquisition of PHA response by various lymphoid organs. The youngest thymus that responded significantly was from a lo-wk fetus (Table 1). Only one thymocyte preparation, that from an 11-wk fetus, failed to respond. A significant response was seen in splenic lymphocytes at 13 wk. The first strongly positive response in the spleen (SI of 4.1) occurred at 1.5 wk of fetal age. Blood lymphocytes were available from fetuses 12.5 wk of fetal age and older. At 14.5 wk, a positive re. 3331
II
.
‘FIG. 1. Maximal responses of fetal lymphoid cells to various doses of phytohemagglutinin after 72 hr of culture. Numbers in parentheses are total numbers of specimens that had significant responses in this study.
CELLULAR
IMMUNITY
IN
THZ
HUMAN
DAYS
IN CULTURE
FETUS
161
CPM 1500 t
500
t
FJG. 2. Time-response of fetal thymic and hepatic cells after stimulation by phytohcmagglutinin (100 pg/ml). 0, Thymus; l , thymus + PHA; D, liver; A, liver + PHA; fetal ape. = 15 wks.
sponseof blood lymphocytes to PHA did occur. With few exceptions there were nap significant positive responses in either liver or marrow cultures. regardless of fetal age. Quantitative aspects of the acquisition of PHA response. Since the stimulation index is the ratio of cpm in stimulated cultures to cpm in control cultures, a high spontaneous incorporation of isotope into control cultures could mask a significant response. A quantitative representation of cpm and stimulation index for the fi\-c fetal organs is detailed in Table 1. Consistently high spontaneous incorporatiotl of [3H]thymidine was seen in blood and bone marrow. In spite of this high background, significant responsesoccurred in most blood cultures; however. with one exception the marrow was nonresponsive. In all responding organs, a tendency for increased responsiveness with incrensing fetal age was noted. This relationship was most striking in spleen, where there was a nearly linear relationship between maximal stimulation index and increasing age. The increase in stimulation index with time was a result of an increased incorporation of isotope into stimulated cultures, not of a coiicornitaiit fall iri the cpm of controls (Table 1). Dose and time relationships foY response to alloge~leic cells (MLR) Selcctetl experiments were performed in thymic and hepatic lymphocytes with dnses of stimulator cells ranging from lo5 to 8 X lo5 and an incubation period of 138 hr. Clear responses were observed over this entire dose range with both thymic ;111(1 hepatic cells (Fig. 3). Employing a ratio of stimulating to responding cells of 1: 1, cultures of thymic and hepatic lymphocytes responding to adult blood stimulator cells were harvested at 4, 6, and 8 days. For thymus and control adult lymphocyte cultures the masimal response occurred on the sixth day (Fig. 4). A response in hepatic cells was also present during the sixth day, but a further increase was detected by the eighth day. Using these dose-time relationships as a guide, all subsequent experiments were performed using 2 x 105 stimulator and 2 x 1V responding cells, nhich were cultured for 138 hr (isotope added for the last 18 hr). Acquisitiom of resfome to nllogerleic cells by fetal lyw@hoid orgatls. The rt’sponsesof lymphoid cells from 23 fetuses from 5 to 18 wk of fetal age were tested in the RILR. A positive response was assigned to those cultures with P 5 0.05 when comparing means of syngeneic to allogeneic pairs. An analysis of positive
a CR, b Not
7.5 10 10 11 11.5 12 12.5 13 13 14 14.5 15 1.5 15.5 15.5 16 16.5 18 18 19
age (wk)
Fetal
crown-rump significantly
34 58 62 70 77 85 93 98 100 110 115 120 125 130 130 140 145 160 160 178
CR* (mm)
length; different
263 459 1081 170 431
SI, stimulation from control
1995 393 530 4177 9200 2462 7459 2007 11,756 9122 12,091 7127 14,734 138,368 3408 13,921 1928 13,224
1195 342 93 273 197 197 337 235 189 276
181
-
PHA kpm>
-
Control (w-d
Thymus
at
P 5 0.05.
index.
917 940 755 1243 430 1396 1695 415 87 1066 686 1767
-
279
-
-
1.7 1.16 5.7 15.3 46.7 12.5 22.1 8.5 62.2 33.1 66.8 27.1 32.1 128.0 20.0 32.3 4.0 58.0
Control (cpm)
OF RESPONSE
SI
ACQUEITION
TABLE
282
1879 1560 1525 5133 2606 8822 9153 12,201 119 9807 32,359 33,926
-
-
--
PHA (wm)
Spleen
l.Ob 2.0 1.7 2.0b 4.1 6.1 6.3 5.4 29.4 1.4b 9.2 47.2 19.2
-
-
SI
1773 34,270 3478 1703 3611
-
27,201 4031 11,783 10,549
-
16,560
-
52,357 62,496 12,520 44,107 109,774
-
88,675 42,325 39,119 76,085
-
35,273
-
PHA (cpm)
Blood
(PHA)
km4
1
Control
TO PHYTOHEMAGGLLJTININ
29.5 1.8 3.6 25.9 30.4
-
3.3" 10.5 3.3b 7.2
2.1b
-
-
SI
OF FETAL
-
2201 621 243 820 908 390
-
1002 1752 1746 511 303 375 1349 615 124 299 254 1690
Control Gw-4
LYMPHOID
242 1 1242 364 1312 1544 546 -
1042 2821 3841 562 485 1050 1753 861 136 508 864 1605 -
PHA (cpm)
Liver
ORGANS
l.lb 2.0* 1.5 1.6 1.7* 1.4b -
1 .O* 1.6* 2.2 1.16 1.6 2.8 1.36 1.4 l.lb 1.7b 3.4 0.9b -
SI
30,414 17,218 30,521
25,345 12,656 14,534
-
-
21,110 -
-
16,888
-
16,397
14,907
-
17,503 49,324
-
-
14,698
-
PHA kpm)
marrow
14,586 32,883
-
-
16,152
-
Control kpm)
Bone
-
-
1.2” 1.46 2.lb
-
-
1.2b
l.lb
1.2” 1.56
0.96
-
SI
CELLULAR
IMMUNITY
IN
THE
HUMAN
FETUS
FIG. 3. MLR response of fetal thymic and hepatic cells to mitomycin-treated (indicated subscript M) stimulator cells. n , Fetal hepatic cells stimulated by adult blood cells; 0, l , fetal thymic cells stimulated hepatic cells stimulated by autologous fetal hepatic cells; adult blood cells; 0, fetal thymic cells stimulated by autologous fetal thymic cells.
263
by fetal by
responses in the MLR is shown in Table 2. Clearly, the first human fetal cells to respond were from the liver at 7.5 wk, and this response was the earliest cellular immune reaction detectable in these experiments. Responding cells were initially detected in the thymus at 12.5 wk of fetal age. Splenic and blood lymphocytes responded at 14.5 wk, these being the youngest specimens available for study. Four marrow specimens were studied, and only one responded. Quantitatizle aspects of crcquisitiow of respone to allogeneic cells. A detailed tabulation of cpm and stimulation index for control and stimulated cultures is shown in Table 2. A strictly quantitative analysis of response in fetal lymphoid organs is not possible because various adult blood donors were employed and responding cells possessed varying histocompatibility antigens with regard to these stimulator cells. Within these limitations, the following observations are made. In liver, 21 of 25 cultures responded (P S 0.05), b u t no consistent age-dependent trend was dis-cerned in hepatic MLR. A moderate response was first noted in fetal thymocytes
FIG. 4. Time response in MLR stimulator cells (Ax). A, Hepatic of fetus, 90 mm (12.5 wk).
of fetal lymphoid cells; 0, thymic
cells to mitomycin-treated adult cells; 0, blood cells; crown-rump
blood length
-
-
-
34
51-
52 58
75
77 85 85 90
7.5
9
9 10 11
11 12 12 12.5
577 355 510 262
-
-
-
-
-
17 30
5 7.5
TTDI~ (wm)
CRO (mm)
Fetal age (wk)
1438 670 760 1027
-
_
--
-
-
-
TAM kpm)
Thymus
RESPONSE
2.5” 1.9” 1.5” 3.9
-
-
--
-
-
-
SI
OF FETAL
-
-
-
--
-
-
-
SSy (cpm)
LYUPHOID
-
-
_
--
-
-
-
SAM bm)
Spleen
CELLS
TABLE
-
-
-
--
-
-
SI
2
-
_
--
-
-
-
_
-
-
-
-
BAM bm)
BBM
Blood
LYMPHOCYTES
kpm)
TO ALLOGENEIC
-
-
-
--
-
-
SI
HHM
1133 280 534 8879
49.5 156 156 1465 39.52
5099 49.5
749 7838
15,620 749
(wm)
6216 794 3738 25,315
2389 807.5 10,389 20,747 14,245
8368 2158
7064 9086
34,220 13,162
(wm)
HAM
Liver
IN THE MIXED-LYMPHOCYTE
5.5 2.8 7.3 2.9
5.2 51.8 66.6 14.2 3.6
1.6” 4.4
9.4 1.2~
2.2~ 17.6
SI
REACTION
MMM
-
-
-
-
(wm)
Bone
-
-
-
-
(cpm)
MAM
marrow
-
-
-
-
SI
3 ; z zi
5
“i
c
2 z “!
216 1112 4X)
135 140 160
6016 35,625 1878
1124 1046 1203 1555 11,144 2124 15,816 7676 6334
262 208 584 172 151 227 7074 332 345
9<1 98 110 115 120 12.5 1.10 130
12.5 13 14 14.5 15 15 15.5 15.5 16 16.5 18
TAM (cpm)
Thyus
TTM” kpm)
CP (mm)
Fetal age (wk)
.__
27.9 32.0 4.4
4.3 5.0 2.1~ 9.0 73.8 9.4 2.2 23.1 18.1
31
360 1446 249
2885 2085 2606 4104 --
SSM (CP)
14,531 43,532 1011
-
22,355 2803 10,779 10,699 40.4 30.1 4.1
7.7 1.3c 4.1 2.6
SI
M&f (CP)
Spleen
11,832
19,066 11,966 29,175
-
BBM (cp$
127,475 -.
80,218 24,186 58,059
-
(cv)
BAM
Blood
10.1
4.2 2.0 2.0
SI
4574 1855 l4.=78
8.9 6.3 4.2
2.7
22,975
8597 511 292 347
2.2 16.8 9.7 3.2 15.9 1.6: -.
__-
SI
19,344 7426 14,597 760 22,562 5646
JiIIY (wm)
8879 443 1512 235 1417 3518
__ HHM (cd
I.iver
ll,l.iS
502.1
‘4,784
11,392
Bone
15,1’7
lO,.ZO.i
40,289
11,690
_
marrow
..-
1.1<
1.7 .-
1.6’
1.P -~
CR” (mm)
17 30
34 34 51
52
58 75 77 85 85 90
Fetal age (wk)
5 7.5
7.5 7.5 9
9
10 11 11 12 12 12.5
197 189 228 674 2782 1382 1041 429 462 1696 4872 212 433 827 390
(cd
Adult control AAM~
-
4.6 0.8” 2.5 l.OC
975 359 2078 396
-
-
-
-
-
ATM (cm)
SI
LYMPHOID
Thymus
FETAL
CELLS
TABLE
-
-
SI
-
AS&f (cpm)
Spleen
AS STIMULATORS
3
-
-
ABm (wm)
Blood
-
-
SI
IN THE MIXED-LY~MPHOCYTE
2469 285 432 650 4253 7459 1129 686 679 13,013 11,682 642 389 1241 357
REACTION Liver
13.5 1.5” 1.9c l.Oc 1.5 6.9 1.70 1.6” 1.2” 7.7 2.4 3.oc 0.9 1.5C 0.9c
SI
-
Bone
marrow
-
SI
3 g CY
s
3
.F +
F
2 z E
93 98 110 115 120 125 130 135 140 160
12.5 13 14 15 15 15.5 16 16.5 16.5 18 3396 5464 7823 3543 954 35,811 12,083
294 137 1238 867 468 600 1578 377 241 1887 539
1.2c 1.5c 3.4 0.7c l.lC 6.9 2.3 6.7 0.7< 3.6 3.1
kpm)
346 199 4156 596 501 1188 3561 2538 172 6778 1686
;\SM (wm)
.%dult control AA&f*
” CR, crowwrump length. * Control culture consisted of adult hlootl lymphorytcs stimulatvd lymphocytes from the same adult stimulated by InitoInycin-tre~ltcd donors. SI, stimulation index; .A, adult cells (see also ‘Table 2). c Sot significantly differrnt from control at P S 0.05.
CR5 (mm)
Fetal age (wk)
7.3 9.1 5.0 9.1” 3.9 19.0 22.4
SI
3 (continued)
by nlitc,mycitl-treated fetal cells. \\‘hrn two
Spleen
T.1BLE
22.8 12.9
13,050 6946
1.3c l.OC 2.4c 0.9c 2.2 3.3 4.2 0.e 5.6 2.5
5210 1583 198 10,482 1347
SI
396 133 2981 819 1043
AHM (wm)
Liver
4885
9.1<
17.2
2936
7.9
-
7.7
marrow
12,176
3738 -
Bone
autologous adult blood Iyml~hocytcs; the experimental culture had test5 were done on cells from the same fetus, different adults served as
4.8 17.1
SI
2240 10,782
.\BM (cpm)
Blood
268
STITES,
CARR,
AND
FUDENBERG
at approximately 12.5 wk of fetal age, and it became stronger at 14.5 weeks. Three of the four younger spleen cultures responded, each moderately, while all three older specimens responded, two showing marked responses. Although all four blood lymphocyte preparations responded, no definite conclusions regarding trend of response with age is possible. In the three youngest available fetal thymuses, MLR reactivity was minimal or absent. However, hepatic cells from these same fetuses had clear-cut responses in the MLR. It was not until approximately 14 wk that MLR reactivity in thymocytes exceeded that seen in hepatic cells. Comfarison of onset of PHA and MLR reactivity. In the thymus, PHA reactivity preceded onset of MLR by 2.5 wk (10 vs 12.5 wk of fetal age). In the spleen and blood (based ‘on limited specimens available), both properties were acquired at approximately the same time of fetal gestation (14.5 wk). Identification of responsive hepatic cells. In order to determine if the hepatic cells that responded in MLR were of fetal, and not maternal, origin, karyotype analyses were performed. Thymocytes from a 12.5wk fetus cultured with PI3.A (100 pg for 72 hr) clearly demonstrated a male (XY) chromosome pattern. Hepatic lymphoid cells from the same fetus cultured in the presence of allogeneic female mitomycin-treated cells were all of male karyotypes. Stiwmlatory ability of fetal lymphoid cells irt MLR. Concurrent with experiments testing response of fetal cells to allogeneic lymphocytes, the ability of fetal cells to stimulate adult lymphocytes was examined in the MLR (Table 3). All fetal lymphoid organs were capable of stimulating DNA synthesis in adult allogeneic lymphocytes. Eight of fourteen thymic preparations and 11 of 21 hepatic cultures were capable of stimulation. All but one spleen (6 of 7), all blood (4 of 4), and all but one marrow (3 of) 4) ‘specimen produced significant stimulation in the MLR. In each case, cells from the youngest available organ were capable of stimulating adult lymphocytes to incorporate DNA. In every case studied, spleen, blood, and marrow cells produced higher stimulation of adult allogeneic lymphocytes than did hepatic and thymic cells. DISCUSSION Human lymphoid tissues from fetuses of various ages exhibit considerable cellular immunocompetence as measured by ,their ‘responses to PHA and to allogeneic cells in vitro. The earliest detectable response in these experiments was to allogeneic cells, and it occurred in hepatic cells from a 7.5-wk fetus (crown-rump length, 30 mm). For the remainder of the observation period, i.e., to 19 wk, hepatic cells responded almost exclusively in the MLR. Reactivity to PHA preceded that to allogeneic cells in fetal thymocytes, occurring at 10 vs 12.5 wk, respectively. Reactivity in both assays was first detected in the spleen and blood at about 14.5 wk. Bone marrow cells generally failed to respond to either stimulus during the entire developmental period under study. Previous studies have shown that the reactivity of fetal thymocytes to PHA begins at approximately 12 wk of gestation (4-7). At this developmental stage a well-demarcated thymic cortex and medulla are present (6). A tendency for DNA synthesis to increase with age in cultures of fetal thymocytes, splenocytes, and blood lymphocytes was noted up to 19 wk. Papiernik (7) has made the same observation and has also demonstrated a progressive diminution of responsiveness
CELLULAR
IRIMUi’iITY
IN
TlIE
HI’MAiX
FliTI-S
‘69
from the 20th week to birth. This age-dependent responsiveness correlated with the appearance of mature lymphocytes in the thymic cortex. Although the precise significance of PHA responsiveness in human lymphoid cells is unclear, studies in patients with congenital absence of the thymus (DiGeorge cells, strongly implies that these syndrome) ( 15)) who have no PHA-responsive cells are thymus-dependent or thymus-derived, Similar unresponsiveness also exists in patients with thymic dysplasia (16) and with hyporesponsiveness in conditions associated with depressed cellular immunity ( 17). Our finding that PHA responsiveness in spleen and peripheral blood follows temporally that in the thymus is also consistent with a thymic origin for these cells. We have recently demonstrated that cells forming rosettes with sheep erythrocytes follow a similar pattern of localization in the thymus and peripheral lymphoid organs (IS), and considerable evidence has been presented that the rosette is a T-cell marker in man (19, 20). Of great interest was the finding that the ability to respond to allogeneic lymphocytes in the MLR was first detectable at a very primitive stage (7.5 wk) in hepatic cells. Evidence that the cells responding in the liver were of fetal and not maternal origin was obtained by use of sex chromosome markers. Because no studies were done trf other functional properties of this population, a positive identification of the responding cells as lymphoid cannot be made. In fact, it is possible that responding cells were hematopoietic stem cells. Lafferty et al. (21) have recently reviem:ed evidence that allogenic cell interactions comprise a unique class of cellular immune reaction distinct from antigenic stimulation. In his studies of graft-vs-host reactivity in chicken embryos, proliferation of thJmic? bursal, or bone marrow cells corresponded temporally with the appearance of stem cells in these organs. If hepatic stem cells were responding in the 311-R. this \vould provide direct evidence exatninatio:l in man for a similar unique type of immune reaction. Norphologic of fetal liver at about 5-7 wk of gestation shows ;t panoply of cell types, some of which do, in fact, resemble mature small lymphocytes. One could conclude that this small population of immunocompetent, mature lymphocytes was actually responsible for allogeneic responsiveness in the early fetal liver. Further studies on the “bone marrow” colony-forming capacity of human fetal hepatic cells in vitro sholtld help clarify this point. 11 number of studies in rodents have shown that cells responding in hll,I< and to PHA can be separated on gradients, suggesting that they are separate functional populations (22). Furthermore, neonatal mouse splenocytes were relativel! insensitive to cytotosic activity (anti-theta), \\-hereas I’HA-responsive cells wer
270
STITES,
CARR,
AND
FUDENBERG
fetal hepatic cells. Tyan (27) demonstrated by means of a two-stage transfer system in viva that the mouse fetal liver gives rise to immunocompetent cells before the appearance of the thymic rudiment. In other experiments (28) he demonstrated the need for a thymic influence for the eventual development of full immunocompetence of transferred fetal hepatic cells in the final host. Furthermore, chromosomally marked (TeT,) fetal hepatic cells (29) can repopulate the thymus and probably eventually migrate to the periphery (30). However, the ,detailed experiments of Stutman et al. (31, 32) clearly demonstrated the need for thymic influence to facilitate the thymic reconstitution of neonatally thymectomized mice given hematopoietic stem cells from fetal liver. Recently, a radiation chimera model was used to demonstrate that allogeneic fetal hepatic cells and a few thymocytes could reconstitute thymectomized, lethally irradiated mice (33). The remarkable absence of graft-vs-host disease suggests that fetal hepatic and thymic transplants, under appropriate conditions, could be used to reconstitute patients with immunodeficiency diseases or bone marrow aplasia. The findings in the present study of hepatic cells responsive in MLR and of a divergence between MLR- and PHA-responsive populations has interesting parallels in certain human immunodeficiency states. In one patient with a variant of thymic aplasia (DiGeorge syndrome), peripheral blood cells were unresponsive to PHA but reacted in one-way MLR (34). T wo additional patients with congenital thymic dysplasia (lymphopenic hypogammaglobulinemia) (16) manifested a similar dichotomy of PHA-unresponsive, MLR-responsive cells. The present report suggests that such individuals are specifically lacking thymic-derived PHA-responsive cells but have liver-derived stem cells which respond in the MLR. In fact, these patients and our ontogenic data taken together add some support to the notion that the fetal liver may be a controlling lymphoid organ which gives rise to a subpopulation of cells that can respond to allogeneic cells. The anatomic proximity of the liver to blood returning from placenta places immunocompetent hepatic cells teleologically in excellent position to ward off infection from foreign cells which might gain access to the developing fetus. From a developmental standpoint, these studies represent the first systematic investigation of the ontogeny of the MLR in man. In the neonatal mouse, Howe an(d Manziello (35) have demonstrated that maximal reactivity in thymic MLR precedes reactivity to PHA. The converse pattern was detected in splenocytes. Knight and Thorbecke (36) have shown that response to allogeneic cells precedes that to xenogeneic cells during immune ontogenesis in the rat. Our study demonstrates that PHA reactivity precedes MLR reactivity in the human fetal thymus. The finding of MLR-reactive cells in fetal liver at a developmental stage before the onset of lymphopoiesis in the thymus raises further questions regarding the nature of the cells that respond to allogeneic cells, and regarding the possible role of the liver in influencing development of this cell population. ACKNOWLEDGMENTS We Rush.
gratefully Dr. Felix
acknowledge the technical assistance of Ms. Janice Perlman Conte, Department of Pediatrics, kindly performed karyotype
and Ms. analyses.
REFERENCES 1. van ‘Furth, R., Schuit, H. R. E., and Hijmans, W., J. Exp. 2. Gitlin, D., and Biasucci, A., J. Cl& Invest. 48, 1433, 1969.
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1173,
1965.
Joanne
CELLULAR
IMMUNITY
IN
THE
HUMAN
FETI:S
271
3. Lawton, A. R., Self, K. S., Royal, S. rl., and Cooper, M. D., Cliu. ~~I~v~I/JI~~~. I~/II~/uH~~patkol. 1, 84, 1972. 4. August, C. S., Berkel, A. I., Driscoil, S., and Merler, E., Prditrtv. 1Zv.v. 5, 5N. 1’171. 5. Kay, H. E. M., Doe, J., and Hockley, A., 1~w~z~~~olug~ 18, 393, 1970. 6. Kyriazis, A. A., and Esterly, J. R., Arclz. P&al. 90, 338, 1970. 7. Papiernik, M., Blood 36, 470, 1970. 8. Carr, M. C., Stites, D. P., and Fudenberg, H. H., Ct./l. Irr~nz~~ol. 5. 21. 1972. 9. Pegrum, G. D., I~w~wtoZog~ 21, 159, 1971. 10. Stites, I>. I’., Carr, M. C., and Fudenherg, H. H., Z’rvc. ilic/f. .1i(ld. s‘c.i. I,-.\‘:1 69. l-l-1(1. 1972. 11. Carr, M. C., Stites, D. P., and Fudcnbcrg, H. H., AVntzfrc .VCIL~ Biol. 241. 279, 1973. 12. Hertig, A. T., “Human Trophoblast,” p. 137. Charles C Thomas, Springfield, 1968. 13. Rijyum, A., Scarad. J. Clin. Lab. Invest. Suppl. 97. I, 1968. 14. Hartzman, R. J., Segall, M., Bach, M. I,., and Bach, F. H., Tmrtsplantation 11. 268, 1971. 15. Kretschmer, R., Say, B., Brown, D., and Rosen, F. S.. A7. Eir{/i. J. dl&. 279. 129.5, 1968. 16. Meuwissen, H. J., Bach, F. H., Hong, R., and Good. Ii. A., J. Peditrtr-. 72. 177, 1968. 17. Gatti, R. A., Garrioch, D. B., and Good, R. A., 1~ “Proceedings of tire Fifth Leukocyte Culture Conference,” p. 339. Academic Press, Xc\<- York, 1970. 18. \Vyhran, J., Carr, M. C., and Fudenberg, H. H., J. c‘lirl. I?zvcsf. 51, 2537, 1972. 1’). Jondal, M., Helm, G., and W?gzelt, H., J. Ext. Jrrd. 136, 207, 1972. 20. Ross, G. I)., Rabellino, E. ?II., Polley, M. J., and Grey, 11. XI.. J. C/in. Iu;v.ct. 52, 377, 1373. 21. Lafferty, K. J., Walker, K. Z., Scollay, R. G., and Killhy, \‘. :1. A., Tj.tm.~bltr~rf Kf,o. 12, 198, 1972. 22. Colley, D. G., Shih Wu, A. Y., and Waksman, B. H., J. S.rp. .l/cd. 132, 1107, 1970. 23. Howe, hf. L., Zlt “Proceedings of the Sixth Leukocyte Culture Conference,” 1’. 475. :\c:idemic Press, New York, 1972. 24. Wagner, II., J. ZVIUZZMOI. 109, 630j 1972. 25. Johnston, J. AI., and W’ilson, D. B., Cell. Iwwtz~~zol. 1, 4.10, 1970. 20. Folch, H., and Waksman, B. E-I., J. Imnzunol. 109, 1046, 1972. 27. Tyan, M. J>.. /. IUZIIULIZO~. 100, 53 j, 1968. 28. Tyan, M. I,., Sricrzcc 145, 934, 1963. 29. Taylor, Ii. B., nl-i/. J. E.r$. I’clllioi. 46, 376, 1964. 30. W~eissman. I. J.., J. E.rP. jllcd. 126, 291, 1967. 31. Stutman, O., Yunis, E. J., and Good, R. A., /. Exp. dlcd. 132, 583, 1970. 32. Stutman, O., Yunis, E. J., and Good, R. A., J. Exp. .V&. 132, 601, 1970. 33. Giller, R. H., Bortin, M. M., Rimm, A. A., Glasspiegel, J. S., and Saltzstein. E. C., [‘,-ni-. Sac. ErP. Bid. Med. 139, 1022, 1972. 34. Gatti, R. A., Gershanhik, J. J., Levkoff, -4. H., Wertelerki, WI:., and (;o(H~, R. A., /. p,,ditr(r. 81, 920, 1972. 3.5. Howe, hf. I,., and blanziello, B., J. I~zrrzm~ol. 109, 534, 1972. 36. Knight, S. C., and Thorbccke, G. J,. Cell. Imm~~nol. 2. 91, 1971.