CEI.LL-L.4R
35, 148-157
IJIMUNOLOGY
Lymphocyte
(1978)
Populations
of Callithrix
jacchus
JOHN \VRIGHT,* LAWREA-CE A. FALK,* LAUREN AND FRIEDRICH \V. ~EIKHARDT~ L)epartwents
of Microbiology,
Rlrsk-PresbSteriarz-St. Centers, Chicago, Received
July
Lrtke’s alrd Illinois 60612
Marmosets G. \VOLFE,
C’nizwsity
of Illinois
Medical
7, 1977
A lymphocyte population of common marmosets (Cal!ithrix jacchm) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 627% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Hrrpesvirrts sairuiri or Herpesvims afe/cs formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C’) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.
INTRODUCTION Human lymphocytes have been divided into two main classes on the basis of surface membrane markers : Thymic-derived or T lymphocytes carry a receptor for sheep erythrocytes (E) ( 1) , while bone marrow-derived B lymphocytes are characterized by expression of membrane-bound immunoglobulin (Ig) (2) and the presence of receptors for the third component of complement (c’3) (3) and the Fc fragment of aggregated Ig (4). Circulating lymphocytes of most nonhuman primates possesssurface membrane properties analogous to those of human lymphocytes (5-7) : 55-65c/o of circulating lymphocytes from cotton-topped (CT) (Sagltiws ocdipw) and while-lipped (\2:L) (Saqzzrinnsfmcicollis, S. nigricollis) nlarmosets (S-12) and 66-80s of squirrel monkey lymphocytes (13, 14) fornl spontaneous rosettes with sheep E while 17-257, of circulating cells from these speciespossessB-cell membrane markers. 11%ile characterizing the circulating lymphocyte populations of common marmosets (Callithvi.r jacclzzrs), we observed that only a small percentage of cells formed sheep E rosettes but 60-70% formed rosettes with African green monkey erythro1 Submitted in partial fulfillment for the Doctor of Philosophy in Microbiology from the University of Illinois at the Medical Center, Chicago, Illinois 60612. *Scholar of the Leukemia Society of America. sPresent address: Max v. Pettenkofer Institute, Pettenkoferstr. 9a, 8000 Muenchen 2, Western Germany. 148 000%8749/78/0351-0148$02.00/O Copyright All rights
8 1978 by of reproduction
Academic Preu, Inc. in
any
form
reserwd.
COMMON
MARMOSET
LYMPHOCYTE
POPULATIONS
149
cytes. The T-lymphocyte populations of common marmosets and galagos, a prosimian species also evaluated in this study, appear to have receptors for African green monkey E but not sheep E, in contrast to other genera of nonhuman primates. MATERIALS
AND
METHODS
Twenty adult colony-born or wild-caught common marmosets (Callithrix jacchus jacchzrs) were used as the source of lymphocytes. Captive animals had been acclimated for over 1 year and none of the animals had been used for experimental studies. Circulating lymphocytes and lymphoid tissues were studied also from CT and WL marmosets, squirrel monkeys, and three prosimian species, galagos (Galago crassicazrdatus), lemurs (Lewur catta), and tupaia (Tupaia glis) . Preparation
of Circulating
Lymphocytes
and Lymphoid
Tissues
Animals were bled by venipuncture and lymphocytes were separated from whole blood on Ficoll-Hypaque (FH) gradients (15). Lymphocytes were washed three times in medium RPM1 1640 and resuspended at a concentration of approximately 1 X loo cells/ml. Spleen and thymus from common, CT, and WL marmosets were removed aseptically ; animals ranged in age from 1 day to 3 months. Individual cell suspensions were prepared either by mincing and trypsinization of the tissues or by mincing and passage through a stainless-steel wire mesh. The cell suspensions were washed and resuspended in medium RPM1 1640 supplemented with 15% fetal calf serum (FCS), glutamine, 100 units of penicillin, and 50 pg of streptomycin/ml. Erythrocytes Blood was obtained from one sheep durin g the entire study and was used within 2 weeks of bleeding. African green monkey erythrocytes (AG-E), in Alsever’s solution, were obtained from Flow Laboratories (Rockville, Md.) . Sheep and African green monkey E were washed in Hanks’ balanced salt solution (HBSS) and adjusted to a 1% suspension in HBSS. Lymphocyte
Surface Membrane
MarKers
(i) E rosettes. E-rosette assays were performed according to a modification of the procedure of Jondal et al. (1). Approximately 3 X lo5 lymphocytes were mixed with 0.3 ml of a 1% suspension of sheep or African green monkey E for an erythrocyte to lymphocyte ratio of approximately 100: 1. Cells were incubated at 37°C for 5 min, centrifuged at 200g for 5 min, and left for 3 hr or overnight at 4°C. Lymphocytes with at least three attached E were considered positive rosette-forming cells (S-RFC or AG-RFC) and at least 200-300 cells from each sample were counted. In several experiments, E-rosette assays were performed as described but with human, gorilla, orangutan, baboon, rhesus monkey, goat, guinea pig, or mouse erythrocytes. (ii) EAC rosettes. Lymphocytes were evaluated for C’3 membrane receptors by a, modification of the procedure described by Mendes et al. (16). Hemolysin-sensi-
150
WRIGHT
ET
AL.
tized sheep erythrocytes (EA) were mixed with a human C’ source for 1 hr at 37°C with periodic shaking. The EAC were washed three times with HBSS and resuspended to 0.5 % concentration. Approximately 2 X lo5 lymphocytes were mixed with 0.2 ml of EAC and incubated for 30 min at 37°C ; the cells then were centrifuged at 200g for 5 min and reincubated at 37°C while the cells were counted. To determine the percentage EAC rosettes, cells were agitated mechanically (Vortex mixer) and diluted with HBSS: At least 200 lymphocytes were counted and cells with three or more attached EAC were considered positive. (iii) Membrane immuno~uorescence. Membrane immunofluorescent antibody assays (FA), performed according to the procedure of Jondal and Klein (17), were used with lymphocyte suspensions for identification of membrane Ig-positive cells and for T lymphocyte-specific membrane antigens. Approximately 1 x lo8 lymphocytes were mixed with 0.2 ml of either: (a) a 1: 10 dilution of fluorescein isothiocyanate (FITC) -conjugated goat anti-human IgM and IgG (Hyland Laboratories, Costa Mesa, Calif.), or (b) a 1: 200 dilution of goat anti-marmoset T-lymphocyte serum (ASTLA) (18) followed by FITC-conjugated rabbit anti-goat IgG (Hyland Laboratories). ASTLA was prepared by hyperimmunization of a goat with a marmoset lymphoblastoid cell line with T-lymphocyte surface 70-N2 cells (19), membrane properties. Serum was absorbed extensively with marmoset B lymphoblasts, marmoset fibroblast cells, and fetal calf serum, and did not react with B lymphocytes at the concentrations used. Virus-Carrying
Lymphoid
Cell Cultures
The surface membrane properties of thymus spleen, and lymph node cells of two common marmosets with Herpes&us sairniri (HVS) -induced lymphoma were studied during a 3-month cultivation period in vitro. The surface membrane markers of lymphoblastoid cell lines derived from common marmoset or CT marmoset cells transformed by HVS, Herpemirus ateles (HVA), or Epstein-Barr virus (EBV) also were evaluated ; CT marmoset cell lines included HVS-transformed 70-N2, HVA-transformed 1022 (20), and EBV-carrying KCM-25 cell lines (21). All lymphoblastoid cell lines were cultivated in medium RPM1 1640 supplemented with 15% FCS, glutamine, and antibiotics. Cytotoxicity
Assay
Circulating lymphocytes from common marmosets were depleted of cell populations reacting with ASTLA by a C-dependent cytotoxicity assay. Lymphocytes from several animals were pooled and approximately 1 X lo6 cells were mixed with 2.5 ml of various dilutions of ASTLA and incubated for 35 min at 4°C; cells were washed three times and 5 ml of a 1: 10 dilution of rabbit C’ was added for 30 min at 37°C. After washing two times in HBSS, the remaining cells were used for E- and EAC- rosette assays. Control samples of lymphocytes alone and lymphocytes treated only with rabbit C’ were used also in rosette assays. RESULTS E-Rosette
Formation
Erythrocytes mon marmoset
by Cowmon
Marvnoset
Circulating
Lymphocytes
from 10 species were assessed for E rosette formation with comcirculating lymphocytes, and results are summarized in Table 1.
COMMON
MARMOSET
LYMPHOCYTE
TABLE E-Rosette
Formation
by Common
Erythrocytes obtained from
Marmoset
1
Lymphocytes Number tested
with
Erythrocytes
B 0
monkey green monkey
pig
2 2 1 6 3 3 5 3 7 2 2
from
Common marmoset rosettes (%) Range
Human Human Gorilla Orangutan Baboon Rhesus African Goat Sheep Guinea Mouse
151
POPULATIONS
other
Species
E
Mean
(lO.(r19.0) (O&1.4) (25.2) (41.8-68.9) (4.3-15.7) (0.9-l .3) (44.2-58.6) (0) (O-3.2) P-w (O-0.5)
14.5 0.7 25.2 64.4 11.6 1.0 53.1 0.0 1.2 0.2 0.2
Means of 53 and 65% of common marmoset lymphocytes formed rosettes with African green monkey and orangutan erythrocytes, respectively. AG-E were used for further study of common marmoset T-lymphocyte surface membrane receptors, due to the unavailability of orangutan erythrocytes. Surface Membrane Properties of Circulating sets and 0 ther Nonhuwma Primates
Lymphocytes
from Coww~on
Marco-
Comparison of the surface membrane markers of circulating lymphocytes from three marmoset species, squirrel monkeys, and three prosimian species is presented in Table 2. The percentage of AG-RFC or S-RFC was evaluated by the standard E-rosette assay with a 3-hr or overnight incubation at 4°C. After 3-hr incubation, an average of 2.3% common marmoset cells formed rosettes with sheep E, while 58.1% of the lymphocytes formed rosettes with African green monkey erythrocytes. With overnight incubation, 7.3% of common marmoset lymphocytes were S-RFC and 62.5% of the cells were AG-RFC. An average of 67.3% of common marmoset lymphocytes was stained in membrane FA assays with a specific antiserum to CT marmoset T lymphocytes (ASTLA) . In contrast, lymphocytes from three other New World monkey species, WL and CT marmosets and squirrel monkeys, possessed receptors for both sheep and African green monkey E. Results of these assays indicated that the numbers of WL and squirrel monkey AG-RFC were usually less than the number of S-RFC. The number of CT AG-RFC or S-RFC was approximately equal. A population of common marmoset circulating lymphocytes was identified as B cells by the presence of immunoglobulin on the cell surface and receptors for C’3 ; as presented in Table 2, 15.4-16.57 o o f common marmoset lymphocytes possessed membrane Ig and/or C’3 receptors. Surface membrane properties of lymphocytes from two galagos were assessed ; more than 80% formed rosettes with AG-E, while < 1% of cells were S-RFC and the B-lymphocyte population was identified by EAC rosettes and Ig staining. Also tested for E-rosette formation with African green monkey and sheep erythrocytes
marmoset)
RFC
NT
0.0
2
rupaia
(&l38.6)
3h
Sheep
0.0
NT
(Zl)
60.0 (42.0-77.2)
47.3 (29.2-68.4)
53.2 (37.9-67.1)
(ye)
other
NT
3.8
NT
71.7 (55.4-83.9)
53.9 (39.5-79.0)
61.6 (46.5-75.1)
7.3 (O-27.8)
Overnight
RFC
Marmosets,
Primates,
NT
12.5
18.0 (11.5-28.7)
22.1 (16.1-26.4)
26.3 (15.7-34.2)
19.5 (9.5-35.0)
15.4 (10.1-25.6)
EAC” rosettes ( 70)
Nonhuman
6 Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC). b Overnight or 3-hr incubation of lymphocytes and erythrocytes at 4°C. c Rosette formation with C/-sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody 0 Not tested.
glis (tupaia)
0.2
NT
NT8
46.6 (38.3-57.1)
52.9 (41.1-61.9)
(41.8-63.5)
50.1
62.5 (34.5-88.7)
2
Common
TABLE from E rosettes@
Overnightb
green
Lymphocytes
1
38.5 (26.1-54.0)
47.0 (39.0-55.7)
35.6 (33&41.0)
58.1 (27.9-68.4)
3h
African
of Circulating
82.5 (80.0-84.5)
3
4
3
17
No. of animals tested
Markers
2
monkey)
marmoset)
marmoset)
Membrane
Galago crassicaudatus kako) Lemur catta (lemur)
Prosimians
(squirrel
Saimiri sciureus
.Yaguinus oed+us (cotton-topped
(white-lipped
Saga&us fuscicollis
(common
Cullithrix jacchus
Species
Surface
assay
9.2 (4.8-15.0)
16.5 (9.7-27.8)
k
Membrane
Prosimians
(FA).
NT
NT
15.7 (12519.0)
15.3
19.9 (17.2-21.7)
and
( 70)
NT
NT
reactivity
No
46.7
64.2 (60.3-70.7)
66.4 (59.8-73.4)
67.3 (39.2-86.5)
ASTLA
FAd
i5 I+ ! 2 .?
\\‘crc circulating I~nipliocytes from one lemur and spleen cells from t\vo tupaia ; cells from both species failed to form rosettes with the erythrocytes tested. Reproducibility of rosette formation. Surface membrane markers of lymphocytes from six colony-born common marmosets were evaluated over a 3-month period; E-rosette assays were incubated overnight at 4°C (Table 3). A mean of 66.1 s of common marmoset cells was AG-RFC, and 68.7% reacted with ASTLA. The B-lymphocyte fraction, as determined by EAC rosettes and membrane Ig staining, represented 15.7% of the cell population. Surface Membrane
Properties
of Lymplzoid
Tissztes
B- and T-lymphocyte markers of cells from lymphoid tissues of common, WL, and CT marmosets are summarized in Table 4. Between 76.2 and 90.9% of thymtls cells from two common marmosets were AG-RFC, while 6.2-18.070 of cells were S-RFC; > 90% of common marmoset thymus cells were stained by ASTLA. In contrast, thymic tissue from six WL or CT marmosets had equivalent percentages of AG-RFC and S-RFC; between 90.6 and 9.2.Oc/o of thymus cells formed AG-E rosettes and S9.1-97.8% were S-RFC. TABLE Study Animal No.
of Lymphocyte Sex
Surface E rosettes*
AG-RFC
Membrane (%)
3 Properties
of Six Common
EAC” rosettes (o/c)
hfarmosets* Membrane
S-RFC
FAd
(c/0)
ASTLA
k
76 AJ-1
D;I
71.7 84.8 59.7
(72.0)”
15.8 4.0 (10.9) 3.7
25.6 22.6 (18.4) 7.0
11.6 22.1 (18.0) 20.4
64.1 83.7
ih AJ-2
RI
63.5 72.7 60.2
(65.4)
8.0 17.0 (10.0) 5.2
19.6 3.5 (12.2) 13.7
14.9 22.1 (21.2) 26.6
74.0 76.2 62.7
(70.9)
76 AO-1
AI
67.5 75.9 63.9
(69.1)
10.5 9.3 2.9
(7.5)
12.1 20.0 8.5
12.8 8.7 (15.2) 24.3
80.5 66.6 66.3
(71.1)
76 AO-2
F
64.9 88.7 53.3
(68.9)
11.5 12.2 5.3
(9.6)
8.0 18.0 (12.2) 10.6
12.8 19.5 11.6
(14.6)
67.1 86.5 39.2
(64.2)
76 M-l
M
65.0 71.4 49.2
(61.8)
5.7 5.2 0.6
(3.8)
17.7 3.6 (13.8) 20.3
12.0 27.8
(19.9)
67.0 71 .a (73.3) 81.2
76 hI-2
&I
54.5 70.3 (59.6) 54.1
15.8 4.0 3.7
(7.8)
15.9 22.9 (16.2) 10.0
22.3 18.5 (19.1) 16.6
(13.5)
75.3 74.2 57.4
(73.9)
(68.9)
a Values obtained from three bleedings of the six animals over a 3-month period. b Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep crythrocytes (S-RFC) with overnight incubation. c Rosette formation with C’sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody assay. e Mean percentage is given in parentheses.
154
WRIGHT
ET
TABLE Surface Membrane Marmoset species
Tissue
Markers
Number tested”
AL.
4
of Marmoset E rosettes” (%)
AG-RFC Callithrix
jacchus
Lymphoid
S-RFC
Cells
EACc rosettes (70)
Membrane FAd (%I k
ASTLA
Thymus Spleen
2 2
83.5 34.9
11.9 1.8
6.0 44.9
0 34.3
92.0 42.1
Thymus Spleen Thymus Spleen
2 3
92.0 31.3
97.8 47.0
34.7 59.0
0.9 35.6
87.9 46.5
4 2
90.6 24.0
89.1 31.6
17.3 59.0
0 52.6
95.1 65.7
Saguinus juscicollis, nigricollis
Saguinus Oedipus
a Number of respective tissue samples obtained ; each tissue was evaluated once and values in table represent means of this number of tests. b Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC) with overnight incubation. c Rosette formation with C’-sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody assay.
The percentage of RFC in common marmoset splenic tissue also was indicative of a population of T cells forming rosettes with African green monkey E (34.9%) with only 1.8% S-RFC; 44.970 of common marmoset spleen cells formed EAC rosettes. Five WL and CT marmoset spleenswere comprised of between 24.0 and 31.3% AG-RFC and 31.6 and 47.0% S-RFC, respectively, and an average of 59.0% of the cells were EAC rosette positive.
Surface Membrane Properties of Virus-Carrying
Cultures
Presented in Table 5 are E- and EAC-rosette characteristics of common and CT marmoset cell cultures carrying HVS, HVA, or EBV. Common marmoset cultures carrying HVS or HVA formed rosettes with African green monkey erythrocytes, and the percentage of S-RFC (2X%6.8%) was insignificant; HVS or HVA CT marmoset cell lines, however, formed rosettes with both African green monkey and sheeperythrocytes. Common marmoset or CT marmoset cell lines, establishedafter transformation of lymphocytes with EBV, had low levels of E-rosette forming cells but expressed properties of B-derived lymphocytes (i.e., surface membrane Ig and/or EAC receptors). C’mediated cytotoxicity assay. To determine if depletion of a T-cell population would eliminate AG-RFC, pooled lymphocytes from four or five common marmosets (73% AG-RFC) were treated with various dilutions of ASTLA and rabbit c’. Results are presented in Table 6. Most AG-RFC were eliminated at ASTLA dilutions of 1: 200 and 1: 400, leaving predominantly EAC rosette-positive cells. A larger percentage of AG-RFC remained after incubation with ASTLA diluted 1: 800 or 1: 1600, and correspondingly the percentage of EAC-RFC decreased.
COikTMON
hJARMOSET
J.YbJPIJOCYTE
TABLE Surface
Membrane
Markers Carrying Cell
Species
Callithrix
jncchus
.%pinus
oedipus
5
of Cell Cultures Lymphotropic
line
and Lymphoblastoid Herpesviruses
Transforming virus
70-N2 1022 KCM-2.5
1.55
POPJJLATJONS
Cell
E rosettesa
Lines
(%)
EAC* rosettes
AG-RFC
S-RFC
HVSc HVA EBV
85.3 41.9 4.9
6.8 2.6 1.2
3.5 3.3 75.2
HVS HVA EBV
85.6 99.1 1.7
88.2 96.0 2.8
10.1 4.8 65.7
(%I
5 Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC) with overnight incubation. 6 Rosette formation with C’-sensitized sheep erythrocytes (see Materials and Methods). ~Lymph node, thymus, and spleen cell cultures from one common marmoset which died of HVS-induced lymphoproliferative disease; cells evaluated over 3-month period.
DISCUSSION Using standard E-rosette assay conditions and overnight incubation of cells at 4°C approximately 62% of common marmoset circulating lymphocytes formed spontaneous E rosettes with African green monkey erythrocytes, but only 7% of cells formed rosettes with sheep erythrocytes. The common marmoset T-lymphocyte population does not form rosettes with sheep erythrocytes, in contrast to T cells of humans and other nonhuman primate species, but appears to possess a surface membrane receptor for African green monkey erythrocytes as: (i) the number of common marmoset circulating AG-RFC (62%) was equivalent to the percentage of circulating T cells in Suguinus marmosets, other nonhuman primates, and man; (ii) 67% of common marmoset lymphocytes were stained with an anti-marmoset T cell serum by immunofluorescence, and this same serum with C’ selectively lysed most African green monkey E rosette-forming common marmoset cells ; (iii) thymus cells from common marmoset neonates were predominantly AG-RFC (83.5%) TABLE C’-Mediated
Lymphocyte
Cytotoxicity
Assay
6 with
Anti-Marmoset AG-RF@
Callithrir
jacchus
lymphocytes
Callithrir
jacchus
lymphocytes
+ C’
Callithrix jacchus lymphocytes +ASTLA (1: 200) (1:400) (1:800)
+ C’
(1: 1600)
0 Spontaneous rosette formation with ) Rosette formation with C/-sensitized 0 Not tested.
African sheep
green monkey erythrocytes.
T Cell (%)
Sera
EAC”
73.2
19.7
83.5
NTC
1.1 4.2 27.8 76.7
85.2 NT 79.3 24.1
erythrocytes.
(ASTLA) (a/o)
\\.itIr Only ;1 small pportio~~
of S-RPC (11.974) ; (iv) 85 ;; Oi cells 1lull tllylllus, spleen, or lymph node of two common marmosets inoculated with HVS, which induces a T-cell lymphoma in CT and WL marmosets, formed rosettes with African green monkey E. The common marmoset cells which formed rosettes with sheep E were not glassadherent cells and may represent a subpopulation of T lymphocytes, perhaps in a stage of differentiation. Results of this study demonstrate that all or a large portion of T lymphocytes of CT and WL marmosets and squirrel monkeys formed rosettes with African green monkey erythrocytes, but in contrast to common marmosets, T lymphocytes of these animals formed rosettes also with sheep erythrocytes. Lymphocytes of three prosimian species were evaluated for rosette-forming properties: galago lymphocytes formed rosettes with AG-E but not with sheep E, whereas lemur and tupaia lymphoid cells failed to rosette with either African green monkey or sheeperythrocytes. The characterization of common marmoset lymphocyte populations was undertaken because common marmosets have been evaluated as potential models for lymphoproliferative diseaseinduced by lymphotropic herpesviruses of humans and nonhuman primates. Animals infected with HVS, HVA, or EBV uniformly develop lymphoproliferative disease (22-24). HVS, HVA, and EBV are lymphotropic for specific lymphocyte populations in their natural (13) and experimental hosts. In experimentally infected CT and WL marmosets, HVS and HVA infect and transform T-derived lymphocytes, while EBV infects and transforms B lymphocytes; lymphoblastoid cell lines established after transformation in viva or in vitro of CT or WL marmoset lymphocytes with HVS or HVA express T-lymphocyte surface membrane markers and EBV-carrying cells possessB-lymphocyte properties. Lymphoid tumor tissues of HVS-infected common marmosets or HVA-carrying common marmoset lymphoblastoid cells are predominantly AG-RFC, indicating that HVS and HVA are T lymphotropic also in common marmosets.
ACKNOWLEDGMENTS This work was supported in part by the U.S. Public Health Service under Contract NO1 CP 33219 from the Division of Cancer Cause and Prevention, National Cancer Institute, Contract CA 5291-02 from the National Cancer Institute, and grant RR 05477 from the Division of Research Resources, National Institutes of Health. The assistance of Dr. Lester Fisher and Dr. Erich Maschgan of Lincoln Park Zoo, Chicago, and Dr. Anita Schwaier, Battelle Institute, Frankfurt, Western Germany, in obtaining blood samples from nonhuman primates is gratefully acknowledged. We thank the Board of Health, City of Chicago, for providing space for housing many of our experimental animals.
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54, 86, Appl.
Ins-
S. Jolmwn, D. Ii., l’cterson, D. A., Wolfe, I,. G., and Deinhardt, F., Frcl. Z’wc. 35, 392, 1976. 9. Johnson, L., Johnson, D. R., Wolfe, L. G., Black, R., Rocchi, J., and Deinhardt, F., Fed. Pror. 35,431, 1976. 10. Wallen, W. C., Neuhaucr, R. H., and Rabin, II., J. d/c*tl. I’rirtrnlol. 3, 41, 1’174. 11. Niblack, G. D., and Gcngozian, N., Clint. Bxb. ZI~~~~~Z~~I.23, 536, 1976. 12. Niblack, G. D., Kately, J. R., and Gengozian, N., Ztr~wumlog_v 32, 257, 1977. 13. Wright, J., Falk, I,. A., Collins, D., and Deinhardt, I;., J. Nat. Cancer Inst. 57, 959, 1976. 14. Martin, L. N., and Allen, W. P., Zpzjcct. Z~rzu~u~z.12, 528, 1975. 15. Thorsby, E., and Bratlic, A., III “Histocompatibility Testing” (P. Terasaki, Ed.), p. 655. Munksgaard, Copenhagen, 1970. 16. Mendes, N. F., Tolnai, M. E., Silveira, N. P., Gilbertsen, R. B., and Metzgar, R. S., J. Z~w~unol. 111, 860, 1973. 17. Jondal, M., and Klein, G., J. Exfi. Med. 138, 1365, 1973. 18. Massey, R., Johnson, D. R., Ogden, J., and Deinhardt, F., Fed. Proc. 33, 811, 1974. 19. Wright, J., Falk, L. A., and Deinhardt, F., J. Nut. Cancer Inst. 53, 271, 1974. 20. Falk, L. A., Wright, J., Wolfe, L. G., and Deinhardt, F., It& J. Cmtccr 14, 244, 1974. 21. Falk, L. A., Wolfe, L. G., Deinhardt, F., Paciga, J., Dombos, L., Klein, G., Henle, W., and Henle, G., I&. J. Cancer 13, 363, 1974. 22. Wright, J., Falk, L. A., Wolfe, L. G., Ogden, J., and Deinhardt, F., J. Nnt. Cn~rrrr I~rst., in press, 1977. 23. Laufs, R., Stcinke, H., Steinke, G., and Petzold, D., J. Nut. Cmzcc~ Inst. 53, 195, 1974. 24. Falk, L. A., Deinhardt, F., Wolfe, L. G., Johnson, D. R., Hilgers, J., and De-The, G., 111f. J. Cancer, 17,785, 1976.
35, 148-157
IJIMUNOLOGY
Lymphocyte
(1978)
Populations
of Callithrix
jacchus
JOHN \VRIGHT,* LAWREA-CE A. FALK,* LAUREN AND FRIEDRICH \V. ~EIKHARDT~ L)epartwents
of Microbiology,
Rlrsk-PresbSteriarz-St. Centers, Chicago, Received
July
Lrtke’s alrd Illinois 60612
Marmosets G. \VOLFE,
C’nizwsity
of Illinois
Medical
7, 1977
A lymphocyte population of common marmosets (Cal!ithrix jacchm) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 627% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Hrrpesvirrts sairuiri or Herpesvims afe/cs formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C’) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.
INTRODUCTION Human lymphocytes have been divided into two main classes on the basis of surface membrane markers : Thymic-derived or T lymphocytes carry a receptor for sheep erythrocytes (E) ( 1) , while bone marrow-derived B lymphocytes are characterized by expression of membrane-bound immunoglobulin (Ig) (2) and the presence of receptors for the third component of complement (c’3) (3) and the Fc fragment of aggregated Ig (4). Circulating lymphocytes of most nonhuman primates possesssurface membrane properties analogous to those of human lymphocytes (5-7) : 55-65c/o of circulating lymphocytes from cotton-topped (CT) (Sagltiws ocdipw) and while-lipped (\2:L) (Saqzzrinnsfmcicollis, S. nigricollis) nlarmosets (S-12) and 66-80s of squirrel monkey lymphocytes (13, 14) fornl spontaneous rosettes with sheep E while 17-257, of circulating cells from these speciespossessB-cell membrane markers. 11%ile characterizing the circulating lymphocyte populations of common marmosets (Callithvi.r jacclzzrs), we observed that only a small percentage of cells formed sheep E rosettes but 60-70% formed rosettes with African green monkey erythro1 Submitted in partial fulfillment for the Doctor of Philosophy in Microbiology from the University of Illinois at the Medical Center, Chicago, Illinois 60612. *Scholar of the Leukemia Society of America. sPresent address: Max v. Pettenkofer Institute, Pettenkoferstr. 9a, 8000 Muenchen 2, Western Germany. 148 000%8749/78/0351-0148$02.00/O Copyright All rights
8 1978 by of reproduction
Academic Preu, Inc. in
any
form
reserwd.
COMMON
MARMOSET
LYMPHOCYTE
POPULATIONS
149
cytes. The T-lymphocyte populations of common marmosets and galagos, a prosimian species also evaluated in this study, appear to have receptors for African green monkey E but not sheep E, in contrast to other genera of nonhuman primates. MATERIALS
AND
METHODS
Twenty adult colony-born or wild-caught common marmosets (Callithrix jacchus jacchzrs) were used as the source of lymphocytes. Captive animals had been acclimated for over 1 year and none of the animals had been used for experimental studies. Circulating lymphocytes and lymphoid tissues were studied also from CT and WL marmosets, squirrel monkeys, and three prosimian species, galagos (Galago crassicazrdatus), lemurs (Lewur catta), and tupaia (Tupaia glis) . Preparation
of Circulating
Lymphocytes
and Lymphoid
Tissues
Animals were bled by venipuncture and lymphocytes were separated from whole blood on Ficoll-Hypaque (FH) gradients (15). Lymphocytes were washed three times in medium RPM1 1640 and resuspended at a concentration of approximately 1 X loo cells/ml. Spleen and thymus from common, CT, and WL marmosets were removed aseptically ; animals ranged in age from 1 day to 3 months. Individual cell suspensions were prepared either by mincing and trypsinization of the tissues or by mincing and passage through a stainless-steel wire mesh. The cell suspensions were washed and resuspended in medium RPM1 1640 supplemented with 15% fetal calf serum (FCS), glutamine, 100 units of penicillin, and 50 pg of streptomycin/ml. Erythrocytes Blood was obtained from one sheep durin g the entire study and was used within 2 weeks of bleeding. African green monkey erythrocytes (AG-E), in Alsever’s solution, were obtained from Flow Laboratories (Rockville, Md.) . Sheep and African green monkey E were washed in Hanks’ balanced salt solution (HBSS) and adjusted to a 1% suspension in HBSS. Lymphocyte
Surface Membrane
MarKers
(i) E rosettes. E-rosette assays were performed according to a modification of the procedure of Jondal et al. (1). Approximately 3 X lo5 lymphocytes were mixed with 0.3 ml of a 1% suspension of sheep or African green monkey E for an erythrocyte to lymphocyte ratio of approximately 100: 1. Cells were incubated at 37°C for 5 min, centrifuged at 200g for 5 min, and left for 3 hr or overnight at 4°C. Lymphocytes with at least three attached E were considered positive rosette-forming cells (S-RFC or AG-RFC) and at least 200-300 cells from each sample were counted. In several experiments, E-rosette assays were performed as described but with human, gorilla, orangutan, baboon, rhesus monkey, goat, guinea pig, or mouse erythrocytes. (ii) EAC rosettes. Lymphocytes were evaluated for C’3 membrane receptors by a, modification of the procedure described by Mendes et al. (16). Hemolysin-sensi-
150
WRIGHT
ET
AL.
tized sheep erythrocytes (EA) were mixed with a human C’ source for 1 hr at 37°C with periodic shaking. The EAC were washed three times with HBSS and resuspended to 0.5 % concentration. Approximately 2 X lo5 lymphocytes were mixed with 0.2 ml of EAC and incubated for 30 min at 37°C ; the cells then were centrifuged at 200g for 5 min and reincubated at 37°C while the cells were counted. To determine the percentage EAC rosettes, cells were agitated mechanically (Vortex mixer) and diluted with HBSS: At least 200 lymphocytes were counted and cells with three or more attached EAC were considered positive. (iii) Membrane immuno~uorescence. Membrane immunofluorescent antibody assays (FA), performed according to the procedure of Jondal and Klein (17), were used with lymphocyte suspensions for identification of membrane Ig-positive cells and for T lymphocyte-specific membrane antigens. Approximately 1 x lo8 lymphocytes were mixed with 0.2 ml of either: (a) a 1: 10 dilution of fluorescein isothiocyanate (FITC) -conjugated goat anti-human IgM and IgG (Hyland Laboratories, Costa Mesa, Calif.), or (b) a 1: 200 dilution of goat anti-marmoset T-lymphocyte serum (ASTLA) (18) followed by FITC-conjugated rabbit anti-goat IgG (Hyland Laboratories). ASTLA was prepared by hyperimmunization of a goat with a marmoset lymphoblastoid cell line with T-lymphocyte surface 70-N2 cells (19), membrane properties. Serum was absorbed extensively with marmoset B lymphoblasts, marmoset fibroblast cells, and fetal calf serum, and did not react with B lymphocytes at the concentrations used. Virus-Carrying
Lymphoid
Cell Cultures
The surface membrane properties of thymus spleen, and lymph node cells of two common marmosets with Herpes&us sairniri (HVS) -induced lymphoma were studied during a 3-month cultivation period in vitro. The surface membrane markers of lymphoblastoid cell lines derived from common marmoset or CT marmoset cells transformed by HVS, Herpemirus ateles (HVA), or Epstein-Barr virus (EBV) also were evaluated ; CT marmoset cell lines included HVS-transformed 70-N2, HVA-transformed 1022 (20), and EBV-carrying KCM-25 cell lines (21). All lymphoblastoid cell lines were cultivated in medium RPM1 1640 supplemented with 15% FCS, glutamine, and antibiotics. Cytotoxicity
Assay
Circulating lymphocytes from common marmosets were depleted of cell populations reacting with ASTLA by a C-dependent cytotoxicity assay. Lymphocytes from several animals were pooled and approximately 1 X lo6 cells were mixed with 2.5 ml of various dilutions of ASTLA and incubated for 35 min at 4°C; cells were washed three times and 5 ml of a 1: 10 dilution of rabbit C’ was added for 30 min at 37°C. After washing two times in HBSS, the remaining cells were used for E- and EAC- rosette assays. Control samples of lymphocytes alone and lymphocytes treated only with rabbit C’ were used also in rosette assays. RESULTS E-Rosette
Formation
Erythrocytes mon marmoset
by Cowmon
Marvnoset
Circulating
Lymphocytes
from 10 species were assessed for E rosette formation with comcirculating lymphocytes, and results are summarized in Table 1.
COMMON
MARMOSET
LYMPHOCYTE
TABLE E-Rosette
Formation
by Common
Erythrocytes obtained from
Marmoset
1
Lymphocytes Number tested
with
Erythrocytes
B 0
monkey green monkey
pig
2 2 1 6 3 3 5 3 7 2 2
from
Common marmoset rosettes (%) Range
Human Human Gorilla Orangutan Baboon Rhesus African Goat Sheep Guinea Mouse
151
POPULATIONS
other
Species
E
Mean
(lO.(r19.0) (O&1.4) (25.2) (41.8-68.9) (4.3-15.7) (0.9-l .3) (44.2-58.6) (0) (O-3.2) P-w (O-0.5)
14.5 0.7 25.2 64.4 11.6 1.0 53.1 0.0 1.2 0.2 0.2
Means of 53 and 65% of common marmoset lymphocytes formed rosettes with African green monkey and orangutan erythrocytes, respectively. AG-E were used for further study of common marmoset T-lymphocyte surface membrane receptors, due to the unavailability of orangutan erythrocytes. Surface Membrane Properties of Circulating sets and 0 ther Nonhuwma Primates
Lymphocytes
from Coww~on
Marco-
Comparison of the surface membrane markers of circulating lymphocytes from three marmoset species, squirrel monkeys, and three prosimian species is presented in Table 2. The percentage of AG-RFC or S-RFC was evaluated by the standard E-rosette assay with a 3-hr or overnight incubation at 4°C. After 3-hr incubation, an average of 2.3% common marmoset cells formed rosettes with sheep E, while 58.1% of the lymphocytes formed rosettes with African green monkey erythrocytes. With overnight incubation, 7.3% of common marmoset lymphocytes were S-RFC and 62.5% of the cells were AG-RFC. An average of 67.3% of common marmoset lymphocytes was stained in membrane FA assays with a specific antiserum to CT marmoset T lymphocytes (ASTLA) . In contrast, lymphocytes from three other New World monkey species, WL and CT marmosets and squirrel monkeys, possessed receptors for both sheep and African green monkey E. Results of these assays indicated that the numbers of WL and squirrel monkey AG-RFC were usually less than the number of S-RFC. The number of CT AG-RFC or S-RFC was approximately equal. A population of common marmoset circulating lymphocytes was identified as B cells by the presence of immunoglobulin on the cell surface and receptors for C’3 ; as presented in Table 2, 15.4-16.57 o o f common marmoset lymphocytes possessed membrane Ig and/or C’3 receptors. Surface membrane properties of lymphocytes from two galagos were assessed ; more than 80% formed rosettes with AG-E, while < 1% of cells were S-RFC and the B-lymphocyte population was identified by EAC rosettes and Ig staining. Also tested for E-rosette formation with African green monkey and sheep erythrocytes
marmoset)
RFC
NT
0.0
2
rupaia
(&l38.6)
3h
Sheep
0.0
NT
(Zl)
60.0 (42.0-77.2)
47.3 (29.2-68.4)
53.2 (37.9-67.1)
(ye)
other
NT
3.8
NT
71.7 (55.4-83.9)
53.9 (39.5-79.0)
61.6 (46.5-75.1)
7.3 (O-27.8)
Overnight
RFC
Marmosets,
Primates,
NT
12.5
18.0 (11.5-28.7)
22.1 (16.1-26.4)
26.3 (15.7-34.2)
19.5 (9.5-35.0)
15.4 (10.1-25.6)
EAC” rosettes ( 70)
Nonhuman
6 Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC). b Overnight or 3-hr incubation of lymphocytes and erythrocytes at 4°C. c Rosette formation with C/-sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody 0 Not tested.
glis (tupaia)
0.2
NT
NT8
46.6 (38.3-57.1)
52.9 (41.1-61.9)
(41.8-63.5)
50.1
62.5 (34.5-88.7)
2
Common
TABLE from E rosettes@
Overnightb
green
Lymphocytes
1
38.5 (26.1-54.0)
47.0 (39.0-55.7)
35.6 (33&41.0)
58.1 (27.9-68.4)
3h
African
of Circulating
82.5 (80.0-84.5)
3
4
3
17
No. of animals tested
Markers
2
monkey)
marmoset)
marmoset)
Membrane
Galago crassicaudatus kako) Lemur catta (lemur)
Prosimians
(squirrel
Saimiri sciureus
.Yaguinus oed+us (cotton-topped
(white-lipped
Saga&us fuscicollis
(common
Cullithrix jacchus
Species
Surface
assay
9.2 (4.8-15.0)
16.5 (9.7-27.8)
k
Membrane
Prosimians
(FA).
NT
NT
15.7 (12519.0)
15.3
19.9 (17.2-21.7)
and
( 70)
NT
NT
reactivity
No
46.7
64.2 (60.3-70.7)
66.4 (59.8-73.4)
67.3 (39.2-86.5)
ASTLA
FAd
i5 I+ ! 2 .?
\\‘crc circulating I~nipliocytes from one lemur and spleen cells from t\vo tupaia ; cells from both species failed to form rosettes with the erythrocytes tested. Reproducibility of rosette formation. Surface membrane markers of lymphocytes from six colony-born common marmosets were evaluated over a 3-month period; E-rosette assays were incubated overnight at 4°C (Table 3). A mean of 66.1 s of common marmoset cells was AG-RFC, and 68.7% reacted with ASTLA. The B-lymphocyte fraction, as determined by EAC rosettes and membrane Ig staining, represented 15.7% of the cell population. Surface Membrane
Properties
of Lymplzoid
Tissztes
B- and T-lymphocyte markers of cells from lymphoid tissues of common, WL, and CT marmosets are summarized in Table 4. Between 76.2 and 90.9% of thymtls cells from two common marmosets were AG-RFC, while 6.2-18.070 of cells were S-RFC; > 90% of common marmoset thymus cells were stained by ASTLA. In contrast, thymic tissue from six WL or CT marmosets had equivalent percentages of AG-RFC and S-RFC; between 90.6 and 9.2.Oc/o of thymus cells formed AG-E rosettes and S9.1-97.8% were S-RFC. TABLE Study Animal No.
of Lymphocyte Sex
Surface E rosettes*
AG-RFC
Membrane (%)
3 Properties
of Six Common
EAC” rosettes (o/c)
hfarmosets* Membrane
S-RFC
FAd
(c/0)
ASTLA
k
76 AJ-1
D;I
71.7 84.8 59.7
(72.0)”
15.8 4.0 (10.9) 3.7
25.6 22.6 (18.4) 7.0
11.6 22.1 (18.0) 20.4
64.1 83.7
ih AJ-2
RI
63.5 72.7 60.2
(65.4)
8.0 17.0 (10.0) 5.2
19.6 3.5 (12.2) 13.7
14.9 22.1 (21.2) 26.6
74.0 76.2 62.7
(70.9)
76 AO-1
AI
67.5 75.9 63.9
(69.1)
10.5 9.3 2.9
(7.5)
12.1 20.0 8.5
12.8 8.7 (15.2) 24.3
80.5 66.6 66.3
(71.1)
76 AO-2
F
64.9 88.7 53.3
(68.9)
11.5 12.2 5.3
(9.6)
8.0 18.0 (12.2) 10.6
12.8 19.5 11.6
(14.6)
67.1 86.5 39.2
(64.2)
76 M-l
M
65.0 71.4 49.2
(61.8)
5.7 5.2 0.6
(3.8)
17.7 3.6 (13.8) 20.3
12.0 27.8
(19.9)
67.0 71 .a (73.3) 81.2
76 hI-2
&I
54.5 70.3 (59.6) 54.1
15.8 4.0 3.7
(7.8)
15.9 22.9 (16.2) 10.0
22.3 18.5 (19.1) 16.6
(13.5)
75.3 74.2 57.4
(73.9)
(68.9)
a Values obtained from three bleedings of the six animals over a 3-month period. b Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep crythrocytes (S-RFC) with overnight incubation. c Rosette formation with C’sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody assay. e Mean percentage is given in parentheses.
154
WRIGHT
ET
TABLE Surface Membrane Marmoset species
Tissue
Markers
Number tested”
AL.
4
of Marmoset E rosettes” (%)
AG-RFC Callithrix
jacchus
Lymphoid
S-RFC
Cells
EACc rosettes (70)
Membrane FAd (%I k
ASTLA
Thymus Spleen
2 2
83.5 34.9
11.9 1.8
6.0 44.9
0 34.3
92.0 42.1
Thymus Spleen Thymus Spleen
2 3
92.0 31.3
97.8 47.0
34.7 59.0
0.9 35.6
87.9 46.5
4 2
90.6 24.0
89.1 31.6
17.3 59.0
0 52.6
95.1 65.7
Saguinus juscicollis, nigricollis
Saguinus Oedipus
a Number of respective tissue samples obtained ; each tissue was evaluated once and values in table represent means of this number of tests. b Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC) with overnight incubation. c Rosette formation with C’-sensitized sheep erythrocytes (see Materials and Methods). d Evaluation of lymphocytes for expression of surface Ig or T lymphocyte-specific antigen by immunofluorescent antibody assay.
The percentage of RFC in common marmoset splenic tissue also was indicative of a population of T cells forming rosettes with African green monkey E (34.9%) with only 1.8% S-RFC; 44.970 of common marmoset spleen cells formed EAC rosettes. Five WL and CT marmoset spleenswere comprised of between 24.0 and 31.3% AG-RFC and 31.6 and 47.0% S-RFC, respectively, and an average of 59.0% of the cells were EAC rosette positive.
Surface Membrane Properties of Virus-Carrying
Cultures
Presented in Table 5 are E- and EAC-rosette characteristics of common and CT marmoset cell cultures carrying HVS, HVA, or EBV. Common marmoset cultures carrying HVS or HVA formed rosettes with African green monkey erythrocytes, and the percentage of S-RFC (2X%6.8%) was insignificant; HVS or HVA CT marmoset cell lines, however, formed rosettes with both African green monkey and sheeperythrocytes. Common marmoset or CT marmoset cell lines, establishedafter transformation of lymphocytes with EBV, had low levels of E-rosette forming cells but expressed properties of B-derived lymphocytes (i.e., surface membrane Ig and/or EAC receptors). C’mediated cytotoxicity assay. To determine if depletion of a T-cell population would eliminate AG-RFC, pooled lymphocytes from four or five common marmosets (73% AG-RFC) were treated with various dilutions of ASTLA and rabbit c’. Results are presented in Table 6. Most AG-RFC were eliminated at ASTLA dilutions of 1: 200 and 1: 400, leaving predominantly EAC rosette-positive cells. A larger percentage of AG-RFC remained after incubation with ASTLA diluted 1: 800 or 1: 1600, and correspondingly the percentage of EAC-RFC decreased.
COikTMON
hJARMOSET
J.YbJPIJOCYTE
TABLE Surface
Membrane
Markers Carrying Cell
Species
Callithrix
jncchus
.%pinus
oedipus
5
of Cell Cultures Lymphotropic
line
and Lymphoblastoid Herpesviruses
Transforming virus
70-N2 1022 KCM-2.5
1.55
POPJJLATJONS
Cell
E rosettesa
Lines
(%)
EAC* rosettes
AG-RFC
S-RFC
HVSc HVA EBV
85.3 41.9 4.9
6.8 2.6 1.2
3.5 3.3 75.2
HVS HVA EBV
85.6 99.1 1.7
88.2 96.0 2.8
10.1 4.8 65.7
(%I
5 Spontaneous rosette formation with African green monkey erythrocytes (AG-RFC) or sheep erythrocytes (S-RFC) with overnight incubation. 6 Rosette formation with C’-sensitized sheep erythrocytes (see Materials and Methods). ~Lymph node, thymus, and spleen cell cultures from one common marmoset which died of HVS-induced lymphoproliferative disease; cells evaluated over 3-month period.
DISCUSSION Using standard E-rosette assay conditions and overnight incubation of cells at 4°C approximately 62% of common marmoset circulating lymphocytes formed spontaneous E rosettes with African green monkey erythrocytes, but only 7% of cells formed rosettes with sheep erythrocytes. The common marmoset T-lymphocyte population does not form rosettes with sheep erythrocytes, in contrast to T cells of humans and other nonhuman primate species, but appears to possess a surface membrane receptor for African green monkey erythrocytes as: (i) the number of common marmoset circulating AG-RFC (62%) was equivalent to the percentage of circulating T cells in Suguinus marmosets, other nonhuman primates, and man; (ii) 67% of common marmoset lymphocytes were stained with an anti-marmoset T cell serum by immunofluorescence, and this same serum with C’ selectively lysed most African green monkey E rosette-forming common marmoset cells ; (iii) thymus cells from common marmoset neonates were predominantly AG-RFC (83.5%) TABLE C’-Mediated
Lymphocyte
Cytotoxicity
Assay
6 with
Anti-Marmoset AG-RF@
Callithrir
jacchus
lymphocytes
Callithrir
jacchus
lymphocytes
+ C’
Callithrix jacchus lymphocytes +ASTLA (1: 200) (1:400) (1:800)
+ C’
(1: 1600)
0 Spontaneous rosette formation with ) Rosette formation with C/-sensitized 0 Not tested.
African sheep
green monkey erythrocytes.
T Cell (%)
Sera
EAC”
73.2
19.7
83.5
NTC
1.1 4.2 27.8 76.7
85.2 NT 79.3 24.1
erythrocytes.
(ASTLA) (a/o)
\\.itIr Only ;1 small pportio~~
of S-RPC (11.974) ; (iv) 85 ;; Oi cells 1lull tllylllus, spleen, or lymph node of two common marmosets inoculated with HVS, which induces a T-cell lymphoma in CT and WL marmosets, formed rosettes with African green monkey E. The common marmoset cells which formed rosettes with sheep E were not glassadherent cells and may represent a subpopulation of T lymphocytes, perhaps in a stage of differentiation. Results of this study demonstrate that all or a large portion of T lymphocytes of CT and WL marmosets and squirrel monkeys formed rosettes with African green monkey erythrocytes, but in contrast to common marmosets, T lymphocytes of these animals formed rosettes also with sheep erythrocytes. Lymphocytes of three prosimian species were evaluated for rosette-forming properties: galago lymphocytes formed rosettes with AG-E but not with sheep E, whereas lemur and tupaia lymphoid cells failed to rosette with either African green monkey or sheeperythrocytes. The characterization of common marmoset lymphocyte populations was undertaken because common marmosets have been evaluated as potential models for lymphoproliferative diseaseinduced by lymphotropic herpesviruses of humans and nonhuman primates. Animals infected with HVS, HVA, or EBV uniformly develop lymphoproliferative disease (22-24). HVS, HVA, and EBV are lymphotropic for specific lymphocyte populations in their natural (13) and experimental hosts. In experimentally infected CT and WL marmosets, HVS and HVA infect and transform T-derived lymphocytes, while EBV infects and transforms B lymphocytes; lymphoblastoid cell lines established after transformation in viva or in vitro of CT or WL marmoset lymphocytes with HVS or HVA express T-lymphocyte surface membrane markers and EBV-carrying cells possessB-lymphocyte properties. Lymphoid tumor tissues of HVS-infected common marmosets or HVA-carrying common marmoset lymphoblastoid cells are predominantly AG-RFC, indicating that HVS and HVA are T lymphotropic also in common marmosets.
ACKNOWLEDGMENTS This work was supported in part by the U.S. Public Health Service under Contract NO1 CP 33219 from the Division of Cancer Cause and Prevention, National Cancer Institute, Contract CA 5291-02 from the National Cancer Institute, and grant RR 05477 from the Division of Research Resources, National Institutes of Health. The assistance of Dr. Lester Fisher and Dr. Erich Maschgan of Lincoln Park Zoo, Chicago, and Dr. Anita Schwaier, Battelle Institute, Frankfurt, Western Germany, in obtaining blood samples from nonhuman primates is gratefully acknowledged. We thank the Board of Health, City of Chicago, for providing space for housing many of our experimental animals.
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Med.
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