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Virus-induced alterations of lymphoid tissues
CELLtiLAR
IMM~SOLOGY
5, 307-317 (1972)
Virus-Induced
Alterations
III. Fate of Radiolabeled Inoculated
JUDITH Department
with
Thoracic
Duct
Newcastle
J. WOODRUFF
of Microbiology
of Lymphoid
and Immunology,
Lymphocytes
Disease
AND JACK
Tissues in Rats
Virus 1
F. WOODRUFF
Downstate
Medical
Center, Brooklyn, College,
New York, 11203 and the Department of Pathology, Cornell University Medical New
York,
Received
New March
York
10021
3, 1972
The influence of Newcastle disease virus (NDV) challenge on the distribution of injected thoracic duct lymphocytes was investigated. NDV was was inoculated into rats immediately after transfer of Wr-labeled lymphocytes. Significantly less radioactivity was recovered in lymph nodes of virustreated animals within 24 hr of cell transfer. By 48 hr, however, lymph nodes of virus-treated recipients contained normal concentrations of radioactivity. It is suggested that the virus transiently impairs the homing of donor lymphocytes into lymph nodes. These results support the proposition that NDV challenge reduces the size of the circulating T-lymphocyte pool by interfering with the normal pattern of lymphocyte migration. Evidence was obtained which indicated that NDV-induced lymphocytopenia was not mediated by abnormalities of the endothelial cells lining the postcapillary venules in the lymph node cortex. INTRODUCTION
The size of the recirculating pool of thymus-dependent (T) lymphocytes decreases rapidly in mice and rats challenged with Newcastle disease virus (ND\*) (I). One day after NDV challenge animals show a deficit of lymphocytes in the blood and thoracic duct lymph: concomitantly there is cellular depletion in those areas of the lymphoid tissues which are the traffic zones for these T-lymphocytes (2-5). These effects of ND\’ may be due to destruction of lymphocytes or an alteration in their pattern of migration in the body. \Ve have investigated this problem by determining the fate of radiolabeled thoracic duct lymphocytes transfused into ND\‘-treated rats. This report presents evidence that viral challenge alters the normal distribution of lymphocytes without killing the cells. In addition, the results indicate that NDV-induced lymphocytopenia is not due to abnormalities nf the endothelial cells lining the postcapillary venules in the lymph nodes. 1 This work was supported in part by National Institutes of Health Grants AI 10080 and GM 00078-11 and PHS Grant FR 05396 from the General Research Support Bureau, Bureau of Health Professions Education and Manpower Training, Division of Research Resources. 307 Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
308
WOODRUFF
AND
MATERIALS AND
WOODRUFF
METHODS
Animals Lewis rats were obtained from Simonsen Laboratory, Gilroy, California and Microbiological Associates, Bethesda, Maryland. Wistar-Furth rats were supplied by Microbiological Associates. All rats were inbred males weighing 180-200 g. Virus Newcastle disease virus (NDV) (Hickman Strain) that was grown in the allantoic fluid of lo-day-old embryonated eggs, was concentrated by ultracentrifugation and resuspended in phosphate buffered saline (PBS) as described previously (1). The infectivity and hemagglutinin (HA) titers of the virus were 10s.? egg infective dose-B,,(EID,,)/O.l ml and 2048 HA units/O.4 ml, respectively. Preparation
of 51Cr-Labeled Thoracic Duct Lymphocytes
The thoracic ducts of rats were cannulated and lymph collected for 12-18 hr at room temperature in flasks containing 20 ml of heparinized saline (20 units heparin/ml) (6). Lymphocytes were separated from lymph by centrifugation (900 rpm X 5 min) at 4°C. The pellet, usually containing 5-8 X lO* lymphocytes, was resuspended in 5 ml saline containing about 10% lymph and incubated at room temperature with 100 &i of radioactive chromium (“‘Cr). 51Cr was supplied as Na,-51 CrO, in isotonic saline at pH 7.0 without preservative by Iso-Serve, Cambridge, Massachusetts. After 1 hr the cells were diluted to 40 ml with saline, centrifuged, washed once and resuspended in saline at a concentration of 2&40 X lo6 lymphocytes/ml. An aliquot (0.5 ml) was removed to determine the amount of radioactivity in the cell suspension. A Packard Automatic Scintillation Gamma Counter was used for all determinations of radioactivity. Experirtzental
Design
51Cr-labeled lymphocytes in 0.5 ml saline were transfused via the lateral tail vein into each of several syngeneic recipients which had been lightly anesthetized with ether. Virus-treated or control rats were injection intravenously (iv) with 0.4 ml NDV or saline, respectively. The dose of NDV inoculated and the time intervals between virus challenge and cell transfer varied and are indicated in the text. At various intervals after cell transfer, recipients were killed and lymph nodes (superficial and deep cervical and mesenteric) and other organs were weighed and assayed for radioactivity as described previously (6). From each recipient 0.3-0.5 g of lymph nodes were obtained and the counts per minute (cpm) per gram lymph node were calculated. Radioactivity in the other organs was calculated as cpm per whole organ. The amount of radioactivity in the aliquot of the injected cell suspension was determined with each tissue assay, and the per cent recovery of injected radioactivity was based on this value. Statistical Methods P values were calculated using a variance technique.
VIRAL
ALTERATION
OF T LYMPHOCYTE
CIRCI-L.\TIOS
304
RESULTS Thoracic duct lymphocytes (TDL), when transfused intravenously, rapidly ant1 selectively accumulate in lymph nodes and spleen. Most of these donor cells localize specifically in the areas which are the traffic zones for T-lymphocytes and subsequently migrate into thoracic duct lymph (2). In order to study the early effects of NDV on lymphocyte recirculation, rats were challenged with virus at varying intervals before and after injection of “‘Cr-labeled TDL: the reco\:ery of radioactivity in lymph nodes was then determined 2 hr after cell transfer. Virusinduced changes at this time must reflect alteration in the rate of migration of donor lymphocytes into the nodes. In other experiments recipients of labeled lymphocytes were killed 1 day after viral challenge so as to determine the distribution of the donor cells when the rats were markedly lymphocytopenic (1). Finally, change> in the distribution of radioactivity in NDV-treated and control rats during thy PC hr after lymphocyte transfer were studied. 2-Hr Recovery of Radioactivity in Lymph Nodes of Recipients of 5JCr-Labeled Thoracic Duct Lymphocytes; Effect of Varying the Time and Dose of -VDl’ Challenge I’revious experiments showed that 24 hr after intravenous inoculation of lo”.” EID,, NDV rats were markedly lymphocytopenic (1). In the current study injection of this dose of virus immediately after labeled lymphocytes significantly reduced the 2 hr recovery of radioactivity in lymph nodes. This effect was also observed when NDV was injected 4 hr before labeled lymphocytes. In contrast. when NDV was injected 24 hr before cell transfer the homing of donor cells into the nodes was normal (Fig. 1). The effect of NDV on donor lymphocytes was dependent on the dose of virus injected. The results of several experiments in which lymphocytes and virus were transfused sequentially (i.e., virus injected immediately after cells) are summarized in Fig. 2. The lymph node concentration of radioactivity was reduced about 70% after chaIlenge with 10*“.3, 50% with 10s.” and 25% with lO”.j EID,, NDV. No reduction in radioactivity was seen after the injection of 109.0 EIl],,, NDV. Distribution of Radioactivity Hr after NDV Challenge
in Recipients
of
51Cr-Labeled Lymphocytes
18-22
The lymphocytopenia found one day after NDV challenge is associated with a deficit of lymphocytes in the lymph and thymus-dependent areas of lymphoid tissues (1). It was thought that by studying the distribution of labeled cells in such animals it might be possible to determine if lymphocytes, unable to enter lymph nodes, preferentially accumulate in any particular site. For this purpose the following experiments were performed. Rats were injected with (i) virus and labeled lymphocytes sequentially, (ii) virus 4 hr before labeled lymphocytes. and (iii) virus 24 hr after labeled lymphocytes. All recipients were killed 18-22 hr after NDV challenge.
310
WOODRUFF
AND
WOODRUFF
120 % z 5 Fq Yg PIsg b&L o.
100
60
60
TIME OF VIRUS CHALLENGE
24-HI?--4 CELL TAANSFER
FIG. 1. Concentration of radioactivity in lymph nodes of virus-treated rats as percentage of control values 2 hr after transfer of 15 X 10s sCr-labeled TDL. Recovery of radioactivity in lymph nodes of control (saline-treated) rats expressed as 100%. NDV (loss EID,,) or saline was injected intravenously 24 or 4 hr before or 5 min after cell transfer. Each point represents mean values of at least four virus-treated and four control recipients. Two-hour recovery was significantly depressed (P < .Ol) when virus given 4 hr before or 5 min after cells.
Virus and labeled lymphocytes injected sequentially. These recipients showed a marked reduction in the concentration of radioactivity in the lymph nodes (Table 1). However, the recovery of radioactivity in the spleens of these rats was normal. These changes were not associated with significantly increased recovery of label in the liver which occurs when donor lymphocytes are killed in vitro by heat or in vivo by anti-lymphocyte serum (6, 7). Also there was no significant difference
rl -
I
,olo.3
IOa8
lOa
DOSE OF NDV INOCULATED .OS p’ co1 <.o I
lOa (ElDso) N.S.
FIG. 2. Two-hour concentration of radioactivity in lymph nodes of rats transfused sequentially with 10 X 10s Wr-labeled lymphocytes and the indicated dose of NDV or saline. Recovery of radioactivity in lymph nodes of control animals expressed as 100%. Each bar represents mean recovery in three to four virus-treated recipients compared to values in three to four control animals.
VIRAL
ALTERATION
OF T LYMPHOCYTE
9.5
48 < .Ol
70 of control P
.8>P>
311
CIRCULATION
90
109
..i>P>
.i
.3
..i>P>
A
“ Itats sacrificed 18 hr after receiving 18 X lo6 Iynphoc>-tes containing 14,635 cpm. immediately after cell3 b Saline (control) or 10”’ 3 EID 50 r\TDV injected intravenously ( XIean \.alrles with rauges in pxmtheses: four ;inirnals ill e:lch gro~~p.
in the recovery of radioactivity virus-treatd animals.
in the lungs, kidney, and thymus of control and
f’hs hjcctrd 4 Izr lwfore labeled lynzplzocytes. In these virus-treated recipients the reduced recovery of radioactivity in lymph nodes was balanced by an increased recovery in the spleen (Table 2). The accumulation of radioactivity in the liver and lungs of the virus-treated animals was normal. Virus injected 24 hr after labeled lyruzphocytes. When labeled lymphocytes are injected rapidly into the bloodstream, as in the current experiments, a substantial proportion of the donor cells are found initially in the lungs and spleen (6). It was possible that this early distribution of transfused lymphocytes was the primary factor which enabled the virus to alter the homing of lymphocytes into the lymph nodes. Therefore, in several experiments recipients were injected with labeled lymphocytes and 24 hr later challenged with NDV. One day later they were killed. The results depicted in Table 3 show that even under these conditions NDV interfered with the recovery of radioactivity in the nodes.
l‘reatnlent rats fd
I
Control
2611
N I)\ V.
of
(2378-2791)
Spleen (cpm/organJ c 1429 (1313-1497) 2645
(2458-3102)
control
66
185
P
<.Ol
<.Ol
I .iver ~CpIn/orgau)
I ,unl: Icpm/organ)
1456 (1199~-16551
266 (o-600)
1034
230 (109.354i
(669-1252) 51
.1>1’>.1
87
.Y>P>.8
“ Rats sacrificed 24 hr after injection of 14 X 1W lymphocytes contaillillg 9836 cpm. b 4 kIr before cell transfer rats injected intravenously with saline ic‘ontrol) or with 10” k El ID,,, N rDv. c Mean values with ranges in parentheses; four animals in each grc~up.
312
WOODRUFF
AND
TABLE
WOODRUFF
3
DISTRIBUTION OF RADIOACTIVITY IN RATS INJECTED WITH NDV 24 HR AFTER TRANSFER OF 51Cr-LABELED LYMPHOCYTES~ Treatment of rats b
Lymph nodes h&d
Control
2639 (2490-2746)
NDV
1998 (1779-2367) % of control
P
75
< .Ol
c
Spleen (cpmbrgan)
Liver (cpmbrgan)
672 (573-757)
365 (211-625)
790 (674-867)
3.50 (113-640)
117
.3>P>
95
.2
0.9
o Radioactivity in organs 48 hr after injection of 10 X lo6 lymphocytes containing 6100 cpm. b 24 Hr after cell transfer rats injected intravenously with saline (control) or with log.* EIDs~ NDV. c Mean values with ranges in parentheses; four animals in each group.
The Distribution of 6’Cr-Labeled TDL in Control and Virus-Treated Intervals of 2-96 Hr After Cell Transfer
Animals a?
The reduced recovery of donor lymphocytes in lymph nodes of virus-treated rats might reflect destruction of transfused cells since dead lymphocytes do not “home” to these tissues (6). Alternatively, some donor lymphocytes might be viable but initially be unable to migrate into the nodes. To distinguish between these two possibilities several experiments were carried out in which the fate of transfused lymphocytes was determined during the 96-hr period after rats were injected with (a) virus and lymphocytes sequentially and (b) virus 4 hr before lymphocytes. Virus and labeled lymphocytes injected sequentially. In the experiment shown in Table 4 each recipient was injected with an aliquot of the same labeled cell suspension and then injected with 10Q.8 EID,, NDV or saline. Although the recovery of radioactivity in lymph nodes was reduced at 2 and 24 hr after cell transfer, by 96 hr the mean concentrations of radioactivity in the nodes of virustreated and control recipients were virtually identical. In contrast, virus-treated animals showed increased recovery of label in the spleen at 2 and 24 hr but not at 96 hr. It was also found that there was less radioactivity than normal in the livers of NDV-treated rats at 24 and 96 hr. In other experiments (Fig. 3) rats were challenged with a larger dose of NDV ( 1O1o*3EID,,). This virus dose reduced even further the recovery of radioactivity in lymph nodes of animals sacrificed within 24 hr of cell transfer. Nonetheless at 48 hr the recovery of radioactivity in the lymph nodes of such rats approximated that detected in control animals. In these animals virus challenge had little if any effect on the accumulation of radioactivity in the spleen, liver or lungs. Virus injected 4 hr before labeled lymphocytes. As shown in Fig. 4 the recovery of radioactivity in lymph nodes of these virus-treated rats was markedly depressed at 2 and 24 hr but not at 96 hr. At each interval studied more radioactivity was found in the spleens of virus-treated rats than in controls. This difference was
VIRAL
I
ALTERATION
Control c \Yru> r’
Control \‘irur
LYMPHOCYTE
57 <.Ol 5784 (5473-5932) 4189 (4086-4387 j
% of control P Oh
T
1660 (1605-1769) 956 (907-1044) % of control P
24
OF
Control Viru.
72 <.Ol 3047 (2874-3355) 3046 (2838-3408)
% or control P
100 >.9
31 3
CIRCIIL.\‘l’lON
c 3339 (2960-36463 4265 r40W 443.3
1016 (992-10.581 016 (744 -I_‘171
118 . 05 > P > .02 1755 (1684-18191 2297 1221lL2397i
90 .7>P>
1106 (10-I-6-1244, 765 i6.32-950 I
I.31 .05>P> .O.! 1031 (969-1096) 1240 11103-14471 I20 .3>P>
.6
69 < .Ol 1018 1961L11741 645 (516-730)
.2
” 14 X 10” lymphocytes containing 11,276 cpm injected intravenously immediately I*Time after cell transfer. e Saline-injected intravenously. CllOQ.* EID,, NDV intravenousl>~. Meal1 valtleh with ranges in parentheseh; four animals ill each group.
6.3 <.Ol before S I )I’.
most pronounced at 24 hr. Although the accumulation of label in the lungs 2 hr after cell transfer was increased in virus-treated rats, at later intervals recovery in this organ was normal. In contrast, recovery of radioactivity in the livers of virus-treated animals was normal except on day 4 when it was slightly reduced. DISCUSSION
NDV inoculated into rats immediately after transfer (Jf “lCr-lal~elecl syngeneic TDL altered the fate of the donor cells. Significantly less radioactivity was recovered in lymph nodes of virus-treated animals 2 and 24 hr after cell transfer. However, by 48 hr recovery of radioactivity in the lymph nodes of these animals had increased and approximated that detected in control animals. The simplest explanation of these results is that in NDV-challenged rats a significant proportion of donor lymphocytes, initially excluded from lymph nodes, later migrated into these tissues. It is not known to what extent, if any, radioactivity in the nodes of virus-treated animals represented labeled progeny of donor lymphocytes. These results support the idea that NDV reduces the size of the circulating T-lymphocyte pool by interfering with the normal pattern of lymphocyte nrigration rather than by destroying the cells. The recovery of radioactivity in lymph nodes was inversely related to the dose of virus injected. This effect was observed in two separate inbred strains of rats
314
WOODRUFF
AND
WOODRUFF
LUNG
SPLEEN
I.
15 IO 5 0
2 92
50-
LYMPH
LIVER
NODES
(L $40 i
30-
3 30 z
205 E IO !$ 20 f-y % 0 2
IO 16
49
72
96
HOURS
AFTER
0'
2
CELL
so 16
46
72
96
TRANSFER
FIG. 3. Recovery of radioactivity in lymph nodes, spleen, liver and lung of control and NDV-treated rats 2-96 hr after injection of 10 X 10s Wr-labeled TDL. A-A 1010.3EID,, NDV or l 0 saline injected immediately after cells. For lymph nodes the values represent percentage of recovery per gram of lymph node; for spleen, liver and lung, percent per whole organ. Each point represents mean value of four recipients. Lymph node values were significantly different at 2 and 16 hr (P < .Ol) only. There was no significant difference in recovery in other organs of virus-treated and control animals.
and was also detected when virus was injected 4 hr before or 24 hr after cell transfer. In contrast, NDV challenge did not impair the homing of transfused lymphocytes into the spleen. However, it is not known if the injected lymphocytes accumulated in the periarteriolar lymphoid sheaths of the spleen as in normal recipients. The virus also caused very little change in recovery of radioactivity in the lungs and liver. The impaired accumulation of label in lymph nodes was in some experiments balanced by an increased recovery in the spleen (Table 4, Fig. 4). This suggests that the lymphocyte population reaching the spleen may contain cells initially excluded from the nodes which later migrate into the nodes. In other experiments, however, particularly when virus was injected after labeled cells there was no evidence that donor cells, unable to enter lymph nodes, were diverted to any single site (Fig. 3). Since the homing of T lymphocytes into lymph nodes depends on their selective affinity for endothelial cells lining postcapillary venules (Z), NDV may achieve its effect by altering the donor lymphocytes and/or the endothelial cells. The present results indicate that the virus does not act on the endothelial cells. Twenty-four hours after viral challenge rats show marked lymphocytopenia and cellular depletion in the paracortical areas of lymph nodes ( 1). Nonetheless, when labeled lymphocytes were injected 24 hr after NDV, the cells homed normally into the lymph nodes (Fig. 1). Thus the deficit of T-lymphocytes in the circulation and in lymphoid
VIRAL
40
OF T LYMPHOCYTE
ALTERATION
CIRCULATION
315
LUNG
SPLEEN
i.2 p.
s
LYMPH
NODES
HOURS
AFTER
CELL
TRANSFER
FIG. 4. Recovery of radioactivity in lymph nodes, spleen, liver and lung of control and NDV-treated rats 2-96 hr after transfer of 20 X 106 51Cr-labeled TDL. A--A 109,s EID,, NDV or l -0 saline injected 4 hr before cells. For lymph nodes, the values represent percentage of recovery per gram of lymph node; for spleen, liver and lung percent per whole organ. Each point represents mean value of four recipients. The lymph node values were significantly different at 2 and 24 hr only (P < .05 and < .Ol, respectively), the spleen values at 24 and 96 hr (P < .Ol), the liver values at 96 hr (P < .Ol) and the lung values at 2 hr I’P <.OS).
tissues of ND\’ challenged rats cannot be attributed tcJ abnormalities of these endothelial cells. The mechanism(s) by which NDV challenge alters the fate of T-lymphocytes is not known. The virus may act directly on the lymphocytes since these cells have receptors for NDV. These receptors apparently terminate in sialic acid which is released from the cells when virus ekes in vitro (8 j Thus viral modification of lymphocyte surface structures (with or without infection of the cells) may l)ia!- a critical role in altering the pattern of lymphocyte migration. This suggestion is consistent with the evidence from previous experiments that sialic acid Containing structures on the lymphocyte surface influence their distribution in the I)ody (6). Moreover, recent studies have shown that the fate of tlonot- TDTbriefly incubated in vitro with NDV 2 is the same as that observed when lymphocyte> are incubated with neuraminidase (T’. clrolcvu) (6). When donor TDL XIX altered by NDV or neuraminidase, each cell population appears to regain the abilit! to migrate normally by 48 hr after injection. This recovery might depend on rexenc-ration of lvmphocyte surface components. Alternat&ely the virus may act indirectly on lymphocytes. The most apparent putative mediators of the viral effect are adrenal steroids, antigenic stimulation, ? Woodruff, J. J. and Woodruff, J. F. Virus-Induced Alterations The effect of Newcastle disease virus on the fate of radiolabeled Manuscript in preparation.
of Lymphoid Tissues IV. thoracic duct lymphocytes.
316
WOODRUFF
AND
WOODRUFF
phagocytosis of the virus, or endotoxin. Previous experiments have shown, though, that adrenal glands are not required for the action of NDV on lymphocytes (1). Antigenic stimulation influences the distribution of transfused lymphocytes (9) but not when antigen is injected immediately after lymphocytes as in the current experiments. Since NDV is rapidly removed from the circulation by macrophages, lymphocytopenia could be mediated by virus-macrophage interaction. Yet, treatment of NDV with specific antiserum, which enhances phagocytosis of the virus in &~o (10) diminishes its lymphocytopenic effect (1). Similarily, the failure of NDV challenge 24 hr before injection of labeled lymphocytes to alter lymphocyte homing suggests that phagocytosis did not play a significant role in this process (Fig. 1). While both endotoxin ( 11) and NDV rapidy produce lymphocytopenia and granulocytosis in mice ulfraviolet light irradiated NDV causes lymphocytopenia but not granulocytosis (1) . Viral alteration of lymphocyte physiology could be immunosuppressive since the induction of an immune response appears to require migration of circulating lymphocytes into the antigen-stimulated lymph node or spleen (12-14). Recent experiments have shown that the survival of skin allografts across a major histocompatibility locus is prolonged by approximately 2 days in NDV-treated mice (unpublished observation). A similar mechanism may account for the immune suppression caused by measles and lactic dehydrogenase (LDH) viruses as well as by oncogenic viruses (15-18). Possibly, enhancement of humoral immunity as observed with LDH and Venezuelan equine encephalitis viruses (19, 20) might also reflect impaired T-lymphocyte function since there is evidence that T-lymphocytes exert, by feedback inhibition, some control over antibody production (21). ACKNOWLEDGMENTS We thank Mrs. Karen technical assistance.
Lefkowitz,
Mrs.
Lynn
Palmer,
and Mr.
Santo Scribani
for excellent
REFERENCES 1. 2. 3. 4. 5. 6. 7. S. 9. 10. 11. 12. 13. 14. 15. 16.
Woodruff, J. F., and Woodruff, J. J., Cell. Immunol. 1, 333, 1970. Gowans, J. L., and Knight, E. J., Proc. Roy. Sot. Biol. Sci. 159,257, 1964. Waksman, B. H., Arnason, B. G., and Jankovic, B. D., J. Exp. Med. 116, 187, 1962. Parrott, D. M. V., desousa, M. A. B., and East, J., J. Exp. Med. 123, 191, 1966. Miller, J. F. A. P., Mitchell, G. F., and Weiss, N. S., Nature (London) 214, 992, 1967. Woodruff, J. J., and Gesner, B. M., J. Exp. Med. 129, 555, 1969. Taub, R. N., and Lance, E. M., Immunology 15, 633, 1968. Woodruff, J. F., and Woodruff, J. J., Cell. Immunol. this issue. Zatz, M. M., and Lance, E. M., J. Exp. Med. 134, 224, 1971. Brunner, K. T., Hurez, D., McCluskey, R. T., and Benacerraf, B., J. ImmunoZ. 65, 99, 1960. Patterson, L. T., Harper, J. M., and Higginbotham, R. D., J. Bacterial. 95, 1375, 1968. Hall, J. G., and Morris, B., Lancet 1, 1077, 1964. Ford, W. L., and Gowans, J. L., Proc. Roy. Sot. Biol. 166, 244, 1967. Ford, W. L., Immunology 15, 609, 1968. Starr, S., and Berkovich, S., N. Engl. J. Med. 270, 386, 1%4. Howard, R. J., Mergenhagen, S. E., Notkins, A. L., and Dougherty, S. F., Transplant. Proc. 1, 586, 1969. 17. Notkins, A. L., Mergenhagen, S. E., and Howard, R. J., Ann. Rev. Microbial. 24, 525, 1970. 18. Snodgrass, M. J., Lowery, D. S.. and Hanna, M. G., Jr., J. Iwwnw~ol. 106, 877, 1972.
VIKAL
10. Notkins.
I\.
L., Mergenhagen,
J. Exj. Med. 20. Howard,
ALTERATION
126,
OF T LYMPHOCYTE
S. E., Rizzo,
.A. A.,
CIRCULATION
Sdrecle.
3I7
c.. ant1 \\‘aldmann.
‘I.
.\
347, 1966.
R. J., Craig,
C. P., Trevinu,
G. S., Dougherty,
S. I;., and Merpenhagen.
S. 1,.
J. Immunol. 103, 699, 1969. 21. Baker, 105,
P. J., Stashak, 1970.
1581,
P. W., Amsbaugh,
D. F., Prescott,
E.. and Barth,
R. F.. J. Immrnol
IMM~SOLOGY
5, 307-317 (1972)
Virus-Induced
Alterations
III. Fate of Radiolabeled Inoculated
JUDITH Department
with
Thoracic
Duct
Newcastle
J. WOODRUFF
of Microbiology
of Lymphoid
and Immunology,
Lymphocytes
Disease
AND JACK
Tissues in Rats
Virus 1
F. WOODRUFF
Downstate
Medical
Center, Brooklyn, College,
New York, 11203 and the Department of Pathology, Cornell University Medical New
York,
Received
New March
York
10021
3, 1972
The influence of Newcastle disease virus (NDV) challenge on the distribution of injected thoracic duct lymphocytes was investigated. NDV was was inoculated into rats immediately after transfer of Wr-labeled lymphocytes. Significantly less radioactivity was recovered in lymph nodes of virustreated animals within 24 hr of cell transfer. By 48 hr, however, lymph nodes of virus-treated recipients contained normal concentrations of radioactivity. It is suggested that the virus transiently impairs the homing of donor lymphocytes into lymph nodes. These results support the proposition that NDV challenge reduces the size of the circulating T-lymphocyte pool by interfering with the normal pattern of lymphocyte migration. Evidence was obtained which indicated that NDV-induced lymphocytopenia was not mediated by abnormalities of the endothelial cells lining the postcapillary venules in the lymph node cortex. INTRODUCTION
The size of the recirculating pool of thymus-dependent (T) lymphocytes decreases rapidly in mice and rats challenged with Newcastle disease virus (ND\*) (I). One day after NDV challenge animals show a deficit of lymphocytes in the blood and thoracic duct lymph: concomitantly there is cellular depletion in those areas of the lymphoid tissues which are the traffic zones for these T-lymphocytes (2-5). These effects of ND\’ may be due to destruction of lymphocytes or an alteration in their pattern of migration in the body. \Ve have investigated this problem by determining the fate of radiolabeled thoracic duct lymphocytes transfused into ND\‘-treated rats. This report presents evidence that viral challenge alters the normal distribution of lymphocytes without killing the cells. In addition, the results indicate that NDV-induced lymphocytopenia is not due to abnormalities nf the endothelial cells lining the postcapillary venules in the lymph nodes. 1 This work was supported in part by National Institutes of Health Grants AI 10080 and GM 00078-11 and PHS Grant FR 05396 from the General Research Support Bureau, Bureau of Health Professions Education and Manpower Training, Division of Research Resources. 307 Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
308
WOODRUFF
AND
MATERIALS AND
WOODRUFF
METHODS
Animals Lewis rats were obtained from Simonsen Laboratory, Gilroy, California and Microbiological Associates, Bethesda, Maryland. Wistar-Furth rats were supplied by Microbiological Associates. All rats were inbred males weighing 180-200 g. Virus Newcastle disease virus (NDV) (Hickman Strain) that was grown in the allantoic fluid of lo-day-old embryonated eggs, was concentrated by ultracentrifugation and resuspended in phosphate buffered saline (PBS) as described previously (1). The infectivity and hemagglutinin (HA) titers of the virus were 10s.? egg infective dose-B,,(EID,,)/O.l ml and 2048 HA units/O.4 ml, respectively. Preparation
of 51Cr-Labeled Thoracic Duct Lymphocytes
The thoracic ducts of rats were cannulated and lymph collected for 12-18 hr at room temperature in flasks containing 20 ml of heparinized saline (20 units heparin/ml) (6). Lymphocytes were separated from lymph by centrifugation (900 rpm X 5 min) at 4°C. The pellet, usually containing 5-8 X lO* lymphocytes, was resuspended in 5 ml saline containing about 10% lymph and incubated at room temperature with 100 &i of radioactive chromium (“‘Cr). 51Cr was supplied as Na,-51 CrO, in isotonic saline at pH 7.0 without preservative by Iso-Serve, Cambridge, Massachusetts. After 1 hr the cells were diluted to 40 ml with saline, centrifuged, washed once and resuspended in saline at a concentration of 2&40 X lo6 lymphocytes/ml. An aliquot (0.5 ml) was removed to determine the amount of radioactivity in the cell suspension. A Packard Automatic Scintillation Gamma Counter was used for all determinations of radioactivity. Experirtzental
Design
51Cr-labeled lymphocytes in 0.5 ml saline were transfused via the lateral tail vein into each of several syngeneic recipients which had been lightly anesthetized with ether. Virus-treated or control rats were injection intravenously (iv) with 0.4 ml NDV or saline, respectively. The dose of NDV inoculated and the time intervals between virus challenge and cell transfer varied and are indicated in the text. At various intervals after cell transfer, recipients were killed and lymph nodes (superficial and deep cervical and mesenteric) and other organs were weighed and assayed for radioactivity as described previously (6). From each recipient 0.3-0.5 g of lymph nodes were obtained and the counts per minute (cpm) per gram lymph node were calculated. Radioactivity in the other organs was calculated as cpm per whole organ. The amount of radioactivity in the aliquot of the injected cell suspension was determined with each tissue assay, and the per cent recovery of injected radioactivity was based on this value. Statistical Methods P values were calculated using a variance technique.
VIRAL
ALTERATION
OF T LYMPHOCYTE
CIRCI-L.\TIOS
304
RESULTS Thoracic duct lymphocytes (TDL), when transfused intravenously, rapidly ant1 selectively accumulate in lymph nodes and spleen. Most of these donor cells localize specifically in the areas which are the traffic zones for T-lymphocytes and subsequently migrate into thoracic duct lymph (2). In order to study the early effects of NDV on lymphocyte recirculation, rats were challenged with virus at varying intervals before and after injection of “‘Cr-labeled TDL: the reco\:ery of radioactivity in lymph nodes was then determined 2 hr after cell transfer. Virusinduced changes at this time must reflect alteration in the rate of migration of donor lymphocytes into the nodes. In other experiments recipients of labeled lymphocytes were killed 1 day after viral challenge so as to determine the distribution of the donor cells when the rats were markedly lymphocytopenic (1). Finally, change> in the distribution of radioactivity in NDV-treated and control rats during thy PC hr after lymphocyte transfer were studied. 2-Hr Recovery of Radioactivity in Lymph Nodes of Recipients of 5JCr-Labeled Thoracic Duct Lymphocytes; Effect of Varying the Time and Dose of -VDl’ Challenge I’revious experiments showed that 24 hr after intravenous inoculation of lo”.” EID,, NDV rats were markedly lymphocytopenic (1). In the current study injection of this dose of virus immediately after labeled lymphocytes significantly reduced the 2 hr recovery of radioactivity in lymph nodes. This effect was also observed when NDV was injected 4 hr before labeled lymphocytes. In contrast. when NDV was injected 24 hr before cell transfer the homing of donor cells into the nodes was normal (Fig. 1). The effect of NDV on donor lymphocytes was dependent on the dose of virus injected. The results of several experiments in which lymphocytes and virus were transfused sequentially (i.e., virus injected immediately after cells) are summarized in Fig. 2. The lymph node concentration of radioactivity was reduced about 70% after chaIlenge with 10*“.3, 50% with 10s.” and 25% with lO”.j EID,, NDV. No reduction in radioactivity was seen after the injection of 109.0 EIl],,, NDV. Distribution of Radioactivity Hr after NDV Challenge
in Recipients
of
51Cr-Labeled Lymphocytes
18-22
The lymphocytopenia found one day after NDV challenge is associated with a deficit of lymphocytes in the lymph and thymus-dependent areas of lymphoid tissues (1). It was thought that by studying the distribution of labeled cells in such animals it might be possible to determine if lymphocytes, unable to enter lymph nodes, preferentially accumulate in any particular site. For this purpose the following experiments were performed. Rats were injected with (i) virus and labeled lymphocytes sequentially, (ii) virus 4 hr before labeled lymphocytes. and (iii) virus 24 hr after labeled lymphocytes. All recipients were killed 18-22 hr after NDV challenge.
310
WOODRUFF
AND
WOODRUFF
120 % z 5 Fq Yg PIsg b&L o.
100
60
60
TIME OF VIRUS CHALLENGE
24-HI?--4 CELL TAANSFER
FIG. 1. Concentration of radioactivity in lymph nodes of virus-treated rats as percentage of control values 2 hr after transfer of 15 X 10s sCr-labeled TDL. Recovery of radioactivity in lymph nodes of control (saline-treated) rats expressed as 100%. NDV (loss EID,,) or saline was injected intravenously 24 or 4 hr before or 5 min after cell transfer. Each point represents mean values of at least four virus-treated and four control recipients. Two-hour recovery was significantly depressed (P < .Ol) when virus given 4 hr before or 5 min after cells.
Virus and labeled lymphocytes injected sequentially. These recipients showed a marked reduction in the concentration of radioactivity in the lymph nodes (Table 1). However, the recovery of radioactivity in the spleens of these rats was normal. These changes were not associated with significantly increased recovery of label in the liver which occurs when donor lymphocytes are killed in vitro by heat or in vivo by anti-lymphocyte serum (6, 7). Also there was no significant difference
rl -
I
,olo.3
IOa8
lOa
DOSE OF NDV INOCULATED .OS p’ co1 <.o I
lOa (ElDso) N.S.
FIG. 2. Two-hour concentration of radioactivity in lymph nodes of rats transfused sequentially with 10 X 10s Wr-labeled lymphocytes and the indicated dose of NDV or saline. Recovery of radioactivity in lymph nodes of control animals expressed as 100%. Each bar represents mean recovery in three to four virus-treated recipients compared to values in three to four control animals.
VIRAL
ALTERATION
OF T LYMPHOCYTE
9.5
48 < .Ol
70 of control P
.8>P>
311
CIRCULATION
90
109
..i>P>
.i
.3
..i>P>
A
“ Itats sacrificed 18 hr after receiving 18 X lo6 Iynphoc>-tes containing 14,635 cpm. immediately after cell3 b Saline (control) or 10”’ 3 EID 50 r\TDV injected intravenously ( XIean \.alrles with rauges in pxmtheses: four ;inirnals ill e:lch gro~~p.
in the recovery of radioactivity virus-treatd animals.
in the lungs, kidney, and thymus of control and
f’hs hjcctrd 4 Izr lwfore labeled lynzplzocytes. In these virus-treated recipients the reduced recovery of radioactivity in lymph nodes was balanced by an increased recovery in the spleen (Table 2). The accumulation of radioactivity in the liver and lungs of the virus-treated animals was normal. Virus injected 24 hr after labeled lyruzphocytes. When labeled lymphocytes are injected rapidly into the bloodstream, as in the current experiments, a substantial proportion of the donor cells are found initially in the lungs and spleen (6). It was possible that this early distribution of transfused lymphocytes was the primary factor which enabled the virus to alter the homing of lymphocytes into the lymph nodes. Therefore, in several experiments recipients were injected with labeled lymphocytes and 24 hr later challenged with NDV. One day later they were killed. The results depicted in Table 3 show that even under these conditions NDV interfered with the recovery of radioactivity in the nodes.
l‘reatnlent rats fd
I
Control
2611
N I)\ V.
of
(2378-2791)
Spleen (cpm/organJ c 1429 (1313-1497) 2645
(2458-3102)
control
66
185
P
<.Ol
<.Ol
I .iver ~CpIn/orgau)
I ,unl: Icpm/organ)
1456 (1199~-16551
266 (o-600)
1034
230 (109.354i
(669-1252) 51
.1>1’>.1
87
.Y>P>.8
“ Rats sacrificed 24 hr after injection of 14 X 1W lymphocytes contaillillg 9836 cpm. b 4 kIr before cell transfer rats injected intravenously with saline ic‘ontrol) or with 10” k El ID,,, N rDv. c Mean values with ranges in parentheses; four animals in each grc~up.
312
WOODRUFF
AND
TABLE
WOODRUFF
3
DISTRIBUTION OF RADIOACTIVITY IN RATS INJECTED WITH NDV 24 HR AFTER TRANSFER OF 51Cr-LABELED LYMPHOCYTES~ Treatment of rats b
Lymph nodes h&d
Control
2639 (2490-2746)
NDV
1998 (1779-2367) % of control
P
75
< .Ol
c
Spleen (cpmbrgan)
Liver (cpmbrgan)
672 (573-757)
365 (211-625)
790 (674-867)
3.50 (113-640)
117
.3>P>
95
.2
0.9
o Radioactivity in organs 48 hr after injection of 10 X lo6 lymphocytes containing 6100 cpm. b 24 Hr after cell transfer rats injected intravenously with saline (control) or with log.* EIDs~ NDV. c Mean values with ranges in parentheses; four animals in each group.
The Distribution of 6’Cr-Labeled TDL in Control and Virus-Treated Intervals of 2-96 Hr After Cell Transfer
Animals a?
The reduced recovery of donor lymphocytes in lymph nodes of virus-treated rats might reflect destruction of transfused cells since dead lymphocytes do not “home” to these tissues (6). Alternatively, some donor lymphocytes might be viable but initially be unable to migrate into the nodes. To distinguish between these two possibilities several experiments were carried out in which the fate of transfused lymphocytes was determined during the 96-hr period after rats were injected with (a) virus and lymphocytes sequentially and (b) virus 4 hr before lymphocytes. Virus and labeled lymphocytes injected sequentially. In the experiment shown in Table 4 each recipient was injected with an aliquot of the same labeled cell suspension and then injected with 10Q.8 EID,, NDV or saline. Although the recovery of radioactivity in lymph nodes was reduced at 2 and 24 hr after cell transfer, by 96 hr the mean concentrations of radioactivity in the nodes of virustreated and control recipients were virtually identical. In contrast, virus-treated animals showed increased recovery of label in the spleen at 2 and 24 hr but not at 96 hr. It was also found that there was less radioactivity than normal in the livers of NDV-treated rats at 24 and 96 hr. In other experiments (Fig. 3) rats were challenged with a larger dose of NDV ( 1O1o*3EID,,). This virus dose reduced even further the recovery of radioactivity in lymph nodes of animals sacrificed within 24 hr of cell transfer. Nonetheless at 48 hr the recovery of radioactivity in the lymph nodes of such rats approximated that detected in control animals. In these animals virus challenge had little if any effect on the accumulation of radioactivity in the spleen, liver or lungs. Virus injected 4 hr before labeled lymphocytes. As shown in Fig. 4 the recovery of radioactivity in lymph nodes of these virus-treated rats was markedly depressed at 2 and 24 hr but not at 96 hr. At each interval studied more radioactivity was found in the spleens of virus-treated rats than in controls. This difference was
VIRAL
I
ALTERATION
Control c \Yru> r’
Control \‘irur
LYMPHOCYTE
57 <.Ol 5784 (5473-5932) 4189 (4086-4387 j
% of control P Oh
T
1660 (1605-1769) 956 (907-1044) % of control P
24
OF
Control Viru.
72 <.Ol 3047 (2874-3355) 3046 (2838-3408)
% or control P
100 >.9
31 3
CIRCIIL.\‘l’lON
c 3339 (2960-36463 4265 r40W 443.3
1016 (992-10.581 016 (744 -I_‘171
118 . 05 > P > .02 1755 (1684-18191 2297 1221lL2397i
90 .7>P>
1106 (10-I-6-1244, 765 i6.32-950 I
I.31 .05>P> .O.! 1031 (969-1096) 1240 11103-14471 I20 .3>P>
.6
69 < .Ol 1018 1961L11741 645 (516-730)
.2
” 14 X 10” lymphocytes containing 11,276 cpm injected intravenously immediately I*Time after cell transfer. e Saline-injected intravenously. CllOQ.* EID,, NDV intravenousl>~. Meal1 valtleh with ranges in parentheseh; four animals ill each group.
6.3 <.Ol before S I )I’.
most pronounced at 24 hr. Although the accumulation of label in the lungs 2 hr after cell transfer was increased in virus-treated rats, at later intervals recovery in this organ was normal. In contrast, recovery of radioactivity in the livers of virus-treated animals was normal except on day 4 when it was slightly reduced. DISCUSSION
NDV inoculated into rats immediately after transfer (Jf “lCr-lal~elecl syngeneic TDL altered the fate of the donor cells. Significantly less radioactivity was recovered in lymph nodes of virus-treated animals 2 and 24 hr after cell transfer. However, by 48 hr recovery of radioactivity in the lymph nodes of these animals had increased and approximated that detected in control animals. The simplest explanation of these results is that in NDV-challenged rats a significant proportion of donor lymphocytes, initially excluded from lymph nodes, later migrated into these tissues. It is not known to what extent, if any, radioactivity in the nodes of virus-treated animals represented labeled progeny of donor lymphocytes. These results support the idea that NDV reduces the size of the circulating T-lymphocyte pool by interfering with the normal pattern of lymphocyte nrigration rather than by destroying the cells. The recovery of radioactivity in lymph nodes was inversely related to the dose of virus injected. This effect was observed in two separate inbred strains of rats
314
WOODRUFF
AND
WOODRUFF
LUNG
SPLEEN
I.
15 IO 5 0
2 92
50-
LYMPH
LIVER
NODES
(L $40 i
30-
3 30 z
205 E IO !$ 20 f-y % 0 2
IO 16
49
72
96
HOURS
AFTER
0'
2
CELL
so 16
46
72
96
TRANSFER
FIG. 3. Recovery of radioactivity in lymph nodes, spleen, liver and lung of control and NDV-treated rats 2-96 hr after injection of 10 X 10s Wr-labeled TDL. A-A 1010.3EID,, NDV or l 0 saline injected immediately after cells. For lymph nodes the values represent percentage of recovery per gram of lymph node; for spleen, liver and lung, percent per whole organ. Each point represents mean value of four recipients. Lymph node values were significantly different at 2 and 16 hr (P < .Ol) only. There was no significant difference in recovery in other organs of virus-treated and control animals.
and was also detected when virus was injected 4 hr before or 24 hr after cell transfer. In contrast, NDV challenge did not impair the homing of transfused lymphocytes into the spleen. However, it is not known if the injected lymphocytes accumulated in the periarteriolar lymphoid sheaths of the spleen as in normal recipients. The virus also caused very little change in recovery of radioactivity in the lungs and liver. The impaired accumulation of label in lymph nodes was in some experiments balanced by an increased recovery in the spleen (Table 4, Fig. 4). This suggests that the lymphocyte population reaching the spleen may contain cells initially excluded from the nodes which later migrate into the nodes. In other experiments, however, particularly when virus was injected after labeled cells there was no evidence that donor cells, unable to enter lymph nodes, were diverted to any single site (Fig. 3). Since the homing of T lymphocytes into lymph nodes depends on their selective affinity for endothelial cells lining postcapillary venules (Z), NDV may achieve its effect by altering the donor lymphocytes and/or the endothelial cells. The present results indicate that the virus does not act on the endothelial cells. Twenty-four hours after viral challenge rats show marked lymphocytopenia and cellular depletion in the paracortical areas of lymph nodes ( 1). Nonetheless, when labeled lymphocytes were injected 24 hr after NDV, the cells homed normally into the lymph nodes (Fig. 1). Thus the deficit of T-lymphocytes in the circulation and in lymphoid
VIRAL
40
OF T LYMPHOCYTE
ALTERATION
CIRCULATION
315
LUNG
SPLEEN
i.2 p.
s
LYMPH
NODES
HOURS
AFTER
CELL
TRANSFER
FIG. 4. Recovery of radioactivity in lymph nodes, spleen, liver and lung of control and NDV-treated rats 2-96 hr after transfer of 20 X 106 51Cr-labeled TDL. A--A 109,s EID,, NDV or l -0 saline injected 4 hr before cells. For lymph nodes, the values represent percentage of recovery per gram of lymph node; for spleen, liver and lung percent per whole organ. Each point represents mean value of four recipients. The lymph node values were significantly different at 2 and 24 hr only (P < .05 and < .Ol, respectively), the spleen values at 24 and 96 hr (P < .Ol), the liver values at 96 hr (P < .Ol) and the lung values at 2 hr I’P <.OS).
tissues of ND\’ challenged rats cannot be attributed tcJ abnormalities of these endothelial cells. The mechanism(s) by which NDV challenge alters the fate of T-lymphocytes is not known. The virus may act directly on the lymphocytes since these cells have receptors for NDV. These receptors apparently terminate in sialic acid which is released from the cells when virus ekes in vitro (8 j Thus viral modification of lymphocyte surface structures (with or without infection of the cells) may l)ia!- a critical role in altering the pattern of lymphocyte migration. This suggestion is consistent with the evidence from previous experiments that sialic acid Containing structures on the lymphocyte surface influence their distribution in the I)ody (6). Moreover, recent studies have shown that the fate of tlonot- TDTbriefly incubated in vitro with NDV 2 is the same as that observed when lymphocyte> are incubated with neuraminidase (T’. clrolcvu) (6). When donor TDL XIX altered by NDV or neuraminidase, each cell population appears to regain the abilit! to migrate normally by 48 hr after injection. This recovery might depend on rexenc-ration of lvmphocyte surface components. Alternat&ely the virus may act indirectly on lymphocytes. The most apparent putative mediators of the viral effect are adrenal steroids, antigenic stimulation, ? Woodruff, J. J. and Woodruff, J. F. Virus-Induced Alterations The effect of Newcastle disease virus on the fate of radiolabeled Manuscript in preparation.
of Lymphoid Tissues IV. thoracic duct lymphocytes.
316
WOODRUFF
AND
WOODRUFF
phagocytosis of the virus, or endotoxin. Previous experiments have shown, though, that adrenal glands are not required for the action of NDV on lymphocytes (1). Antigenic stimulation influences the distribution of transfused lymphocytes (9) but not when antigen is injected immediately after lymphocytes as in the current experiments. Since NDV is rapidly removed from the circulation by macrophages, lymphocytopenia could be mediated by virus-macrophage interaction. Yet, treatment of NDV with specific antiserum, which enhances phagocytosis of the virus in &~o (10) diminishes its lymphocytopenic effect (1). Similarily, the failure of NDV challenge 24 hr before injection of labeled lymphocytes to alter lymphocyte homing suggests that phagocytosis did not play a significant role in this process (Fig. 1). While both endotoxin ( 11) and NDV rapidy produce lymphocytopenia and granulocytosis in mice ulfraviolet light irradiated NDV causes lymphocytopenia but not granulocytosis (1) . Viral alteration of lymphocyte physiology could be immunosuppressive since the induction of an immune response appears to require migration of circulating lymphocytes into the antigen-stimulated lymph node or spleen (12-14). Recent experiments have shown that the survival of skin allografts across a major histocompatibility locus is prolonged by approximately 2 days in NDV-treated mice (unpublished observation). A similar mechanism may account for the immune suppression caused by measles and lactic dehydrogenase (LDH) viruses as well as by oncogenic viruses (15-18). Possibly, enhancement of humoral immunity as observed with LDH and Venezuelan equine encephalitis viruses (19, 20) might also reflect impaired T-lymphocyte function since there is evidence that T-lymphocytes exert, by feedback inhibition, some control over antibody production (21). ACKNOWLEDGMENTS We thank Mrs. Karen technical assistance.
Lefkowitz,
Mrs.
Lynn
Palmer,
and Mr.
Santo Scribani
for excellent
REFERENCES 1. 2. 3. 4. 5. 6. 7. S. 9. 10. 11. 12. 13. 14. 15. 16.
Woodruff, J. F., and Woodruff, J. J., Cell. Immunol. 1, 333, 1970. Gowans, J. L., and Knight, E. J., Proc. Roy. Sot. Biol. Sci. 159,257, 1964. Waksman, B. H., Arnason, B. G., and Jankovic, B. D., J. Exp. Med. 116, 187, 1962. Parrott, D. M. V., desousa, M. A. B., and East, J., J. Exp. Med. 123, 191, 1966. Miller, J. F. A. P., Mitchell, G. F., and Weiss, N. S., Nature (London) 214, 992, 1967. Woodruff, J. J., and Gesner, B. M., J. Exp. Med. 129, 555, 1969. Taub, R. N., and Lance, E. M., Immunology 15, 633, 1968. Woodruff, J. F., and Woodruff, J. J., Cell. Immunol. this issue. Zatz, M. M., and Lance, E. M., J. Exp. Med. 134, 224, 1971. Brunner, K. T., Hurez, D., McCluskey, R. T., and Benacerraf, B., J. ImmunoZ. 65, 99, 1960. Patterson, L. T., Harper, J. M., and Higginbotham, R. D., J. Bacterial. 95, 1375, 1968. Hall, J. G., and Morris, B., Lancet 1, 1077, 1964. Ford, W. L., and Gowans, J. L., Proc. Roy. Sot. Biol. 166, 244, 1967. Ford, W. L., Immunology 15, 609, 1968. Starr, S., and Berkovich, S., N. Engl. J. Med. 270, 386, 1%4. Howard, R. J., Mergenhagen, S. E., Notkins, A. L., and Dougherty, S. F., Transplant. Proc. 1, 586, 1969. 17. Notkins, A. L., Mergenhagen, S. E., and Howard, R. J., Ann. Rev. Microbial. 24, 525, 1970. 18. Snodgrass, M. J., Lowery, D. S.. and Hanna, M. G., Jr., J. Iwwnw~ol. 106, 877, 1972.
VIKAL
10. Notkins.
I\.
L., Mergenhagen,
J. Exj. Med. 20. Howard,
ALTERATION
126,
OF T LYMPHOCYTE
S. E., Rizzo,
.A. A.,
CIRCULATION
Sdrecle.
3I7
c.. ant1 \\‘aldmann.
‘I.
.\
347, 1966.
R. J., Craig,
C. P., Trevinu,
G. S., Dougherty,
S. I;., and Merpenhagen.
S. 1,.
J. Immunol. 103, 699, 1969. 21. Baker, 105,
P. J., Stashak, 1970.
1581,
P. W., Amsbaugh,
D. F., Prescott,
E.. and Barth,
R. F.. J. Immrnol