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CONF-117-1
CONCEING THE CAPACITY OF LYIPHOCYTES TO GIVE KISE TO ERYTHROPOIESIS. * Joan
:right Goodman, Biology Division, Oak Ridge National Laboratory, Oak Ridge,
Tennessee, 37831, U.S.A.
ak Ridge: MASTER
The hcavily irradiated mouse has provided an excellent medium for the
study ci differentiation and proliferation of transplanted hemopoietic cells.
In this apparently ideal environment, not only transplanted bone marrow, but
her.cpoictic stem cells found among peripheral blood leukocytes and peritoneal
fluid cells are stimulated to differentiate into all mature formed elements
normally found in blood and lymphatic tissues. Recent data on lymphocyte
transiormation, as well as attempts to correlate "marrow lymphocyte" numbers
with hematopoietic potential, have stimulated interest in the small lymphocyte
as a pluripotential stem cell. Heavily irradiated mice were transfused with
large numbers of lymph-node lymphocytes, and histological and 59fe-uptake data
were collected during the second week after transplantation to determine the
capacity of lymphocytes to support red cell formation. Good regeneration of
lymphatic tissues, including many plasma cells and germinal centers, was seen
in lymphocyte-transfused mice but not in radiation controls. Marrow, spleen
red pulp, and thymus were depleted of cells in both experimental and control
groups. Only an occasional mouse from these experiments showed small areas
of erythropoiesis or moderate thymus regeneration. 595e-uptake data from
lymphocyte-treated mice substartiated the histological finding that lymphocytes
have no significant capacity to give rise to erythropoiesis.
* Research sponsored 'oy the U. S. Atomic Energy Commission under contract with
the Union Carbide Corporation.
Abstract for C. N. R. S. Discussions on the Transplantation of Allogenic
Hematopoietic Cells, September 7-9, 1964, Paris, France.
--LEOAL NOTICE -

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CONCERNING THE CAPACITY OF LYMPHOCYTES TO GIVE RISE TO ERYTHROPOIESIS
Joan Wright Goodman
Biology Division, Oak Ridge, National Laboratory, Oak Ridge, Tennessee
contenimientri mintimitatom
Research sponsored by the U. S. Atomic Energy Commission under contract with the
Union Carbide Corporation.
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Now that the lymphocyte is in vogue and its ability to transform
1.8 doubted only by a few intransigients, it might be in order to investigate
the capacity of this cell type to differentiate. There is little doubt
that cells taken from lymph nodes, presumably lymphocytes, can take part
in nonograft reactions. In the transplantation field, for example, it is'
well known that isologous cells given to irradiated recipients will
prevent successful homologous marrow takes. Thoracic duct cells, again
presumably lymphocytes, have the same ability (Gesner and Gowans, 1962),
as have blood leukocytes (Goodman, 1962). Nobody would dispute the ability
of lymph node cells to give rise to antibody formation (
although the exact role played by the lymphocyte in the many complex changes
taking place in lymphatic tissue after antigenic stimulation is far from
well defined. There is, however, considerable disagreement as to whether
lymphocytes are pluripotential stem cells and can give rise to granulocytes,
erythrocytes, and megakaryocytes. This disagreement actually goes back to
the description of small, darkly-staining cells found in marrow, called
variously "small round cells" (Schooley and Giger, 19), "marrow-lymphocytes"
(Everett, 19 ), "lymphocyte-like cells" (Cudkowicz, 19 ),
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"small lymphocytes" (Yoffee, 19 ), and undoubtedly other things. Even if
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these small, round, darkly-staining cells could be shown to be morphologically
ädentical with small lymphocytes found in lymphatic tissues and peripheral
circulation, the task would still remain of showing that they are truly
,
pluripotential, hemopoietic stem cells.
a down
The following experiments were done to test the ability of lymphocytes
to give rise specifically to red cells in the mouse. Lymph node cell
suspensions, predominantly small and medium lymphocytes, were administered
intravenously to heavily irradiated recipient mice, and a week or so later
24-hour Fe% uptake by their spleens and red cells was measured. It was
thought that these conditions vould provide an environment optimal for the
transfused Lymphocytes to differentiate, if it were possible, into red cells.
METHODS
Lymphocyte donor and irradiated recipient mice of the following types
were used: (C57L/Cum 4 X AVHECum )F,, hereafter called LAF, ;
: (C57BL/6. 4 X DBA/2 . )Fq, or B6D2F2; (C57BL/Cum 4 X C3H/Anf Cum . Ey, or
wired
BC3F; and the inbred strain C57BL/10.D2, or B10.02. Donors and recipients
:
:
were always of the same sex and differed in age by no inore than 4 weeks,
irradiated recipients, from 12-16 weeks old, being the younger in each
case.
Mice were irradiated in a revolving lucite cage that held up to
12 animals at a time. F. hybrids were given a single, whole body exposure of
950 r, B10.D2's 900 r. Physical parameters for the x-fadiation were
250 Kv, 15 ma, 1 mm Al added filtration, HVL of 0.5 mm Cu, and a dose rate
in air of approximately 160 r/min.
Lymph nodes were resected from decapitated donors and were collected
in a small amount of cold physiological saline. The nodes regularly used
included cervical, brachial, inguinal, and mesenteric. Other, smaller nodes
were occasionally taken. Cell suspensions were prepared by pressing lymph
nodes through a stainless steel screen with a glass rod flattened at the
end. Cells were further dispersed by passage through a 24-gauge needle
into the syringe from which injections were to be made. Sometimes cell
suspensions were gently spun down in a clinical centrifuge, debris and fat
were removed along with the supernatant, and cells were resuspended in
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saline. On other occasions suspensions were injected unwashed. In all
cases cell counts were made in a hemocytometer before dilution for injection.
Intravenous injections of lymph node cell juwpensions frequently
produced a shock-like state in recipients, presumably from pulmonary
embolism, and sometimes death ensued. To improve chances for survival, the
intended dose for one day was administered in two separate inocula
administered 1/2 to 2 hours apart. In all experiments reported here,
cells were injected on the same day (2-6 hours after) recipients were
irradiated, and in some cases an additional cell dose was injected on the
following day.
Mice were checked daily for mortality, and Fe> was injected as many
days after irradiation as it was thought possible to obtain results, before
death occurred, from a particular group of recipients. Details of
preparation and injection of Fe” were the same as those described previously
(Goodman and Hodgson, 1962). Mice were analyzed by standard methods for
EPP
Fes uptake by spleen and red cells 24 hours after Fe? injection.
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Tissues taken from experimental and control mice at autopsy were
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were stained with hematoxylin and eosin.
RESULTS
Table 1 outlines the five experiments to be presented and shows mortality
results. In experiment A, all mive died, and no Fe-uptake data were
,
obtained. In the other experiments, however, at least some mice that
survived 244 hours after Fe'injection were killed for spleen counts and/or
histological study.
In no case was survival recorded beyond the second
week after X rays and cellular treatment.
At autopsy the major difference noted between X-ray controls and
lymphocyte-transfused mice was that lymph nodes were larger in those that
had received cells. Spleens were small in all cases, and nodules were
not seen.
Microscopically, lymph nodes were dramatically different, transfused
mice showing extensive regeneration with plasma cell formation and active
germinal centers (Fig. la), whereas untreated controls presented the
usual picture of almost complete emptiness (Fig. 20). This lymphatic
regeneration in lymphocyte recipients was also quite evident in spleen white
pulp and 18 1llustrated in Figure 2. Red pulp in these recipients was
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usually bare (Fig. 2a) and did *** differ from that of controls (Fig. 26).
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However, foci of erythropoiesis and a few megakaryocytes were occasionally
seen in spleen and marrow sections, notably in mice from experiment ------
(see Table 1). In general, marrow spaces, of lymphocyte-transfused mice
were as empty as those of controls (Figure 3).
Iron”
uptake data are shown in Table 2, and for the sake of
comparison, peritoneal cell and bone marrow transplantation data taken
from published work have been included. It can be seen that Fes uptake
values for lymphocyte-transfused mice are higher than for controls in
experiment C, and slightly higher in experiment D, but no real differences
between control and transfused animals are seen in the other two. The
individual C3H control, taken from a separate experiment, shows a spleen
Fe” uptake similar to that of lymphocyte-transfused mice of experiment C
and an erythrocyte Fe% uptake actually higher. However, when any of these
values are compared with those of recipients of relatively low doses of
bone marrow or of comparable doses of peritoneal fluid cells, it
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is immediately evident that they are indeed very low, and erythropoiesis
almost negligible.
DISCUSSION AND CONCLUSION
Lymphocyte transplantation in the present experiments was not able
to promote survival of heavily irradiated isologous recipient mice. Similar
findings and related ones for thoracic duct cells and thymocytes have been
reported by others (Micklem and Ford, 1960; Gesner and Gowans, 1962).
Regeneration of lymph nodes and spleen of white pulp of recipients was
IS
.
well advanced by the second week after lymphocyte administration. That
the transfused cells bad not simply settled out in lymphatic tissue but were
proliferating there was evident from the presence of mitotic figures, many
plasma cells, and active germinal centers.
Bone marrow and spleen red pulp, on the other hand, were as empty in
transfused mo.ce as in untreated controls. There were a few exceptions to
this general statement in which foci of red cell formation and a few
megakaryocytes could be found. Erythropoiesis in these few mice waso
confirmed by Fes uptake values which were greater than those of comparable
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No red cell counts of the lymph node cell inocula were made, so that it
is impossible to estimate the amount of peripheral blood leukocyte
contamination that may have been present.
One cannot discount the
possibility that pluripotential stem cells recently delivered to the
nodes from the blood, but not yet committed to lymphopoiesis, could
give rise to the small amount of erythropoiesis seen in the present
experiments.
.
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controls. However, an occasional untreated, control mouse, as illustrated
in Table 2, can show erythropoiesis in the same 7e59-uptake range. This
suggests the possibility that "tiding over" nay - ave been promoted by
lymphocytes in experiment C, so that host cells could have begun to
recover and to proliferate. Not enough data are at hand to speculate
further on this. When one compares the highest Fe59-uptake value obtained
from a lymphocyte-treated mouse with values from peritoneal fluid cell
or bone marrow cell transplantation, it is clear that lymphocytes have
very Uttle, if any, capacity to give rise to erythropoiesis.
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TABLE 1
NUMBERS OF MACE DYING ON SPECIFIC DAYS AFTER X IRRADIATION
POLLOWED BY INTRAVENOUS INJECTION OF ISOLOGOUS LYMPH NODE CELLS
Days after irradiation
:
Sex
and
strain
Millions of
lympha
node cells
Numbers of
m ice alive
after injection
Expt.
6* 7
8
9
10
11
12
13
A
B10.De ene
B20.D2
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80
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4
2
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100+
212**
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200+
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125
*No mice died before this time.
* tice killed for Pe 59-uptake of histological studies.
Divided into 2 doses (4 injections) administered an days 0 and 1.
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Table 2. 70% uptake by spleens and erythrocytes of irradiated, isologous-lymphocyte.
transtused mice.

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Percentage of injected po59 dose* found in:
Lymphocyte No.
(millions)
Days after X-rays
BAJS 6504
spleen
erythrocytes
Expt.**
950 r mice
Ave. value
range
Ave. value
range

LAP, H
50
0.040-0.080
0.110-0.190
0.556-1.544
0.004-0.110
B6D2F4 na 100
100
11
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..........0.060(3)..
0.157(3)
0.500-2.300
1.249(4)
0.130-0.400 0.041(3)
0.239-0.722 0.180(3)
0.289-0.482 0.008(2)
0.004-0.466 0.004(5)
0.093-0.613 0.000(3)
BC3F, son
$1.267(3)
| 0.263(3)
0.459(3)
0.386(2)
10.165(714
? 0.250(4)
200
0.030-0.410
0.007-0.008
0.000-0.018
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BODEF, 44
225
7
Isolated Irradiation controlla
C3H$
8
0.593(1)
2.680(1)
Isologous bone marrow transplantation
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LAF Mod
BCF, pod
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29.969)
15.3(11)
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Peritoneal fluid cell transplantation"
DBA/2 nd 40. 7
12.73(2)
BLO.D2 46 125. 9
30.47(5)
*0.5 uc Fe" as citrate intravenously in 0.25 ml buffered sodium citrate.
**See table 1 for other details.
Numbers of mice represented in avera, in parentheses.
From a peritoneal fluid cell experiment unrelated to others presented here.
3
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"Taken from J. W. Goodman: Transplantation 1: 334-346, 1963.
LI
FIGURE LEGENDS
Figure 1. Lymph node sections from
B6D2F, females 8 days after 950 r.
a. recipient of 125 x 10° 1sologous lymph node cells. X 125.
b. untreated control. . x 150.
Pigure 2. Spleen sections from B6D2F, females 8 days after 950 r.
'
&. recipient of 125 X 200 180logous lymph node cells. X 125.
b. untreated control. X 150.
Figure 3. Sternal bone marrow sections from B6D2F, females 8 days after
950 r.
8. recipient of 125 x 10° isologous lymph node cells. X 125.
b. untreated control. X 150.
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DATE FILMED
12/ 1 /64
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