A critical analysis of bone marrow-spleen cell interaction in the immune response
by Donna Gail Sieckmann
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements of the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Donna Gail Sieckmann (1970)
Abstract:
The spleen-bone marrow cell interaction phenomenon was studied in three strains of mice, using
various experimental parameters. Lethally irradiated mice were reconstituted with either spleen,
thymus, bone marrow, or combinations of these cell suspensions and immunized with sheep
erythrocytes or bovine gamma globulin. The immune response was measured by Jerne plaque spleen
assay or immune elimination.
The immune response in spleen or bone marrow reconstituted animals was dependent upon two cell
interactions taking place. However, low doses of spleen and bone marrow alone were not able to
immediately reconstitute an animal's immune potential.
Spleen dilutions from normal SPF mice could not interact synergistically with bone marrow, but could
interact with normal thymus cells. Spleen dilutions from immune mice or older conventionalized mice
were able to interact synergistically with bone marrow. The appearance of the synergistic cell in the
immunized spleen followed that of the memory cell population. It was concluded that the size of the
antigen reactive cell population of the spleen could be.- altered by immunization, exposure to
cross-reacting antigens and/or aging of the animal. In presenting this thesis in partial fulfillment of the
requirements for an advanced degree at Montana State University,
I agree that the Library shall make it freely available for inspection.
I further agree that permission for extensive copying of this thesis
for scholarly purposes may he granted by my major professor, or, in
his absence, by the Director of Libraries=
It is understood that
any copying or publication of this thesis for financial gain shall
not be allowed.without my written permission=
Date
A CRITICAL ANALYSIS OF
BONE MARROW-SPLEEN CELL INTERACTION
IN THE IMMUNE RESPONSE
by
DONNA GAIL S BECKMANN
A thesis submitted to the Graduate Faculty in partial
fulfillment of the requirements of the degree
of
MASTER OF SCIENCE
in
Microbiology
Approved;
Head, Major Department
Chairman, Examining Committee
Graduate/ Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
August, 1970
iii
ACKNOWLEDGEMENT
I wish to express my sincere appreciation to my advisor, Dr.
Norman D= Reed, for his inspiration and guidance, and for generously
giving of his time and talents during the course of my studies.
I would also like to thank the faculty of the Department of
Microbiology at the University of Nebraska and the Department of
Botany and Microbiology, Montana State University, for their
cooperation.
I wish also to express my gratitude to Drs. E. 0= Jones,
University of Nebraska Medical Center; John McGreer Jr., Lincoln
General Hospital; and L. Brewton and L= Hammer, St. James Hospital,
for the irradiation of experimental animals.
Financial support for the author and research materials were
provided by NIH Grants 7 ROI AI09862 and 7 EOI AI09859 and by NIH
Training Grant.No.
AI131-09.
TABLE OF CONTENTS
Page
VITA c
o
o
e
o
o
c
ACKNOWLEDGEMENT' c O
e
C
o
O
TABLE OF CONTENTS,
i
Q
LIST OF TABLES.
e
e
O
o
e
C
o
o
O
o
e
O
o
O
e
e
O
o
e
G
e
e
o
O
O
O
O
o
c
o
e
ii
e
iii
a
o
o
o
o
o
o
e
O
o
iv
o
e
®
vii
o
o
F
®
o
o
o
e
e
e
»
®
e
e
o
o
o
e
LIST OF FIGURES o
o
o
p
o
o
o
o
o
o
e
o
o
e
e
*
»
®
o
viii
ABSTRACT
ix
e
o
INTRODUCTION
e
Q
O
o
o
o
o
o
o
o
o
o
o
o
e
e
o
o
®
®
®
®
®
0
0
0
0
0
9
0
0
0
0
0
«
®
0
0
«
e
»
0
0
o
e
I
MATERIALS AND METHODS. . . . . . . . . . . . . . . .
Mice.
o
»
Antigens.
5
e
5
e e e e o o e e e e o o e e e . ' e e o e o e a o e
5
o
Irradiation
o
o
0
o
0
o
0
o
0
o
0
o
.
0
e
0
e
0
o
0
o
0
o
0
o
0
0
o
o
e
+
0
e
0
e
0
e
0
e
0
o
0
o
^
6
0
Preparation of Reconstituting Cell Suspensions
Reconstitution of Irradiated Mice .
o
Jerne Placpie Assay .
.
.
.
.
.
.
T
.
.
.
©
.
e
.
®
.
»
7
o
.
p
o
o
.
Assay of Immunity to BGG by Immune Eliminations
Preparation of Labled Protein . o © o « o o o
o
o
o
©
©
o
o
o
o
8
e
e
8
10
o
Immune EIimx ns ~bion p o o o o o o ® e o o o e e o
11
Serum TltrstIOns
11
RESULTS O O O B O O O O
e
o
e
o
e
o
o
o
o
»
o
o
e
»
e
0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Irradiation and Hematopoietic Reconstitution o
O
O
0
O
O
13
O
13
V
Page
13
Assay of Immunity to BGG by Immune Elimination:
A0
Immune and Nonimmune Elimination, . . . . . . . . .
13
Bo
Correct Assay Time, 0
0
0
16
Co
Tolerance Induction .
o
o
0
0
o
0
o
*
e
0
e
e
o
e
o
o
e
e
o
19
*
Time of Recovery of Immune Responsiveness After
Irradiation and Reconstitution With Bone Harrow
19
A Dose-Response Curve for Bone Marrow Transplantation
22
A Dose-Response Curve for Spleen Cell Reconstitutions
The Dilution Effect With SRBC Antigen
2k
The Spleen Dilution Effect With BGG Antigen ,
0
Absence of Synergism Between Normal
Spleen and Normal, Bone Marrow in SPE Mice
*
0
0
0
0
p
0
Variation in Experimental Parameters Affecting the
Immune Respqnse in Reconstituted Mice 0 0 0 0 0 0
e
0
e,
Zk
0
0
0
0
28
0
0
0
0
32
Absence of Normal Spleen-Bone. Marrow Synergism in
in C57B1 with BGG Antigen a . , . . . . . . . . . , , . .
36
The Effect of the Immune State of the Donor
Cell Population Ipon Synergism o o o o e o o ,
36
The Time of Appearance of the Synergistic
Cell in the Spleen After Antigenic Stimulation
The Specificity of Primed Spleen Cell-Bone
Marrow Cell Synergism 0 0 0 0 0 0 0 0
Spleen^Thymus Synergism,0
0
0
*
Natural, Hemagglutinins to SRBC
0
0
0
0
0
0
0
0
0
9
0
0
o
0
0
Normal Spleen-Bone Marrow Synergism in 15
Week Old ..Conyentiqnal LAE^/J Mice,.
Op
o
O
o
O
o
O
o
o
O
o
O
O
kl
p
o
kk
0
0
0
0
0
0
0
0
kk
0
0
0
0
0
0
0
0
k?
O Av o< O
0
0
0
0 . 0
vi
Page
DISCUSSION
e
e
o
o
p
o
e
e
o
o
e
e
a
e
t
i
o
e
e
e
e
o
e
o
e
e
e
o
52
S U M M A R Y
o
o
o
o
o
o
o
o
o
o
o
o
o
a
<
i
o
o
o
«
o
»
*
*
9
a
o
«
o
^ 1O
APPENDIX
o
o
.
o
*
o
o
e
e
e
62
e
63
'
LITERATURE CITED
i
o
e
a
e
e
»
o
*
6
e
o
e
e
*
e
e
e
e
e
*
e
o
o
*
e
e
®
e
e
e
®
o
e
e
#
*
»
e
e
e
e
*
vii
LIST OF TABLES
Page
TABLE
I
TABLE
II
TABLE
III
TABLE
TABLE
TABLE
TABLE
IV
V
VI
VII
TABLE
VIII
TABLE
IX
TABLE
TABLE
X
XI
Effect of Irradiation and
Hematopoietic Reconstitution. „ „ „ „ ........
,
14
The Spleen Dilution Effect.:'.
,
26
Absence of Normal Spleen-Bone Marrow
Synergism in C57B1 Mice: .Low Cell Dose . . . . ,
30
Absence of Normal Spleen-Bone Marrow.
Synergism in C57B1 Mice: High Cell Dose. „ . . ■
31
Absence of Normal Spleen-Bone Marrow. .
Synergism in Balb/C Mice. . . . . . . . . . . .
.
33
Absence of Normal Spleen-Bone Marrow
Synergism in LAF_/j Mice. . . . . . .
■
34
The Effect of Delayed Reconstitution on
Subsequent Normal Spleen-Bone Marrow
Interaction, Oi- &r> o * Ofr O*
o> e» e o. ©i oi o*1 o»
^.
35
Effect of Time of Jerne Assay on PFC Response . .
37
Immune Spleen-Bone Marrow Synergism
in C57B1 Mic e *. P.,,.
^
.
4o
Immune Spleen-Bone Marrow
Synergism in LAF^/J Mice9. . . . . . . . . . . .
,
42
The Specificity of Immune Spleen Cell-Bone
Marrow Synergism. . . . . . . . . . . . . . . .
.
45
o« O'i
o
TABLE
. XII
Spleen-Thymus Synergism in LAF^/J Mice. . . . .
TABLE
XIII
Natural SEBC Hemagglutinins in C57B1 Mice . . .
TABLE
XIV
Natural SRBC Hemaglutinins in LAF^/J Mice . . .
TABLE
XV
.
.
.
Normal Spleen-Bone Marrow Synergism in
15 Week Old Conventional LAF^/J Mice. . . . . . .
46
48
49
50
Tiii
LIST OF FIGURES
Page
Figure
Figure
Figure
I
2
3
Immune and nonimmune elimination of
—
■O 6 e ® o < i o e o o o e o o e
#eeeee-e
15
Assay for immune elimination at various
times during the immune response • ' ' . • • • • • • • «
17
Assay for immune elimination at various
times during the immune response
18
20
Figure
4
Tolerance induction in C57B1 adults
Figure
5
Tolerance induction in adult C57B1 which
had been treated with soluble BGG at birth
Figure
Figure
Figure
Figure
6
7
8
9
Figure 10
Figure 11
»««>«-»
21
The apperance of an immune response to SHBC
in irradiated, bone marrow reconstituted
C57B1 miceu-j 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 ® ® ® ® ® ° ° -
23
A dose-response curve for reconstitution
with bone marrow o o ® ® ® ® ® ® ® ® ® ® ® ® ® ® ® ®
25
A dose-response curve for reconstitution
with spleen cells
^7
Anti-BGG response in spleen cell
reconstituted C57B1 mice® ® o » ® o ® o » ® ® ° = » 9
29
Immune elimination in normal spleen and bone
marrow cell reconstituted 057B 1 . « ® » ® ® ® ® « ° °
38
The appearance of the synergistic cell in the
spleen after immunization ® o . ® ® ® ® ® ® ® » « « » ®
^3
ABSTRACT
The spleen-bone marrow cell interaction phenomenon was studied
in three strains of mice, using various experimental parameters„
Lethhlly irradiated mice were reconstituted with either spleen, thymus,
bone marrow, or combinations of these cell suspensions and immunized
with sheep erythrocytes or bovine gamma globulin=, The immune response
was measured by Jerne plaque spleen assay or immune elimination.
The immune response in spleen or bone marrow reconstituted
animals was dependent upon two cell interactions taking place=. However %
low doses of spleen and bone marrow alone were not able to immediately
reconstitute an animal--iS :•immune potential»
Spleen dilutions from normal SPF mice could not interact
synergistieally with bone marrow, but could interact with normal thymus
cellso Spleen dilutions from immune mice or older conventionalized
mice were able to interact synergistieally with bone marrow= The
appearance of the synergistic cell in the'immunized spleen followed
that of the memory cell population= It was concluded that the size of
the antigen reactive cell population of the spleen could be.- altered
by immunization, exposure to cross-reacting antigens and/or aging of
the animal=
INTRODUCTION
The role of lymphoid cells in the development and main­
tenance of an animal's immunelogical competence has been well docu­
mented during the last two decades 0
However, data which reveal the
complexity of the mechanism of antibody formation at the cellular
level have only recently been provided*
There has been evidence
suggesting that cell-cell interactions are involved in the development
of an immune response (l6 , 21, 11, 12)„
Fishman (11) was first to
show that macrophages, which had ingested bacteriophages, produced an
RNA species that could induce neutralizing antibody formation in
nonimmune lymphocytes =
Ford, Gowans, and McCullagh (12) showed that
peritonial macrophages, which had ingested sheep erythrocytes (SRBC),
could induce specific antibody formation by culturing them with
thoracic duct lymphocytes»
In 1966, Clamen, Chaperon, and Triplett (4) presented more
direct evidence for the involvement of two cell types in antibody
production*
They showed that when the immune potential of lethally
irradiated mice was reconstituted with both thymus and bone marrow
cells, the cellular response to SRBC, as measured by foci of anti­
body production in the spleen, was several fold greater than the
response which would have been expected had the reconstituting effect
of thymus and marrow cells been only additive =
The results clearly
I
2
showed that a synefglstic interaction between the two cell populations
had taken place=,
This phenomenon of thymus-bone marrow synergism has
sinpe been confirmed repeatedly with SHBC as antigen (10,21) and also
with protein antigens (3 ,31)=
With the -development of the Mishell-Dutton technique for im­
munization of dissociated spleen cells in vitro (24), it was soon
discovered that three cell types were involved in the in vitro res­
ponse to SEBGs
a glass-adherent cell, now thought to be the
macrophage, and two nonadherent celld (23)°
,
t
The adherent cell was
found to be necessary for an in vitro primary response to SRBCe
How­
ever, this cell is present and functional in the lethally-irradiated
animals used in in vivo studies of cell Interaction 0
The exact role
of the adherent cell is presently being debated=
The functions of the nonadherent cells have been more suc­
cessfully investigatedo
By use of
hybrids and anti-H2 isoantisera,
it has been demonstrated that the bone marrow cell or cells derived
from the marrow produce antibody (8 , 22, 30)»
These are the plaque
forming cells (PFC) of the Jerne hemolysis-in-gel assay for antibodyproducing cells and are also the cells responsible for the different
classes of antibodies produced during the immune response (7 )®
The other nonadherent cell, the thymocyte or a cell derived
from the thymus, has now been termed the antigen reactive cell (ARC)o
3
It has been shown (21, 29) that this cell proliferates after contact
with the antigen and then interacts with antigen and a percursor of
the PFC (P=PFC) to cause differentiation of the P-PFC into the PFC»
By statistical analysis, it has been shown (29) that after the initial
contact with antigen, the AEC undergoes 6 to 10 cell divisions,
producing 80 to 800 progeny ARC, each of which is able"to interact
with approximately 150 PFC»
These figures, which depict a mushrooming
of the PFC population as the result of antigen contact with only one
ARC, help to explain the magnitude of the response obtained after the
synergistic interaction between these two cel] populations«,
Cell-cell interactions can be most profitably studied by use
of thymus and bone marrow, which contain relatively pure populations
of the ARC and P-PFC 9 respectively=
The premium effect, shown by
Celada (2), or the dilution effect, as it is called by Talmage and
co-workers (30), and studies by Hosier and Coppleson (25) indicate
that cell interactions can also take place between two lymphoid cells
in the spleen, namely, a thymus-derived ARC and a bone marrow-derived
P-PFC=
These investigators showed that the response to antigen was
a nonlinear function of the spleen cell dose given to lethallyirradiated anumals or in in vitro culture with antigen=
A 50 fold
increase in the munber of spleen cells given was accompanied by a
2500 fold increase in. the response of the irradiated animal,(26)=
4
Talmage and co-workers furthermore demonstrated that a subtraction
of unprimed spleen gave no response alone, but could give a
synergistic response to SEBC if given with bone marrow (26).
On the other had, Clamen and co-workers (5) did not detect
any enhancement of the immune response of spleen cells by bone
marrow.
Radovich (27) also reports that spleen-bone marrow synergism
does not occur in all strains of mice tested.
In our laboratory, preliminary experiments were run in
attempt to show spleen cell-marrow cell synergism in order that it
might later be applied to the study of immunologic tolerance.
synergistic effect could not be shown.
A
The experiments which
followed, and that are reported below, sought to determine the
cause for the discrepancies between reports in the literature on
spleen-bone marrow cell interactions.
MATERIALS AND METHODS
Miceo
Young adult C57B1 (H-2h ), Balb/C (H-2d ), and LAF^/J
(C57L/J x A/HeJ ) inbred mice, 6 to 12 weeks old, were used in the
experimentso
Specific pathogen free (SPF) C57B1 and Balb/C stock
breeders were obtained from Dr= J» J= Trentin, Baylor University Medi­
cal School, Houston, Texas, in 1968, and have since been maintained
by brother-sister matings under SPF conditions»
LAF^/J mice were
obtained at the age of 6 weeks from Re B 0 Jackson Memorial
Laboratories, Bar Harbor, Maine, and were housed in the SPF animal
quarters upon arrival 0
All cages, watering bottles, and San-i-cell
bedding were autoclaved before use 0
Mice received Wayne Sterilizable
Lab Chow or Autoelavable Puyina Laboratory Chow 5010, which had been
sterilized, and acidified-ehlorinated water (20) ad Iibitum 0 All
mice remained in the SPF animal quarters until removal for experi­
mental use=,
Antigens 0
Sheep (SRBC) and horse (HRBC) blood in Alsever1s
solution were obtained from Colorado Serum C o 0, Denver, Colorado,
and washed three times in sterile O 085$ NaCl (saline) before use in
immunization, serum titrations, or Jerne plaque assay=,
Lyophilised bovine gamma globulin (BGG, Cohn Fraction IV) was
obtained from Immunology, Inc, Glyn Ellyn, Illinois =, The BGG was
dissolved in saline and used for immunization either in an aggregated
form, prepared by heating for 20 min at 70° C, or as an emulsion
6
consisting of equal volumes of Freund’s Complete Adjuvant (FCA, Difco,
Detroit) and BGG solution®
BGG solutions used for isotopic labeling
or to induce tolerance (soluble BGG) were freed of aggregated mole­
cules by centrifuging at 40,000 x G for 30 min in a Spinco Model L
refrigerated ultracentrifuge with an SW25°1 rotor®
BGG solutions were quantitated by optical density analysis at
280 nm in a Beckman DB Spectrophotometer®,
The
^
was found to be
1®040 in 0o03M sodium phosphate buffer, pH 7®0, using a solution
whose concentration was determined by micro-Kjeldahl nitrogen
analysis ®
Irradiation®
Mice were given 900 rad whole body gamma
irradiation using Co-60 Teletherapy units with the following dose
rates:
162 R/min at the University of Nebraska Medical Center,
Omaha, Nebraska, by Dr= E® 0® Jones? 7b R/min at Lincoln General
Hospital, Lincoln, Nebraska, by Dr® John MeGfeer, Jr=? ?2 rad/min at
St® James Hospital, Butte, Montana, by Drs= L= C= Brewton and L=
Hammer®
Twelve mice were held fast in a round perforated plexiglass
cage (H&H Plastics, Lincoln, Nebraska), designed for irradiation
purposes=
This cage was positioned in a 19 x 19 cm irradiation field,
60 cm below the source, oh top of a turntable (Sieckman Const= Co®,
Lincoln, Nebraska)®
In order to achieve uniform irradiation of all
animals, the mice were rotated at 2rpm during irradiation®
Maximum
7
backscatter conditions were produced by placing a five-inch thickness
of masonite under the cage on the turntable<> The mice were held in
the cage only for the duration of the irradiation time, which varied
between 5 and 15 min, depending upon the source used=
In several experiments a Model M Cs-137 Gammator (M34-1-1074,
Radiation Machinery Corporation, Parsippany, Illinois) was used*
The
mice were irradiated individually at a rate of 195R/min«
The type of source used in any particular experiment will be
recorded within the experimental procedure =
Donor and
recipient mi.ee were generally pre-bled, and when possible, were
selected from mice showing no natural SBBC hemagglutinins„
Donqr spleens were removed aseptieally, placed in Hank’s
balanced salt solution (HESS, Grand Island Biological Co=, Grand
Island, No To), teased apart with forceps, and pipetted gently with
a Pasture pipette to produce a single-cell suspension0
large
particles were allowed to settle out and the supernate was brought
up to 15 ml, quantitated with a hemacytometer, centrifuged at 500 x G
in the cold for 10 min, and resuspended in fresh BBSS to the proper
cell concentration=
Bone marrow was obtained from femurs and tibias of donor mice=
Bones were removed and cleaned of tissue=
The marrow was removed by
8
cutting off the epiphyses and injecting HBSS into the marrow cavity
thereby forcing the marrow out of the other end of the bone®
A
single cell suspension was produced by pipetting gently with a Pas­
ture pipette=
Large particles were allowed to settle out and the
supernate was quantitated with a hemacytometer, centrifuged in
the cold at 500 x 6 for 5 min, and resuspended in fresh HBSS to the
proper cell concentration=
Thymus cell suspensions were prepared by mashing the thymus
in HBSS against a fine mesh wire screen with the flat end of a glass
syringe plunger=
Further processing was the same as that for bone
marrow=
Reconstitution of Irradiated Mice=
reconstituted at 5 hrs after irradiation=
Mice were generally
Cell suspensions were
mixed and injected intravenously (IV) in a lateral tail vein=
In
cases of immunization to SRBC or HRBC, the antigen was mixed with
and injected with the cell suspension=
When BGG was used for
immunization, the aggregated form was given intraperitonealiy (IP)
after reconstitution of the mouse =
Experimental mice were main­
tained on sterile feed and acidified-chlorinated water ad libitum=
Mice to be assayed by immune elimination were started on 0=1% KI in
tap water at the time of reconstitution=
Jerne Plaque Assay=
Spleens were assayed for antibody
9
producing cells.by the method of Jerne and Nordin (17)«,
Spleens were
removed aseptically, and placed in cold Eagle’s minimal essential
medium. Hank’s base (MEM, Grand Island Biological C o 0)»
After the
spleen was teased apart with forceps, cell aggregates were further
broken down by gently pipetting with a Pasture pipette *
Coarse
material was allowed to settle out in a centrifuge tube, and the
supernate was removed and plated immediatly thereafter?
Either 0=,1
or O 02 ml of the resulting cell suspension or a I;10 dilution of the
suspension was added to an overlay agar mixture at 47°C«,
The over­
lay was composed of I ml of 1°/o agarose (L 0Industrie Biologique
Frangaise 8 «, A 0), I ml of 2x MEM, and 0=9% NaCl 0
The overlay was
spread in a plastic petri disk, which contained a base agar layer
composed of 10 ml of 1 04$> Ionagar, No= 2 (Oxoid, Colab Laboratories)
in HBSS, to which 500 ug of DEAE-dextran was added per ml of media
after autoclaving='
Both the overlay and the base agar solutions
were filtered after sterilization and adjustment to pH 7 with 7=5%
NaHGCyo
Double distilled water was used for all solutions =
The plates were incubated at 37°C in a humidified 5% C0_-95%
air incubator for 3 hrs=
Development of plaques was accomplished
by addition of 1=5 ml of 1:10 guinea pig ebmplement (Difco, Detroit,
or Colorado Serum Co=, Denver) and incubation at 37°C for I hr=
Plaques were counted under a dissecting microscope at IOOx magni­
10
fication.
Spleen, cell suspensions were quantitated with a hemacyto­
meter=
Assay of Immunity to BGG by Immune Elimination:
of Labled Protein=
The Chloramine T method of McConahey and Dixon
(19) was used to lahle BGG with
Na
131
Preparation
Iodine-131 was obtained as
I 9.biochemical grade 9 in carrier-free form from Mallinckrodt
Nuclear«> Chloramine T 9 200 yug in 0=05 ml sodium phosphate buffer,
pH 7=0 (Eastman Organic Chemicals) was injected into a small
stoppered vaccine bottle containing 200 ^ig soluble BGG in 0,4 ml
buffer and 200 ^zC Na"^"*"I in 0=1 ml buffer=
agitated on crushed ice for 10 min=
The vial was gently
Then 0=05 ml (400 jag) sodium
meta-bisulfite (Fisher Scientific Go=,) was added with a syringe to
stop the reaction and to reduce any remaining Chloramine T and free
iodine =
The BGG was separated from the salts on a 0=9 x 15 cm Sep-
hadex G-50 (superfine) column (15)°
The column had been prewashed
with nonlabled BGG and phosphate buffer=
Ten-drop fractions were
collected.and assayed for radioactivity with a Nuclear Chicago
Scintillation Counter-Analyser Computer (Model 132A) in a well-type
sodium iodide crystal Scintillation Detector (Model DS5)=
Active
fractions at the void volume were pooled, and the protein content
was assayed by optical density at 280 nm=
The pooled fractions were
diluted to a concentration of ItiOjig protein N/ml=
protein was generally used immediately=
The labled
11
Immune Elimination.
Each mouse, which had previously been
maintained on Ool^ KI in tap water, Was given 10 p g ^ ^ I - B G G (approx.
Oo? ^aC) IV.
Whole body counts were taken immediately and at various
times thereafter.
The mouse was held in a small cup, which was
placed directly on top of the NaI crystal detector.
At least 6400
counts were taken to give values with no more than 3% error.
Back-
round activity was measured with the empty mouse holder in place.
Correction
for daily variation in machine efficiency and for decay
of isotope still present in the animal was made by counting a stan­
dard, consisting of a stoppered test tube containing a solution of
I^l
Na ^ I.
Correction:
for counting efficiency was necessary, due to
x
I
fluctuations in line voltage.
Results are expressed as the per­
centage of initial activity remaining in the animal at a certain
time after injection of the labled material.
For sample calcu­
lations, see the appendix.
Serum Titrations.
Sera were titrated using a microtitrator
system (CookeEngineering Co., Alexandria, Va.).
Twenty-five jal of
serum was diluted in two-fold dilutions, 1.2 to I;4096, in modified
barbital buffer (I).
Then 2 5 yil of Q a3% SRBC in 1% normal, absorbed,
heat inactivated mouse serum in buffer was added.
Hemagglutinin
assays were incubated at room temperature for I hr and then over­
night at 40C.
Hemolysin assays were held 10 min at room temperature
12
before adding 25 ^al of a 1:20 dilution of guinea pig complement in
buffer.
Incubation for I hr at 37°C and overnight at 4°C completed
the hemolysin assay.
RESULTS
.Irradiation and Hematopoietic Reconstitutions
A preliminary
experiment was conducted to test the effectiveness of irradiation and
reconstitution of the strain
of mice to be used in these Studies„
C 57B 1 female mice were given either 600 or 900 rad whole body irradi­
ation at the University of Nebraska Medical Center=,
Syngeneic bone
marrow was administered to several of the irradiated mice either IV or
IP=,
All mice receiving 600 rads survived and showed very little weight
loss (Table I)=
A dose of 900 rad proved lethal for nontreated mice*
The time of death indicated that it was due to hematopoietic failure
and not damage of the intestial tracts
reconstitution with 2 x 10
7
Death could be prevented by
syngeneic bone marrow cells»
Either route
of injection proved satisfactory for this number of bone marrow cells®
A dose of 900 rad was used in subsequent experiments =
Assay of Immunity to rite Toy Immune Elimination®
In antici­
pation of studying cell interactions involving a soluble protein anti­
gen, preliminary experiments were run to determine the practicality of
using immune elimination of
A0
131
I-BGG as an assay for immunity to BGG=.
Immune and Nonimmune Elimination®
The average elimination
pattern of 8 normal C57B1 mice is shown in Fig® I by curve A 0
The 10
jig ‘^■'t -BGG given IV was eliminated with a half-life of 3°2 days in
the linear portion of the curve®
Curve B represents the average of three mice which had been
TABLE I.
Mouse
Number
The Effect of Irradiation and Hematopoietic Reconstitution
Dose
(rad)
Body Weight(gms)
Day 2
Day 7
3
4
5
6
7
8
10
11
900
900
900
900
900
900
900
900
BM IV
BM IV
BM IP
BM IV
none
none
none
none
17.9
15.2
17.8
17.9
19.4
16.3
18.8
13.5
15.3
15.6
15.8
15.4
17.6
13.5
14.5
I
2
3
4
5
6
600
600
600
600
600
600
BM IV
BM IV
BM IV
none
none
none
17.3
18.9
16.4
22.5
22.2
16.1
18.3
18.9
16.5
22.2
21.6
15.3
BH = 2 x 10
b
„
a
Treatment
syngeneic bone marrow cells
Post-irradiation
-
Time of Death
Due to Radiation
(days)
9b
11
9
10
4
H
-Fr
4-
u
I
2
DAYS
3
A FT E R
4
I-BGG
5
6
7
INJECTIO N
Figure I. Immune and nonimmune-elimination of
^I-BGG. Curve
A represents the elimination of 10 Jig
I-BGG given IV ^n^day 0 to 8
normal C57B1 mice. Curve B shows immune elimination of ^ I-BGG by 3
mice previously immunized 23 days before with 8 mg BGG in FCA subcu­
taneously. Verticle bars represent the range of individual curves.
Ir
16
immunized 23 days before with 8 mg of BGG in FCA subcutaneously,
A
rapid elimination of 60# of the injected protein was most probably due
to circulating antibody»
Most of the remaining protein was then eli­
minated with a half-life of 0*88 days, which reflects upon the actual
rate of synthesis of antibody in the animal»
The remaining 3% was
eliminated slowly with a rate almost approaching natural biologic
decayo
This portion of the curve may represent (i) elimination of
"^"*"1 re-utalized by the animal in its own metabolism, (ii) elimination
of
131
I-BGG molecules which have acquired a certain foreignness due to
denaturation during the labeling process, or (iii) antigen-antibody
aggregates of labled BGG in the tissues of the animal„
Bo
Correct Assay Time 0
The purpose of the following
experiment was to determine the Manliest time in the immune response
at which immune elimination could be detected=
Adult C57B1 female
mice were immunized with 2 mg BGG in FCA subcutaneously=
trol mice received saline-FCA subcutaneously =
Three conr
On days 6 , 8 , 10, 12,
and 14 several mice were given tracer doses of labled BGG=
Controls
received labled BGQ on day 6 =. The results of this experiment are
shown in Figs= 2 and 3°
The 6 day immune mice did not show immune
elimination until one day after injection of labeled BGG=
All groups
assayed beyond day 7 showed immediate immune elimination, the rate of
which increased M t h time after immunization=
It was concluded, that
mice in subsequent experiments could be assayed at least 7 days
Control
6 DAY IMMUNE
8 DAY IMMUNE
DAYS
A FTER
131I-BGG
IN JE C T IO N
Figure 2. Assay for immune elimination at various times
during the immune response. Fiers• 2 and 3 show elimination patterns
of groups of mice given 10 j i g a t
6 , 8 , 10, 12, and 14 days
after immunization with 2 mg BGG in FCA subcutaneously. Three con­
trols received saline-FCA. All other groups consisted of 2 adult
female C57B1.
A C TIVITY
REMAINING
18
IO DAY IMMUNE
IN ITIA L
12 DAY IM M UNE
14 DAY IM M U N E
DAYS
AFTER
13I-BGG
INJECTION
Figure 3* Assay for immune elimination at various times
during the immune response.
19
post-immunization,
Co
Tolerance Induetiono
Adult C57B1 mice were given 2»5 mg
soluble BGG IP and 10 days later challenged with 8 mg of BGG in FCA
subcutaneously»
^ I-BGG was given 12 days later®
the results of this tolerance inducing regimen®
Fig. 4 displays
The animals appeared
partially toleraht, showing an average elimination half-life of 1.19
days.
Fig. 5 shows a half-life of 2=75 days for BGG in adult G57B1
which had been made tolerant by injection of 50 jfxg of soluble BGG IP
onbe each week after birth for 8 weekso
The mice were challenged with
I mg of BGG. in FCA at 10 weeks and given the tracer BGG 20 days later.
Time of Recovery of Immune Responsiveness After Irradiation, and
Reconstitution With Bone Marrow.
Preliminary to running experiments
involving cell transplantations and cell interactions, it was thought
necessary to determine the time at which bone marrow alone could "iully
reconstitute the.immune potential of a lethally irradiated animal.
Bone marrow has been shown to contain precursor cells for all types of
cells involved in the immune response.
Since in later experiments we
wished to repopulate the animal with only the P-PFC (precursor of
plaque forming cell) by giving bone marrow, it was important to know
the length of the latent period preceding the appearance of the ARC
(antigen reactive cell) being derived from the bone marrow cell pre-
20
Nontreated
Immune
DAYS
A FTER
131I - BGG
INJECTION
Figure 4. Tolerance induction in C57B1 adults. Adult C57B1
were given 2=5 mg soluble BGG IP and 10 days later challenged with
8 mg BGG in FCA. Solid lines represent individual elimination curves.
Broken lines show nonimmune nontreated controls and immune controls.
21
Nontreoted
Immune
DAYS AFTER 131I-BGG INJECTION
Figure 5 . Tolerance induction in adult C57B1 which had been
treated with soluble BGG at birth. The solid line represents the
average elimination curve of four C57B1 mice challenged with I mg of
BGG in FCA 20 days before giving 10 ug labled BGG. The mice had been
given 50 JJg soluble BGG IP once every week for 8 weeks after birth.
Superimposed on the graph are immune and nonimmune elimination
curves. The vertical bars represent the range of individual curves.
22
cursors, which pass through the thymus 0
Adult female C57B1 mice were given 900 rad whole body irradi­
ation at Lincoln General Hospital and reconstituted the same day with
rp
2»65 x IOf syngeneic bone marrow cells=
Immediately, and at weekly
intervals thereafter, subgroups were immunized with I x 10° SRBC IP=
Mice were bled from the retro-orbital sinus at days 7 and 14 after
immunization=
Individual sera were titrated for hemolysins and
hemagglutinins to SRBC 0
It can be seen from Fig= 6 that a full res­
ponsiveness had not developed until the second and third week after
reconstitution=
This implied that any response measured up to 2
weeks after reconstitution was due to interactions between cells which
were fully differentiated at the time of transplantation and not due
to differentiation of transplanted precursor cells from the bone
marrow=
A Dose-Response Curve for Bone Marrow Transplantation=
The
next question to be answered was, how many cell types are present in
bone marrow= ..It was important to know if transplantation of 5 x I O^,
cells would involve only one or both of the nonadherant cells neces­
sary for a response, since this dose would be used in later cell
interaction experiments =
C57B1 male mice given 900 rad at St= James Hospital were
reconstituted with 5 , 10, 50, or 100 million syngeneic bone marrow
23
F
256
SRBC
— Hemolysins
- - - Hem agglutinins
256I28TSRBC
W EEKS
A F T E R IR R A D IA TIO N
Figure 6 . The appearance of an immune response to SRBC in
irradiated, bone marrow reconstituted C57B1 mice. Groups of mice (2
mice per group), given 900 rad and 2.65 x IO? syngeneic bone marrow
cells at time 0 , were immunized immediately and at I, 2 , 3i and k
weeks thereafter. The graphs show hemolysin and hemmagglutinin titers
of individual mice within each group. The response in nonirradiated
immune controls approximated the response of mice injected at 4
weeks.
24
O
cells and a constant dose of 4 x 10° SEBC IV0
assayed for antibody-producing cells on day 8»
Their spleens were
The results of this
experiment are shown in Fig, 7 as a plot of log response vs» cell
dose given.
The curve displays a slope of 1«60, indicating that two ■
cell populations are interacting to give the response (6)«
A Dose-Response Curve for Spleen Cell Reconstitution:
Dilution Effect With SRBC Antigen.
The
Groups of C57B1 female mice which
had been given lethal doses of irradiation at Lincoln General Hos­
pital were reconstituted with either I x IOb 9 10 x 10 „ or 50 x 10
syngeneic spleen cells*
Several mice in each group were also immu-
nized on the day of irradiation with I x 10
8 SRBC®
To determine the
cellular response, their spleens were assayed on day 5 for antibodyproducing cellso
A marked dilution effect was seen, in that an
average of 958 times more PFC were present in the spleen of mice when
50 million spleen cells were injected than when one million spleen
cells were injected (Table II)*
This dilution effect was also seen if
results were transformed into number of PFC per million recipient
spleen cells*
The dose-response curve (6) for this data shows a slope
of 1*40, indicating that two cell types are present in the spleen
being used for reconstitution and that they are interacting to give
the response (Fig* 8)*
The Spleen Dilution Effect With BGG Antigen*
C57B1 male mice
25
Slope = 1.60
Log BONE MARROW CELL DOSE
Figure 7» A dose-response curve for reconstitution with bone
marrow. The graph shows a plot of log FFC response per recipient
spleen to 4 x 10° SRBC on day 8 vs. the log bone marrow cell dose
given to the recipients on day 0 after 900 rad irradiation. Each
point is average of 5 C57B1 male mice.
TABLE TI.
Treatment
The Spleen Dilution Effect
a
No. of
Mice
Spleen Cells
Transferred
Nucleated
Cells/Spleen
x 10 "6
PFC/Spleen
PFC/IO6
Spleen Cells
NONEfe
SRBC
I
3
50 x
50 x IO6
89.0
41.7
0
4792
NONE
SRBC
I
3
10 x
10 x 10°
17.8
18.1
10
36
0.56
4
NONE
SRBC
I
2
0
0
0.29
I
I
X
X
IO6
5.80
11.3
5
0
79.
All mice received 900 rad.
b I x IO8 SRBC
C
Assayed by Jerne plaque on day 5. Nonirradiated immunized control spleens
contained an average of 20,738 PEG.
27
CL 4 .0
Slope = 1.40
Log SPLEEN
CELL DOSE
Figure 8, A dose-response curve for reconstitution with
spleen cells„ The graph shows a plot of log PFC response per ICr
recipient spleen cells to I x 10° SRBC on day 5 vs. the log spleen
cell dose given to irradiated C57B1 female mice. Each point is the
average of 3 mice.
28
were given 900 rad whole body irradiation with the Gammator, recon­
stituted with I, 10, or 50 million syngeneic spleen cells, and
immunized with I mg of aggregated BGG=
131
Eight days later 10 jfig
I-BGG was given IV, and the elimination was monitored by whole-body
counting.
The rate of elimination was shown to increase with increasing
dose of spleen cells given (Fig. 9)»
The half-life of BGG in the
immunized mice decreased from 2.7 days when one million cells were
given to 0.63 days when 50 million spleen cells were given.
However,
transformation of the data into a dose-response curve did not display
the spleen dilution effect nor the presence of cell interaction.
Absence of Synergism Between Normal Spleen and Bone Marrow in
SBF Mice.
G57B1 male mice were irradiated at St. James Hospital and
reconstituted with I x 10° normal syngeneic spleen cells and/or 5 x
/■
10° bone marrow cells. Their spleens were assayed for PFG on day 7»
The results (Table III), recorded as PFC per spleen, reveal no in­
creased response in mice receiving both spleen and bone marrow over
the response in mice receiving only marrow.
A notable increase in
the spleen size of groups of mice which had received bone marrow
over those that did not was evident.
The data in Table IV show the absence of synergism in mice
receiving larger doses of nonpal spleen, 10 and 50 million cells,
29
Figure 9. Anti-BGG response in spleen cell reconstituted
C57B1 mice. The graph shows the average elimination patterns
beginning on day 8 of groups of mice given I, 10, or 50 million
spleen cells and I mg aggregated BGG after irradiation. All points
are the average of 2 or 3 mice. Control curve represents elimination
in normal nonirradiated C57B1 mice.
TABLE III,
Absence of Normal Spleen-Bone Marrow Synergism in C57B1 Mice:
Low Cell Dose
Treatment
a
No. of
Mice
Nucleated
Cells/Spleen
x 10 -7
PFC/Spleen*3
Spleen
Spleen + SRBC
2C
5C
Bone Marrow
Bone Marrow + SRBC
2
5
20.80
19.95
9
35
Spleen + Bone Marrow + SRBC
4
15.29
31
Control
Control + SRBC
2
2
10.15
7.750
1.059
-
6
-
15
36,625
All mice except controls received 900 rad at St. James Hospital. Cell
doses were I x IO^ spleen cells and 5 x IO^ bone marrow cells.
Immunized mice received I x 10® SRBC.
Assayed by Jerne plaque on day 7
c
All mice in this group died before day of assay
TABLE IV.
Absence of Normal Spleen-Bone Marrow Synergism in C57B1 Mice:
High Cell Dose
Treatment
C
Experiment A3
10 x IO0 Spleen + SRBC
5 x IO^ Bone Marrow + SRBC
Spleen + Bone Marrow + SRBC
No. of
Mice
Nucleated
Cells/Spleen
x 10"7
5
4
4
1.62
5.87
6.20
PFC/ Spleen
330
81
516
b
Experiment
50 x IOb
5 x IO^
Spleen +
B
Spleen + SRBC
Bone Marrow + SRBC
Bone Marrow + SRBC
I
I
I
30.5
31.4
16.6
220,500
200
111,000
Mice received 900 rad in the Gammator and were assayed on day 5.
Mice received 900 rad at Lincoln General Hospital and were assayed
on day 8.
c
g
All mice were immunized with I x 10
SRBC.
VJ
H
32
with a constant number of bene marrow cells=
Similar experiments
were also conducted using male and female Balb/C and male LAF./J
-L
mice, irradiated at St= James Hospital=
The results in Table V
show that no synergistic effect was observed in the Balb/C strain
of mice=
An attempt to show synergism in LAF^/J mice resulted in very
low numbers of PFG per spleen in all groups (Table VI)=
synergism was seen=
No impressive
Only one out of three mice showed synergism=
Variation in Experimental Parameters Affecting the Immune
Response in Reconstituted M c e =
The lack of synergism between spleen
and bone marrow cell populations may have been due to the toxic envi­
ronment of the irradiated host immediately after irradiation=
To
test this hypothesis, C57B1 female mice were Irradiated at St= James
Hospital dnd reconstituted 20 hrs Iater9 at a time when the toxic sub®
stances produced in the host's irradiated tissues could be suf­
ficiently avoided= . Table VII shows that delayed reconstitution
produced results similar to those obtained for immediate reconsti­
tution=
Since the synergistic effect observed by Talmage et al= (26)
might have been dependent upon the time of assay of the host Spleen9
an experiment was conducted to determine the time of maximum res­
ponse in the host animal=
Female mice of the C57B1 strain irradiated
at St= James-Hospital, were reconstituted with one million spleen
x
TABLE V.
Absence of Normal Spleen-Bone Marrow Synergism
in Balb/C Mice
Treatment
a
No. of
Mice
PFC/Spleenb
Spleen + SRBC
2
13
Bone Marrow + SRBC
2
58
Spleen + Bone Marrow + SRBC
3
35
All mice received 900 rad. Cell doses were: I x IO^
spleen cells, 5 x IO6 bone marrow cells, and
I x 10 SRBC.
b
Assayed on day 6
TABLE VI.
Absence of Normal Spleen-Bone Marrow Synergism in LAF^/J Mice
Treatment3
Nucleated
Cells/Spleen
x 10 "6
No. of
Mice
Spleen + SRBC
2
Bone Marrow + SRBC
2
81.9
5
Spleen + Bone Marrow + SRBC
3
90.4
25
SRBC
2
SRBC Control0
2
a
6.25
PFCZSpleenb
5.74
173.
2
15,600
All mice except controls received 900 rad. Cell doses were: I x 10
spleen cells, 5 x 10° bone marrow cells , and 4 x IO8 SRBC.
Assayed on day 6
c
3
g
Nonirradiated mice were given 4 x 10
SRBC IV.
6
TABLE VII.
The Effect of Delayed Reconstitution on Subsequent Normal
Spleen-Bone Marrow Interaction
Treatment3
No. of
Mice
Nucleated
Cells/Spleen
x 10 7
PFC/Spleenb
Spleen + SRBC
2
Bone Marrow + SRBC
2
10.4
30
Spleen + Bone Marrow + SRBC
I
20.5
87
2:95
85
All mice were reconstituted and immunized^20 hr after being letjaally
irradiated. Cell doses were:g5 x 10 spleen cells, 5 x 1 0
bone marrow cells, and I x 10 SRBC.
b
Assayed by Jerne plaque on day 7 after irradiation
36
cells and 5 million bone marrow cells, immunized with I x 10
SRBG,
and assayed for PFC at days 4, 5» 6, and 7 after reconstitution®
The
daily variation in the efficiency of the Jerne assay was controlled by
assaying a 4 day immune mouse on each day of Jerne assay®
The results
of this experiment, compiled in Table VIII, show that the response
increased with time after reconstitution®
The time of maximum
response was not seen in this experiment ®
However, for convenience
of running experiments simultaneously, day 8 was the chosen time of
assay for all subsequent experiments®
There was little variation
in the efficiency of the Jerne assay, and hence, results obtained with
experimental mice within., this experiment may be compared®
Absence of Normal Spleen-Bone Marrow Synergism in C57B1 With
BGG Antigen®
C57B1 male mice were irradiated in the Gammator and
reconstituted with 10 million normal spleen cells and/or 5 million
bone marrow cells. ‘ Several mice in each group were immunized with Img
of aggregated BGG IB.
immune elimination®
All mice were assayed for immunity on day 11 by
Fig® 10 shows the absence of synergism in mice
receiving both spleen c&lls and bone marrow.
The magnitude of the
response of spleen cells alone was even seen to be diminished^ by the
addition of bone marrow.
The Effect of the Immune State of the Donor Cell Population
Upon Synergism®
Experiments described thus far have shown a lack of
TABLE VIII.
Effect of the Time of Jerne Assay on the PFC Response
No. of
Mice
Day of
Assay
Nucleated
Cells/Spleen
x 10 "7
PFC/Spleen
PFC/106
Spleen Cells
Experimental
Control
2
2
4
4
0.975
39.4
17
117,450
1.75
291.
Experimental
Control
2
2
5
5
4.74
28.5
75
81,500
1.59
260.
Experimental
Control
2
2
6
6
19.2
21.4
285
62,500
1.48
292.
Experimental
Control
2
2
7
7
20.1
20.8
708
73,875
3.53
355.
38
O - IM M UNIZED
------NONIM M UNIZED
» - IM M U N IZE D
.......NON IM M UNIZED
Ar IM M UNIZED
' ---------N ONIMM UNIZED
m- IM M U N IZE D
I -------- N O N IM M U N IZE D
IMM UNE CONTROL
SPLEEN +BG G
BONEMAR ROW + BGG
S P L E E N + BONE MARROW + BGG
DAYS
AFTER
I-BGG
INJECTION
Figure 10» Immune elimination in normal spleen and bone
marrow cell reconstituted C57B1. Mice were irradiated, reconstituted
with 10 x 10& spleen cells and/or 5 x 10° bone marrow cells, and
immunized with I mg aggregated BGG IP. Labled BGG was given IV on
day 11 after reconstitution. Points are the average of 2 mice.
39
synergism between dilutions of normal spleen cells and the bone
marrow P-PFGe
Furthermore, this inactivity was shown to be inde­
pendent of mouse strain, time of reconstitution, and time of assay„
It was thus proposed that the concentration of the AEG in the spleen
was too low for purposes of showing a synergistic effect with bone
marrowo
This suggested that synergism might be shown by use of an
immune donor spleen, which supposedly contains an increased number of
AEG due to ABC proliferation and formation of memory cells during the
immune responsee
"
a
C57B1 mice were irradiated at S t e James Hospital and recon­
stituted with normal or immune bone marrow, normal or immune spleen,
or combinations of thesee
Immune cells were harvested from two 657B1
Q
males which had received 4 X 10
SBBG IV 8 days previouslye
The PFC
response of recipients 8 days after reconstitution and immunization
O
with 4 x 10° SBBC are presented in Table IXe The combined effect of
normal bone marrow and normal spleen should have given 55 plus 28 or
83 PFC per spleen with normal cellse Whereas the combined effect of
immune spleen and normal bone marrow should have been 34 plus 55 or
89 PFC per spleen, the actual response was l?o7 times greater, or
1575 PFC per spleen, revealing impressive synergism between the two
cell populations 0
In contrast, no synergism was seen when immune bone
marrow was combined with normal Spleen0
The same type of experiment was conducted using LAF^/J male
TABLE IX.
Immune Spleen-Bone Marrow Synergism in C57B1 Mice
Treatment3
No. of
Mice
Nucleated ^
Cells/Spleen
x IO'7
PFC/Spleen'
Normal Spleen + SRBC
2
Normal Bone Marrow + SRBC
2
Immune Spleen + SRBC
2
Immune Bone Marrow + SRBC
I
13.7
350
Normal Spleen + Normal Bone Marrow + SRBC
2
13.4
81
Immune Spleen + Normal Bone Marrow + SRBC
2
10.2
1575
Normal Spleen + Immune Bone Marrow + SRBC
I
12.8
HO
SRBC Control
2
10.5
2572
All mice received 900^rad and 4 x IO^ SRBC IV.
cells and 5 x 10 bone marrow cells.
3.74
12.5
4.86
55
34
Cell doses were I x IO^ spleen
Cell suspensions were counted using a Coulter Counter.
Assayed on day 8
28
41
mice irradiated at St. James Hospital and reconstituted with 5 dayimmune cells.
Similar results were obtained (Table X).
The Time of Appearance of the Synergistic Cell in the Spleen
After Antigenic Stimulation.
In order to define the c&ll type res­
ponsible for synergism in systems using immune spleen* it was
necessary to determine the time of appearance and longevity of this
cell in the immune spleen.
The experimental design called for donor
mice in various stages of the immune response.
Donor C57B1 were
immunized with 4 x IO^ SEBC IV I, 3» 5* 7* and 15 days before transfer
of their spleen to recipients irradiated at St. James Hospital.
PFC
to SEBC were assayed on day 8.
11.
The results are plotted in Fig.
Each point is an average of 2 or 3 mice.
Mice receiving one million
immune spleen cells only* without further immunization* showed very
little response, indicating negligible transfer of antibody-producing
cells from dohors undergoing a primary response.
The Secondary res­
ponse shown by immune spleen cells alone remained fairly constant
after reaching, a plateau on day 5°
was found to appear ground day 3°
The cell responsible for synergism
This is shown by an increase in
the response of recipients of both cell types over the added responses
of either cell type alone.
to near maximum by day 5°
with 15 day immune spleen.
The active cell increases in concentration
The- greatest amount of synergism was seen
TABLE X.
Immune Spleen-Bone Marrow Synergism in LAF^/J Mice
No. of
Mice
Treatment3
Nucleated
Cells/Spleen
x IO'7
PFC/Spleenb
Normal Spleen + SRBC
2
1.975
122
Immune Spleen + SRBC
2
1.737
690
Normal Bone Marrow + SRBC
2
19.13
380
Immune Bone Marrow + SRBC
2
23.19
372
Normal Spleen + Normal Bone Marrow + SRBC
3
20.13
260
Immune Spleen + Normal Bone Marrow + SRBC
3
15.20
2347
Normal Spleen + Immune Bone Marrow + SRBC
2
20.53
173
SRBC
2
1.000
42
SRBC Control0
2
8.925
11,000
All mice received 900 rad; cell doses were I x IO^ spleen cells, 5 x IO^ bone
marrow cells, and 4 x 10 SRBC IV,
Assayed on day 8
g
Nonirradiated mice given 4 x 10
SRBC IV
43
8000
6000
SRBC + SPLEEN + BONE MARROW
4000
2000
SRBC + S P L E E N
SRBC+BONE MARROW
DAY OF CELL
TRANSFER
AFTER
ANTIGEN
Figure 11= The appearance of the synergistic cell in the
spleen after immunization. The abscissa represents the time after
immunization at which donor spleens were removed and used to
reconstitute irradiated C57B1 males with I x IO^ spleen cells. In
addition, some mice were also given 5 million bone marrow cells and
4 x 10° SRBC= Points are average of 2 or 3 mice measured on day 8.
44
The Specificity of Primed Spleen Cell - Bone Marrow Cell
Synergism.
In dealing with primed spleen cells in synergism
experiments, it was important to know if the effect of priming was
specific for the antigen or whether priming was only a nonspecific
sti^mlus o
If the later were true then synergism should occur using
donor spleen primed with a non-crossreacting antigen.
C57B1 male mice were given 900 rad at St. James Hospital and
reconstituted with normal bone marrow and/or 7 day HEBC (horse
erythrocytes) immune spleen with either SEBC or HBBC.
On day 8 Jerne
plaque assay was performed using either SEBC or HBBC.
The results, shown in Table XI, indicate that priming of
donor spleen is antigen specific.
While synergism did not occur using
HEBC-primed donor spleen cells in mice immunized with SEBC, synergism
was demonstrated in mice that were immunized with HEBC after receiving
HEBC-primed spleen and normal bone marrow.
Spleen - Thymus Synergism.
Since synergism between normal
spleen and the bone marrow P-PFC did not occur, it1 was questioned
whether or not normal spleen could react sy&ergistically with the
AEC of the thymus.
Thus, LAF^/J male mice, irradiated at St= James
Hospital, were reconstituted with 50 million thymus cells and/or I or
10 million normal spleen cells.
The results of Jerne plaque spleen
assay on day 8 (Table XII) confirmed that one million spleen cells
TABLE XI.
The Specificity of Immune Spleen Cell-Bone
Marrow Synergism
NO. of
Mice
Nucleated
Cells/Spleen
x 10-7
IS + SRBC
BM + SRBC
IS + BM + SRBC
2
2
2
1.044
8.853
5.275
IS + HRBC
BM + HRBC
IS + BM + HRBC
2
2
2
1.153
10.84
9.778
HRBC Control
SRBC Control
2
2
10.06
5.913
Treatment
a
6
PFC/Spleen to
HRBC
SRBC
8
15
20
-
-
383
80
3370
2720
8000
2005
35
_
6
IS = I x 10 HRBC immune spleen cells • BM = 5 x 10 nogmal
bone marrow cells; SRBC = 4 x 10 ; HRBC = 4 x 10 HRBC
TABLE XII.
Spleen-Thymus Synergism in LAF^/J Mice
Treatment
Thymus
a
b
+ SRBC
No. of
Mice
Nucleated
Cells/Spleen
x !Cr?
PFC/SpleenC
2
2.300
30
I x IO6 Spleen f SRBC
Thymus + I x 10 Spleen + SRBC
2
3
2.713
3.083
35
230
10 x IO6 Spleen £ SRBC
Thymus + 10 x 10 Spleen + SRBC
2
3
SRBC Control
2
5 x 10
normal syngeneic thymus cells
b 4 x IO8 SRBC
c
Assayed on day 8
10.58
10.84
6.918
880
850
9895
47
could interact synergistically with thymus cells.
Synergism could
not be seen at the higher spleen cell dose, however, indicating, per­
haps, a selective interaction between splenic ABC and splenic P-PFC.
Natural Hemagglutinins to SBBC.
During the course of these
experiments all C57B1, and in several cases, LAF^/J mice were
screened for natural hemagglutinins to SBBC before use in experiments.
The results of these titrations are compiled in Tables XIII and XIV.
The average titer was found to increase '^Significantly when
C57B1 mice were 10 weeks old.
This increase in titer was mainly due
to an increase in the number of mice showing a naturally acquired
titer and not to an increase in the magnitude of individual titers.
Natural titers as high as 1:128 were found.
Normal Spleen - Bone Marrow Synergism in 1$ Week Old Con­
ventional LAFj/J Mice.
LAF^/J male mice were removed from the SPF
animal room as young adults and fed on non-sterile feed and tap water
until 15 weeks of age.
Twelve of these mice were given 900 rad at
St. James Hospital and later reconstituted with I x 10
5 x 10
6
spleen and/or
bone marrow cells from non-irradiated 15 week old mice and
immunized with 4 x 10
8
SEBC.
Their PFC response was measured on day
8.
The results, as recorded in Table XV, show that mice receiving
both normal spleen and bone marrow gave a response that was 8 times
48
TABLE XIII.
Natural SRBC Hemagglutinins in C57B1 Mice
No. of
Mice
Age
(weeks)
Average
Titera
10
6
0.4
1:4
90
19
7
2.7
1:32
74
19
7
1.2
1:8
68
10
7
2.9
1:8
30
16
6-8
2.3
1:16
63
36
7-8
6.7
1:128
31
16
7-8
2.0
1:128
63
11
8
4.3
1:32
45
22
8
7.5
1:64
50
27
8-9
1.7
1:16
74
20
8-9
2.7
1:16
55
11
8-9
0.0
0
100
19
8-10
12.4
1:64
31
9
11
23.6
1:128
19
12
13.5
1:32
11
3
> 12
13.3
1:32
0
a
Reciprocal of titer
Highest
Titer
% Animals
With No Titer
0
TABLE XIV.
Age
(weeks)
Average
Titer3
5
7
0.4
1:2
80
20
10
2.0
1:16
80
16
15
2.8
1:16
50
No. of
Mice
a
Natural SRBC Hemagglutinins in LAF1ZJ Mice
Reciprocal of titer
Highest
Titer
% Animals
With No Titer
TABLE XV.
Normal Spleen-Bone Marrow Synergism in 15 Week Old
Conventional LAF^/J Mice
No. of
Mice
Nucleated
Cells/Spleen
x 10"6
SRBC
2
7.39
8
Spleen + SRBC
3
23.6
60
Bone Marrow + SRBC
3
311.
17
Spleen + Bone Marrow + SRBC
4
256.
643
Treatment
a
PFC/Spleen^
All mice received 900 rad at St. James Hospital. Cell doses were
I x 10 spleen cells, 5 x IO^ bone marrow cells, and 4 x 10®
SRBC.
b
Assayed by Jerne plaque on day 8
■51
larger than the added reponses of either cell type alone.
All four
mice in this group showed a positive synergistic response«, Such a
response was never seen in young adult mice.
DISCUSSION
The immune response has been recently shown to be mechanis­
tically complex, involving the interactions of several different cell
populations0
In this study of immunocompetent cell populations with­
in the spleen, lethally irradiated, and thus immunologically incom­
petent, animals were used to determine the reactions of various cell
types to antigen.
It was shown (Table I) that 900 rad gamma radi­
ation was lethal for mice if not compensated for by resupplying the
animal with hematopoietic stem cells from the bone marrow.
This
dose also proved efficient for eliminating the natural responsiveness
of mice to SEBC (Tables VI, X, XV)d
Thus, the lethally irradiated
mouse could serve as an ideal living "test-tube" for studying the
reactions of lymphoid cells injected into it.
Bone marrow has been shown to repopulate an animal's
hematopoietic system as well as i^s immune potential.
However, the
later is a delayed reaction, presumably due to the need for certain
cell types to migrate through the thymus to produce the thyitiasderived antigen reactive cells (AEG) (23), which are the necessary
initiators of an immune response.
It was shown (Fig. 6) that the
immune responsiveness had not recovered until the second and third
weeks after irradiation and reconstitution with 20 million bone
marrow cells.
This confirms the work of Gregory and Lajtha (14),
who demonstrated that full responsiveness, as measured by the PFC
53
response to SKBC9 did not occur until 2 weeks after irradiation and
reconstitution with one million bone marrow cells.
They hypothesized
that recovery was due to a renewal of the ARC population, since ad­
dition of thymus cells hastened the recovery of the mice.
Thus, with­
in the subsequent experiments of this study, it can be assumed that
the reactions studied were due to cells which were fully dif­
ferentiated at the time of transplantation.
Coppleson and Michie (6) showed that the slope of a I o g ^
cell dose-log^Q response curve could be used to indicate the number
of cell interactions taking place.
Hosier and Coppleson (25) later
utalized this method in determining the number of cells which were
interacting to give an immune response in in vitro spleen cell
cultures.
In the present studies, it has been shown (Fig. 7) by use
of the Iog10 cell dose ■>* Iogl0 plaque forming cell (PFC) response
plot, that two cell types are interacting to give a response in a ■
bone
marrow-reconstituted animal.
This implies that although the
bone marrow has been used in numerous
experiments as a rich source
of PFC precursors (P-PFC), it also contains small numbers of fully
differentiated ARC.
However, if small doses of one to five million
\
cells are given, the predominant cell received in the host spleen
will be the P-PFC, as can be seen by the low response (10 PFC/spleen)
obtained using these low doses.
54
Spleen cell preparations will also reconstitute an animal's
hematopoietic and immune systems,
Talmage et a l . (26), Celada (2),
and Gregory et al, (13) have shown that the responses of irradiated
animals was not a linear function of spleen cell dose used for
reconstitution (spleen dilution effect or premium effect).
In the
present studies, a I o g ^ dose - I o g ^ response representation of data
showing the spleen dilution effect (Table II) indicates that the
spleen also contains two cells which are interacting to give a res­
ponse (Fig, 8),
Proof that the increased response is not due to an
increased probability of cells lodging in the spleen as the dose
increased is provided by Kennedy et al,
(18),
They demonstrated
that the relationship between the number of hemolytic spleen foci
(caused by one ARC) and the number of cells injected is linear be­
tween doses of O to 8 x IO^ spleen cells injected.
By double-host
trasfer, it was calculated that Vy% of the ARC injected had lodged in
the spleen after 2 hr (l8),
Talmage and co-workers (30) reasoned that even though the
spleen contained both P-PFC and. ARC, these two cell populations were
not of equal size.
Thus, various dilutions of the spleen of 16-17
week old normal LAF^/J male mice were used, which lacked one cell type
(P-PFC), but retained enough of the other cell type (ARC) to give an
enhanced response when combined in vivo with P-PFC of the bone marrow.
This synergistic effect was masked when a larger number, of spleen
55
cells was given, due to the increased presence of the splenic P-^PFC in
these doses.
The present studies show that the response of similar Apleen
cell numbers from normal young adult SPF mice is not enhanced by
the addition of marrow.
Attempts to show a synergistic interaction .in
the SPF Balb/C, C57B1, and LAF^/J mouse strains failed ( Tables III,
IV, V, VI).
Lack of interaction was furthermore shown to be inde­
pendent of the time of reconstitution (Table VII) and assay of the
PFC response.
Assay times of 5s 6, 7s and 8 days after reconstitution
gave little indication of cell interaction.
It was concluded that
such low numbers of spleen cells did not contain sufficient numbers
of ARC to show a synergistic response with bone marrow.
In contrast to the above, it was demonstrated that low doses
of immune spleen could synergize with normal bone marrow (Table Et)'=
This finding, coupled with the fact that immune bone marrow did not
enhance the activity of normal spleen, indicates that through im­
munization the ARC population increases in size, such that addition
of P-PFC caused an increased response.
According to Cdtikowicz (29)
the PFC population is greater t M h the ARC population during the
immune response.
Thus, any fraction of spleen taken during this time
would contain both cell types.
This experiment therefore implies
that the ARC formed immediately after immunization (including both
functional ARC and memory cells) are potentially able to react with
56
more P-PFC than are present in the spleen.
Such mas shown by the 17-
fold increase in the PFC response of immune spleen by addition of
normal bone marrow (Table IX)0
These results confirm similar ex­
periments by Talmage and co-workers (26) and Cunningham (9)»
Additional evidence supporting the hypothesis that immuni­
zation had caused a specific proliferation of SEBC-AEC is given in
Table XI, where it was shown that a non-crossreacting antigen, HEBC,
was unable to induce the appearance of the SEBC synergistic cell in
the spleen.
This specificity of the priming antigen has also been
shown by Cunningham (8),
In these studies, the synergistic population of ABC was
shown to have appeared in the spleen by the third day after im­
munization (Fig, 11),
The size of this cell population increased
significantly on day 5 after immunization.
Either the size or the
potential of this population was, of all times tested, greatest at
15 days after immunization.
These findings seem to indicate that the
primary type of ARC responding to the second antigenic stimulus in
the irradiated host is an IgM (or direct Jerne) memory cell.
Sup­
portive evidence comes from Sercarz and Byers (28), who claimed that
IgM memory cells to SEBC began to appear one day after priming.
Cunningham (8) has given evidence for the memory cell being separate
from the PFC cell line, and thus possibly related to or formed from
the AEC of the primary response.
By use of adoptive transfer
57
systems, he has shown that IgM memory to SHBC was maintained with a
near constant PFC inducing potential for at least 5 months.
The
finding in these studies, that synergism was maximal on day 15, a
time at which the primary IgM PFC rsponse has waned to background
levels, supports the hypothesis that the observed synergistic inter­
action between immune spleen and bone marrow involved primarily a
form of memory cell-derived ARC.
An experiment showing that limiting numbers of spleen not ,
synergizing with bone marrow were able to interact synergistically
with thymus, supported the earlier conclusion that the splenic ARC
population of normal young adult mice was smaller than the splenic
P-PFC population of such mice.
Data displayed in Table XII show that
low doses of spleen could interact with thymic ARC, indicating that
the splenic ARC could be removed from cell suspensions fey dilution,
leaving a population of P-PFC0
A final experiment (Table XV) demonstrated that older animals,
removed from SPF conditions, develop a spleen in which the ARC out­
numbers the P-PFC, such that appropriate dilutions of spleen cell
suspensions are able to synergize with bone marrow in vivo.
This re­
sult can be explained by ^evidence (Tables XIII and XIV) for an
increased probability of natural immunization to the antigen, used
in these and other experiments (26) with the age of the animal.
nature immunization is an uncontrollable processo
An animal's
In
58
immunologic defense, units are constantly being challenged by foreign
antigens in the environment»
Among the most important stimuli are
the microbial flora of the skin and intestional tract and respiratory
tract, and foreign material in food=
It is important that the
possibility of intervening crossreacting antigens in immunological
experiments be avoided=
In these experiments, this factor was con­
trolled by use of young adult animals maintained under SPF conditions=
An attempt was made to eliminate from experiments all animals having
already been exposed to SBBC crossreaeting antigens =
In conclusion, these studies have shown that bone marrowspleen synergism is dependent upon the age and immune state of the
animal=
In young adult mice, the P-PFC outnumbers the smaller ARC
population=
This cell type ratio seems logical from a knowledge of
the proliferative events which follow antigenic stimulation=
Natural
or controlled immunization to SRBC causes formation tif a large'memory
cell population, which is able to interact with bone marrow P-PFC to
give an enhanced secondary response=
These studies do not rule out
the possibility that with the increase in the age of the animal a
proliferation of splenic stem cells occurs without antigenic stimulus
or that more stem cells migrate into the spleen from the thymus as
the animal ages=
However, the information presented above indicate
that inadvertent immunization accompanied by the formation Cf radio­
resistant (32) memory cells is an important factor influencing the
59
siz,e of the lymphoid cell populations in the spleen
SUMMARY
Cell-cell interactions involving the spleen in its immunologic
response to sheep erythrocytes (SRBC) and bovine ghmma globulin (BGG)
were studied by adoptive transfer of either spleen, thymus, bone
marrow, or combinations of these cell preparations to lethally
irradiated mice=
The response of cells in the host animal to anti­
genic stimulation was followed by the hemolytic plaque forming res­
ponse to SRBC or the ability to eliminate iodine-131 labled BGG=
A response in the irradiated, reconstituted, and immunized
animals resulted from the interaction between antigen, an antigen
reactive cell (ARC), and a precursor of the antibody forming cell
(P-PFC)=
Synergism between the two cell populations was detected by
a significantly large increase in the number of antibody producing
cells formed from P-PFC as a result of this interaction=
Both bone marrow and spleen of a normal animal contained
unequal numbers of both ARC and P-PFC=
In the young adult animal the
P-PFC population of both bone marrow and spleen outnumbered the ARC,
such that appropriate dilutions of these cell preparations were
unable to supply the host spleen with ARC=
Such host spleens, which
had received dilutions of young adult normal spleen and thus P-PFC but
not ARC, could respond to antigen only if thymus cells, containg ARC
only, were also given to the host=
During immunization the ARC population of the spleen
6l
proliferates and transforms into memory cellso
-
Dilutions of immune
.
donor spleen were thus able to interact synergistically with bone
c
'
marrowo The spleens of older conventionalized mice also had an
enlarged ARC population in the spleen*
This is most probably due
to inadvertent immunization of the animal by exposure to environmental
crossreacting antigens*
Natural non-antigenically stimulated pro­
liferation of Atem cells may also be a contributing factor*
APPENDIX
Calculations for Deriving Immune Elimination Curves
Data corrected for Background:
Whole body count of mouse - day 0
Whole body count of mouse - day 3
3.46 x
1.08 x 105
IO5
cpm
cpm
Standard - day 0
Standard - day 3
5-79 x
4.70 X IO^
cpm
cpm
Calculations:
Standard-day 3
Standard-day 0
Ic
_
~
4700
5790
0.812
2*
Corrected cpm of _ cpm of mouse-day 3 _ 1.08 x IO^ _ ^
33 x 10'
F
"
0.812
mouse on day 3
3.
% Initial Activity
Remaining - Day 3
Corrected cpm-day 3
cpm of mouse-day 0
1.33 x IO^
3.46 x IC k
38.4*
100
LITERATURE CITED
1.
Campbell, D 0H . J.S. Garvey, N.E» Cremes, and P<,H. Sussdorf.
1964. Methods in Immunology. W.A. Benjamin. New York,
p. 246.
2.
Celada, F., 1967« Quantitative studies of the adoptive
immunological memory in mice. II Linear transmission of
cellular memory. J, Exp.Med. 125:199«
3«
Chiller, J.M., G.S. Habicht, and W.O. Weigle. 1970. Cellular
sites of immunologic unresponsiveness. Proc. U.S. Nat. Acad.
Sci0 65:551«
4.
Clamen9 H.N., E 0A. Chaperon, and R.F. Triplett. 1966. Thymusmarrow cell combinations. Synergism in antibody production.
Proco Soc, Exp, Biol, Med. 122:1167.
5«
Clamen, H.N,, E.A. Chaperon, and R.F. Triplett. 1966. Immunocompetence of transferred thymus-marrow cell combinations«
J. Immunol. 97:828,
6«
Copple^on, L 0W, and D, Michie, 1966, A, quantitative study.of
the chdrioallantoic membrane reaction in the chick embryo,
Proc, Roy. Sec, (London) B 1 6 3 :555°
7«
Cudkowicz, G o 9 G.M. Shearer, and R.L, Priore, 1969» Cellular
differentiation of the immune system of mice. V Class
differentiation in marrow precursors of plaque forming cells,
J. Exp. Med. 130:481.
8.
Cunningham, A.JJp. 1969.
immunological memory.
9.
Cunningham, A ,J.* 1969, Studies on the cellular basis of IgM
immunological memory. The induction of antibody formation
in bone marrow cells by primed spleen cells. Immunology
17:933,
10.
11.,
Studies on the cellular basis of IgM
Immunology 16)621.
Davjj-S9 WoEo9 L=J. Cole9 and W.T, Schaffer® 1970. Studies on
synergistic thymus-bone marrow cell interactions in
immunological responses, Proc, Soc. Exp, Biol. Med. 133:144.
Fishman9 M. and F.L. Adler. 1967« Macrophage RNA and anti­
body synthesis.
In Immunity9 Cancer, and Chemotherapy. Edt.
by Eo Mikich. Academic Press. New York. p. 177«
Sk
12o
Foz6Cl, W.L,, J.Lo Gowans, and P»J«, McCullagh0 1966. The origin
and function of lymphocytes = In Ciba Foundation Symposium
on the Thymus» Edt, by G 0E 0W 0 Wolstenholme and R. Porter,
Little, Brown, and Co., Boston, Mass. p. 58«
13»
Gregory, C.J., and L.G. Lajtha. 1968, Kinetic study of the
production of antibody forming cells from their precursors.
Nature 218:1079»
Ika Gregory, C.J. and L.G. Lajtha.
1970. Recovery of immune
responsiveness in lethally irradiated mice protected with
syngeneic marrow cells. Internat„ J 0 Rad. Biol. 17:117«
15»
Hunter, W 0M 0 1967» The preparation of radioiodinated proteins
of high activity, their reaction with antibody in vitro:
The radioimmuno-assay„ In Handbook of Experimental Immu­
nology. E d t . by D.M. Wier . F.A. Davis Co. Philadelphia„
p. 608o
. 16.
Inchley, C.J. 1970. Requirement for cellular interaction in
the antibody response to bacteriophage T, in mice. J 0
Immunol. 104:14.
17»
Jerne, N.K. and A.A. Nordin9 1963» Plaque formation in agar
by single antibody producing cells. Science 140:405»
18.
Kennedy, J.C., J.E. Till, L. Siminovitch, and E.A. McCulloch.
1966. The proliferative capacity of antigen-sensitive
precursors of hemolytic plaque-forming cells. J. Immunol.
96:973»
19»
McConahey, P.J. and F.J. Dixon. 1966. A method of trace
iodination of proteins for immunologic studies. Int. Arch.
Allergy 29:185.
20.
McPherson, C.W. .1963» Reduction of Pseudomonas aeruginosa
and coliform bacteria in mouse drinking water following
treatment with hydrochloric acid or chlorine. Laboratory
Animal Care 13:737«
21.
Miller, J.F.A.P. and G.F. Mitchell. .1967» The thymus and
the precursors of antigen reactive cells. Nature 216:659»
65 .
22o
Miller, J«F.A.P» and G.F. Mitchello 1968«. Immunological
activity of thymus and thoracic duct cells= Proc= TJoS=
Nat= Acad= Sci= 59,; 296=
25=
Miller, J.F.A.P. and G.F« Mitchell* 1969=
reactive cells= Transplant= Rev= I;3®
24=
Mishill, R=I= and R=W= Dutton= 1967« Immunization of dis­
sociated spleen cell culture from normal mice= J= Exp= Med=
126:423«
25=
Hosier, D=E= and L.W. Coppleson= 1968= A three cell inter­
action required for the interaction of the primary immune
response in vitro = Proc= U=S= Nat= Acad= Sci= 61:542=
26=
Radovich, J = , H= Hemmingsen, and D=W= Talmage = 1968= The
enhancing effect of bone marrow cells on the immune response
of irradiated mice reconstituted with spleen cells from
normal and immunized donors = J= Immunol, 100:756=
27=
Radovich, J=
28=
Sercarz, E=E= and V=S= Byers = 1967= The X - Y t Z scheme of
immunocyte maturation= III Early IgM memory and the
nature of the memory cell= J= Immunol= 98:836=
29=
Shearer, G=M= and G= Cudkowicz= 1969® Distinct events in
the immune response elicited by transferred marrow and
thymus cells= I Antigen requirements and proliferation of
thymic antigen-reactive cells= J= Exp= Med= 130:1243=
30=
Talmage, D=W=, J= Radovich, H= Hemmingsen. 1969= Cell inter­
action in antibody synthesis= J= Allergy 43:323°
31°
Taylor, R=B= 1969° Cellular cooperation in the antibody
response of mice to two serum albumins: Specific function
of thymus cells= Transplant = Rev= I^114=
32=
Zaretskaya, Yu= M=, E=I= Panteleev, and R=V= Petrov= 1969»
Accumulation of antibody-forming cells in spleens of
pre-immunized irradiated mice after transplantation of
syngeneic bone marrow= Nature 221:567»
1970=
Thymus and antigen
Personal communication=
MONTANA STATE UNIVERSITY LIBRARIES
762 100 5484 6
N378
SiiU
cop. 2
Sieckmann, Donna G
A critical analysis of
bone marrow-spleen cell
interaction in the immune
response
NAMK
AND
AD O RKKK
JV /</
r