Differential recovery of antibody responses to various antigens after sublethal whole-body irradiation
by Edward Lee Newman
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in MICROBIOLOGY
Montana State University
© Copyright by Edward Lee Newman (1981)
Abstract:
Experiments were performed that showed a differential in the recovery of BALB/c mouse plaque
forming cell (PFC) responses against various antigens, following 550r 60Co whole-body irradiation. It
was found that PFC responses against a thymus-dependent antigen, sheep erythrocytes, and two
thymus-independent type 1 antigens, trinitrophenol-Brucella abortus and butanol-extracted Eshericia
coli lipo-polysaccharide, rebounded to normal levels within 30 days post-irradiation whereas PFC
responses against two thymus-independent type 2 antigens, polyvinylpyrrolidone (PVP) and
phenol-extracted E. coli lipopolysaccharide, remained well suppressed 30 days post-irradiation.
Radiation-induced suppression of murine anti-PVP PFC responses lasted for at least 120 days, and was
not due to a radiation-induced shift of the dose response curve against PVP. The anti-PVP responses of
DBA/2 and C57B1/6 mice were also shown to be suppressed 30 days post-irradiation. In addition, the
anti-PVP PFC responses of adult athymic (nude) mice, but riot of young (14 day old) euthymic mice,
were shown to remain well suppressed 30 days post-irradiation. Radiation-induced suppression of the
anti-PVP PFC responses of euthymic was abrogated by adoptive repair with normal spleen cells or fetal
liver cells, but not by repair with normal serum or spleen cell lysates. Adoptively transferred spleen
cells, capable of repairing the anti-PVP PFC responses of irradiated mice, were resitant to anti-theta
and complement (C'), sensitive to anti-mu and C', and adherent to anti-mu coated petri dishes. STATEMENT OF PERMISSION TO COPY
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Signature
Date
S
/ g* I
DIFFERENTIAL RECOVERY OF ANTIBODY RESPONSES
TO VARIOUS ANTIGENS AFTER SUBLETHAL
WHOLE-BODY IRRADIATION
by
EDWARD. LEE NEWMAN
A thesis submitted in partial fulfillment
of the requirements for the degree
of
MASTER OF SCIENCE
in
■ MICROBIOLOGY
Approved:
ItMi
Chairman!, 'Examining 'Committee
Head, Major Department
'dL
Graduate Dedn
MONTANA.STATE UNIVERSITY
Bozeman, Montana
May,'1981
iii ■
■
ACKNOWLEDGMENTS
I-would like to express my sincere appreciation to Dr.
N . D . Reed for the guidance, financial support, and labora­
tory space given me while a graduate student under his
direction. . I would also like to thank Dr. N . D . Reed, D r .
J . E . Cutler, and Dr. M . H . Smith for serving on my graduate
committee and offering me consultation and technical advice.
Thanks are also offered to Dr. Ray Daynes and Dr.. J . A. Rudbach for providing me with anti-theta and LP S , respectively.
I would like to give a special thanks to my wife, Kim, for
much needed moral support.
Last, but not least, I would
like to thank thousands of furry little creatyres who cour­
ageously
gave their lives for the advance of science and '
the education of Ed Newman. '
TABLE OF CONTENTS
Page
VITA .
11
........................ ..
■ACKNOWLEDGMENT'S
LIST OF.TABLES
.
. .' .. A
.
................
. .
... .. .
'. . ,
... :
iii
. .
'.V
LIST OF FIGURES
ABSTRACT
vi
. . . . . . . . . . . . . . . . . . . .
INTRODUCTION ...................... .
MATERIALS AND METHODS
..' .
.' . .'
Vvii
.'.........
..
.
... . .
A n i m a l s ........... . ........... ..........
I r r a d i a t i o n ................. .
.Passive Transfers ............... . . . . .
Adoptive Transfers
. .................. .. .
Antigens and Immunizations
.............
Plaque Assays
.■ .'.. . . . ... . •.......
S t a t i s t i c s ........... •
................. .. .
I
'
8
8
9
9
10
11
12
. 13
. .
14
PVP and SE Responses in Irradiated Mice .. ■.
Duration of Radiation-Induced Suppression ■
of Anti-PVP Responses ..... ...........
Dose Response Curve to. PVP
. ........... .
Radiation-Induced Suppression of the AntiPVP Responses in BALB/c, DBA/2, and
C57B1/6 Mice .
Irradiation of Young Mice . . . ........... •
PFC Responses of Irradiated Mice.to TI-I
Antigens
.................
Irradiation of Nude Mice
.........
Repair of Irradiated Mice With'Whole Cell
P r e p a r a t i o n s ............... .
■ Passive Repair of Irradiated Mice . .■■ ; . .
Repair of Irradiated Mice With Fractionated
Cell Preparation . . . . ; . . . . . . .
■14
RESULTS
............... ..
'16
18
21
21
24
31
32
36.
38
DISCUSSION
47
•REFERENCES
58
' LIST OF TABLES
Table
Page
I.. Direct PFC responses'of mice to SE or ■
PVP 30 days post-irradiation . . . . . . .
. 2.
3.
15
Long term, suppression of anti-PVP. re- .
sponses by sublethal irradiation . . . .
17
Responses to.PVP of normal and irradiated
young (14.days old) and adult (7-9
weeks old) mice
. . . . . . . . . . . . .
25
4. ' Response's of normal and Irradiated mice to
PVP and LPS 011.3 . . .................... . 2 7
5.
6.
7.
8.
9.
Responses of normal and irradiated mice to
phenol- or butanol-extracted LPS K235
.
29
Responses of normal and irradiated mice to
PVP and TNP-B. abortus (TNP-BA)
. . . .
30
Responses of normal and irradiated a'thymic •
(Nude) mice and their euthymic littermates (ELM) to P V P ......................
33
Anti-PVP responses of irradiated mice re­
paired, w i t h .adoptively transferred normal
adult spleen (AS') or fetal liver cells
(FL)
................... . . . .
37
Anti-PVP responses of irradiated mice re­
paired with serum from normal or irradi- •
ated mice
. . . . .
. . . . . . . . .
39
10. . Anti-PVP responses of irradiated mice re­
paired with spleen cells or spleen cell
lysates from normal or irratiated mice .
40
11.
50
Properties of B^-I and.B^-2 cells
LIST OF FIGURES
Figure
1.
2.
3.
4.
5.
Page
Dose response curves of normal and irradi­
ated mice to PVP ' ..........................
20
Responses of normal and irradiated BALB/c,
. DBA/2, and C57B1/6 mice to P V P ...........
23
Repair of the responses to PVP of irradiated
mice with normal spleen c e l l s .............
35
Repair of the responses to PVP of irradiated
mice with normal spleen cells treated with
a n t i s e r u m .................................
435
Repair of the responses to PVP of irradiated
mice with fractionated normal spleen cells
46'
vii
ABSTRACT
Experiments were performed that showed a differential
in the recovery of BALB/c mouse plaque forming cell (PFC)
responses against various antigens , following ■550r-6-0q o
whole-body irradiation. It was found that PFC responses
against a thymus-dependent antigen, sheep erythrocytes, and
two thymus-independent type I antigens., trinitrophenolBrucella abortus a n d 'butanol-extracted Eshericia coli Iipo.polysaccharide, rebounded to normal levels within 30 days
post-irradiation whereas PFC responses against two thymus-.
independent type 2 antigens, polyvinylpyrrolidone (PVP) and
phenol-extracted E . coli lipopolysaccharide, remained well
suppressed 30 days post-irradiation.
Radiation-induced sup­
pression of murine anti-PVP PFC responses lasted for at
least 120 days, and was not due to a radiation-induced shift
of the dose response curve against P V P . The anti-PVP re­
sponses of DBA/2 and C57B1/6 mice were also shown to be sup­
pressed 30 days post-irradiation. In addition, the anti-PVP
PFC responses of adult athymic (nude) mice, but riot of young
(14 day old) euthymic mice, were shown to remain well sup­
pressed 30 days post-irradiation.
Radi at ion-induced suppres­
sion of the anti-PVP PFC responses of euthymic was abrogated
by adoptive repair with normal spleen cells or fetal liver
cells, but not by repair with normal serum or spleen cell
lysates.
Adoptively transferred spleen cells, capable of
repairing.the anti-PVP PFC responses of irradiated mice,
were resitant to. anti-theta and .complement (C 1) , sensitive
to anti-mu and C 1, and adherent to anti-mu coated pe.tri
dishes.
INTRODUCTION
In 1966, Claman (11) and co-workers showed that the
anti-sheep erythrocyte (SE) response of irradiated mice could
be restored with the injection of both bone marrow and thymus
cells from syngenic donors.
Injection of syngepic bone mar­
row or thymus cells alone did not completely restore the re­
sponse.
This observation was evidence that at least two
different cell types were necessary for a normal antibody
response to SE:
One that could be obtained from the bone
marrow, and one that could be obtained from the thymus.
It .
was shown that the bone marrow cells (B cells), were respon­
sible for forming anti-SE antibodies while the thymus cells
(T cells) acted as helper cells, somehow inducing the B cells
to produce more antibodies than they would without the help
of T cells (13,42,45).
Since those initial observations, researchers have tried
to characterize B and T cells. B and T cells have been shown
to vary from each other on the basis of their cell surface
antigens (22,28,31,35,57,72), their responses to various
mitogens (10,23,66,71),
and the nature of their, receptors for
antigens (5,12,38,70).
T and B cells have also been shown to
differ in sensitivity to ionizing radiation (4) and in non­
specific adherence to nylon fiber (i.e. most B cells stick to
nylon fiber while most T cells do not)(27).
2
Further investigation has now made it possible' to iden­
tify separate subpopulations within.both T and B cells.
T
cell subpopulations can be described by differences.in re­
sponses to mitogens (23,66), in recirculation after adult
thymectomy (.9), and in antigenic moieties found on the cell
surface (8,9, 58,64).
Delineation of B cell subpopulations is much less a d - .
vanced, .which is why I decided to study this particular area..
There is evidence to divide B cells into two major subgroups:
Bg cells which respond against thumus-dependent (TD) anti­
gens, such as SE-; ■and require T cell help for antibody for­
mation or tolerance induction;
and B^ cells which respond
against thymus-independent.. (T I ) antigens,' such as lipopolysaccharide (LPS), and require no T cell cooperation for
either process (20,28,54)..
The evidence for two B cell sub-
populations includes the fact that the antibody response
against a.hapten coupled to a TD carrier is additive to the
antibody response against the same hapten coupled to a TI
carrier, but is not additive to.the antibody response against
the same.hapten coupled to another TD carrier (26,55).
Also,
limiting dilution experiments indicate the existence of dif­
ferent precursors for B^ and B^ cells (35).
■In addition, only -B0 cells require complement (C3) for
3.
a normal antibody response and m o s t . c e l l s
cells are complement-receptor
receptor positive while most
negative (35,36).
are complement-
Erythrocyte-complement complexes will
attach to the complement receptor found 'on B - cells, thus
allowing.separation of B^ cells from B^ cells by velocity
sedimentation (35).
Alternatively (21), because B^ and B^
cells differ in size they can be separated, to some extent,
by velocity sedimentation alone.
In 1978, in this laboratory, Brooks (7) made the initial
observation that.mice give 550 rads (r) whole body irradia­
tion would recover their anti-SE plaque forming cell (PFC)
response within 30 days but would not recover their an.tipolyvinylpyrrolidope '(PVP) PEG response within the same time
period.
Since SE is a TD antigen and PVP is a TI antigen,'
Brooks decided to test irradiated mice, which had been
.
allowed to recover for 30 days, against other TD and TI anti-.
gens. -He tried dinitrophenol—ficoll (DNP-f icoll)', Type 3
pneumococcal polysaccharide,
and phenol-extracted Eshericia-
coli DPS, all TI antigens, and DNP-ovalbumin,
a TD antigen.
He found that.irradiated mice had recovered their anti-DNPovalbumin response within 30 days but showed marked suppres­
sion of their PEG responses against all 3 TI antigens 30
days post-irradiation.
These experiments'indicated that sub-
4
lethally irradiated mice may offer an excellent system for
the study of B cell■subpopulations, and were the foundation
of the work reported in' this thesis paper.
Recently,
a great deal of evidence has been published
that suggests that
groups.
.
cells can be subdivided into two
Immature mice and immunologically defective CBA/N
mice mount normal responses against some TI antigens, desig­
nated as TI-I antigens, but are deficient in their responses.,
against other TI.antigens, designated as TI- 2 .antigens (2,
16,46,47,51).
Spleen cells from normal adult mice have a
higher surfac.e IgD to surface IgM ratio, than, do spleen cells
from normal immature-, mice or CBA/N. mice (52,62,65a, 65b, 67)
and spleen cells' from normal mature mice bear surface anti­
gens, Lyb-3, Lybr-5, Lyb-7, not found on normal immature or
CBA/N mice' (1,52,67)'.
Also, .spleen cells treated with anti-r
LybrB and complement cannot mount an antibody response against
TI-2 antigens,
in vitro, (1,6,47).
This evidence leads to the conclusion that CBA/N mice
are. analogous to normal immature mice -in that both groups
are deficient in a ■subpopulation of B^ cells, found in normal
adult mice, which produces antibodies against TI-2 antigens,
has more surface IgD than surface IgM, and bears some or all
of the following antigens:
Lyb-3, Lyb-5, and Lyb-7 (52).
5
Therefore, in the light of these new developments
I chose
to investigate- the ■possibility that there may in fact be a
difference in recovery from sublethal radiation, not only
between B, and Bn .cells, b u t , perhaps also between the two
different subpopulations of B^ cells.
, '
In an attempt to be.as succinct.as possible I will,
henceforth, refer to those Bn cells reactive to TI-I antigens .as B^-I cells and those B^ cells' reactive to TI-2 anti­
gens as B^-2 cells. .A plausible explanation for the appear­
ance of B 1-2 cells only in adult normal mice (i.e. not in
■immature or CBA/N mice) is that B^-2 cells develop later ■
than B 1-I cells and that the-CBA/N mouse has a B cell developmental defect.
This defect found, in CBA/N mice has been
determined to be X-Iinked (2).
The CBA/N mouse is an attractive model system for study­
ing B cell subpopuiations and their development., .and has been
used extensively for this purpose (.1,41,61) . However, CBA/N
mice exhibit additional deficiencies,
including a less than
normal response to SE (2), defective B cell colony formation
in agarose (30) , and other abnormalities (52-)-.
It would
.therefore be beneficial to B cell subpopulation identifica­
tion and development .studies to find a supplementary B^-2
'
cell deficient murine system to corroborate the data derived
from CBA/N mouse studies.
Work done in this lab indicates.
■
6
•
that a sublethally irradiated mouse may be such a system.
Whole body irradiation causes numerous sequelae and the
immyne system appears to be particularly' radio-sensitive (4).
Ionizing radiation-in sublethal doses may depress (25,56,68,
69), or augment (3,14,15,68,69) specific antibody responses
depending on the antigen tested,
the time of irradiation,
the age of the irradiated animal, and the rate, dose, and
type of radiation.used (4).
,
- •
'
The affected cells may consist
'
.
. -I
of one or several cell types depending on the previously men­
tioned parameters (4).
Despite the difficulties involved in using ionizing
radiation,
there are definite advantages to the sublethally
irradiated mouse system over the CBA/N mouse system.
The
sublethally irradiated mouse is not unique to a specific
strain of mouse and can be related to several mouse strains '
without interbreeding.
In addition, sublethally irradiated
mice are deficient in their responses to TI-2 antigens, but
normal in their responses to TI-I and TD antigens.
The primary objective of my thesis work was to more
thor,oughly define the sublethally irradiated mouse system,
including identification of the cell type that is necessary
for a normal PFC response to PVP and does not recover from
550r whole body irradiation; and I feel that I have accom-
7
plished that objective..
Evidence presented in this paper
will show that mice that are sublethally irradiated, arid
allowed to recover for at. least 30 days, afford a simple,
inexpensive, and promising system for the' study o f 'B 1 cell
subpopulations.
MATERIALS AND METHODS
Animals:
Inbred male and female BALB/c mice were used in most
experiments.
Breeding stock was originally obtained in 1966
from Baylor Medical School (Houston, T X ) and mice of this
strain have since been maintained by random mating in our
laboratory.
Other' inbred mice were purchased from Cumberland
View Farms (Clinton, T N ).
Congenitally thymus-deficient
(nude) mice and their euthymic littermates (ELM) were pro­
duced from heterzygous mating pairs from a line made congenic
with BALB/c by cross-intercross mating.
Nude and ELM mice
used in these experiments were the offspring of .heterozygous
parents of the 13th generation.
Adolescent mice were 13 days old and nude and ELM mice
were 4-6 weeks old at the beginning of experiments.
All
other mice were 7-12 weeks old at the beginning of experi­
ments.
Groups of mice were sex and age matched.
Nude and ELM mice were raised in a clean room, in cages
with filter caps, and maintained on sterilized Sterilizable
Wayne Lab Blox (Allied Mills, Inc., Chicago,. IL) and acidi­
fied water;
Upon irradiation, nude and ELM mice were given
sterilized water containing tetracycline (60 mg/ml of Tetra D
■Soluble Powder Concentrate S-Diamond Laboratories, Inc., Des
Moines, I A ) until the day of immunization at which time they
.9
■were maintained- on sterilized water without tetracycline.
All other mice were raised in .conventional conditions and
maintained on Wayne Lab Blox (Allied Mills, Inc., Chicago,
I L ) and acidified water.
Sanicell Bedding (Paxton Process­
ing Co., Paxton, IL) was sterilized before u s e . ■
Irradiation:
■ Mice were exposed to gamma rays' from a Picker V4-M60
with a
60
Co source.
'
Plastic holders with individual compart­
ments were used to immobilize mice during exposure to ^ C o .
Skin to source distance was either 70cm or 60cm.
Approxi- .
mate radiation delivery rate was 70 r/min.
Passive Transfers:*
2
Whole blood obtained from normal or irradiated mice was
allowed to- clot at '5°C for 2h- before centrifugation at 200G
(also at 5°C) for 10 min. . Recipient mice received 0.2B ml
of undiluted serum intravenously (I .V .).
Cell lysates- were prepared from spleen cell suspensions
from normal or irradiated mice by placing a suspension of
o
2 x 10 splenocytes/ml-phosphate buffered saline (PBS) into
a Sonicator Cbll Disrupter (Heat Systems-Ultrasonic ■
, Inc.,
Plainview, NY) for 2 min.
-Sonicated suspension's were .thdn.
centrifuged for 30 min. a t '10,000 G ,
Recipients received
10
0.25 ml of the supernatant liquid I .V .
Adoptive Transfers:
A. )
Whole- cell suspensions.
Single cell suspensions were prepared from the spleens
or bone marrow of adult male and female BALB/c mice (43).
Alternatively, single cell suspensions were prepared from
fetal livers (60) harvested from 14-day old fetal BALB/c
donors.
All cells were washed once in chilled PBS and resus­
pended in PBS to the desired concentration.
Cells.were
counted in a Neubauer Hemacytometer and assayed for viability
by a trypan blue exclusion test (18).
B.)
'■
Fractionated cell suspenions. .■
Some spleen cells were treated in various ways to either
enrich or deplete a certain cell type.
Depletion.of B cells
was done by treatment of the cell suspension with goat anti-,
mouse itiu heavy chain (CappeI Labs' Inc., Cpchranville, P A ) :
T cells, were depleted by treatment of the cell, suspension
with goat anti-mouse theta (obtained through the courtesy.of
Dr. Ray Daynes, University of Utah, Salt Lake City, UT).
In
both cases, cells were incubated with, a 1:20 dilution, of
antiserum at 4°C for 60 miri,- followed by extensive washing
and then,addition of a 1:10 dilution of rabbit complement at
37°C for 60 min.
Percent killing was determined by, a trypan
11-
blue exclusion test (18).
Enrichment of B cells (37) was done by allowing spleen
■
cell suspensions to settle onto polystyrene petri dishes
coated- with goat anti-mouse mu chain (Cappel Labs Inc.,
Cochranville-) PA) .
Noriadherent cells were siphoned off and
adherent cells (including B cells) were detached from the ■
petri dish surface by treatment with xylocaine.
Various tests were used to evaluate the success.of each
enrichment or depletion
procedure.
An indirect immunefIour-
escent stain (19) using fluorescein isothiocyanate conjugated
-goat anti-rabbit immunoglobulin,
and either rabbit anti-mouse
brain or rabbit anti-mouse immunoglobulin (Cappel Labs,
Cochranville,. PA), was used to assess the percentage of T or
B cells, respectively, iri a cell population.
A technique
■described by Morrison and Cutler (50), utilizing a Giemsa'
stain to identify those cells which had phagocytized killed
Candida albicans', was used to assess the percentage, of phago­
cytes- in a cell population.■
Antigens and Immunizations:
Sheep erythrocytes (SE) were obtained from the ColoradoSerum Co., Denver, CO.
Mice were immunized I .V . with 0.25
ml of a 10% suspension of washed SE in PBS. .
Polyvinylpyrrolidone (PVP) K90 (GAP Corporation, New
[
12
Y o r k , NY) used for. immunization, had an average molecular' 1i
weight of 360,000 d. . PVP K90 was dissolved in PBS at a con­
centration of I microgram/ml and m i c e .received 0.25 ml (0.25
micro grams of P V P ) of the, suspension I .V .
PhenoI-extracted lipopolysaccharide
(LPS) from E . coli
0113 and phenol and butanol-extracted- LPS from E . coli K235
were obtained through the courtesy of D r . 'J . A. Rudbach, De­
partment of Microbiology, University of Montana, Missoula,
MT.
Mice were immunized'I .V . with 10 micrograms LPS in 0.25
ml PBS.
'
Brucella abortus atraih 19, purchased from Jensen-Salsbury Laboratories (Kansas Ci t y ,; MO), was heat-killed (29)
.and coupled to 2,4 ,'6-trini trophenol "
■(TN P ) by the method
described by Mond _et aJ (46).
Washed TNP-B. abortus .was
suspended in PBS at a concentration of 5 x 10® cells/ml and.
0.20 ml;of..this suspension was injected I .V. into mice,
.Plaque Assays:
Plaque-forming cells (PFC) specific for SE, P1V P , LP S ,
or TNP were detected by a slide modification of the localized
hemolysis-in-gel technique (44).
The P E G ■response specific
for SE, P V P , or LPS was measured on day 5. post-immunization,
while.the PFC response elicited by TNP-B. abortus and speci-'
■fid for TNP was measured on day 4, post-immunization.
13
The procedures for coating SE with PVP' (32) or LPS (53)
have- been described elsewhere.
SE coated with TNP by the
method described by Mishell (43) were used to test the mag­
nitude of the anti-TNP response in mice' immunized with.-TNPB . abortus.
Statistics:
Results were expressed as the arithmetic mean of PFC/
spleen or PFC/106 splenocytes.
Differences in arithmetic
means were determined to be significant only if the P values
were less than 0.05 as assessed by the Student's, t test.
RESULTS
PVP and' SE Responses in Irradiated Mice
Before expanding on the work done by Brooks (7), in. '
this lab', it was first necessary to confirm his original ■
observation.
He found that, mice showed a dichotomy in the.
recovery from irradiation in their immune responses to vari
ous TD and TI antigens,.
His experiments showed that the.
immune responses of irradiated mice to SE and DNP-ova'lbumin
(TD antigens) recovered to,normal levels.within 30 days
whereas the immune responses to PVP-, type 3 pneumococcal
polysaccharide, and DNP-f icoll (TI-2 antigens) did not re-­
cover to normal levels,within 30 days.
As did Brooks, I irradiated mice with 550r, allowed
them to.recover for 30 days, immunized them with either SE
or P V P , and assayed their spleen cell PFC responses 5 days
later with a hemolysis-in-agar technique.
Table I illus­
trates 2 experiments which show that the PFC responses of
irradiated mice against SE, 30 days post-irradiation, had
recovered to normal values, but irradiated mice immunized
with,P V P , 30 days post-irradiation, did not show normal PFC
responses against P V P .■
I
15
Table I.
Direct PFC responses of mice to SE or PVP 30 days
■ post-irradiation
Immunogen ■ .■550ra
.Experiment'#1.
. . '.
V
SE
+
SE
PFC/Spleenb
%
of
Normal
PFC/lO-6b
96,562.5
v
(16,365.3)
100
119.8 ,
(30.4)d
68,916.7
(53,204.5)
' 71.4
134.8
, '112.5
(101.1) '
7036.3
(1923.4)
PVP
+
2270.8° ,
(732.0)
-
107)500
(38,167.9)
+
74,887.5.
(24,731.3)
PVP'
■—
10602.5■ '
(2724.2)-
PVP .
+
PVP
' '100
%
of .
Normal
27.8 .
(9.8)d
32.3
100
100
37.5
Experiment #2.
SE
SE
-.
'‘
'
100
69.5'
100
11.5
1223.3 .
(995.7)
376.4
(194.4)°
100 ■
474.4
,
(208.9)
126.0
57.1
(17.D d
100
19.0
..
(19.6)d
.33.2
aMice received 550r 6<^Co whole-body irradiation on day 0.
h)
'
'
Arithmetic means of 5 mice determined 5 days after immuni­
zation .
^Significant .difference.from normal (p less' tha,n .05).
d
Standard deviation.
-
16
Duration o f ■Radiation-Induced Suppression of Anti-PVP'
Responses
Brooks (7) showed that the anti-PVP responses of mice
could be suppressed for at least 60 days by administration
of 550r whole-body irradiation.
I w i p h e d to see i f 550r
could suppress the anti-PVP responses of mice for even longer
periods of time.
•
1
Table 2 shows 2 experiments in which the
anti-PVP responses .of normal mice were compared to the antiPVP responses of mice irradiated either 30 or 120-135 days
before immunization.
In experiment #1, all mice were the
same age at the time of the.plaque assay, and therefore at
different ages at the time of irradiation.
In experiment #2,
ail mice were 8 weeks old at the time of irradiation, and
therefore at different ages at the time of the plaque assay.
This experiment was done both ways because the magnitude of
the responses to TI antigens has been shown to vary with the
age of the mouse (16,46,47,51)', and radiation may not have .
the same effect on mice of different ages (56).'
However,
Table 2 shows that'regardless of the experimental design, the
anti-PVP responses' of irradiated mice remain suppressed at
least 120 days .after irradiation.
•Table 2.
Long term suppression o f anti-PVP responses by
sublethal irradiation.
Treatmenta
Group-'
PFC/Spleenb
%
of • PFC/IQ6b
N o r m a l ,,
#
of
Normal
d
Experiment #1•
Normal
550r day-30
550r day-120•
21,435.4
(5963.0)
3922.9C
.(2625.4)
7454.2C
(3157.1)
TOO
18.3 1
116.9
(59.7)
20.5°
. (14.5)
' . 34.8
37.5C
(22.8)
100
88.5
(31.9)
'■ 100
17.5
32.1 .
Experiment #2e
Normal-30
550r day-30
20,575 1
(6 361 .0) ..
■ 6405.8
(2204.2
Normal-I35
31,982.5 ■
(6088.7)
550r day-135
9603.3
(2381.5)
31.1
100
30.0
51.8
8.3
144.1
' (42.4)
57.9
(15.4)
100 ■
58.5
TOO
.
40.2
aIrradiated mice, received 5 5Or ^ C o who Iembody irradiation
either 3Q or 120 days before immunization with P V P .
b'’
•
.
Significant difference from unirradiated mice (p less than
.05).
0All mice were the same age at the time of the plaque assay.
aAll mice were the same age at the t i m e .of irradiation.
18
Dose Response Curve to PVP
Because radiation has been s h o w n .to sometimes, lengthen ■
the lag phase of antibody production (4), Brooks ran an ex­
periment to establish that day 5' post-immunization is the
day .of optimal anti-PVP response for both irradiated, and
.
normal mice.
It was also possible that irradiated mice were not being
immunized with the optimal dose of P V P .
Lake and Reed (33)
showed that mice were responsive only within a narrow dose
range of P V P .
injection of 0.1 micrograms to 1.0 micrograms
of P V P :produced a good anti-PVP PFC response whereas injec­
tion of doses of PVP outside this range resulted in paraly­
sis of the anti-pyP- PFC .response.'
To test the possibility
that radiation may.have caused a shift in the optimal dose
'.of PVP for immunization,
Figure I.
I ran the experiment illustrated in
Irradiated or control mice received O'.01, 0.25,
or 10 micrograms op P V P . . The dose response curves to PVP for
irradiated and normal mice were similar; the optimal dose.of
PVP for immunization, was 0.25 micrograms ,for both normal and
irradiated mice.'
This experiment was repeated once and the
results were comparable.
Therefore, I have concluded that
550r whole-body.irradiation did not cause a shift in the
optimal dose of PVP for immunization.
19
'
-
Figure I.
Dose response curves of normal (NC) and irrad­
iated (550r) mice to P V P , immunized 30 days after receiv­
ing 550r 60Cb whole-body irradiation.
Each value repre­
sents the arithmetic means of 5 mice determined 5 days
post-immunization.
a
PFC
□ NC
DOSE
OF
PVP
(10 6 g)
SSOr
21
Radiation-Induced Suppression of the- Antl-PVP Responses in
BALB/c, DBA/2, and C57B1/6 Mice
Having shown that the lack of recovery from irradiationof the anti-PVP responses of mice was a very real and long
term phenomenon,, I next decided to show that this phenomenon
was not unique to one strain of mice.
All of Brooks work
(7), and most of my work, using radiation to suppress speci­
fic antibody responses was done using BALB/c mice.
Figure 2
illustrates .that prolonged suppression by 5 5 0 r ,whole-body
irradiation of the responses to PVP is not unique to BALB/c
mi c e .
BALB/c, DBA/2, and C57B1/6 mice were irradiated with
550r and allowed to recover for .30 days.
Irradiated and
normal mice were then immunized with PVP., and their anti-PVP
PFC' responses were compared 5 days later.
Irradiated mice
o f , a l l '3 strains exhibited significant"(p less than .05) sup­
pression of their anti-PVP PFC responses 30 days post-irrad­
iation, as compared to the unirradiated controls.
The ex­
periment ,illustrated in Figure 2 was repeated once with com­
parable results.
Irradiation of Young Mice
Since the variety of the mice had little effect on the
recovery of their immune responses to PVP following radia­
tion, I next decided to see if the age of the mice at the
22
:-1
-
:'
,■
Figure 2. ' Responses .of normal and irradiated BALB/c,
DBA/2, and C57B1/6 mice to P V P .
days after receiving 550r
60
Mice were immunized 30
Co whole-body irradiation.
Open (PFC/lO^ splenocytes) and closed (PFC/spleen) bars
represent the arithmetic means of 5 mice determined 5
days ,,post-immunization, expressed as the percent of the
arithmetic mean of 5.unirradiated control mice of the
corresponding strain.
PFC/S
□
PFC/10
OF CONTROL
■
BA L B /c
D BA /2
MICE
C 57B I/6
24
time of irradiation could be a factor.
Quintans and Kaplan
(56) reported that young mice (13 days old), unlike adult
mice '(.6-12 weeks did) , fully recovered their PFC responses
against TNP-ficoll, TNP-dextran, and PnCs (a phosphorylcholine containing C-polysaccharide of Cs-encapsulated
■
Streptococcus pneumoniae strain), all TI-2 antigens, 6 days
after 500r whole-body irradiation.
In an attempt to demon­
strate, a similar phenomenon in our system, I irradiated
young (13.days old) and adult (7-9 weeks Old) mice with 550r
and tested their immune responses against PVP 30 days later.
Table 3 illustrates 2 experiments which show that young mice
did, indeed, recover from radiation better than adult mice.
Young Irradiated mice showed anti-PVP PFC levels equivalent
to normal mice of their age while adult irradiated mice ex­
hibited the characteristic suppression of their ant L-PVP PFC
responses.
,
PFC Responses of Irradiated Mice to TI-I Antigens
Collectively,' the experiments done by Brooks and myself
describe a distinct difference in the.'abilities of murine
PFC responses.to TI and TD antigens to recover following 550r
■whole-body irradiation.
However,
all of the TI. antigens
tested by Brooks and myself up to this time had been TI-2
antigens.. Therefore,* it was necessary to test the PFC
25
Table- 3.' Responses to PVP of normal and irradiated young
. (14 days old) .and adult (7-9 weeks old) mice .
Group
550r a
PFC/Spleenb
%
of
Normal
PFCZlO6b
%
of
Normal
Experiment. #1
Adult
-
Adult
+■
12,130 ■ ■
(3624.6)
.
2367.5a
(1170.8)
Young
—
4913.3
(2220.2)
Young
+
5594.2
(4741.3)
100
19.5
94.5
(45(6)'
100 . .
34.4^
(15.9)
36.4
100
41.0
(12.8).
- 113.9
37.3
(21.4)
: 100
64.6
(36.1)
'
100
90.1
Experiment #2
Adul t
-
7388.8
(2667.7)
Adult ■
+
2515:0
(1337.5)
Young
-
3660
(873.5)
Young
2560
(1483.8)
34.0
■loo
16.2
(5.9)
26.0
(14.9)
69.9 . 17.7
(13.4)
100
25.0 '
100
68.1
aMice received 550r ^^Co whole-body irradiation- on day 0.
b
'
Arithmetic means of 5 mice determined 5 days after immuni­
zation with P V P . .Mice were immunized on day 30. Numbers
in parentheses represent standard deviation.
^Significant difference from unirradiated mice of same age
(p less than .05) .'
'■ '
26
responses of irradiated mice, 30 days post-r-irradiation,
against TI-I antigens.
Brooks (7) immunized normal and
irradiated mice, 30 days post-irradiation, with LPS 0113
and reported that the anti-LPS PFC responses of irradiated
mice recovered within 30 days.
Since he believed his LPS
preparation to be a TI-I antigen, he concluded that there
was a dichotomy in recovery of PFC responses to TI-I and
TI-2 antigens after irradiation.
However, I was unable to
confirm this observation using the same LPS preparation
(0113).
Table 4 illustrates 2 experiments using LPS 0113
to immunize normal and irradiated mice 30 days post-irrad­
iation.
I found the anti-LPS 0113 PFC responses
of irrad­
iated mice to be significantly (p less than .05) lower than
normal mice in both experiments.
I believe my interpreta­
tion of the data to be correct because I have since learned
that the LPS 0113, used by Brooks and myself, was extracted
with phenol, and phenol extraction of LPS removed lipidassociated protein (LAP), thus making phenol extracted LPS
a TI-2 antigen (16).
I therefore decided to test the responses of irradiated
mice against other LPS preparations.
butanol extracted E . .coll K235 L P S .
I used phenol and
Butanol extraction,
does not remove LAP from L P S , thus butanol-extrapted LPS is
27
Table '4.
Immunogen
Responses of normal and irradiated mice to PVP
and LPS 0113.
SSOra PFC/Spleenb
Experiment #1
—
PVP
+. .
LPS 0113
9255.0
■
(1825.0)
'• 100
.2000.O^
(1989.4)
21.6
19540
(19,791.1)
'+ •
2750.0°'
(1640.5)
•
Experiment #2.
%
of
Normal
100
14.1
..
47.6
(14.6)
100
10.6°
(8.0)
22:2
100.9 ■'
(94.0)
19.1°
(10.9)
100 •
18.9
.
10,700
(5218.2)'
PVP
PVP
■PFC/I06b
..
PVP .
LPS 0113
' of
Normal
3984.4° '
(1024.2)
■+
LPS 0113
—
12662.5
(6673.1),
LPS 0113'
+
2537,5° ,
(823.7)
100
37.2
' 100 '
20.0
249.5
(114.7)
100
21.3°
(5.9)
8.5
79.5
(30.7)
16.6°
(7.3)
100
-
20,.9
^Mice received SSOr ^?Co whole-body irradiation on day 0.
b
Arithmetic means.of 4-5 mice determined 5 days after im­
munisation with PVP or LPS 0113 (on day 30 post-irradia­
tion) . Numbers in parentheses represent standard devia­
tion.
^Significant difference from unirradiated mice
.05) .
,
■
(p less than
'
28
a TI-I antigen (17).
Table 5 illustrates 2 experiments
testing the PFC responses of irradiated and normal mice
against phenol— extracted LPS (a TI-2 antigen), and against
butanol-extracted LPS (a TI-I antigen),
iation.
30 days post-irrad­
Both experiments showed that the PFC responses
against butanol-extracted LPS rebounded to normal levels
within 30 days post-irradiation while the PFC responses
against phenol-extracted LPS was still suppressed 30 days
post-irradiation.
To support the LPS data, I decided to test the PFC
responses of irradiated mice against another TI-I antigen,
namely, TNP-Brucella abortus.
Table 6 illustrates.2 experi­
ments in which normal and irradiated mice were immunized
with P V P , 30 days post-irradiation, or TNP-B. abortus 31
days post-irradiation.
As a control, in experiment #1, some
irradiated mice were immunized with B . abortus without TNP
31 days post-irradiation.
Plaque assays against either PVP
or TNP were done on all mice 35 days post-irradiation.
Mice
immunized with just B . abortus, were plaqued against TNP..
Table 6 shows that, unlike the PFC responses against P V P ,
the PFC responses illicted by TNP-B. abortus (against TNP)
did recover within 30 days post-irradiation.
Irradiated
mice immunized with B . abortus had much lower anti-TNP
29
Table 5.
Responses of normal and' irradiated mice to phenpl■ or butanol-extracted LPS K235.
'550ra PFC/Spleehb
LPS ' •
Extraction
of
Normal
PFC/106b
Experiment #1 .
Phenol
.-
Phenol
+
#
of
Normal
.
'
1715 ' .
(922.9)
' 647.5°
(505.1)
100
37.8
.
13.6
(8.5)
' 100
4.'5°
(3.5)
31.6
Butanol
—
'27558.8
(15,551.1)
100
196.2’
(122.7)
100
Butanol
+
45,459.4
(26,523.0)
165.0
320.3 '
(283.5)
163.3
2082.5.
(1279.8).
100
11.2.
(5.2)
100
Experiment #2'
Phenol
. -
517.5
(169.5)
Phenol
Butanol
Butanol
4"
15,125.0
(8903.5) 9795
(7641.9)
■ 24.8
100
. •-.
64.8
5.3
(2.9)
73.0
(39.5)
152.8
(122.5)
47.9
100
209.4
aMice received-' 550r
Co whole-body irradiation on day 0.
b
■■
Arithmetic means of 4-5 mice determined 5 days after
'.
immunization.
Mice were.immunized with phenol-.or butanolextracted L P S .K 2 3 5 .on -day 30. Numbers in parentheses repre­
sent standard deviation. .
.
^Significant difference 'from unirradiated mice
.0 5 ).
(p less than
30
Table 6.
..Responses of normal and irradiated mice to PVP
and TNP-B. abortus (TNP-BA)
Immunogen .550ra PFC/Spleenb .
#
of.
Normal
PFCZlO6b ' .%
of
Normal
■
Experiment #1
PVP'
-
10,153.1.
(1377,2)
PVP
+
2777.5°
(1274.1)
TNP-BA
—
163,210
(58,103
TNP-BA
■+
122,165
(37,525)
+
2970.
(1163.4)
BA
Experiment. #2 .
PVP
■
PVP
27.4 .18.0° •
(11.6)
100 '
74.9
21.3
1115.8'
.(718.3)
100
1862.4,
(1803.3) •
169.9
.
12.0
(7.9)
"
10,602.5
(2724.2)
+
1223.3
(995.7)
: 11 ;5 ■ 19.0
(19.6)
99,070
(59,598)
100
+
100
84.6
(17.1)
-
TNP-BA
TNP-BA
100 .
63,490
(15,405.4)
. 100
: . 64.1
57.1
(17.1)
■616.4
(319.0)
464.7.
(117.7)
■ 100
33.2
' 100
.
75.4
aTWice received 550r ^ C o whole-body irradiation on day 0.
^Arithmetic means of 5 'mice immunized with PVP on day 30 .or
with TNP-BA on day 31.' PFC responses against■PVP and TNP
■ were determined on .day 35. Numbers'in parentheses repre­
sent standard deviation.
’
^Significant.difference from unirradiated mice (p less than'
.05).
'
.
31
responses than did irradiated mice immunized with TNP-B.
abortus.
This confirms the fact that the, anti-TNP PPG re­
sponses of irradiated mice immunized with TNP-B. abortus
were a demonstration of immunity against T N P , and not just
measures of polyclonal activation.by B ..abortus.
The findings that the 'PFC responses of irradiated mice
against TNP-B.' abortus and butanol-extracted LPS rebound
.
within 30 days post-irradiation, while the PFC responses of'
irradiated mice against PVP and phenol-extracted LPS remain
suppressed 30 days post-irradiation, indicate that .there is
a dichotomy in recovery of PFC responses to TI-I and TI-2
antigens.following irradiation. ' This contention is sup­
p o r t e d Wy Brooks' work (?) which showed that irradiated
mice did not:recover their PFC responses against type 3
pneumococcal polysaccharide or DNP-ficolI (both TI-2 anti-r
gens) within 30- days posb-irradlat:ion,.
•
Irradiation of Nude Mice
‘
The inability to recover from sublethal irradiationwithin 30 d a y s .is then unique to PFC responses -against TI-2
antigens ,(as opposed to PFC responses against TD or TI-I
antigens') . -The next question .to ask is then:
What is the
mechanism of this ihability of anti-PVP responses to recover
post-irradiation?
T cells have been shown to have some
32
effect on the PFC responses of mice against TI antigens ('6
33,47-49,59),
and so radiation-induced evolution of suppres­
sor T cells was. a possibility.
Tb investigate this possi­
bility, nude and ELM mice were given either 550r (experiment
#1) .or 400r .(experiment #2} whole-body irradiation and their
anti-PVP PFC responses were tested 30' days after irradiation.
The experiments illustrated in Table 7 show that the antiPVP responses,of both ELM and nude mice did not recover
within 30 days.
These experiments indicate a lack of T cell
involvement in the inability of the mouse's anti-PV? response
to recover from radiation.
Repair of Irradiated Mice With Whole Cell Preparations
Brooks (7) showed that the anti-PVP responses of irrad­
iated mice could be restored to normal levels if they, were
reconstituted with spleen or bone marrow cells 7 days after
irradiation,
that Is, 23 days before immunization with P V P .
Figure 3 illustrates an experiment in which I attempted to
repair the anti-PVP responses of irradiated mice with adop­
tively transferred,normal spleen cells at a later time.,
allowing less time between irradiation and immunization.
7
Irradiated and normal mice received 5 x 1 0
normal spleen
cells (in 0.25 ml P B S , I .V .) either 3 dr 5 days before
33
Tablo 7.
Mouse
Group
Rcnponses of normal and irradiated atbymio (Nude)
mice and.their euthymic littermates (ELM) to P V P .
550ra
PEG./ Spleenb
#
of
Normal
PFC/I06b
%
of
Normal.
Experiment #1
Nude
-
6932.5
(3468:9)
Nude
+
1265.6°
(576.2)
ELM
—
8055
. (2308.9)
ELM
2220.0
(722.3)
100
23.6
(14.3)
100
32.2
18.3 '7.6°
(6.4)
100
27.6
42.9
(22,2)
• 100
17.5
(9.7)
40.8
Experiment #2
Nude
-
3156.3
(414.6) ■
Nude
+■
356.3
(125.4)
ELM
-
1921.9
(567.2)
ELM
+
828.1
(272.4)
100
11.3
100
43.1
22.4 •
(16.9) '
100
3.0
(.1.9)
'13.2
17.3
(5.0)
5.0
7.0
(1.2)
40.5
aMice'received 550r (Exp. #1) or 400r (Exp. #2)
body irradiation on day O .
b
60 ■
Co whole-
Arithmetic mean of 5 mice determined 5 days after immuni­
zation.
Mice were immunized with PVP on day 30. Numbers
in parentheses represent standard deviation.
^Significant difference from non-irradiated mice
than .05).
(p less
34
Figure 3.
Repair of the responses to PVP of irradiated
mice with normal spleen cells.
■
'
"
■ ■ ■■•
Normal control (C) and
..
irradiated (I) mice were repaired (R) with 5 x 1 0
7
nor-
mal spleen cells dither 3 or 5 days before immunization.
Mice were immunized with PVP 30 days after receiving
550r ^ C o whole-body irradiation.
Open (PFC/10® spleno-
cytes) and closed (PFC/spleen) bars represent the arith­
metic means of 5. m i c e , determined 5 days' post-immuniza­
tion as percent of the arithmetic mean of 5 unrepaired ■
■normal control m i c e .
GROUPS
36
■immunization with P V P , 30 days post-irradiation.
seen in Figure 3, 5 x 1 0
7
As can be
normal spleen cells, given 5 days •
before immunization, repaired the anti-PVP responses of
irradiated mice to hear normal levels.
5x10
7
Adoptive transfer of
■
normal spleen cells 3.days before immunization, also
produced significant (p less than .05) repair, although not
quite as impressively as when transferred 5 days before
immunization.
The fact that normal spleen cells can repair the a n t i - ■
PVP PFC responses of irradiated mice when given only 5 days
before antigenic challenge.is even more impressive when one
looks.at the experiments illustrated in Table 8.
In these
7
experiments, normal and irradiated•mice received 5 x 10
live spleen cells 5 days before immunization with. P V P , or
7
alternatively, received 5 x IQ live fetal liver cells, from
■14 day old fetuses j either 21 or 5 days before immunization
with PVP (30 days post-irradiation).
Fetal liver cells were
found to be every bit as capable as adult spleen cells of
repairing the ahti-PVP responses of irradiated mice.
■Passive Repair of Irradiated Mice
,It was possible that donor cells repaired th e ,anti-PVP
PFC responses of irradiated mice by elaboration of some fac­
tor, whi c h .m a y •have induced the maturation.of resident.B
37
Table 8.
Anti-PVP'responses of irradiated mice repaired
with adoptively transferred normal adult spleen
(AS) or fetal liver cells(F L ).
Donor
550r
Cells (Day)
PFC/Spieena
#
of
Normal
PFC/IO6a
%
of
Normal
Experiment #1
Nonrepaired
—
Nonrepaired
9232.5
(2079.1)
100
2635b
(1213.4)
28.5
FL (6)
+
12,665.8
(7407.7)
137.2
FL (22)
+
6309.4
(1548.9)
AS (22)
-
54.2
(15.9)
100
23.4b
(10.5)
43.1
154.6
(103.4)
285.2
68.3
112.1
(57.9)
206.7
11,869.8
(2191.1)
128.6
57.7
(15.8)
+
50250
(2726.2)
54.4
.Nonrepaired
-
10,550
(3457.9)
Nonrepaired
+ • 2345.I
(1193.4)
22.2
18.7
(5.2)
FL (22)
+
7713.5
(3102.9)
73.0
52.6 ‘
(18.1)
AS (22)
4-
7900.4
(2735.8)
74.9
51.0
(20.6)
AS (22)
46.2
(22.1)
106.5
'
85.3
Experiment ,#2
100
65.6
(12.2)
100
28.5
. 80.2
77.8
^Arithmetic means of 5 mice determined 5 days after immuni­
zation with PVP (on day 27).
Irradiated mice received 550r
60 q o whole-body irradiation on day 0.
Some mice received
5 x 10? fetal'liver or adult spleen cells on day 6 or day
22. Numbers shown in parentheses represent standard devia­
tion.
^Significantly less than (p less than .05) normal Unrepaired
mice.'
38
cell precursors, or which may have, somehow, repaired radia­
tion damage .to resident mature B cells.
possibility,
To investigate this
I attempted to reconstitute irradiated mice
with serum or spleen cell lysates from normal mice.
Irradi­
ated mice received 0.25 ml of undiluted serum or 0.25 ml of
the cell lysate from 7.5 x 10
7
spleen cells in PBS, from
normal or irradiated mice, 5 days before immunization with
P V P . ' Tables 9 and 10 illustrate 2 experiments, which exem­
plify 6 experiments (4 with serum and 2 with cell lysates), ■
in which I tested the anti-PVP responses of irradiated mice
that had received serum or spleen cell lysates from normal
or irradiated mice.
Although I observed some repair of the
anti-PVP responses from serum and cell lysate, the repair
Was not consistent and never as much as that bestowed by
live spleen cells."
These data do not necessarily disprove
the existence of some type of repair or maturation factor,
but they do indicate that if such a factor does exist, then
its half-life is too short to make it readily detectable.
Repair, of Irradiated Mice With Fractionated Cell Preparation
Therefore,
it appeared that live cells were needed to
repair the anti-PVP responses of irradiated mice..
I next
attempted to' characterize- the cell type that was responsible
for repairing the response to PVP or irradiated mice. ' I
39
Table 9.
Anti-PVP responses of irradiated mice repaired
with serum from normal or irradiated mice.-
TreatmentReceived
PFC/Spleenb
Normal
Unrepaired
7745.Oc
(1329.1)
550ra
Unrepaired
1060.0
(453.7)
96
of
Normal
. 100
'
PFC/106b
41.7°
(9.2)
%
of
Normal
100
13.7
10.3
(3.6)
24.7
SSOra-Serum from 2265.0
(1895.0)
normal mice
29.2
25.6
(25.9)
61.4
SSOra-Serum from 2535.0
irradiated mice
(1885.7)
32.7
24.0C
(16.1)
57.5
2057.5
(1358.6)
26.6
19.3
(15.4)
46.3
SSOra-PBS
aMice received 550r ^ C o whole-body irradiation o n .day O .
^Arithmetic means of 5 mice determined 5 days after immuni­
zation.
Some mice received 0.25 ml P B S , or 0.25 ml of
serum from normal or irradiated mice on day 26. Mice were
immunized with PVP on day 31. Numbers in parentheses
represented standard deviation.
cSignificantly .(p less than .05) more than irradiated non­
repaired mice.
40
Table '10.'
Anti-PVP responses of irradiated mice repaired
with spleen cells- or spleen cell lysates from
normal or irradiated mice.
TreatmentReceived
NormalNonrepaired-.
550ra
Nonrepaired
• EEC/Spleenb
%
of
Normal
8657.5
(2736.9)
.2242.5
(557.6)
550ra-Normal
spleen cells ■
4410° .
(1512.8)
SSOra-Lysate
from normal
spleen cells
962.5
(313.1)
SSOra-Lysate
. 2415.0
from irradiated
(1632.3)
spleen cells
100
.'
.
PFC/I06b
51.2
'(9.6) .
%
of
Normal '
100
. 25.9
18.8
(5.4)
36.7
50.9
36.5C
(8.4)
71.3
11.1
9.3
(2.4)
18.2
27.9
14.7
(9.8)
28.8
'
aMice received SSOr b^Co whole-body irradiation on day 0.
^Arithmetic means of 5 mice determined S days after immunization with P V P . Mice were immunized on day 30.
Some mice
received 5 -x 10? normal spleen cells or the lysate of 5 x
IO^ normal .spleen cells on day 25. Numbers in parenthesesrepresent standard deviation.
cSignificantly (p■less t h a n -.05) more than irradiated non­
repaired mi c e .
41
attempted this using both enrichment and depletion tech­
niques.
Spleen cell suspensions were depleted of T or B
cells using'anti-theta and complement (Ct) or anti-mu and
C 1, respectively.. Irradiated mice received 5 x 1 0
7
live
spleen cells that were depleted of T cells., depleted of B
cells, or non-treated.
In the experiment illustrated in
Figure 4, non-treated spleen cells and spleen cells treated
with anti-theta and C 1 were able to repair the anti-PVP
responses of irradiated mice, but spleen cells treated with
anti-mu and C 1 were n o t .
This experiment was duplicated
once and the results were comparable.
Since the cell type
responsible for repairing the anti-PVP responses of irradi­
ated.mice was sensitive to anti-mu and C 1, I deduced the
probable identity of this cell type to be a B cell.
To corrobate this deduction,
I enriched for the cell
type responsible for the repair of the anti-PVP response
using a technique known as panning (37,39,73).
In this
case, panning means to allow a suspension of spleen cells
to rest in a petri dish that has been coated with goat anti
mouse mu chain.
After Ih at room temperature,
the non­
adherent cells can'be siphoned off and the adherent cells ■
can then be removed by treatment, with xylocaine H C l . ■ Pan­
ning, then, when used in this manner, would select for mu-
42
Figurev. 4. /. Repair of the responses to PVP of irradiated
mice with normal spleen cells treated with antiserum.".
Irradiated mice,
25 days after receiving 550r
60
Co whole-
body irradiation, received no cells (I), or received 5' x
7
IQ normal spleen cells that were not treated (NS), de­
pleted of B cells by treatment with anti-mu and C 1 (S-B),
or depleted of T cells by treatment with anti-theta and
C 1.
Open (PFC/106 splenocytes) and closed (PFC/spleen)
bars represent the arithmetic means of 4-5 mice, deter­
mined 5 days post-immunization,
expressed as percent of
the arithmetic mean of 5 normal unrepaired control mice.
■ PFC/S
□ R F C /10
GROUP
44
bearing cells, such as B cells..
Figure 5 illustrates an example of an experiment in
which irradiated mice were reconstituted with spleen cells
that were:
non-fractionated, non-adherent to non-coated
petri dishes, adherent, to anti-mu coated petri dishes, or''
non-adherent to anti-mu coated petri dishes.
As seen, in"
Figure 5, the irradiated mice reconstituted with spleen
cells that were non-fractionated, non-adherent to n.on-coated
petri dishes, or adherent to anti-mu coated petri dishes had
anti-PVP PFC responses Significantly higher (p less than
0.05) than irradiated non-repaired mice or irradiated mice
that received cells that were non-adherent to anti-mu coated
petri dishes.
This experiment was repeated once and the
results were comparable.
Therefore, the cells that were
responsible for repairing, the. anti-PVP responses of irradi­
ated mice bound to anti-mu a n d .Were probably B cells.
■ Figure 5.
Repair of the responses to PVP of irradiated
. mice with fractionated normal spleen cells.
Irradiated
mice, 25 days after receiving 550r 60Co whole-body irradiation, received no cells (I), or received 5 x 10
nor­
mal spleen cells that were non-fractionated (S) , nonadherent to uncoated petri dishes (NA-P), adherent to
■ant !/-mu' boated petri dishes (A-MU) , or non-adherent to
anti-mu coated petri dishes (A-MU) .
S consisted of
87.4% immunoglobin (Ig) + cells, 31.7% theta + cells,
and 12. 1 % -phagocytes!; NA-P consisted of 38.1% Ig +, ■
-cells, 35\2% theta* + cells, and 8.8% phagocytes.
A-MU
consisted of 87.4% Ig + cells, 0.8% theta + cells, and
4.9% phagocytes.
NA-MU consisted of 1.0% Ig + cells,
71.3% theta > cells, and 5,.'8% phagocytes.
Open (PFC/10®
splenocytes) and closed (PFC/spleen) bars represent the
arithmetic means of 4-5 mice, determined 5 days postimmunization ,' expressed as percent of the arithmetic
r.
mean of 5 normal unrepaired control mice.
100
■P FC /S
□ R F C /10
80
60
O
O
Li. 4 0 O
*
20
0
-
NA-P A-MU
GROUP
NA-MU
DISCUSSION
Brooks (7) made the original observation that mice
given 550r whole-body irradiation and allowed to recover for
30 days responded with normal PFC responses when immunized
with SE or DNP-ovalbumin (TD antigens) but responded with .
significantly .suppressed (p less than .05) PFC responses
when immunized with P V P , DNP-ficoll, or type 3 pneumococcal
polysaccharide .(TI antigens).
In this thesis research, I first confirmed (Table I)
the original observation made by B r o o k s A
dichotomy in the
recovery following irradiation of the PFC responses against
SE and P V P .
It was then necessary to show that this dichot­
omy in recovery from irradiation was an actual phenomenon
and not an experimental artifact. . Brooks (7) showed that
his plaque assays of the PFC responses of irradiated mice
against P V P , which were performed on day 5 post-immunization
were done on the optimum.day post-immunization. 'My work
(Figure I) has shown that the dose of PVP (0.25 micrograms) •
used by both Brooks and myself was the optimum dose of PVP
for immunization even in irradiated mice.
Brooks (7) showed
that the inability- of irradiated mice to regain normal antiPVP PFC responses lasted for at least 60 days and I have
extended this observation by showing that the irradiated
mice have, still not regained normal levels in their anti-
48
PVP PFC responses by 120 days post-irradiation (Table 2).
Therefore,
the. radiation-induced suppression of the anti-
PVP responses in mice is certainly not a short term phenom­
enon.
In addition, I have shown (Figure 2) that this phe­
nomenon was not unique to the strain used by Brooks (BALB/c)
Secure.in the knowledge that the dichotomy in the re­
covery following irradiation of the PFC responses to the TD
a.nd the TI antigens tested was a very real phenomenon, I
decided to expand these observations.
Since all 3 TI anti­
gens tested by Brooks were TI-2 antigens,' I decided to in­
vestigate the recovery of PFC responses, to TI-I antigens
following irradiation.
Tables 4 and 5 illustrate experi­
ments showing that the PFC responses against butanol ex­
tracted LPS and TNP-B. abortus (TI-I antigens) rebounded to
normal levels within 30 days post-irradiation, but the PFC
responses against phenol-extracted LPS and PVP (TI-2 anti­
gens) remained suppressed 30.days post-irradiation.
The observation that sublethally irradiated mice re­
sponded normally to TD and TI-I antigens is reminiscent of
normal immature and CBA/N mice.
Normal immature and CBA/N
mice mount antibody responses comparable to those produced
by normal mature mice against TI-I antigens but not against
TI-2 antigens (2,16,46,47,51,52).
Normal immature and CBA/N
49
mice also vary.from normal mature mice in the antigenic
moieties found on their spleen cell surfaces. .■ Spleen cells
from normal adult mice have a higher surface IgD to surface
IgM ratio than do spleen cells from normal immature or CBA/N
mice (52,62,65a,67), and spleen cells from normal immature
and CBA/N mice lack antigens (Lyb-3, Lyb-5, Lyb-7) found on
the spleen cells of normal mature mice (1,6,47,52,67).
This evidence leads to the conclusion that there exist
two subpbpulations of B^ cells.
One subpopulation (B^-2)
is found only in normal adult, mice, responds to TI-2 anti­
gens, has a high surface IgD to surface IgM.ratio, and bears
Lyb-3, Lyb-5, Lyb-7; the other 'subpopulation (B^-I) is found
in normal adult, normal immature, and CBA/N mice, responds
to TI-I antigens, has a low surface IgD to surface IgM ratio
and does not bear Lyb-3,'Lybi-S, or Lyb-7.
The lack of B^-2
cells found in CBA/N mice, has been shown to be an X-Iinked
defect. • The properties of B^-I and B^-2 cells are summar­
ized in Table 11.
Sublethally irradiated mice, 30 days post-irradiation,
were analogous to CBA/N mice in their normal responses to
TD and TI-I antigens;
therefore, it seemed logical.that the
lack of responsiveness' to TI-2 antigens, found in suble-
50
Table 11.
Properties of B^-I and B^-2 .cells.
• Characteristic
Found in:
Normal adult mice
• ■
Normal immature mice .
CBA/N mice
Responded to:
'TI-I antigens
TI-2 antigens
Spleen cells bear:
Lyb-3
■. Lyb-5
Lyb-7
B 1-I Cells .
I
+ ■
+
.+ ■
B -T2 Cells
•I
+
-
■
+
-
—*
+
•
—
-
+ '
+•
■+
—
51
thally irradiated mice, might also'have been due to a B cell
defect.
An alternative explanation could be that 'sublethal
irradiation somehow activated regulatory cells wp^cp pup-pressed the anti-PVP PFC responses of irradiated, m i c e .
How­
ever, the fact that the anti-PVP PFC responses of nude mice
were unable to.recover 30 days post-irradiation (Table 7),
and that adoptively transferred normal spleen, cells were
able to repair the anti+PVP PFC responses of irradiated
mice (Figure 3), discredit this explanation.
Since the anti-PVP PFC responses of sublethally irradi­
ated mice were repaired by adoptive transfer of spleen cells
from normal animals, but not by spleen cell lysates or serum
from normal animals, sublethally irradiated mice indeed.,
appeared to lack some population of cells.
However, the B
cell was not the only possible cell type that was necessary
for a nornjal PFC response against T I - 2 'antigens and that.'was
unable to recover from irradiation within 30 days. • Radia­
tion-induced damage to T cells and/or macrophages could con­
ceivable have caused an abnormal response to TI-2 hntigens.
T cells are radiosensitive (4) and do exercise some
regulation over the PFC responses against TI-2 antigens (6,
33,47-49,59).
HoWever,. nude mice showed suppression of
52
anti-PVP PFC responses, 30 days post-irradiation,, comparable
to that found in their euthymi'c littermates (Table 7). ■ In
addition,
adoptive transfers of spleen cell preparations,
that had been depleted of T cells by treatment with anti­
theta and complement, effectively repaired the .anti-PVP re­
sponses of irradiated recipient mice (Figure 5), whereas
adoptive transfers, of spleen cell preparations,
that had
been enriched for T cells by panning, did not repair the
anti-PVP PFC responses of irradiated recipient mice (Figure
5).
Therefore, a lack of T cells was a very improbable
mechanism for the lack.of recovery of the immune responses
to PVP after sublethal irradiation.
Macrophages are somewhat radiosensitive (22,48) and are
necessary for a normal antibody response against at least
some TI-2 antigens' (6,40)..
However in the experiment illus­
trated in Figure 5 the spleen cell fraction that was adherent
to anti-mu coated petri dishes was much better (significant
difference p less than .05) at. repairing the anti-PVP re­
sponses of irradiated mice than was the non-adherent spleen
cell fraction;
furthermore ,• the non-adherent spleen cell
fraction had a higher percent of phagocytes than did, the
adherent spleen cell fraction.
Also, macrophages are known
to be much less radiosensitive than lymphocytes (4).
There-
53
fore, the cell type required for a normal anti-PVP response,
that did not;.rebound within' 30 days post-irradiation, was
probably not a macrophage.
From my observations, I suggest that the immune defect
found in sublethally.irradiated mice, 30 days post-irradia­
tion, was due to radiation-induced damage to B cells.
Adop­
tive transfers of spleen cell preparations, that had been
depleted of mu-bearing cells by treatment with anti-mu and
complement, could, not repair the anti-PVP responses of ir­
radiated recipient mice.
On the other han d , adoptive trans­
fers of spleen ceil preparations, that had been enriched for
mu-bearing cells by panning, could quite adequately repair
the anti-PVP responses of irradiated mice.
can say, with some degree of confidence,
Therefore, I
that t h e .cells that
failed to recover within 30 days post-irradiation were prob­
ably B cells, and since the irradiated mices1 responses
against TD and TI-I- antigens wbre normal 30 days post­
irradiation, the cells that- failed to recover 30 days post­
irradiation were probably B -2 cells. ■
The inability of B^-2 cells to rebound 30 days post­
irradiation may have been due .ter the loss of the precursors
for B^-2 cells or the loss of some factor-that induces the
maturation of. these precursors.
Sherr et. ad (62) found that
54
fetal liver cells, adoptively transferred into lethally
irradiated hosts, matured to antibody producing cells much
faster in the irradiated recipient than in the fetal donor. .
This led them to conclude that the reason neonatal mice do
not produce antibody responses, equivalent to those of adults
is not something intrinsic to their B cells, but rather a
lack of some factor, found in adult mice, that induces the
•maturation of' B cell precursors.
I have shown that adop­
tively transferred fetal.liver cells were capable of restor­
ing the anti-PVP PFC responses of sublethally irradiated
mice to normal adult levels when given 5 or 21 days, before
immunization,
and this has led me to conclude that the rea­
son the anti-PVP responses of sublethally irradiated mice
do riot rebound within 30 days is due to a loss of the pre­
cursors for B^-2 cells rather than the factor that induces
the maturation of these precursors.
It is not known if B 1-I and B -2 cells arise from dif-L
I . ferent precursors, or if B^-2 cells are simply a further
maturation stage of B^-I cells.
However, the observation
that resident B cell precursors, in the sublethally irradi­
ated m i c e ,. differentiated only to the B-^-I stage whereas
transplanted fetal liver donor cells -differentiated to the
B^-2 stage ,. suggests -that B1-I .
'arid B^-2 cells do come from
55different precursors.
-
Observations presented in this paper showed that.male
and female mice given 550r
6
Co irradiation, and allowed to
recover for 30 days, were deficient in B^-2 cells*, but,
apparently, had normal levels of.all other components that
were necessary for optimum PFC responses against various TD
and TI antigens.
This B^-2 cell deficiency lasted for at
least 120 days post-irradiation and could be abrogated by
adoptive repair with normal adult B cells or normal fetal
liver cells.
In addition, B^-2 cell deficiency could be
induced in various mouse strains,
it was only required that
the experimental mice be of the proper age —
old —
7 to 12 weeks
upon irradiation (Table 3 and Figure 2).
Therefore,
sublethally irradiated mice 30 days post-irradiation, com­
prised an excellent,
model.
.
in vi v o , B 1-2' .cell deficient mouse
.
There are several additional experiments that could be
done with this system.
For instance,
it would be interest­
ing to compare the mitogenic responses and fluorescence pro­
files of sublethally irradiated■to those of normal immature
and CBA/N mice.
"
Besides their lack.of response to TI-2 antigens, their
low surface IgD to.surface DgM ratio, and their lack of
56
Lyb-3, Lyb-5, and Lyb-7, spleen cells from normal immature
and CBA/N mine, vary from spleen cells from ,normal mature
mice in other.ways.
Spleen cells from normal immature and
CBA/N mice produce mitogenic responses comparative to -phose
of spleen cells from normal mature mice when stimulated with
concanavalin A and phytohemagglutinin, but not when stimu­
lated with LPS or anti-mu (42a,42b,61).
Also,
spleen cells
from normal immature and CBA/N mice show similar fluores-■
cence profiles,
from a Fluorescent Activated Cell Sorter
(FACS), when labelled with fluorescein conjugated anti-mu,
which differ from fluorescence profiles shown by spleen ■
cells from normal adult mice when labelled with fluorescein
conjugated anti-mu (62,63).
To investigate other possible analogies between sub- ■
lethally irradiated mice and normal immature and CBA/N mice,
I have communicated by findings to Donna G . Sieckmann and
her associates (Naval Medical Research Institute, Bethesda,
MD), and they have agreed to compare Sublethally irradiated
mice, 30 days post-irradiation, with normal mice in their
responses to various mitogens and antigens, in vit r o ,■and
in their FACS analyses.
These experiments are currently
underway.
Another interesting experiment would be an attempt to
57
identify the factor in adult mice that induces maturation
of the precursors for B^-2 cells by adoptive transfer of
spleen cells from sublethalIy irradiated mice into nbrmal
immature mice.
The experiments 'I have already performed,, the experi­
ments I have suggested, and many other possible experiments,
illustrate the value of sublethally irradiated mice as an
in vivo B^-2 cell deficient mouse system.
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MONTANA STATE UNIVERSITY LIBRARIES
stks N378.N463@Theses
Differential recovery o f antibody respon
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N378
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DATE
Newman, Edward L
Differential recovery
of antibody responses to
various antigens ...
IS S U E D T O
„!
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