Genetic Control of Immune Response to Sperm Whale
Myoglobin in Mice : II. T Lymphocyte Proliferative
Response to the Synthetic Antigenic Sites
This information is current asof April 25, 2011

Kenji Okuda, Sally S. Twining, Chella S. David and M. Zouhair
AtassiJ Immunol 1979;123;182-188

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Copyright ©1979 by The American Association of
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0022-1767/79/1231-0182502.00/0
THE JOURNAL OF IMMUNOLOGY

Copyright© 1979by The Williams&WilkinsCo.

GENE

Vol. 123,No. 1, July 1979
Printed in U.S.A.

C CONTROL OF IMMUNE RESPONSE TO SPERM WHALE
MYOGLOBIN IN MICE

II. T L y m p h o c y t e Proliferative R e s p o n s e to the Synthetic Antigenic Sites I
KENJI OKUDA, SALLY S. TWINING, CHELLA S. DAVID, AND M. ZOUHAIR ATASSI
From the Department of Immunology, Mayo Clinic and Medical School, Rochester, Minnesota 55901

Studies in a variety of species have revealed that the immune
response to various antigens are under control of immune
response (Ir) 2genes (1-4). Immune response to thymus-dependent antigens are controlled by genes in the I region of the major
histocompatibility complex (MHC) of mice (1). The specificityof Ix gene control for synthetic antigens and native proteins canbe distinguished at the primary sequence level of the antigen(5-8). Ix-gene defects may be expressed on macrophages and B
cells as well as T cells (9). Most of the antigens studied to datefor Ir-gene control have been synthetic polymers of a few aminoacids. Only recently, genetic studies were extended to include
several natural protein antigens such as staphylococcal nuclease(10), myoglobin (11, 12), and cytochrome c (13).

In our laboratories we have initiated studies on the genetic
control of the immune response in mice to sperm whale myoglobin and to the antigenic sites on this protein. Sperm-whale
myoglobin is made up of a single polypeptide chain (14) (153
residues, m.w. 17,816), and its entire antigenic structure has
been determined (15). The native protein has five antigenic
sites located in exposed conformationally sensitive continuous
portions of its polypeptide chain (Fig. 1). The five antigenic
sites occupy the following regions: Site 1: sequence 15 through22; Site 2: sequence 56 through 62; Site 3: sequence 94 through99; Site 4: sequence 113 through 119; Site 5: sequence 145
through 151 (15). These antigenic sites were determined byusing antibodies made against whole sperm whale myoglobin in
rabbits and goats. We wanted to know whether mice would
recognize the same antigenic sites. If they do, the availability ofcongenic and recombinant strains of mice would enable us tomore definitively study and map the gene(s) controlling theimmune response to individual antigenic sites.
In previous studies, we found that T lymphocyte proliferative
response to the intact myoglobin molecule was under H-2linked Ir-gene control (12). We identified at least two genes
mapping in I-A and I-C subregions. Our present experiments
revealed that different mouse strains recognize different determinants on the molecule, and the T cell proliferative responses
to sites 1, 2, 4, and 5 are controlled by genes mapping in the IA subregion. Additional Ix genes for sites 1 and 2 also mappedin the I-C subregion. This study does not rule out the possibility
that mice might recognize antigenic sites in other parts of themolecule not recognized by rabbit or goats. However, preliminary studies have shown that the antibodies to myoglobin madein three strains of mice (H-2 d, 1t-2 ~, and H-2 f) bind to the
expected antigenic sites. The total binding activity of the reacting sites accounted for almost 100% of the mouse antimyoglobin
antibody (Twining et al., in preparation).
MATERIALS AND METHODS

Mice. The inbred strains, the recombinants, and the F1 hyReceived for publication September 25, 1978.
brids used in this study were produced in our immunogenetics
Accepted for publication April 9, 1979.
mouse colony at Mayo Clinic. Some of the inbred strains were
The costs of publication of this article were defrayed in part by the purchased from Jackson Laboratories, Bar Harbor, Maine. All
payment of page charges. This article must therefore be hereby marked mice used in these experiments were between 8 and 16 weeks
advertisement in accordance with 18 U.S.C. Section 1734 solely toof age at the start of immunization.
indicate this fact.
Antigens. Myoglobin used in these studies was the major
Supported by grants from the National Institute of Allergy and
Infectious Diseases (AI 14764), National Cancer Institute (CA 24473), chromatographic component No. 10 (MbX) obtained by chroNational Institute of Arthritis and Metabolic Diseases (AM 18920), and matography of the cryst~llir~e protein on CM-cellulose (16).
MbX was homogeneous by starch gel and by polyacrylamide
the Mayo Foundation.
2Abbreviations used in this paper: Ir, immune response; MHC, major disc electrophoresis. The peptides synthesized and studied inhistocompatibility complex;PPD, purified protein derivative; PETLES, the present work are shown in Table I. They comprise the five
peritoneal exudate T lymphocyte-enriched cells.
antigenic sites of myoglobin (15}, two reference or control
182

Downloaded from www.jimmunol.org on April 25, 2011

The genetic control o f T l y m p h o c y t e proliferative res p o n s e to the five synthetic antigenic sites o f m y o g l o b i n ,t w o synthetic n o n a n t i g e n i c control peptides, and one"nonsense" peptide w a s determined in i n d e p e n d e n t andr e c o m b i n a n t strains o f mice. In all the strains examined,
the n o n a n t i g e n i c control peptides and the " n o n s e n s e "peptide did n o t i n v o k e a r e s p o n s e in m y o g l o b i n - p r i m e dmice. Further, w h e n m i c e w e r e n o t primed w i t h w h o l em y o g l o b i n , no r e s p o n s e w a s obtained w i t h a n y o f theantigenic sites. H a p l o t y p e s U-2 d, H - 2 f a n d H - 2 " a r ehigher r e s p o n d e r s to sites 1 and 2, w h e r e a s h a p l o t y p e s
1-1-2 d a n d t l - 2 ~ a r e high r e s p o n d e r s to site 5. R e s p o n s e tosite 3 m a y be controlled by a non-H-2-linked gene. Site
4 can stimulate 1 t - 2 b and 1 t - 2 k h a p l o t y p e s that are nonr e s p o n d e r s to the w h o l e m y o g l o b i n . Studies w i t h ther e c o m b i n a n t strains s u g g e s t e d that Ir genes to sites 1
and 2 m a p in the I-A s u b r e g i o n and I-C subregion andw e r e d e s i g n a t e d Ir-Mb-l,2(A) and Ir-Mb-l,2(C). Ir g e n e s
to sites 4 and 5 m a p p e d only in the I-A s u b r e g i o n andw e r e d e s i g n a t e d Ir-Mb-4(A) and Ir-Mb-5(A). T h e s e studies s u g g e s t that individual antigenic sites in a m o l e c u l e
are controlled by unique Ir genes.

1979]183

T LYMPHOCYTE PROLIFERATIVE RESPONSE TO ANTIGENIC SITES OF MYOGLOBIN

Boston Mass.) 16 hr before termination of the cultures. EachP E T L E S experiment was run in triplicate culture wells and the
P E T L E S assay on each strain was tested three to six times.
The data are expressed as mean counts per minute (cpm) plus
or minus standard deviation (S.D.).
RESULTS

peptides representing nonantigenic parts of the myoglobin molecule (15}, and a control "nonsense" peptide whose sequence
was designed so that it does not resemble any part of the
myogtobin molecule, even at the dipeptide level. The synthesis
and purification of the myoglobin peptides 15 through 22 (17),56 through 62 (18), 94 through 99 (19), 113 through 120 (20),and 145 through 151 (21) have already been described in detail.
T h e control nonantigenic peptide 1 through 6 was synthesized
and purified as previously described (19). Synthesis and purification of the control nonantigenic peptide 121 through 127 andof the "nonsense" peptide were performed by similar procedures. All peptides used in this work were homogeneous in
heavily loaded peptide "maps" showing purity of 99% or better.
The amino acid composition of each peptide was in excellent
agreement with that expected from its structure (Fig. 1). Purified protein derivative of tuberculin (PPD) was purchased from
Connaught Medical Research Labs., Willowdale, Ontario, Canada.
Immunization. Dose response studies were fh-~st initiated todetermine the optimum amount of myoglobin for priming. A
dose of 50 pg gave an optimum proliferative response (12). Eightmice of each strain were immunized with 50 pg of myoglobin
mixed 1:1 in complete Freund's adjuvant containing 1 mg/ml ofMicrobacterium tuberculosis, strain H37Ra (Difco Labs., Detroit, Mich.). The mixture was injected in the two hind foot/pads, and the animals were sacrificed 3 weeks later.
Cultures. The preparation of peritoneal exudate T-lymphocyte-enriched cells (PETLES) and their in vitro culture havebeen described in detail by Schwartz et al. (22). In brief,
thioglycolate-induced peritoneal exudate cells were passed
through nylon-wool columns. The cells were cultured at 2.5 ×
l0 s per well in U-type microtiter plates in 0.2 ml of a modified
Eagles-Hanks' media (23) containing 10% heat-inactivated fetal
calf serum for 72 hr in the presence or absence of antigen at the
final concentration of 40 #g/ml. The stimulation was assessedby measuring the incorporation of a 2 pCi pulse of tritiatedthymidine (specific activity 1.9 Ci/mM, New England Nuclear,

15
13
12
11

10

F----o

9

."

.~ 6

.-7"

I~./
,g

_...,~....... m-""

2o

40

8b

'-"""~.pg/mlFigure 2. Dose response curve of T-lymphocyte proliferative response to myoglobin antigenic sites. BALB/c by PETLES sensitizedwith whole myoglobin were cocultured with each concentration of
antigenic sites; site 1 (O-.-.-O); site 2 (O-----C));site 3 (A~ A), site
4 (A..... Z~), site 5 (x ..... x) and nonsense peptide (xx) as well as
whole molecule ( H ) .
Each point represents the mean of three
replicates.

Downloaded from www.jimmunol.org on April 25, 2011

Figure 1. A schematic diagram showing the mode of folding of Mband its antigenic structure. The solid black portions represent segments
which have been shown to comprise accurately entire antigenic sites.
The striped parts, each corresponding to one amino acid residue only,
can be part of the antigenic site with some antisera. The dotted portions
represent parts of the molecule which have been shown exhaustively toreside outside antigenic sites. (From Atassi 05); reproduced with permission from Pergamon Press).

Dose-response studies. Studies with whole myoglobin showedthat 50 ~g of the protein with Freund's complete adjuvant was
the optimum dose for the T lymphocyte proliferative response(12). The stimulation indices obtained with this optimum dosewere, however, only a fraction of that obtained with PPD. T h edose of each site that would optimize the proliferative responseof sensitized P E T L E S in the culture wells was also determined.
Strains B A L B / c (H-2d), B10.K (H-2k), B10.S (H-2S), and B10.M(/-/-2 f) were challenged with each site at the dosage level of 10,
20, 40, 80, and 160 t~g/ml. The stimulation was measured at 48,
72, and 96 hr. The results on B A L B / c and B10.K are shown in
Figures 2 and 3. All our experiments represent a 72-h culture
period since the 96-hr cultures gave high backgrounds. Varying
the whole myoglobin dose 1/200th to '/2 on a molar basis relative
to that of the peptide did not significantly change the cpm.
Response of independent haplotypes. The T lymphocyte
proliferative response to individual antigenic sites was tested inmouse strains of all independent haplotypes. The mice wereprimed in vivo with the whole myoglobin and then challenged
in vitro with each of the synthetic peptides of the antigenic
sites 1 through 5, the two control sites, and the "nonsense"
peptide. P P D was included in each experiment, and response ineach strain varied between 15,000 to 130,000 cpm. The data are
not included in the table to conserve space. Each strain wastested approximately six times in the P E T L E S assay. Sinceoccasionally we obtained either no stimulation or extremely
high stimulation, the lowest and highest counts were discarded.
Experiments involving very low P P D responses were repeated.
The data shown represent the median cpm and stimulation

184

K. OKUDA, S+ S, TWINING, C. S. DAVID, AND M. Z. ATASSI
1o[VOL. 123

m+
N+++

9
8

N

+
m~mm

N N ~ N ~

7

i+
2,
0

. .+++Jm.++.~.:++

~o

20

tm

. . . . . . . . . . . . . ~4*. . . . . . . . . +, .

~bom~++N++m++ +~: i~JagimlFigure 3. Dose response curve of T-lymphocyte proliferative re-sponse to myoglobin antigenic sites. B10.K PETLES sensitized withwhole myoglobin were cocultured with whole myoglobin (©..... ©), site
1 (x×), site 2 (X..... ×), and site 4 ( H ) .
Each point represents
the mean of three replicates.
TABLE I
Synthetic

Antigenic

Sites

Site 1

15
22
Ala-Lys-Val-Glu-Ala-Asp-Val-Ala

Site 2

56
62
Lys+Ala-Ser-Glu-Asp-Leu-Lys

Site 3

94
100
Ala-Thr-Lys-His-Lys-Ile-Pro

Site 4

113
120
His-Val-Leu-His-Ser-Arg-His-Pro

Site 5.a~++++m++o+.

~mmmm~+
i+-ll~m++~

.+
,.m

145
151
Lys-Tyr-Lys-Glu-Leu-Gly-Tyr
Synthetic Control Non-AntigenicRegions

Control 1

1
6
Val-Leu-Ser-Glu-Gly-Glu

Control 2

221
127
Gly-Asn-Phe-Gly-Ala-Asp-Ala
Synthetic"Nonsense"

PeptideGlu-Ser-Ser-Gly-Thr-Gly-Ile

++

++++

++ + + o + . ~ + ® + + +index of the middle four values and the range of stimulation
indices. Stimulation indices were calculated by equating the
response to "nonsense" peptide as 1.00. A strain was classified
as a responder if the cpm was twice as much as seen with thenonsense peptide.
As shown in Tables I, II, and III, of the H - 2 ~ haplotype
strains tested (B10, B6, and A.BY), B10 gave a weak responseto the whole myoglobin whereas B6 and A.BY did not responda t all. Among the sites, site 4 stimulated, whereas sites 1, 2, and5 failed to stimulate, in all three H - 2 ~ haptotypes. Even though
the SI for site 4 in H - 2 ~ haplotypes was not very high, this was
highly repeatable and confirmed in several other strains (B6K1, B6-K2, B6(TL+), and AKR-H-2b). Among the H - 2 d haplotypes, B10.D2, BALB/c, and D B A / 2 responded substantiallyto the whole myoglobin as well as sites 1, 2, and 5. None of theH - 2 ~ mice tested responded to site 4. B A L B / c gave a substantial
response to site 3 whereas D B A / 2 gave only a weak response.
T h e H+2 f haplotype mice of strain B10.M responded to thewhole molecule as well as sites 1, 2, and 3, but failed to respondto sites 4 and 5. T h e H - 2 ~ haplotype strains, B10.BR and B10.K,++.......
m+Nmm+N+-+
++
.mm++

~M+m+m++++®o~ m+

I '~2~ ¢+''m "+ 'e+

Downloaded from www.jimmunol.org on April 25, 2011

Sequence of synthetic peptides

1979]T LYMPHOCYTE PROLIFERATIVE RESPONSE TO ANTIGENIC SITES OF MYOGLOBIN

like the H-2 h haplotype strains, failed to respond to the wholemyoglobin and to sites 1, 2, 3, and 5, but did give a significant
response to site 4. This was again conf~med in other H - 2 kstrains (C3H, CBA, C58, B6-H-2 k, AKR, and BRVR). Strains
B10.WB (H-2i), B10.P (H-2P), B10.G (H-2q), and B10.RIII (H2") failed to respond to the whole myoglobin as well as to anyof the sites. The H-2 s haplotype strains B10.S and ASW responded to the whole myoglobin as well as sites 1, 2, and 5.
Strain ASW, in addition, responded to site 3. The results
obtained on the independent haplotypes indicate that: 1) haplotypes H-2 d, H-2 f, and H-2 s are high responders to site 1 and2. In addition, haplotypes H - 2 d and H-2" are also high respond-

185

ers to site 5; 2) response to site 3 may be controlled by a non-H2 linked gene; and 3) site 4 can stimulate H-2 b and 11-2 k
haplotypes, which are nonresponders to the whole myoglobin.
In all the strains examined, the nonantigenic peptides did not
stimulate a T lymphocyte proliferative response in myoglobinprimed mice. Unprimed mice did not give T lymphocyte proliferative response when challenged with any of the antigenic
sites.
R e s p o n s e in H-2 recombinants. In order to map the genes
controlling immune responses to individual antigenic sites ofsperm whale myoglobin, we tested selected H-2 recombinants
involving high and low responder swains (Table IV). Strain

TABLE III
Stimulation indices of independent haplotypes to myoglobin antigenic sites ~
B10

B6

AIBY

B10.K

B10.P

B10.G

B10.RIII

B10.S

b

b

b

B10.D2 BALB/cBY DBA/2
d

d

d

B10.M
f

B10.WB I B10.BR
j

k

k

p

q

r

s

s

Nonsense

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

t.0

1.0

1.0

1.0

1.0

Whole Mb

1.8
(1.5-2.7)

0.9(0.5-1.3)

1.5(1.0-2.0)

4.3
11.6
2.6
(2.4-6.3) (11.4-12.0)(2.2-2.9)

5.0
1.7~i 1 0(2.6-9.7) (1.5-2.3)(0.8'-1.2)

1.3(1.1-1.7)

1.6
1.2(1.3-t.7) (1.2-1.3)

1.5
3.4{1.4-1.8) (2.1-4.4)

4.8(3.9-5.8)

Site 11,1(0.9-1.5)

1.0(0,7-1.1)

1.1(1.1-1.3)

4.5(3.0-5.6)

4.6(4.1-4.9)

3.4(2.5-4.3)

2.3
1.6
1.0
(1.6-3,1)(1.0-2.6)(0.6-1,1)

1.0
1.4
1.0
(0.8-1.2)(1.2-1.7)(0.9-1.1)

1.2
4.3(0,9-1.6) (0.7-9.5)

3,3(2.6-4.1)

Site2

0.9
(0.5-1.1)

0.9(0.6-1.3)

0.9(0.8-1.1)

5.4(1.~9.8)

4.3{1.2-5.8)

3.2
2.4(2.9-3.5) (1.2-2.7)

1.3(1.0-1.9)

1.1
1.4(0.9-1.5) (1.2-2.0)

1.3(0.9-1.5)

1.1
3.9(0.8-1.4) (1.5-6.5)

3,3(2.6-3.9)

Site3

0.8
(0.7-1.0)

1.6(0.9-2.0)

1.3(1.0-1.5)

4.2(2.3-8.0)

3.8{3.2-4.2)

2.3
2.0
1.7
{1.4-3.3) (1.6-2.7) (1.0-2.6)

0.9
1.3(0.7-1.3) (0.6-2.1)

1.2
0.8(0.6-1.5) (0.7-1.5)

1.1(0.9-1.2)

Site4

2.2
2.0
(1.7~.1) (1.5-2.5)

2.0(1.7-2.3)

0.8(0.7~.8)

1.4(1.0-1.6)

I.I
1.1
1.4 ] 2.0
(1.1-t.2) (0.6-1.5) (0.~2.3)~(1.7-2.1)

1.3(1.0-1.4)

0.8(0.6-1.0)

I.I
1.4
1.7
(0.9-1.2) (0.7-1.9) i(1.2-2.3)

Site 50.7(0.6-0.7)

0.8(0.7-1.1)

1.3(1.1-1.4)

2.5(1.4-4.3)

5.2(4.1-7.2)

4.6(4.5-4.7)

1.0
1.4
1.0
(0.9-1.1) (L1-2.1) (0.6-1.2)

1.2(1.1-1.3)

1.0(0.9-1.2)

2.1(1.0.3.1)

Control 1

0.7(0.7-0,7)

1.0(0.6-1.6)

0.9(0.7-1.7)

0.7(0.6-0.8)

1.0(0.7-1.2)

1.1
0.8
0.8
(1.1-1.2) (0.7-0.9) (0.6-1.5)

0.9
1.2(0.6-1.4) (1.0-1.4)

1.2(1.0-1.4)

1.0
0.9(0.7-1.3) i(0.9-0.9)

Control 2

1.0(0.9-1.1)

1.0(0.7-1.2)

1.1(1.1-1.2)

0.8(0.4-1.2)

1.0(0.9-1.1)

1.1
0.7(1.0-1.2) (0.7-0.7)

0.9
1.2(0.7-1.2) (1.1-1.3)

0.8(0.6-1.0)

1,4
0.9(1.0-1.4) (0.7-1.0)0.8
1.1(0.5-1.0) (0.6-1.8)

2.4(2.0-3.2)

0.8
1.1(0.7-L0) (0.9-1.3)

1.3
0.8(0.6-2.0) i (0.6-0.8)

1.3(0.7-2.3)

1.3(1.3-1A)

A.SW

1.8
3.8
(1.6-2.6) 1(0.7-3.7)

2.5(1.0-3.5)

Each strain was tested at least three times. The median stimulation index and the range for each strain is shown. The stimulation indices were calculated bytest antigen cpm
making the cpm for nonsense peptides as 1.00. SI
SI with PPD varied between 15 and 100.
nonsense peptide cpm"

TABLE IV
Stimulation indices of selected recombinants and F, to myoglobin antigenic sites"(B10.S x B10.K)F,(B6 x
ATFR.5 B10.BR)F,

StrainH-2 haplotype

A.TL

A.TH

B10.HTT

D2.GD

B10.A

t2

t3

g2

a

B10.A
(2R)
h2

B10.A
(5R)
i5

B10.S
(8R)
as

B10.A
(4R)
h4

B10.S
(9R)
t4

C3H.OL

tl

o'

ap5

Nonsense

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1,0

1.0

Whole Mb

1.1
(1.0-1.2)

3.0(2.1-3.7)

7.1(3.5-11)3.0(2.3-4,2)

3.4(1.4-4.7)

2.8(2.7-2.9)

3.9(2.2-5.7)

1.3(1.0-1.9)

1.8(1.5-1.8)

Site 1

1.1(0.9-1.3)

2.8(1.6-3.0)

3.6(1.0-7.3)

2.2(1.6-3.0)

2.1(1.5-2.4)

2.5(1.8-3.6)

5.0(3.3-8.9)

1.3(0.8-1.7)

1.5(1.3-1.6)

1.0(1.0-1.0)

2.5
3.2(1.0-3.0) (1.5-6.3)

3.3(1.7-5.4)

2.7(1.2-5.3)

2.4(1.7-3.5)

1.9(1.2-2.4)

1.4

t.4

(0.6-2.3)

1.1(1,0-1.2)

1.1
1.1(0.8-1,4) (1,0-t,2)

1.1(1,0-1,3)

2.2(1.2-4,0)

1.7(1.1-2,7)t (1.7-3.0)

1.1(0.9-1.4)

1.0(0.9-1.0)

0.9(0.8-1.1)

2.4(1.5-3.3)

0.8(0.6-0.9)

2.2(1.9-2.4)

2.9(1.6-4.6)

2.6(1.0-4.0)

1.0(0.7-1A)

sx k

bx k

1.0

1.0

1,0

8.0
3.1
(3.2-11.2) (2.6-3.8)

3.2(2.6-4.7)

2.5(2.0-2.7)

1.1(1.0-1.2)

4.0(1.6-6.1)

2.0(1.7-2.4)

3.1(1.7-4,5)

3.1(2.0-4,5)

1.2(1.0-1.5)(1.3-1.5)

4.9(3.4-7.9)

2.4(1.2-2.9)

3.2(1.9-6.4)

2.7(1.8-4.5)

1.2(1.0-1.4)

2.8(1,7-3,9)

1.2
1.3(0.7-1.6) 11.3-1.4)i

1.6(0.8-2.3)

2.4(2.0-3.0)

2.9(2.0-4.3)

2.4(2.1-3.2)

1.0(0.9-1.0)

2.5(1.7-3.5)

2.1(1.2-2.7)

3.0(1.1-5.3)

2.2
i
0.6
(2.0-2.4) (0.6-0.7)

1.6(0.9-2.2)

1.3(1.1-1.8)

2.3(1.9-3.0)

2.0(1.9-2.2)

0.9(0.8-0.9)

1.2(1.0-2.9)

1.2

1.7

(0.8-1.6)(1.3-2.0)

3.0(0.7-5.6)

2.6(2.1-3.3)

1.1(0.9-1.3

3.0(2.2-3.6)

1.4(0.8-1.7)

Control I

0.8
1.0
1,0
1.3
I.I
I.I
I.I
I.I
(0.8-1,1) (1.0-1,0) (0.6-1.4) i (0.9-1,2) (0.5-1,7) (0.7-1,5) (I.0-1.2) (0.7-1.0)

1.3(L2-1.3)

1.2(0.6-2.2)

1.6(0.9-2.7)

1.4(1.1-2.0)

1.3(1.0-1.6)

1.0(0.8-1,4)

Control 2

1.1(0.9-1.2)

1.4
1.3(1.1-1.6) i {0.9-1.6)

1.3

I.I

(1.2-2.6)

Site 2t

Site 3I
Site 4Site 5I

2.9

1.4(1.3-1.4)

t.0(0.9-1.2)

1.3(0.9-2.0)

1.1(0.8-1.6)

1.2(0.9-1.6)

1.4(1.1-1.7)

I,I(0.7-1.3)i

1.6(0.9-1.5)(1.0-1.2)

I.I(1.0-1.1)" E a c h strainwas tested at least three times. The median stimulation index and the range for each strain is shown. The stimulation indiceswere calculatedby
test antigen c p m, SI with P P D varied between 15 and 100,
nonsense peptide c p mmaking the c p m for nonsense peptides as L00. SI

Downloaded from www.jimmunol.org on April 25, 2011

StrainHaplotype

186

K. OKUDA, S. S. TWINING, C. S. DAVID, AND M. Z. ATASSIDISCUSSION

T h e present studies clearly showed t h a t T lymphocyte proliferative response to sperm whale myoglobin and four of thefive antigenic sites are under control of H-2-1inked I r genes. In
T a b l e V, we have used the key strains to summarize the

TABLE V
Summary o f response to myoglobin antigenic sites in selected
recombinants
Strain

K

A

B

J

E

C

S

G

D

Response

B10
BI0.D2
D2.GD
C3H.OL
B10.K
A.TL
B10.A
B10.A(2R)
B10.A(5R)
B10.S
B10.S(9R)
B10.HTT
B10.S(8R)

b
d
d
d
k
s
k
k
b
s
s
s
k

b
d
d
d
k
k
k
k
b
s
s
s
k

b
d
b
d
k
k
k
k
b
s
?
s
~

b
d
b
d
k
k
k
k
k
s
k
s
9

b
d
b
d
k
k
k
k
k
s
k
k
s

b
d
b
d
k
k
d
d
d
s
d
k
s

b
d
b
k
k
k
d
d
d
s
d
k
s

b
d
b
k
k
k
d
?
d
s
d
k
s

b
d
b
k
k
d
d
b
d
s
d
d
s

4
I, 2, 3, 5
1, 2, 5
1, 2, 3, 5
4
4
I, 2, 3, 4
1, 2, 4
1, 2, 3, 4
1, 2, 5
1, 2, 5
1, 2, 5
4

H-2 Gene Complex
Site
1.2
3

Non-H-2

K A B J E C S G D
H
I-"-4

4
5

Figure 4. lx gene mapping for individual antigenic sites of sperm

whale myoglobin.
mapping of the IX genes to myoglobin antigenic sites. Figure 4
depicts the mapping of these Ir genes. So far, we have been
unable to separate the IX genes to sites 1 and 2, which havealways been expressed together. T h e IX genes to the myoglobinsites will be designated as follows: 1) I r - M b - l , 2 ( A ) for the gene
responding to sites 1 and 2, mapping in the I - A subregion; 2) IrM b - I , 2 ( C ) for the gene responding to sites 1 and 2 mapping in
the I - C subregion; 3) I r - M b - 3 for the gene(s) responding to site
3, which is non-H-2-1inked; 4) I r - M b - 4 ( A ) for the gene responding to site 4, mapping in the I - A subregion; and 5) I r - M b - 5 ( A )for t h e gene responding to site 5, mapping in the I - A subregion.
One interesting point is the response of recombinant B10.S(8R),
which carries the I - C subregion from H - 2 s haplotype, a highresponder, failed to respond to sites 1, 2, and 5. This suggests
t h a t H - 2 d haplotype expresses both I r - M b - l , 2 ( A ) and I r - M b 1,2(C),whereas H - 2 s haplotype expresses only the I r - M b - l , 2 ( A ) .
T h e I - E / I - C gene products have not been detected in H - 2 ~haplotype.
T h e expression of either I r - M b - l , 2 ( A ) or I r - M b - l , 2 ( C ) issufficient for response to sites 1 and 2. This suggests t h a t
individual lx genes can respond to distinct antigenic sites on themolecule. Gene complementation could occur by additive response of two Ix genes responding to different sites on themolecule. In the parent strain, response generated by one of
the IX genes m a y not reach the threshold value to be classified
as a responder. Another area of interest is the response of H - 2 k
and H - 2 b haplotypes to site 4, although they failed to respondto t h e whole myoglobin molecule. T h e r e are two possible explanations: 1) Dosage effect: when the primed lymphocytes of
H - 2 k a n d H - 2 b haplotypes are challenged in vitro with the site
4, t h e y are exposed at optimum dose to approximately 18 times
more antigen compared to challenge with the whole molecule.
If this were true, increasing the dosage of myoglobin 10- to 20fold during challenge should m a k e H - 2 b and H - 2 k high responders. It is significant t h a t these strains remained low responders

Downloaded from www.jimmunol.org on April 25, 2011

A.TL responded only to site 4 as did the H - 2 k haplotype,whereas A.TH strain mice responded to sites 1, 2, and 5 as theH - 2 ~ haplotype. Since A.TL and A.TH differ only between H2 K and H - 2 D the Ix genes for myogiobin sites 1, 2, 4, and 5 m a p
i n / , S, or G regions. Strain B10.HTT, which carries the highresponder H-28 alleles in I - A , I - B , and I - J , responded to sites 1,
2, and 5. Since B10.S(9R) ( i . j k ) also gave similar results,
mapping of IX genes for sites 1, 2, and 5 was further narrowed
down to the I - A a n d / o r I - B subregions. T h e gene controlling
responses to site 1, 2, and 5 specifically m a p p e d to the I - A
subregion since recombinant D2.GD responded to these sites
similarly to H - 2 d haplotype. D2.GD carries the H - 2 b haplotype
genes to the right of I - A , and mice of this haplotype do notrespond to these sites. Since D2.GD failed to respond to site 4
to which H - 2 b responds; the IX gene to site 4 can also be m a p p e dto the I - A subregion. Recombinants B10.A, B10.A(2R), andB10.A(5R) responded to sites 1 and 2, whereas B10.A(4R) and
B10.S(8R) failed to respond. Since H - 2 k haplotype also does
not respond to sites 1 and 2, a second IX gene to sites 1 and 2
can be m a p p e d between the I . E subregion and the H - 2 D
subregion. T h e recombinants C3H.OL and C3H.OH, which
express the S and G regions from low and high responders,
respectively, gave similar responses to sites 1 and 2; the influence of these regions must be minimal. On the basis of theserecombinants, a second Ix gene to myoglobin sites 1 and 2 canbe tentatively m a p p e d to the I - C subregion. T h e results with
the H-2 congenic strains suggested t h a t the Ir genes to myoglobin site 3 might be associated with non-H-2 linked gene. Among
the recombinants, B10.A and B10.A(5R) responded to site 3,
whereas B10.A(2R) failed to respond. This suggests that response to site 3 m a y be influenced by some genes mapping in
the T L region.
Several F1 crosses were made involving combination of responders and nonresponders. Crosses between responders andnonresponders always responded to the whole myoglobin as
well as to the sites suggesting dominant Ix-gene control. In
matings involving two nonresponder strains, we did not see any
evidence for complementation of Ix genes for response to theantigenic sites.
Results of experiments testing several other recombinantstrains confirmed the mapping of Ix genes to the myoglobin
antigenic sites. B10.HTG and B10.BDR (id) gave results similar
to the H - 2 d haplotype mice. B10.A(18R), B10.A(21R),
B10.BAR(5),B10.D2(R106),
B10.D2(R107),
A-BTR-1,
B10.F(14R), and H T I (ib) gave results similar to the H - 2 b
haplotype mice, in t h a t they responded only to site 4.
B10.M(17R) and B10.AQR (/k/d) gave results similar to the H2 ~ haplotype. A.TFR-1 (ik), A.AL, A.AKR, B10.AM, andB10.AKM responded to site 4 similar to A.TL. B 1 0 . M ( l l R ) and
A.TFR-5 (I f) gave results similar to the H - 2 r haplotype.
B10.T(6R) (Iq) gave results similar to H-2 q haplotype, and
B10.S(7R) and B10.S(24R) (P) gave results similar to H - 2 sstrains. All the mutants t h a t were typed gave results t h a t werecharacteristic of the parental strains. F o r example, B6-H-2 bg~,
B6-H-2 bh, and B10.M505 responded to site 4 similar to the B6(H-2b). M504 gave results s'unilar to B10.D2 (H-2d), and M523
responded to site 4 similar to H - 2 k haplotypes.

[VOL. 123

1979]T LYMPHOCYTE PROLIFERATIVE RESPONSE TO ANTIGENIC SITES OF MYOGLOBIN

phages phagocytize antigens and p u t out antigen fragmentst h a t bind to their surface membranes. Recently, Benacerraf(28) has proposed t h a t specific Ir-gene function dictates theability of the Ia molecule on macrophages and B cells to interact
specifically with unique amino acid sequences on the antigen
concerned. This would generate the determinants specific for T
cell recognition and would suggest t h a t the Ia molecules possessbinding sites t h a t are complementary to an antigen segment ofa specific amino acid sequence. Applying this idea to our studies,
it would mean t h a t the Ia molecules generated by genes codedin the IoA subregion of H - 2 d haplotype can bind to sites 1, 2,
and 5, whereas the Ia molecule from H - 2 k haplotype havecomplementary surface structure for binding only site 4. Thishypothesis is being experimentally tested in our laboratories.
A c k n o w l e d g m e n t s . T h e authors t h a n k Suresh Savarirayan
for superb mouse husbandry, and Sharon Ames and Connie
B a t t for skiUflfl preparation of the manuscript.

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Downloaded from www.jimmunol.org on April 25, 2011

when the myoglobin dose was varied from 1/200th to 1/2relative
to t h a t of the peptide. T h u s the dosage effect m a y be ruled out.
2) Suppressive site: there might be a site on the myoglobin
molecule t h a t gives suppressive signals in H - 2 k and H - 2 b h a p lotypes. W h e n the mice are exposed to site 4 only, in theabsence of this putative suppressive signal, t h e y are able toproliferate. This hypothesis can also be experimentally verified
by challenging with couplets of site 4 and the other sites.
Studies with the sites in the priming process might also reveal
some interesting information.
T h e entire antigenic structure of sperm whale myoglobin wasdetermined originally by Atassi on the basis of the antibody
response in rabbits and goats. An initial question was whether
the mice would recognize the same antigenic sites. T h e T cellproliferation studies indicate t h a t the mice recognize the samesites. We also considered the possibility t h a t the mice mightrecognize additional sites on the molecule. However, preliminary studies have also shown t h a t the antibodies to myoglobin
made in three strains of mice bind to the expected antigenic
sites. T h e total binding activity of the reacting sites accounted
for almost 100% of the mouse antimyoglobin antibody {Twining
et al., in preparation).
Studies with synthetic amino acid polymers have suggestedt h a t Ir-gene control at the T cell level was carrier dependent.
However, the present studies suggest that this need not be
n e c e s s a r y true and t h a t the recognition could be at the antigenic site level. Presumably Ir-gene control can be mediatedthrough several avenues. Even though studies with amino acidpolymers have yielded very important information on the immune response gene phenomena, there are also certain pitfalls.
For example, synthetic amino acid polymers prepared from Ncarboxy-amino acid anhydrides would be expected to differ
from one preparation to another, and also a given preparation
does not represent a discrete molecular species. These chemicaldifferences would lead to changes in the antigenic properties ofthe molecule and the mechanism by which it is recognized.
Studies with protein antigens should generate new informationon the Irogene mechanisms.
T h e T cell proliferation and the antibody response to thewhole molecule gave similar results. Studies in several laboratories {24-26) have suggested t h a t T cells recognize different
determinants from those recognized by B cells. T h e T cell
responses are believed to be specific for amino acid sequenceon fragments of native protein antigens, whereas antibodies are
directed against the tertiary structure of the antigenic sites on
the native antigen. It is therefore significant to point out t h a t
the control synthetic peptides, representing nonantigenic partsof the molecule, consistently failed to stimulate a T cell proliferative response in all the numerous strains examined. I t would
be interesting now to see whether the antibody response to thefree synthetic sites {which would be expected to be completely
unfolded in solution) in appropriate strains would give similar
results as shown in this paper for the T cell proliferation. Can
antibodies be produced to the synthetic antigenic sites in thehigh responder mice by immunization with each of these sites
or is the tertiary structure required for antibody production?
Of course, it should not be ruled out t h a t binding receptors
could induce the free peptide into the appropriate conformation
as do antibodies (15). Further, at the antigenic determinantlevel, are the T cell repertoire and B cell repertoire similar ordifferent? These are questions t h a t can be answered by doing
antibody-binding assays and the T cell proliferative assays inthe same strains of mice by using the synthetic antigenic sites.
Unanue and Cerottini (27) have established t h a t macro-

187

188

K. OKUDA, S. S. TWINING, C. S. DAVID, AND M. Z. ATASSI

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