Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin

CELLULAR

IMMUNOLOGY

46,

384-397 (1979)

Evidence for Cellular but not Serologic Cross-Reactivity between Keyhole Limpet Hemocyanin and Sperm-Whale Myoglobinl G. T. GOOCH,~

Department 44106;

A. B. STAVITSKY,~ W. W. HAROLD, G. MANDERINO, AND M. Z. ATASSI~

of Microbiology, School of Medicine, Department of Immunology, Mayo Received

Case Western Reserve University, Cleveland, Medical School, Rochester, Minnesota 55901

November

Ohio

9, 1978

The additionof keyholelimpethemocyanin (KLH) to culturesof rabbitlymphnodecells (LNC) primedwith KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund’s adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed. ’ This research was supported by Research Grants AI-l 1420 and CA-18648 to A.B.S., Training Grant STI-GM-171 to the Department of Microbiology, and AM-13389 to M.Z.A. from the United States Public Health Service. 2 United States Public Health Service Postdoctoral Fellow. Present address: Department of Microbiology, Texas Tech School of Medicine, Lubbock, Tex. 79409. n To whom reprint requests should be sent. 4 Department of Immunology, Mayo Medical School, Rochester, Minn. 55901. 384 000%8749/79/100384-14$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

HEMOCYANIN/MYOGLOBIN

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385

INTRODUCTION In a previous study (1) rabbits were immunized with alum-KLH and alum-HSA in the hind footpads. Some weeks or months later the draining lymph nodes were removed for cell culture. The addition of 1 pg KLH to these lymph node cells (LNC)5 induced an anamnestic anti-KLH antibody response. Adding 100 pg KLH to these LNC evoked anamnestic anti-HSA antibody production. It was shown that KLH and HSA did not cross-react serologically. Therefore, it was postulated that the 100 ,ug KLH induced KLH-reactive T helper LNC to produce a nonspecific soluble factor which enabled HSA-reactive B memory cells to respond to residual HSA immunogen in the lymph node. Evidence for the presence of residual HSA immunogen associated with dendritic cells in the node has been presented in recent studies (2-4). The results of a number of other studies presented at about the same time and subsequently are in agreement with the proposed nonspecific stimulation of T helper activity (5 10). The present study was an outgrowth of attempts to develop another system for heterologous immunogenic induction of antibody synthesis. It was desirable to employ at least one antigen whose antigenic structure was known so that eventually one or more of its synthetic antigenic sites could be employed more incisively to answer questions of the relationship between structure and function of antigenic determinants in cell-mediated immune responses. Sperm-whale myoglobin (Mb) was selected because its complete antigenic structure is known (11) and we have shown that Mb, its five synthetic antigenic sites, and a nonantigenic Mb peptide (sequence l-6) in vitro induce Mb-primed rabbit LNC to produce antibody, macrophage inhibitory factor (MIF), protein, IgG, DNA, and RNA (12, 13). It was soon found (as indicated in Table 1) that added KLH induces rabbit LNC educated with KLH and Mb to synthesize increased amounts of antibody to Mb. This finding raised questions about the mechanism of this apparently heterologous stimulation. Three mechanisms were considered: (a) KLH is a nonspecific polyclonal activator of T and/or B cells; (b) KLH and Mb cross-react serologically, implying at least cross-reaction at the B-cell level; and (c) KLH and Mb cross-react cellularly, implying a possible cross-reaction at the T-cell level. These experiments have provided evidence for a cross-reaction at the cellular level between KLH and Mb and between KLH and several synthetic Mb antigenic sites. MATERIALS

AND METHODS

Animals. Random-bred albino rabbits (2-3 kg) of either sex were obtained from a local supplier. They were maintained on a diet of Purina chow and water. Antigens. Keyhole limpet hemocyanin (KLH) was obtained from Schwarz/Mann Laboratories, Orangeburg, N.Y. This preparation showed a single line of precipitation by immunoelectrophoresis or double diffusion against a hyperimmune rabbit antiserum against crude KLH (Pacific Biomarine Supply Co., Venice, Calif.). Sperm-whale myoglobin (Mb) was the major chromatographic component 5 Abbreviations serum albumin; inhibitory factor; A, concanavahn

used: Mb, sperm-whale myoglobin; KLH, keyhole limpet hemocyanin; HSA, human LNC, lymph node cells; CFA, complete Freund’s adjuvant; MIF, macrophage ATG, goat anti-rabbit thymocyte globulin; AFab’, goat anti-rabbit Fab’ globulin; Con A; SIg, surface immunoglobulin.

386

GOOCH

ET AL.

No. 10 obtained by chromatography of twice-crystallized Mb on CM-cellulose (14). The Mb was homogeneous by starch gel, acrylamide, and disc electrophoresis. The amino acid sequence, molecular weight, and molar excess (to cause maximum inhibition of precipitation) of each synthetic and antigenic Mb peptide were summarized previously (12). The methods of synthesis and purification of these peptides were as follows: 15-22 (15); 56-62 (16); 146-151 (17). Following exhaustive purification, each peptide possessed purity of 99% or better as determined by elution and 570-nm absorption of the ninhydrin-positive spots from heavily loaded peptide maps. Complete characterization of these peptides is given in the aforementioned references. Antisera. The hyperimmune antisera to KLH and Mb utilized in the assay of radioactive antibody were prepared by 7 to 10 injections of 2 mg alum-antigen intravenously and into the hind footpads of rabbits over a period of months. Seven days after the last injection, the rabbits were bled by cardiac puncture. The goat IgG antibody to rabbit thymocytes (ATG) and normal goat IgG were prepared as previously described (18). By a variety of rigorous criteria (18-20) this ATG was specific for rabbit T lymphocytes. Zmmunizati~n of rabbits. Rabbits were primed by the injection into the hind footpads of 5 mg Mb-complete Freund’s adjuvant (CFA), 1 mg alum-Mb, or 1 mg alum-KLH. At various times postimmunization popliteal lymph nodes were removed for culture. Cell cultures. LNC cultures were prepared as previously described (19). KLH, Mb, Mb synthetic antigenic sites, or a mixture of KLH and Mb synthetic antigenic sites were added to the LNC for the first 24 hr and then removed by washing the cells twice in Hanks’ balanced salt solution. The cells were resuspended in fresh medium for another 48 hr when L-[14C]leucine (3 12 mCi/ml) was added for the final 48 hr to radiolabel newly synthesized antibody IgG and protein. At the termination of culture the cells were removed by centrifugation and the culture fluid utilized for assays of radioactive antibody and protein. For induction of DNA synthesis the LNC were treated with antigen as above, washed, and then resuspended in fresh medium containing 2.5 &i [3H]thymidine during 24-48 hr of culture, the period of maximal incorporation when the cells were harvested for assay (19). For the enhancement of cyclic [3H]AMP uptake, the LNC were treated with antigen for O-24 hr, washed, and then incubated in fresh medium with 1.25 &i cyclic [3H]AMP for 24-48 hr when the cells were harvested for assay (21). Assays of antibody, protein, andZgG syntheses. De novo synthesis of antibody to KLH and Mb was determined by an immunosorbent procedure previously described (22). Each sample was assayed at least in duplicate and the range differed by less than 10% from the mean. IgG synthesis was assayed by a coprecipitation method. The radioactive IgG in the culture medium was coprecipitated by the addition of a specific goat anti-rabbit IgG serum. Nonspecific radioactivity was assayed by subjecting the medium to coprecipitation by added ovalbumin and antiserum to ovalbumin. The difference between the specifically and nonspecifically precipitated radioactivity provided an assay of IgG-associated radioactivity. Determinations were made in duplicate analyses and always varied by 210% or less. The average value is reported. Protein synthesis was assayed as described earlier (19). Serological assays. Serum antibody was sometimes assayed by the passive

HEMOCYANIN/MYOGLOBIN

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387

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hemagglutination procedure (23). Serological cross-reactivity was also assayed by the Farr method (24) and an immunosorbent procedure (22). Preparation and characterization of rabbit T and B LNC. Popliteal lymph node cells from unprimed rabbits were separated into two populations by passage through goat anti-rabbit Fab’ (AFab’)-Sepharose 6 Mb affinity columns (25). The adherent cells, eluted with rabbit IgG, were 90%+ surface immunoglobulin (SIg) positive and enriched for complement receptor lymphocytes, properties of rabbit B-cell populations (26). These adherent LNC did not incorporate r3H]thymidine when challenged with concanavalin A (Con A), but did when AFab’ was added. In contrast, the nonadherent LNC contained 1% or fewer SIg-positive cells and were stimulated to incorporate [3H]thymidine by Con A, but not by AFab’. Thus by a variety of criteria, the nonadherent LNC were almost exclusively T cells and the adherent cells almost exclusively B cells. Both the adherent amd nonadherent cell preparations contained about 5% macrophages. RESULTS KLH-Induced, Thymus-Dependent Synthesis of Antibody Primed Rabbit Lymph Node Cells

to Mb by KLHIMb-

Table 1 presents the results of a typical experiment in which KLH or Mb was added to cultures of rabbit LNC primed with alum-KLH/Mb. Added Mb usually did not induce LNC primed with alum-Mb to produce anti-Mb antibody, as found previously (12, 13). However, the addition of 10 to 100 pg KLH to alumKLH/Mb-primed LNC consistently raised the level of synthesis of antibody to Mb as well as anti-KLH antibody many-fold over the spontaneous level. This heterologous enhancement of antibody synthesis was thymus dependent: The addition of a highly specific goat IgG antibody to rabbit thymocytes (18-20) reduced the level of both anti-KLH and anti-Mb antibody syntheses induced by TABLE KLH-Induced

Thymus-Dependent Synthesis KLH-Mb-Primed Rabbit Lymph

Additions Mb

of Antibody Node Cells”

(pg)

KLH 0

1

cpm/lO’ ATG

0

0

1 10 100 1 10 100 1 10

100

to Mb by

600 600 600

Anti-KLH

LNC Anti-Mb

1.25 0.80 0.88 1.4 I.? 16.1 13.2 1.2 1.0 0.9

a Rabbit was injected in each hind footpad with 1 mg alum-KLH and The popliteal lymph nodes were removed for culture on Day 40. * Underlined data are significant at least at the 5% level by the I test.

0.96 1.2 1.2 1.4 1.7 3.5 ~ 3.7 1.5 2.1 1.5 1 mg alum-Mb

on Day

1.

388

GOOCH ET AL.

KLH. As found earlier (19), the addition of 1 to 100 pg KLH to alum-KLH-primed LNC elevated by severalfold antibody production to KLH. KLH

as a Polyclonal

T- or B-Cell Activator

The possibility that KLH nonspecifically activates rabbit T or B cells was tested by exposing normal unfractionated LNC as well as purified T and B LNC to various concentrations of KLH. As shown in Table 2 (Expt 1) even 100 pg KLH did not induce whole, T or B, unprimed rabbit LNC to incorporate C3H]thymidine over the basal levels. The data in Expt 2 indicate that 100 pg KLH did not promote the synthesis of antibody to KLH or Mb or of protein and IgG by purified T cells. Nor did purified B cells (data not shown) in that experiment show enhanced synthesis of these macromolecules upon challenge with 100 pg KLH. In none of these experiments did the addition of Mb induce these unfractionated, T or B LNC populations to incorporate [3H]thymidine or to produce increased amounts of antibodies to KLH or Mb, or protein or IgG (data not shown). Determinations

of Serological

Cross-Reactivity

between KLH

and Mb

Several highly specific and sensitive techniques were employed to determine whether KLH and Mb cross-react serologically. Hyperimmune rabbit anti-KLH serum yielded a passive hemagglutination titer of 81,920 with KLH-conjugated tannic acid-treated sheep erythrocytes (23). This titer was reduced to 20 by 200 pg KLH, but was not lowered at all by 200 pg Mb. Figure 1 shows the results of analysis of cross-reactivity by the Farr method (24). Radioiodinated Mb was bound by hyperimmune anti-Mb antibody. Unlabeled Mb competitively inhibited the binding of lz51-labeled Mb by anti-Mb. However, up to 100 ng of KLH failed to TABLE Failure of KLH Nonspecifically

2

to Activate Unprimed Rabbit T and B Lymph Node Cells Incorporation

Expt No. 1

2

KLH-pg added

Cell prep.”

0 1 loo 0 1 loo 0 1 100

Whole

0 1 100

T cells B cells

T cells

[3H]Tdr

(cpm X 10-3)b

AntiKLH

AntiMb

Protein

W

0.575 0.547 0.722

1.18 0.97 0.97

10.82 10.12 12.18

4.09 4.35 4.95

8.9 9.0 7.5 90.9 95.8 86.6 6.42 6.37 6.91

a 5 X lo6 LNC. Cells prepared as described under Materials and Methods. b [3H]Thymidine incorporation during 24-48 hr; anti-KLH, anti-Mb, protein, and IgG syntheses during 96-120 hr of culture.

HEMOCYANIN/MYOGLOBIN

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CROSS-REACTIVITY

389

390

GOOCH

ET AL.

inhibit the binding of this radioiodinated Mb by anti-Mb. As a matter of fact, even 100 pg of KLH did not inhibit this binding (data not shown). Conversely, unlabeled Mb failed to inhibit the formation of a radioactive complex between anti-KLH and 1251-labeled KLH (data not shown). Finally, the immunosorbent bromacetyl cellulose (BAC)-KLH bound [‘4C]leucine-labeled antibody to KLH, but not [14C]leucine-labeled antibody to Mb. These radiolabeled antibodies were produced by challenge of Mb-primed LNC (12) with Mb and KLH-sensitized LNC with KLH (19) followed by inclusion of L-[14C]leucine in the culture medium. Conversely, BAC-Mb bound radioactive antibody to Mb, but not radioactive antibody to KLH. Determinations

of Cellular

Cross-Reactivity

between

KLH

and Mb

The last series of experiments examined the question of cellular cross-reactivity between KLH and Mb by utilizing a wide variety of criteria of specific lymphocyte activation, including antigen-induced nucleotide transport (21), thymidine uptake (19), antibody synthesis (12, 13, 19) and protein synthesis (13, 19). These experiments were done against a background of repeated failure to induce these responses by addition of KLH, Mb, or Mb synthetic antigenic peptides to unprimed LNC ((12, 13); Table 2, this study). Induction of Heterologous Antibody Synthesis upon Addition Synthetic Antigenic Sites to LNC Primed with Mb-CFA

of KLH,

Mb, or Mb

Table 3 summarizes typical results of numerous experiments in which these antigens were added to LNC primed for 1 or 2 weeks with Mb-CFA. A short period of priming with Mb-CFA was chosen because previous experiments revealed that such LNC did not frequently synthesize anti-Mb antibody upon challenge with Mb (12). Several findings are noteworthy. First, added KLH frequently enhanced the anti-Mb response as well as anti-KLH antibody synthesis by these Mb-primed LNC (8301).‘j Second, the addition of Mb sometimes elevated anti-KLH antibody production as well as anti-Mb antibody response (8301, 8271). Third, three synthetic antigenic sites of Mb (15-22,56-62,145151) influenced the KLH as well as Mb antibody responses of these Mb-educated LNC in three ways: (a) enhanced anti-KLH antibody synthesis by 7-day-primed LNC (8301); (b) inhibited anti-KLH antibody responses of lCday-primed LNC (8271); and (c)inhibited enhancement of anti-Mb antibody synthesis induced by KLH (8254). Induction of Heterologous Antibody Responses upon Addition Mb Synthetic Sites to LNC Primed with Alum-Mb

of KLH,

Mb, and

The next series of experiments employed three additional criteria of lymphocyte activation based on our previous demonstration that the addition of KLH to KLH-educated LNC enhanced the uptake of cyclic nucleotides (21), the incorporation of tritiated thymidine, and protein synthesis (19). LNC sensitized with alum-Mb were employed because it has been observed that such cells challenged with Mb usually do not show enhanced cyclic nucleotide uptake, thymidine incorporation, and protein or anti-Mb antibody production (A. B. 6 In vitro challenge by KLH of splenic cells of CeH/HEJ mice primed with alum-Mb frequently enhanced anti-Mb antibody synthesis (A. B. Stavitsky and W. W. Harold, unpublished observations).

HEMOCYANIN/MYOGLOBIN

CELLULAR TABLE

391

CROSS-REACTIVITY

3

Induction of Various Types of Antibody Responses upon Addition of KLH, Mb, and Mb Peptides to Rabbit Lymph Node Cells Primed with MB-Complete Freund’s Adjuvanr Additions (r.qg) Rabbit No. 8253

Interval (days) 6

KLH

Mb

0

0

15-22 0

56-62 0

145- 151

Antibody KLH

0

0.37 0.49 0.36 0.61 1.11 0.848 0.494

1 100 100

100 1 1 8301

7

0

100

0 10 loo

0

0

0

1

10 100 1 10 100 8271

14

0

0 10

2.96 4.68 88 2

1 10 100

0

0

0

50 100

cpm x lo+ to Mb

6.07 6.85 4.52 6.12 5.90 5.54 4.97 675 A 7.27 1.25 -2.4 0.54 0.52

1.3 3.9 9.57 2.53 2.42 2.26 0.80 2.8 3.6 2.0 4.8 33 A 5.43 10.6 5.25 4.8

a Rabbits were injected with 5 mg Mb in complete Freund’s adjuvant and lymph nodes removed for culture on the designated days.

Stavitsky and W. W. Harold, unpublished observations). Table 4 presents typical results of these experiments. Added Mb did not enhance any immune responses; in fact, anti-KLH antibody synthesis was often inhibited (8366). However, the addition of KLH to these LNC consistently enhanced cyclic [14C]AMP uptake (8366), thymidine incorporation (8122), and anti-Mb antibody and protein synthesis (8122). The LNC from rabbit 8122 were also challenged with HSA and did not show any immune responses. A number of other experiments were done in which Mb-primed LNC challenged with other proteins, including bovine globulin, lysozyme, thyroglobulin, and fibrinogen also did not manifest any of these responses (data not shown). induction of Mb Antibody Synthesis and Thymidine Incorporation upon Challenge with KLH or Mb of LNC Primed with Mb-CFA and Alum-Mb

The final series of experiments with Mb-primed LNC employed cells primed both with Mb-CFA and alum-Mb. Typical results are shown in Table 5. The addition of KLH to these LNC enhanced their incorporation of r3H]thymidine and of synthesis

392

GOOCH ET AL. TABLE

4

Induction of Various Types of Immune Responses upon Addition of KLH and Mb to Rabbit Lymph Node Cells Primed with Alum-Mb” Immune responses (cpm X 10e3) Additions (&* Rabbit No. 8122

Interval (days) 6

Antibodye

KLH

Mb

HSA

0

0 10

0

CAMP uptakec

Tdr uptaked

KLH

11.3 14.2 12.4 101.86 178.42

100 10 100 10 100 8366

30

0

0

1 10 100 1 10 100

0

2.9 2.2 2.6 2.7 2.7

Mb

ProteitF cpm

0.30 0.33 0.43 0.86

1.26 1.12 1.87 4.5

1.43 0.25

63 2 1.1

0.34

0.74

0.38 0.146 0.088 0.305

6.1 219

a Rabbits were injected with 1 mg alum-Mb in each hind footpad were removed at the indicated intervals for culture. b O-24 hr of culture. c Cyclic [W]AMP added to cells during 24-48 hr of culture; cells counting. d [3H]Thymidine added to cells during 24-48 hr of cultures; cells counting. e [W]Leucine in medium during 72-120 hr of culture, after which

and the popliteal lymph nodes then harvested for washing and then harvested for washing and collected for assay.

of Mb antibody. As expected (12), added Mb induced anti-Mb Added HSA did not promote anti-Mb antibody synthesis. lnakction of Various Types ofAntibody Responses upon Addition Mb Synthetic Antigenic Sites to Alum-KLH-Primed LNC

antibody

of KLH,

synthesis.

Mb, and

If cross-reactivity between KLH and Mb is demonstrable by challenge of Mb-primed LNC with KLH, this cross-reactivity might also be shown by challenge of KLH-primed LNC with Mb and/or Mb sites. Table 6 presents typical data from these types of antigenic challenges. In this type of system added KLH either did not enhance anti-Mb antibody synthesis (8266,8429) or inhibited this response (8298). The addition of Mb inhibited (8298) or enhanced (8428, 8429) anti-KLH antibody synthesis. Mb synthetic sites 15-22 and 56-62 inhibited anti-Mb antibody synthesis consistently (8298,8429) and anti-KLH antibody production in about half of the experiments (cf. 8298, 8429). The Mb site 145- 151 consistently inhibited anti-Mb antibody responses of alum-KLH-primed LNC, but suppressed anti-KLH antibody synthesis in about half of the experiments (cf. 8298, 8429). Finally, the

HEMOCYANIN/MYOGLOBIN

CELLULAR TABLE

393

CROSS-REACTIVITY

5

Induction of Mb Antibody Synthesis and Thymidine Incorporation upon Challenge with Mb of Rabbit Lymph Node Cells Primed with Mb-Complete Freund’s Adjuvant and Alum-Mb” Additions (pg) Mb

KLH

HSA

0

0

0

Thymidine uptake (cpm x 10-3)

1 10 100 1 10 100 1 10 100

Mb antibody (cpm X 10m3M)

15.7 15.16 36.48 57.52 18.46 30.59 SO.58 16.86 15.46 19.01

0.509 0.56 0.9% 2.292 1.01 1.29 2.49

(1This rabbit was injected with 5 mg Mb in complete Freund’s adjuvant in each hind footpad on Day 1, with 1 mg alum-Mb in each hind footpad on Day 23, and the popliteal lymph nodes removed for culture on Day 52.

addition of peptide 145-151 plus KLH to these cells consistently antibody synthesis relative to KLH per se (8266, 8429).

inhibited

KLH

DISCUSSION This study was suggested by the finding (Table 1) that the addition of KLH to KLH/Mb-primed LNC induced the synthesis of Mb antibody. Similar observations with other antigens in murine (5-7,9, 10) and rabbit (8) systems were attributed to production by antigen-stimulated T cells of soluble factors which promote the antibody response of B cells to heterologous antigen. However, not excluded were alternative explanations such as polyclonal activation of T and/or B lymphocytes by heterologous antigen or serological (B cell) or cellular (T and/or B cell) crossreactivity. Therefore, these alternative possibilities were investigated with the KLH/Mb rabbit LNC systems. One hundred micrograms KLH, which induced KLH/Mb-primed LNC to synthesize Mb antibody, did not cause nonspecific activation of unprimed T or B cells to incorporate [3H]thymidine or to synthesize protein, IgG, or antibodies to KLH or Mb. By several sensitive and specific serological assays KLH and Mb did not cross-react (Table 2, Fig. 1). However, abundant and diverse evidence was obtained of cellular cross-reactivity between KLH and Mb and between KLH and three synthetic antigenic sites of Mb: 15-22, 56-62, and 145- 15 1. Experiments were not done with the two other Mb antigenic sites, 94- 100 and 113-119, which induced production by Mb-primed LNC of antibody and MIF (12), protein, IgG, DNA, and RNA (13, 27) and peptide l-6 (representing a nonantigenic region) which stimulates all of these responses except MIF (12). Adding KLH to Mb-primed LNC enhanced anti-Mb antibody synthesis. KLH plus KLH-educated LNC caused a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either elevated or suppressed anti-KLH antibody production, depending on whether Mb-CFA or alum-Mb was used for

394

GOOCH ET AL. TABLE

6

Induction of Various Types of Immune Responses upon Addition of KLH, Mb, and Mb Peptides to Rabbit Lymph Node Cells Primed with Alum-KLH” Additions (pg) Rabbit No.

KLH 0

Mb

15-22

0

0

cpm x 10m3

56-62

145-151 0

0.1 1 10 100 10 10 10

0.1 1 8298

0 1 10 100

0

0

0

1 10 1 1 8428

0

0 10 100

0

0

Anti-KLH

Anti-Mb

10.1 47.1 55. I 19.% 10.8 8.1 26.85 30.2

2.1 2.1 2.28 2.03 2.58

2.6 18.65 8.61 3.59 0.98 0.94 1.14 1.38

3.3 0.36 0.24 0.20 0.70 0.46 0A 0.62

0.687 6.22 5.24

0.385 0.600 0.643

100 1 100 8429

0 1 100

0

0

0

0

1 10 100 10 100 1

100 100

1.76 29.53 15.34 3.5 1.58 1.87 1.60 1.61 1.48 13.7

Protein

IgG

21.57 39.71 50.04 35.70 36.01 36.87

1.26 9.14 11.59

1.15 0.73 1.03 0.37 0.63 0.50 0.36 0.56 0.59 0.51

a These rabbits were injected with 1 mg alum-KLH in all four footpads on Day 1 and the draining axillary and popliteal lymph nodes removed on Day 7 for culture.

immunization. The addition of Mb to KLH-primed LNC suppressed or enhanced Mb antibody synthesis; anti-KLH antibody formation was unaffected or inhibited by added KLH. Several types of experiments indicated that KLH cross-reacts at the cellular level with the three Mb antigenic sites studied: Mb site plus Mb-sensitized LNC elevated or inhibited the anti-KLH antibody synthesis, depending on the length of time between priming and in vitro challenge. Challenge of Mb-educated LNC with a combination of KLH and Mb site lowered the anti-Mb antibody response compared

HEMOCYANIN/MYOGLOBIN

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395

to KLH alone. This mixture of KLH and Mb peptide also reduced the anti-KLH antibody synthesis by KLH-primed LNC compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced several types of cellular immune responses when KLH was added to KLH-primed LNC. These responses included the uptake (transport) of cyclic nucleotides (21), the uptake of thymidine into DNA (19), and protein synthesis (21). Finally, the addition of KLH, Mb, Mb antigenic sites, or combinations of KLH and Mb antigenic sites to KLH-primed LNC provided additional evidence for cellular cross-reactivity (Table 6). Mb induces these cells to produce antibody to KLH, protein, and IgG. Mb antigenic sites induced protein synthesis, but inhibited anti-KLH antibody production. Whereas KLH induced the production of antibody to KLH, the combination of antigenic site 146-151 plus KLH inhibited this antibody synthesis relative to KLH per se. These results do not elucidate the mechanisms of these cross-reactive immune responses, especially the inhibition. However, at least two features are noteworthy and may ultimately lead to more knowledge of the mechanisms. First, special conditions are required for these responses. The challenge of Mb-CFA-primed LNC with Mb enhanced anti-KLH antibody synthesis, whereas challenge of alum-Mb-educated cells suppressed this response. Second, the interval between in viva priming and in vitra challenge was also crucial: Mb synthetic sites plus LNC sensitized with CFA-Mb for 1 week enhanced KLH antibody synthesis. Addition of peptide to LNC sensitized 2 weeks earlier inhibited this antibody response. The interval between priming and antigenic challenge influenced help and suppression in other systems. Enhancement of antibody synthesis to the trinitrophenyl (TNP) hapten was noted when fowl y-globulin (FGG) was added to TNP-FGG-primed spleen cells from mice primed 5 weeks earlier, but inhibition when this antigen was added to murine splenocytes primed 14 weeks previously (IO). Early suppression of humoral immunity was noted in a hapten-carrier system (28). Optimum suppression of anti-TNP antibody synthesis resulted from the transfer of spleen cells from mice primed 3 days previously with 100 pg /3-galactosidase and challenge of recipients with TNP-galactosidase. Intervals of priming shorter than 7 days and longer than 14 days have thus far not been employed in our experiments on cellular cross-reactivity, except when rabbits were primed with both CFA-Mb and alum-Mb (Table 5). The enhanced cellular cross-reactive immune responses presumably depend upon the reaction of discrete antigenic sites with antigen-primed T and/or B memory cells, most likely T helper cells.’ This conclusion depends upon the following evidence: First, challenge of unprimed LNC with KLH, Mb, or Mb synthetic antigenic sites does not elicit any of these responses (12, 13; A. B. Stavitsky and W. W. Harold, unpublished observations). Second, challenge of KLH- or Mb-primed LNC with other proteins, including HSA (Tables 4 and 5), bovine y-globulin (BGG), ovalbumin, fibrinogen, and egg white lysozyme (A. B. Stavitsky and W. W. Harold, unpublished observations) does not elicit any of these immune responses. Third, addition of KLH, Mb, or Mb peptides to LNC primed with other proteins, including HSA and BGG, does not evoke any of these immune ’ Recently, clear evidence has been obtained for the cross-reactivity between KLH and Mb at the rabbit T-cell level (A. B. Stavitsky and W. W., Harold, unpublished observations).

396

GOOCH

ET AL.

responses (26). Fourth, enhancement of anti-MB antibod,y synthesis upon addition of KLH to KLH/Mb-sensitized LNC is thymus dependent. Fifth, KLH and Mb do not cross-react serologically. By analogy with the suggestion that the enhanced immune responses involve cross-reactivity at the T helper cell level, the suppressed immune responses may involve cross-reactive T suppressor cells. Recently, evidence has been presented for suppressor cells in the rabbit (29, 30). Each of the five Mb antigenic sites defined by Atassi (11.) induced Mb-primed LNC to produce MIF and antibody to Mb (12) as well as protein, IgG, DNA, and RNA (13). It was postulated (12, 13) that a Mb peptide triggered peptide-specific T cells to produce soluble factors that provide an essential signal to B cells; the other signal for these macromolecular syntheses presumably is provided by residual immunogen on dendritic cells (2-4). In the current study (Tables 3 and 6), addition of Mb sites enhanced or inhibited the anti-KLH antibody response of Mb-sensitized LNC; lowered the anti-Mb antibody response evoked by KLH compared to KLH; and reduced the anti-KLH of KLH-primed LNC compared to KLH alone. Possibly the Mb sites effected these results by reactions with T helper and/or suppressor cells, the outcome depending on the relative numbers of these cells. Two types of T helper cells specific for KLH have been found in the mouse (31). One helps B cells to respond to TNP coupled to KLH through the participation of KLH-specific T-cell factor(s). The other upon stimulation with KLH helps B cells to respond to sheep erythrocytic antigens-but not TNP-through the production of non-antigen-specific factors. It is not known whether the same or different populations of T helper cells respond to KLH and Mb. Striking differences have been observed in the in vitro antibody and other immune responses to KLH, Mb, and Mb sites of LNC from rabbits from different sources. The results in Table 6 provide examples of wide variability in spontaneous anti-KLH antibody responses and in KLH- and Mb-induced antibody responses by alum-KLH-primed LNC. This variability has prompted us to investigate the genetic control of the antibody response to Mb in mice (32) and that to Mb and KLH in rabbits (33). The immune response to Mb has been found (32,34) to be under H-2 Ir-gene control. These experiments raise many questions: Is there cross-reactivity at the T-, but not the B-, cell level? If so, how to explain this: Are the specificities of T- and B-cell receptors identical? How common is this type of cross-reactivity? What are its implications with respect to autoimmune, protective, and pathogenic immune processes, including the termination of tolerance and apparent nonspecific resistance to tumors and infections (e.g., (35))? Is the suppression due to suppressor cells? How do such small peptides induce such a variety of immune responses? What structural features of peptides are involved in these phenomena? Recently, highly purified, and immunologically active populations of antigen-primed rabbit T and B LNC have been prepared (36, 37). The combination of these populations, single Mb antigenic sites, and the systems described here should permit incisive study of some of these questions. REFERENCES 1. Stavitsky, A. B., and Self, C. H., Immunol. Commun. 1, 491, 1972. 2. Tew, J. G., Self, C. H., Harold, W. W., and Stavitsky, A. B., J. Zmmunol. 111,416,

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