Studies on functional subpopulations of B cells in mice

CELLULAR

61,386-396

IMMUNOLOGY

(198 1)

Studies on Functional Correction

Subpopulations

of B Cells in Mice

of the Immune Defect of CBA/N Mice by Transfer of C3 Receptor-Bearing B Cells

TULLIA

LINDSTEN

AND BIRGER ANDERSON

Department of Tumor Biology, Karolinska Institutet, S-104 01. Stockholm 60, Sweden Received January 21. 1981; accepted January 28, I%!?/ The present paper is an attempt to investigate closer the role of the C3 receptor as a surface marker for functionally different subpopulations of B cells. It was shown that B cells of normal mice can be divided into C3 receptor-positive and C3 receptor-negative cells. The former subpopulation was further shown to be significantly smaller in CBA/N, a mutant mouse strain of CBA with an X-linked immune defect at the B-cell level. Spleen cells from normal mice were fractionated into CRL-enriched and CRL-depleted populations and characterized functionally. The CRL-enriched subpopulation was shown to give a higher antibody response to PVP, the TI antigen tested, than the CRL-depleted subpopulation. The CRL-enriched subpopulation of spleen cells from normal (A X CBA/N)F, female mice could alone reconstitute the antibody response to PVP in immunodeficient (A X CBA/N)F, male mice. We thus conclude that the subpopulation lacking in CBA/N is a C3 receptor-positive B cell.

INTRODUCTION Some antigens can be classified as thymus dependent (TD),’ requiring helper T cells to trigger B cells into antibody synthesis. Other antigens can alone activate B cells and are accordingly called thymus independent (TI). TI antigens can also be further divided into a TI-1 subclass and a TI-2 subclass (for definition see (1)). It has been suggested that two different subpopulations of B cells are responsible for antibody responses to TI and TD antigens (for review see (2)). The surface characteristics of these postulated subpopulations are not known. CBA/N mice, a mutant strain of CBA, have an X-linked immunodeficiency at the B-cell level. This immune defect makes the CBA/N mice unable to respond to various TI antigens of the TI-2 subclass (3-8). Antibody production to the TI-1 antigens (9, 10) and TD antigens (5) are normal, as well as T-cell-mediated functions (4). In an earlier paper we have proposed a model for the ontogeny of the B cell in the CBA/N mice (3). It was suggested that a deviation of development occurs so that in the adult CBA/N mice, the subpopulation of B cells responsible ’ Abbreviations used: Ig, immunoglobulin; C3, complement factor 3; CRL, complement receptor lymphocytes; HRBC, horse red blood cells; SRBC, sheep red blood cells; EAC, erythrocytes coated with antibody and complement; EA, erythrocytes coated with antibody; SBA, soybean agglutinin; BSA, bovine serum albumin; BSS, balanced salt solution; TI, thymus independent; TD, thymus dependent; PVP, polyvinyl pyrrolidone; ABC, antigen binding capacity of antisera. 386 0008-8749/81/100386-l I$O2.00/0 Copyright D 1981 by Academic Press, Inc. All rights of reproduclion in any form reserved.

FUNCTIONAL

B CELL SUBPOPULATIONS

387

for antibody responsesto TI antigens is lacking. Analysis of the surface properties of CBA/N B cells could be a useful tool to study the differences between TI and TD subpopulations of B cells. So far it has been shown that CBA/N B cells fail to develop minor lymphocytestimulating determinants (1 l), lack cells which bear low to intermediate amounts of Ig on their surfaces (12, 13), have an increased ratio of IgM to IgD chains on their cell membranes (14, 15), and lack the differentiation antigens Lyb3, Lyb5, and Lyb7 (16-18). It has also been reported that CBA/N B cells express low surface C3 receptors ( 19). The present paper confirms the finding that a majority of CBA/N B cells lack C3 receptors. Also normal mice were shown to have a minor subpopulation of B cells devoid of C3 receptors. An attempt was made to characterize functionally a CRL-enriched subpopulation of spleeen cells from normal mice as well as a CRLdepleted subpopulation, by applying a separation technique developed by Parish and Hayward (20, 21). The functional studies were also extended using CBA/N as a model. MATERIALS

AND METHODS

Mice. CBA/N mice were obtained from NIH Rodent and Rabbit Production Section, Bethesda, Maryland. A/Sri and A.CA mice were taken from our own animal department. (A/Sri 8 X CBA/N 0) F, mice were used. The males of these hybrids are immunodeficient whereas the females can be used as normal controls. A/Sri and A.CA mice possessa dominant gene(s) that determines high response to PVP (22). Antigens and immunizations. Polyvinyl pyrrolidone (PVP) with molecular weights of 10,000 and 360,000 were obtained from Fluka AG, Switzerland. Immunization was performed with 0.1 pg of the 360,000 preparation given dissolved in BSS intravenously together with cells in cell transfer experiments. Serologic tests. Determination of hemolytic antibody to HRBC and PVP-labeled sheep erythrocytes as well as antigen binding capacity of antisera using ‘251-PVP were performed as previously described (23). Irradiation of mice and preparation of cell suspensions. Performed as previously described (23). Antisera. For preparation of rabbit anti-SRBC IgM antibody, a rabbit was immunized with 2 ml of a 10% solution of SRBC intravenously. Five days later the rabbit was bled and the serum collected. A monoclonal anti-Thy 1.2 serum was a kind gift from Dr. E. A. Clark, University of Washington, Seattle, Washington. Determination of Ig-bearing cells. Cells, 0.5 X 106, were incubated at 4°C for 30 min with 50 ~1of an 1:10 dilution of FITC-coupled goat anti-mouse Ig (Hyland Division Travenol Laboratories Inc., Costa Mesa, Calif.). After washing the number of fluorescent cells was determined using a uv-microscope. Preparation of EAC. A preparation of 0.5% SRBC was incubated with rabbit anti-SRBC at a final dilution of 1:40 at 4°C for 30 min. After washing the antibodycoated SRBC (EA) were incubated with fresh A/Sri mouse serum, as a source of complement, at 37°C for 30 min, at a final dilution of 1:4 if not indicated differently in individual experiments. EAC prepared in this way reacts both with CRL carrying C3b receptors and those carrying C3d receptors (24).

388

LINDSTEN

AND ANDERSSON

Determination of CRL. Cells, 0.5 X 106, were mixed with an equal volume of a 0.5% solution of EAC, and incubated at 37°C for 30 min and the number of CRL, i.e., cells carrying two or more red blood cells, was determined. In some experiments a two-step labeling technique was applied. Cells, 0.5 x 106,were first incubated with FITC-coupled goat anti-mouse Ig as described above. After washing the cells were allowed to react with an equal volume of EAC at 37°C for 30 min. The cells were inspected in a uv-microscope. Four different cell types were scored separately, Ig+CRL-, Ig+CRL+, IgCRL+, and Ig-CRL- cells, In all experiments cells were also allowed to react with EA, as a control, under the same conditions as with EAC. If not stated in individual experiments cells never formed rosettes with EA. Rosette sedimentation. The procedure used followed essentially the method described by Parish and Hayward (20, 21). Accordingly, EAC was prepared by mixing a 5% solution of SRBC with rabbit anti-SRBC at a final dilution of 1:40. After incubation at 4°C for 30 min the antibody-coated SRBC were washed and fresh A/Sri serum at a final dilution of 1:lO was added. After incubation at 37°C for 30 min the EAC was diluted to a 20% solution in BSS with 10% FCS. To 2.5 ml of the EAC preparation an equal volume of cells at a dilution of 4 X 10’ cells/ ml was added. The number of CRL was determined after incubation at 37OC for 30 min. The EAC-lymphocyte mixture was then layered on 4 ml of 14% FicollIsopaque. The tubes were spun at 1200g for 30 min at room temperature. After centrifugation the interphase and the pellet were collected separately. The number of CRL was determined immediately in the pellet. This fraction of cells make up the CRL-enriched subpopulation. The number of CRL in the interphase was determined after de nova incubation with EAC as described above. The interphase cells constitute the CRL-depleted subpopulation of cells. Before cell transfer of the fractionated cells the SRBC in the pellet were lysed by adding four parts of distilled water followed by addition of one part of 4.5% NaCl. The sera from all mice receiving fractionated cells were also extensively absorbed with SRBC before titration of sera by hemolysis. SBA agglutination. Performed essentially as described by Reisner et al. (25). Cells, 4 X 108, were allowed to react with an equal volume of SBA at a dilution of 2.5 mg/ml. After 5 min at room temperature the cells were layered on 40 ml of 2.5% BSA. Cells were allowed to sediment for 15 min at room temperature. The agglutinated fraction in the pellet and the nonagglutinated cells in the supernatant were collected and allowed to react for 10 min with 0.2 M D-galactose at room temperature and were then extensively washed in D-galactose followed by BSS. SBA was a generous gift from Dr. Nathan Sharon, Weizman Institute of Science, Rehovot, Israel. Treatment of cells with anti-Thy 1.2 serum. One milliliter of an 1:100 dilution of a monoclonal anti-Thy 1.2 serum was added to 2 X 10’ cells. After incubation at 4°C for 30 min the cells were washed and mixed with 2 ml of a 1:lO dilution of Low-Tox Rabbit Complement obtained from Cedarlane Laboratories Ltd., Hornby, Ontario, Canada. RESULTS Frequency of CRL in normal CBA mice and immunodeficient CBA/N mice, using EACprepared with different dilutions of the complement containing serum.

FUNCTIONAL

389

B CELL SUBPOPULATIONS

The frequency of CRL in spleens of normal CBA and immunodeficient CBA/N mice was determined by enumerating the number of rosettes formed between lymphocytes and EAC. The EAC was prepared with various concentrations of the complement-containing serum. As can be seen in Table 1, the percentage CRL in CBA/N is lower than that in normal CBA at high concentrations of complementcontaining serum. At a dilution of 1:4, 27% CRL were detected in CBA/N whereas the frequency in CBA was 38%. Upon dilution of the complement-containing serum the difference in frequency of CRL between CBA/N and CBA is even more pronounced. At a serum dilution of 1:40 only 13% of the CBA/N spleen cells form rosettes, compared to 37% of the control spleen cells. Thus the density of the C3 receptors on CBA/N spleen cells seemsto be lower than that in normal mice since the CBA/N CRL are more sensitive to dilution of the complement than the CBA CRL. In conclusion, CBA/N spleens lack a substantial part of a C3 receptorpositive lymphocyte subpopulation which is present in normal mice. In addition, the density of the C3 receptor on those cells that do possessthe receptor is lower in CBA/N. It should be pointed out that our indicator does not discriminate between C3b and C3d receptors, but measuresvirtually the total surface C3 receptors. Purijed B cells in normal CBA and immunodejicient CBA/N mice: Frequency of Ig-positive cells with C3 receptors. In order to directly demonstrate the existence

of B cells lacking C3 receptors in normal and immunodeficient CBA/N mice the following experiment was done. Spleen cells from CBA and CBA/N mice were allowed to react with SBA. The agglutinated cells were collected in the pellet whereas the nonagglutinated cells were found in the supernatant. The different subgroups of cells were then subjected to a two-step surface labeling. First the cells were labeled with FITC-coupled goat anti-mouse Ig, washed, and thereafter mixed with EAC, prepared with a dilution of 1:40 of the complement-containing serum. As can be seen in Fig. la, the cells in the pellet consist mainly of Ig-positive cells, i.e., B cells. It further shows the existence of a minority, 23%, of B cells lacking C3 receptors in normal CBA mice. In CBA/N mice, 50% of the Ig-positive cells lack C3 receptors as can be seen in Fig. lb. The same result was obtained when B cells were purified from spleens using a monoclonal anti-Thy 1.2 serum followed by lysis with rabbit complement, as can be seen in Figs. 2a and b. It is thus shown that in normal mice two subpopulations of Ig-positive cells exist, one which also TABLE 1 Frequency of CRL in Spleens of Normal CBA and Immunodeficient CBA/N Using EAC Prepared with Different Dilutions of Complement-Containing Serum Percentage CRL” Serum dilution

CBA

No serum I:4 1:20 1:40 1:lOO

0 38 36 31 I5

’ Mean of four mice.

CBA/N 0 21 21 13 16

390

LINDSTEN

AND

ANDERSSON CBA

Spleen

I

SBA-aqqlutination

Pbllet

Ig+CRL20%

Ig+CRL+

Ig-CRL+

66%

2%

Ig-CRL-

Ig+CRL-

12%

Ig+CRL+

Ig-CRL+

8%

0%

16%

Ig-CRL76%

a

CBA/N

SBA-agglutination

FIGS.

CBA/N

la AND b. Frequency of Ig-positive cells with C3 receptors mice, after treatment of spleen cells with SBA.

in normal

CBA

and immunodeficient

carries C3 receptors and another which lacks C3 receptors. The former subpopulation makes up the majority of the Ig-positive cells in normal mice. CBA/N mice, however, to a large extent lack the subpopulation of Ig-positive cells possessingC3 receptors. The method used does not show if the subpopulation of B cells in CBA/N mice that do have the C3 receptor contain cells with low-density C3 receptors. The antibody response to PVP and HRBC after transfer of A.CA spleen cell subpopulations enriched for or depleted of CRL, to lethally irradiated A. CA mice. In order to functionally characterize the CRL and the non-CRL in spleens of normal mice the following experiment was done. Spleen cells from normal A.CA were separated into one subpopulation enriched for CRL and one subpopulation depleted of CRL, by rosette sedimentation. As can be seen in Table 2, the CRLenriched subpopulation contained 60-96% CRL while the CRL-depleted subpopulation contained virtually no CRL at all. Unseparated spleen cells were 30-65% CRL positive. The separated subpopulations as well as unseparated spleen cells

FUNCTIONAL

B CELL

391

SUBPOPULATIONS

CBA

Spleen CRL+ 43%

1g+ 52%

anti-Thy-l.2

+ rabbit

complement

anti-Thy-l.2

+ rabbit

complement

i

CBA/N

Spleen

I,

I +CRL- Ig+CRL+ 39%

34%

Ig-CRL+ 0%

Ig-CRL-

I

26%

b

FIGS.2a AND b. CBA/N

mice after

Frequency treatment

of Ig-positive cells with C3 receptors in normal CBA of spleen cells with anti-Thy I .2 serum and rabbit

and immunodeficient complement.

were then transferred to lethally irradiated A.CA mice, together with the antigens, PVP and HRBC. The mice were bled at Day 20 and hemolytic antibody titers and antigen binding capacity were determined. The results from two different experiments are shown in Table 2. The CRL-enriched subpopulation gave a higher antibody response to PVP than the CRL depleted one. In Experiment 1, the antibody response to HRBC was higher in the CRL-depleted subpopulation than in the CRL-enriched one. Thus, a subpopulation of spleen cells enriched for CRL gives a better antibody response to the TI antigen used, than a subpopulation depleted of CRL. This latter subpopulation of spleen cells on the other hand gives a better response to a TD antigen. The antibody response to PVP in immunode$cient, nonirradiated (A X CBA/N)-

392

LINDSTEN

AND ANDERSSON TABLE 2

The Antibody Response to PVP and HRBC after Transfer of ACA Spleen Cell Subpopulations, Enriched for or Depleted of CRL, to Lethally Irradiated ACA Mice Antibody response Day 20b Log, hemolytic titer’ %CRL

PVP

HRBC

log,, ABC + SE PVP

Experiment 1 CRL enriched CRL depleted Unseparated

60 0 30

3.5 (46.8) 2.0 (9.0) 1.0 (3.0)

1.5 (5.2) 2.8 (21.7)
N.D. N.D. N.D.

Experiment 2 CRL enriched CRL depleted Unseparated

96 1 65

5.0 (243.0) 3.7 (58.3) 3.4 (41.9)

N.D. N.D. N.D.

2.6 + 0.08 (426) 2.3 -t 0.07 (177) 2.5 k 0.08 (275)

Cells transferreda

’ l-2 X 10’ cells were given iv. ’ Mean of 4-10 mice. (-Arithmetic values given within parentheses.

F, 6 mice after transfer of spleen cell subpopulations from A X CBA/N)F, P mice enriched for or depleted of CRL. It has earlier been shown that after transfer of spleen cells from normal female F, hybrids of CBA/N mice to immunodeficient male littermates, these mice can mount normal antibody responsesto TI antigens (26). In order to elucidate whether transfer of a spleen cell subpopulation enriched for CRL would alone be able to restore the antibody response to TI antigens, to the same degree as unseparated spleen cells, the following experiment was done. Spleen cells from normal (A X CBA/N)F, P mice were fractionated into CRLenriched and CRL-depleted subpopulations by rosette sedimentation. The separated subpopulations were transferred to immunodeficient, nonirradiated (A X CBA/ N)F, dmice. A control group of mice received unseparated spleen cells. The mice were immunized at the same time with PVP. Twelve days later the mice were bled TABLE 3 The Antibody Response to PVP in Immunodeficient, Nonirradiated (A X CBA/N)F, d after Transfer of (A X CBA/N)F, 9 Spleen Cell Subpopulations Enriched for or Depleted of CRL

Cells transferred”

Percentage CRL

Antibody response Day 12’ Log, hemolytic titer< PVP

CRL enriched CRL depleted Unseparated No cells

87 3 45 0

2.4 (14.0) 1.0 (3.0) 2.0 (9.0)
a 2 X 10’ cells were given iv. b Mean of five mice in each group. ’ Arithmetic values given within parentheses.

FUNCTIONAL

393

B CELL SUBPOPULATIONS

and hemolytic antibody titers were determined. As shown in Table 3, the group of mice that received the CRL-enriched subpopulation of spleen cells had antibody titers in the same range as the mice given unseparated spleen cells. The mice given CRL-depleted subpopulation had a reduced antibody response to PVP. It has been ruled out in an earlier paper (3) that the antibody response seen is the effect of helper T cells making the CBA/N mice able to respond to PVP. This experiment shows that the C3 receptor-positive cell present in the spleens of normal mice which is responsible for antibody responses to TI antigens can completely restore the immune defect of CBA/N mice. The antibody response to PVP after transfer of (A X CBA/N)F, P puriJied B cells enriched for or depleted of CRL, to immunodejicient, nonirradiated (A X CBA/N)F, 6. In order to determine whether the low antibody response to PVP

seen after transfer of a CRL-depleted subpopulation of normal spleen cells to deficient mice, really was due to the inability of a B-cell subpopulation to respond to PVP or whether it was an effect of contaminating T cells the following experiment was done. Spleen cells from (A X CBA/N)F, 0 were separated into CRL-enriched and CRL-depleted subpopulations by rosette sedimentation. As shown in Fig. 3, double staining with FITC-coupled goat anti-mouse Ig and EAC revealed 83% Ig+CRL+ cells in the pellet, i.e., the CRL-enriched subpopulation. The CRL-depleted subpopulation contained only 5% Ig+CRL+ cells whereas the number of Ig+CRL- mounted to 25%. The CRL-depleted cell population was then subjected to SBA agglutination which increased the number of Ig+ cells to 55% in the agglutinated cell fraction. As indicated in Fig. 3, Group A consisting of unseparated (AxCBA/N)F~

p

Spleen Iq+CRL8%

A

Ig+CRL+ Ig-CRL+ 40% 0%

I

Ig-CRL52%

I

I

Rosette-sedimentation

B

Ig+CRL6%

Ig+CRL+ Ig-CRL+ 83%

0%

I¶-CRL-

Ig+CRL-

11%

25%

Ig+CRL+ Ig-CRL+ 5%

0%

I C

FIG. 3. Separation scheme for results shown in Table 4.

Ig-CRL70"

SBA-aqqlutination

N.D.

394

LINDSTEN

AND

ANDERSON

spleen cells, Group B containing C3 receptor-positive B cells, and Group C containing C3 receptor-negative B cells were transferred to immunodeficient nonirradiated (A X CBA/N)F, 8 mice together with PVP. At Day 14, the mice were bled and hemolytic antibody titers were determined. The results are shown in Table 4. It is clear that there is a close to complete restoration of the antibody response to PVP in the mice given C3 receptor-positive B cells as compared to mice given unseparated spleen cells. Still there is a low antibody response to PVP in the group of mice which received the C3 receptor-negative B-cell subpopulation. Our interpretation is that a subpopulation of B cells lacking the C3 receptor gives a low antibody response to TI antigens. The subpopulation of B cells responsible for antibody response to TI antigens is C3 receptor positive. This cell population can correct the immune defect of CBA/N and is therefore presumably absent in these mice. DISCUSSION This paper deals with the surface characteristics of functional B-cell subpopulations in the mouse. CBA/N mice with an X-linked immune defect at the B-cell level are used as models in these studies. Our initial finding was that the frequency of CRL in spleens of CBA/N mice was greatly reduced at a limiting dilution of the complement-containing serum, as compared to normal CBA mice. It was also concluded that the density of C3 receptors on the surface of spleen cells was lower in the CBA/N mouse than in normal controls as the sensitivity to dilution of the complement-containing serum was greater in CBA/N than in CBA. Thus, CBA/N mice lack a substantial part of a C3 receptor-positive spleen cell subpopulation and those cells that do possessC3 receptors carry it at a lower density. To directly demonstrate the frequency of B cells carrying the C3 receptors in normal and immunodeficient mice, B cells were purified from spleens by agglutination with SBA or by treatment with an anti-Thy 1.2 serum and complement. Normal mice were shown to have 23-35s B cells lacking C3 receptors whereas in CBA/N mice 50-53s of the B cells were C3 receptor negative, using a twostep surface labeling procedure. No attempt was made to determine the density of the C3 receptors on those B cells in CBA/N that did carry the receptor. We TABLE

4

The Antibody Response to PVP in Immunodeficient, Nonirradiated (A X CBA/N)F, Transfer of (A X CBA/N)F, Q B Cells Enriched for or Depleted of CRL

Antibody response Day 14h Log, hemolytic titer’ PVP

Cells transferred”

2.4 2.2 1.0 0.8

Unseparated spleen cells (A) C3 receptor-positive B cells (B) C3 receptor-negative B cells (C) No cells O2 x IO’ cells were given iv. For explanation b Mean of S-10 mice. ’ Arithmetic values given within parentheses.

d after

see Fig. 3.

( 14.0) (11.2) (3.0) (2.4)

FUNCTIONAL

B CELL SUBPOPULATIONS

395

thus have determined the surface characteristics of two subsets of B cells in the normal mouse, the Ig+CRL- and the Ig+CRL+ cell type. CBA/N was shown to be deficient in the latter of these cell types. In order to functionally characterize the two different spleen cell subpopulations in normal mice, a separation procedure described by Parish and Hayward was used. Thus a CRL-enriched subpopulation of spleen cells from normal mice was shown to give a higher antibody response to PVP, a TI antigen, than the CRL-depleted subpopulation, when tested in vivo. The CRL-depleted subpopulation on the other hand gave a better antibody response to HRBC, a TD antigen. We conclude that spleen cells carrying C3 receptors are responsible for antibody production to TI antigens. This was further stressed by the fact that when the CRL-enriched spleen cell subpopulation from normal (A X CBA/N)F, P was transferred to nonirradiated, immunodeficient (A X CBA/N)F, 6, a complete restoration of the antibody response to PVP occurred. The low antibody response to PVP in immunodeficient mice given the CRL-depleted subpopulation persisted after elimination of the majority of the T cells after agglutination with SBA. SBA, per se, does not alter the ability of cells to respond to antigenic stimulus (25). The low antibody response seen thus seemed to be caused by inability of the B cells in the CRL-depleted fraction to respond to PVP. It is suggested that the TI subpopulation of B cells in the normal mouse carry C3 receptors and that the TD subpopulation is a C3 receptor-negative cell. The CBA/N mouse lacks a major part of the C3 receptor-positive B-cell subpopulation and is thus unable to respond to TI antigens. The possibility exists that in fact the TI subpopulation of B cells in normal mice can further be divided into a TI- I subset characterized by low-density C3 receptors, and a TI-2 subset characterized by high-density C3 receptors. The TI-1 subsets of cells should then be present in CBA/N mice and could explain the fact that the Ig+CRL+ subpopulation is not totally absent. Experiments determining the functional properties of Ig+CRL+ cells from CBA/N mice have to be performed, to investigate this possibility. Thus, C3 receptors seem to be the surface characteristic of the TI-2 subset of B cells. The C3 receptors do not, however, seem to be involved in triggering of these cells, as it has been shown that the antibody response to PVP, a TI-2 antigen, is normal in mice depleted of circulating C3 (27). Further experimentation is also required in order to determine the C3b and C3d receptors on functionally different subsets of B cells responding to TI-1 and TI-2 antigens. In accordance with our results, it has recently been shown by others that B-cell populations enriched for CRL respond better to two other different TI-2 antigens, Fc fragments and DNP-Ficoll, respectively, as compared to cell populations depleted of CRL (28, 29). ACKNOWLEDGMENTS This work was supported by the Swedish Cancer Society, the Swedish Medical Research Council, and by funds from the Karolinska Institutet.

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2279, 1979. 19. Sher, I., Ahmed, A., Sharrow, S. O., and Paul, W. E., In “Development of Host Defenses” (M. D. Cooper and D. H. Dayton, Eds.), p. 55. Raven Press, New York, 1977. 20. Parish, C. R., and Hayward, J. A., Proc. R. Sot. Lond. B 187, 47, 1974. 21. Parish, C. R., and Hayward, J. A., Proc. R. Sot. Lond. B 187, 65, 1974. 22. Andersson, B., and Blomgren, H. In “Immune Reactivity of Lymphocytes” (M. Feldman and A. Globerson, Eds.), p. 283. Plenum, New York, 1976. 23. Andersson, B., and Blomgren, H., Cell. Immunol. 2, 41 I, 197 I. 24. Ross, G. D., and Polley, M. J., Sand. J. Immunol. lS(Suppl. 5), 99, 1976. 25. Reisner, Y., Ravid, A., and Sharon, N., Biochem. Biophys. Res. Commun. 72, 1585, 1976. 26. Sher, I., Steinberg, A. D., Bernig, A. K., and Paul, W. E., J. Exp. Med. 142, 637, 1975. 27. Pepys, M. B., J. Exp. Med. 140, 126, 1974. 28. Berman, M. A., Morgan, E. L., and Weigle, W. 0.. Cell. Immunol. 52, 341, 1980. 29. Nariuchi, H., and Kachiuchi, T., Cell. Immunol. 54, 264, 1980.