EXPERI~IENTAT.

PARASITOI.OGY

41, 141-159

( 1977)

Ascaris SUUM: Immune Response in the Guinea Pig

Laboratory

I. Lymphoid

Cell

PHILIP

B.

Responses during KHOURY

AND

of Parasitology,

Primary

Infections

E. J. L. SOULSBY

School of Veterinary Philadelphia, Pennsylvania

Medicine, University 19174, U.S.A.

(Accepted

12 April

for publication

of Pennsylvania,

1976)

E. J. L. 1977. Ascaris suum: Immune response in the KHOURY, PHILIP B., AND SOULSBY, guinea pig. I. Lymphoid cell responses during primary infections. Experimental Parasitology 41, 141-159. The lymphoid cell responses of the mesenteric, hepatic, and mediastinal lymph nodes, of the spleen, and of the Peyer’s patches of normal guinea pigs that (a) were orally infected with 10,000 infective eggs of Ascaris suum (Group I), (b) received 10,000 artificially hatched second-stage larvae of A. suum via the mesenteric vein (Group II), or (c) received 1500 third-stage larvae of A. suum via the saphenous vein (Group III) were assessed by antigen-induced lymphocyte-transformation, rosette-formation, and rosette-plaquing techniques. Antigen-induced lymphocyte transformation measured qualitatively the antigen-sensitive cell population, rosette formation measured quantitatively the immunoglobulin-receptored cell population and rosette-plaquing measured quantitatively the receptored antibody-producing and the nonreceptored antibody-producing cell populations. Antigen-induced lymphocyte-transformation and rosette-formation studies showed that the immune response to A. suum was local in character at least for the first 11-12 days of infection as exemplified by the marked responses of the draining lymph nodes at the time the parasite was migrating through the tissues of the host. Rosette-plaquing studies in conjunction with the rosette-inhibition studies showed that the lymphoid cell responses of the draining lymph nodes progressed from an antigen-sensitive state to an immunoglobulin-receptored state, then to a receptored state capable of antibody production and finally to a nonreceptored antibody-producing state. In animals of Group I, the lymphoid cells of the mesenteric, hepatic, and mediastinal lymph nodes formed predominantly IgM and IgE antigen-specific rosettes (peak responses at Days 2, 5, and 7 for the mesenteric, hepatic, and mediastinal nodes, respectively), rosette-plaques (peak responses at Days 5, 7, and 9 for the mesenteric, hepatic, and mediastinal nodes, respectively), and plaques (peak responses at Days 7, 9, and 12 for the mesenteric, hepatic, and mediastinal nodes, respectively). The IgE-responsive cells were appreciably higher in the mediastinal lymph nodes than in the other lymphoid organs studied. The responses of the spleen were predominantly IgM in character and they increased gradually as the infection progressed without showing detectable peak responses. The lymphocytes of the Peyer’s patches did not exhibit antigen-specific rosetting, rosette-plaquing, or plaquing responses. In Group II animals (elimination of the bowel phase of the infection) only the hepatic and mediastinal nodes exhibited peak responses and these were similar to those of Group I animals in the type and pattern of the responses and in the time of their sequential appearance. In animals of Group III (elimination of both the bowel and the hepatic phase of the infection) only the mediastinal nodes exhibited peak responses and these were similar to those of Groups I and II in the type (predominantly IgM and IgE) and their sequential Peak antigen-specific rosettes, rosettepattern but differed in the time of their appearance: plaques, and plaques occurred in the mediastinal nodes of Group III animals at Days 2, 5, and 7 after infection, respectively. In each of the responsive lymphoid organs studied, 141 Copyright All rights

1977 by Academic o? reproduction in any

Press, Inc. form reserved.

ISSN

0014-4894

l-1-2

KHOURY AND SOULSBY there occurred a certain population of lymphoid cells which was producing imlnunoglobuli~ls that were not necessarily specific for the A. suum antigen. INDEX DESCRIPTORS: Ascaris suum; Nematode; Guinea pig; Lymphocyte transformation; Rosette formation; Rosette-plaque formation; Plaque formation; Receptors; IgM-, IgA-, IgGa-, and IgE-forming cells; Lymphoid cell kinetics; Thymidine, tritiated; Rosette inhibilion; Immunity.

Various serological techniques, including complement fixation (Jezioranska and Dombrowolska 1956)) precipitin ( Coventry 1929; Oliver-Gonz?dez 1946), and conglutinating complement absorption ( Taff s 1958) tests, have been used to determine the antibody response in primary and secondary infections with Ascaris suum. These studies characterized the general nature of the response but could not delineate the contribution of each developmental stage to this general response. It was suggested by Dobson et al. (1971) that IgE-type antibodies were produced by local lymphoid tissue draining parasitized organs in guinea pigs infected with A. suum, and a sequential response of lymph nodes of parasitized organs was demonstrated by Soulsby ( 1972). The latter author showed by antigen-induced blastogenesis that cells of the hepatic and mediastinal nodes responded when larvae had migrated to the liver and lungs, respectively, but the response by cells from the spleen was negligible until some 10 days after infection. Such studies would suggest that antigen derived from the various developmental stages of the parasite, at least in the early stages of the infection, does not become generally distributed in the body. The present study extends these previous observations using infections with A. suum variously abbreviated to confirm that the location of the developmental stage of A. suum determines which lymphoid center will respond. In addition, this study is concerned with a quantitative and qualitative characterization of the lymphoid cell populations of the lymphoid tissues draining, and distant to, the site of a primary infection with A. suum. This was accomplished

by the use of in vitro antigen-induced lymphocyte-transformation, rosette-formation, and rosette-plaquing techniques. Such methods assessed antigen sensitivity, antigen binding, and antibody production by lymphocytes, respectively, and the use of inhibition techniques permitted a determination of the progression of local lymphoid cell responses in the lymphoid organs draining parasitized tissues. These responses were related to the migration of the parasite in the host. MATERIALSANDMETHODS

Animals Outbred Hartley-strain guinea pigs Perfection Breeders, Douglasville, Pa.) with an initial weight of 450-500 g and an age of 5 weeks were used for infection. New Zealand white rabbits, 3-4 months of age (Perfection Breeders, Douglasville, Pa. ), were used in the preparation of antisera. Infections with Ascaris suum Three experimental groups each of 35 guinea pigs were used. Group I. Animals were orally infected with 10,000 infective eggs of A. suum. Group ZZ. Animals were infected by injection of 10,000 artificially hatched secondstage larvae (2L) of A. suum into the mesenteric vein, after laparotomy under halothane ( Fluothane, Ayerst Laboratories ) anesthesia. Infective 2L were obtained by the hatching technique of Jaskoski and Collucci (1964) and its modification by Williams and Soulsby (1970). Larvae were cleared of debris by passage through a pad of cotton in a Baermann apparatus and were sterilized in an antibiotic solution

LYMPHOID

(Morseth and Soulsby 1969). They then were suspended in sterile saline to a concentration of 10,000 larvae/ml. Group III. Animals were infected by injcction of 1500 third-stage larvae (3L) of A. suum into the saphenous veiu. These larvae were obtained by the Baermann technique from the lungs of guinea pigs that had received infective eggs 7 days previously. After washing in sterile saline and sterilization in an antibiotic solution (Morseth and Soulsby 1969), harvested larvae were suspended in sterile saline to a concentration of 1500 larvae/ml. Control

143

CELL RESPONSES IN ASCARIASIS

Animals

Group IV. This group was comprised of three subgroups each of 35 animals, which served as controls for each of the experimental groups. Five animals from each experimental group, together with an equal number from a control subgroup were sacrificed at Day 0 and at Days 1, 2, 5, 7, 9, and 12 after infection, The mesenteric. hepatic, and mediastinnl lymph nodes, the spleen, and the Peycr’s patches of each animal were removed under sterile conditions and individually processed for studies of in vitro antigen-induced lymphocyte transformation, rosette formation, and rosette plaquing. Antigen Soluble antigen was prepared from the adult stages of A. suum ( WWAg) as described by Williams and Soulsby ( 1970). Protein was estimated using a Beckman DG-B grating spectrophotometer and using 260- and 280-nm wavelengths with standards prepared against rabbit serum albumin. The protein concentration of the antigen was adjusted to 0.5 mg/ml for antigen-induced lymphocyte-transformation studies and to 5 mg of protein/ml for rosette-plaquing rosette-formation and studies.

Processing of Lymphoid

Cells

Each lymphoid organ was placed in a sterile petri dish containing Eagle’s minimal essential medium ( MEM) (GIBCO, Grand Island, N.Y.) in combination with 300 U of penicillin/ml and 0.3 mg of streptomycin/ml. The lymphoid cells were teased out in MEM and were passed through a sterile fine-mesh stainless-steel sieve. Macrophages were removed from the lymphoid cell populations using carbonyl iron according to the method of Coombs et al. (1970). The remaining cells were washed by centrifugation three times in MEM and were then adjusted to a concentration of lo6 lymphocytes/ml for in &To antigen-induced lymphocyte-transformation and lo7 lymphocytes/ml for rosette-formation and rosette-plaquing studies. In Vitro Antigen-Znduced Transformation

Lymphocyte

The technique used was basically that described by Dobson and So&by ( 1974). One milliliter of lymphoid cell suspension containing 10c lymphocytes was dispensed into each of nine sterile siliconized 13 x loo-mm Pyrex test tubes (Bellco, Vineland, N.J.). In addition, sets of three tubes received either 0.1 ml of antigen (0.5 mg of A. suum protein/ml) or 0.1 ml of culture medium. All tubes were then incubated at 37 C in an incubator containing 10% COn in air. Cultures containing antigen or medium were pulsed with &i of tritriated thymidine ( [“H]Tdr) (sp act 2 Ci/mmole) (International Chemical and Nuclear Corporation, Irvine, Calif.) per culture tube 16 hr prior to harvest at 5 days. Subsequently, the lymphocytes were washed three times in an excess sterile saline, solubilized in Nuclear Chicago solubilizer (Nuclear Chicago), and processed for liquid scintillation spectrometry. The results were expressed as the ratio of the mean distintegrations of tritium per minute per lo6 lymphocytes (dpm 3H/10g cells) from antigen-

144

KHOURY

stimulated cultures to lymphocytes control nonstimulated cultures.

AND SOULSBY

from

Red Blood Cells Sheep erythrocytes (SRBC) were collected weekly from the same animal into an equal volume of Alsever’s solution. Red blood cells were separated by centrifugation, washed three times in sterile saline, and stored as a 50% suspension in MEM at 4 C in a siliconized flask for 24 hr prior to use. When required, they were washed once in sterile saline. Fractions Preparation of lmmunoglobulin of Guinea Pig Serum and Antisera Immunoglobulins (Igs) and their subfractions were isolated from guinea pig serum by chromatography and assessed for their purity by immunoelectrophoresis (IEP). Antisera to these fractions were prepared in rabbits and the antisera were then adsorbed with Igs crosslinked with glutaraldehyde to render them specific. Their specificity was assessed by IEP and gel diffusion (GD). Gel Chromatography Sixty grams of Sephadex G-200 and Sephadex G-25 (Pharmacia, Sweden) were swollen for 3 days and 1 day, respectively, in an excess of 0.1 M phosphate-buffered saline (PBS ), pH 7.2. Approximately 0.2% sodium azide was added in order to prevent bacterial contamination. Three K25/100 Sephadex laboratory columns (Pharmacia, Sweden) were mounted vertically and were filled with PBS containing 0.2% sodium azide. Approximately 5 g of swollen Sephadex G-25 were added to each column and were allowed to settle. The columns were then packed over a period of 48 hr with Sephadex G-200, from a large funnel, under gravity pressure of 15 cm. After the columns were packed, flow adaptors were inserted at the top of each column. The columns were then connected

in a series of three and cycled by upward flow from an eluant reservoir at a flow rate of 12-15 ml/hr. For application of the sample to the column, the eluant reservoir was disconnected and the sample was allowed to feed into the base of the first column by gravity. The eluant reservoir was then connected to the column. After the passage of the void volume (360 ml) of the columns, collection of the fractions was commenced. Ion-Exchange

Chromatography

DEAE-Sephadex A-25 (Pharmacia, Sweden) was swollen for 2 days at room temperature in an excess of 0.01 M Tris-HCl buffer, pH 8.6, and then washed with 2-3 liters of buffer using a Buchner funnel. A 25 x 2.5-cm Chromaflex column (Kontes Biomedical Products, Vineland, N. J. ) was packed with DEAE-Sephadex A-25. The sample was dialyzed against the starting buffer and was then applied to the column. One hundred milliliters of starting buffer was then passed through the column and was followed by a nonlinear continuous gradient of buffer consisting of 300 ml of 0.01 M Tris-HCl buffer, pH 8.6, and 300 ml of 0.3 M. Tris-HCl buffer, pH 7.2, mixed in a gradient maker. Collection

of Fractions

The outlet tubing from the columns (Sephadex G-200 and DEAE-Sephadex A-25) was connected to a photoelectric drop counter (National Instrument Laboratories, Washington, D. C. ) and the eluant was recovered in a fraction collector, fractions of 200 drops being collected. The optical density of the fractions was determined at 280 nm in a Beckman DG-B grating spectrophotometer. Concentration

of Fractions

Selected fractions from the chromatogram were pooled and concentrated by dialysis against Aquacide III (Calbiochem,

LYMPHOID

La Jolla, Calif.)

to a volume of 1-2 ml. These were stored at -20 C until needed. 7ross-Linking of Proteins with Xutaraldehyde Ten milliliters of guinea pig Ig, or Ig sub‘raction, containing 8 mg of protein/ml, were adjusted to pH 4.5 by the addition of l:l M HCl; 0.5 ml of 0.1% solution of {lutaraldehyde (Glutaraldehyde Solution, iO% w/w, Fisher Scientific Company, Fair -,awn, N.J.) was added to the sample, and -he resulting mixture was stirred continunlsly for 2 hr at room temperature. One nilliliter of 1 M lysine was then added, md stirring was continued further for LO min. The mixture was then centrifuged bt 1500g for 10 min and the resulting pre:ipitate (gel) was washed twice with saline 10.1 M, pH 6.8), once with glycine-HCl luffer (0.1 N, pH 2.0), and again twice with saline. The gel was then stored at 4 C. Idsorption

of Antisera

The globulins were precipitated at room zmperature from the rabbit antisera by the addition of saturated ammonium sulfate ‘SAS), pH 7.5, to a final concentration of fO% ammonium sulfate (Stelos 1967). Two nilliliters of globulin solution were inacIvated for 30 min at 56 C and were then added to the appropriate gel (approxmately 2 ml containing about 35 mg of ?rotein/ml) obtained by cross-linkage of guinea pig Igs with glutaraldehyde. The nixture was stirred for 1 hr at 37 C and igain for 1 hr at 4 C. The mixture was then :entrifuged at 1500g and the supernatant luid was adsorbed twice more with the gel. ?he supernatant from this represented ad:orbed antiserum which was then tested ‘or purity by IEP and CD, using the Igs jolated from guinea pig serum. ‘mmunization

145

CELL RESPONSES IN ASCARIASIS

of Rabbits

Rabbits were injected intramuscularly at veekly intervals for a period of 4 weeks

with 0.5-ml (containing 4.1 mg of protein of IgM, 3.4 mg of protein of IgA, or 3.1 mg of protein of IgGp) quantities of each protein emulsified with an equal volume of complete or incomplete Freund’s adjuvant (CFA or IFA). Each rabbit was then boosted 4 weeks after the last injection. When a test serum sample revealed precipitating antibodies on IEP, the animal was bled for serum. Preparation

of Light Chains (L Chains)

Three volumes of a 4% solution of Ethodin (Winthrop Laboratories, New York, N.Y.) were added to 1 vol of guinea pig serum and the pH of the solution was adjusted to 7.6. The solution was incubated overnight at 4 C and was then centrifuged at 2000g. The resulting precipitate was discarded and the supernatant fluid was clarified by the addition of activated charcoal (Atlas Chemical Industries, Wilmington, Del.) and centrifugation. SAS was added to the resulting supematant fluid to a final concentration of 40% ammonium sulfate (Stelos 1976)) and the precipitate formed was dissolved in PBS and was applied to a Sephadex G-200 (Pharmacia, Sweden) column. The IgG peak was collected and concentrated. The L chains were then prepared from the IgG globulins as described by Fudenberg ( 1967). Anti-Guinea

Pig Serum ( Anti-gp)

Guinea pig serum was injected with an equal volume of IFA into rabbits. The animals were bled and the serum was separated and stored at -20 C. Anti-Guinea

Pig Colostral

Whey

Guinea pig colostrum was collected for the first 4 days after parturition and was defatted by centrifugation at 2000g for 30 min at 4 C. One-tenth milligram of rennin was added to 1 ml of colostrum and the mixture was centrifuged at 2000g for 30 min at 4 C; the resulting casein precipitate

146

KJSOURY

AND

was removed and the remaining colostral whey was injected with IFA into a rabbit. The rabbit was bled and the serum was collected and stored at -20 C. Guinea Pig IgA

SOULSBY

IgM peak and then with cross-Iinkcd fcta guinea pig serum in order to remove an) antibodies present against a2-macroglobu lins. The resulting antiserum was tested fd. purity by IEP and GD against whole guinea pig serum, guinea pig IgM, and I chains.

Guinea pig colostral whey was chromatographed on Sephadex G-200 and the IgA peaks formed on the descending arms Guinea Pig IgG, of the 19s proteins were pooled, concenNormal guinea pig serum was chromate trated, and rechromatographed on DEAE- graphed on Sephadex G-200 columns Sephadex A-25 (Pharmacia, Sweden). IEP Those fractions forming the 7s peak wer\ was performed on each fraction. Those pooled, concentrated, and passed througl fractions which formed an IgA band ex- DEAE-Sephadex A-25. The breakthrougl clusively were pooled and concentrated. formed with 0.01 M Tris-HCl buffer, pl 8.6, was pooled, concentrated, and rechro Anti-Guinea Pig a Chains (Anti-a) matographed twice more on DEAE-Sepha Guinea pig IgA (13.6 mg of protein) was dex A-25. injected into a rabbit. The animal was bled, and the antiserum was adsorbed with a Anti-Guinea Pig yz Chains (Anti-y,) glutaraldehyde cross-linked protein comGuinea pig IgG2 (12.4 mg of protein posed of guinea pig IgM and those fractions was injected with CFA into a rabbit. Thd of colostral whey that were eluted after animal was bled and the antiserum oE the IgA peak, The adsorbed antiserum was tained was electrophoresed with guine; tested for purity by IEP and GD against pig IgG,. The resulting IgGz bands wen colostra1 whey, whoIe guinea pig serum, cut from the agar and were injected wit1 IgA, and L chains. CFA into a rabbit. The rabbit was blec

and the antiserum obtained was adsorbe; with a glutaraldehyde cross-linked protei! Normal guinea pig serum was chro- composed of all those proteins present il matographed on Sephadex G-200. The as- the guinea pig serum that were eluted froi-, cending arm of the first 19s peak was DEAE-Sephadex A-25 between 0.08 fi, pooIed, concentrated, and reapplied to the Tris-HCI buffer, pH 7.2, and 0.04 M Tris Sephadex G-200 columns. Again, the as- HCl buffer, pH 7.2. The adsorbed ant’? cending arm of the 19s peak was collected serum was tested for purity by IEP and GI and concentrated. This was applied further against guinea pig IgG,, whole guinea pi: to the Sephadex G-200 column and the serum, and L chains. latter procedure of collection and concentration was repeated. Anti-Human E Chains (Anti-,) Guinea Pig 7gM

Anti-Guinea Pig p Chains (Anti-p) Guinea pig IgM (16.4 mg of protein) was injected with CFA into a rabbit. The animal was bled, and the antiserum obtained was adsorbed first with a glutaraldehyde cross-linked protein consisting of all Senhadex G-200 fractions other than the

This antiserum was obtained from Hy land Division Travenol Laboratories, In& Costa Mesa, Calif. It was tested for puri$ by the inhibition of indirect agglutinatio: systems (Stavitsky and Ingraham 19641 This antiserum was found to cross-rear with guinea pig IgE by Dobson et a f 1971).

LYMPHOID

Coupling of Antigen Glutaraldehyde

CELL

RESPONSES

to SRBC with

Antigen coupled to SRBC with glutaraldehyde was used as the rosette target-cell in both the rosette-formation and rosetteof the plaquing assays. A modification methods of Onkelinx et ab. (1969) and Eskenazy and Petrunov (1971) was used. Three milliliters of 0.15 M phospbate-buffered saline (pH 7.2), 0.5 ml of WWAg (5.0 mg of protein/ml), 0.2 ml of 50% SRBC, and 0.05 ml of glutaraldehyde were mixed. The mixture was incubated for 1 hr at room temperature and was then centrifuged at 2000g for 10 min at 4 C. The ;upernatant was discarded and the antigencoupled SRBC were washed three times in saline and twice in MEM and then SUSpended in MEM to a concentration of 1 x 10” SRBC/ml. Coupling Chromic

of Antigen Chloride

or Antisera

with

WWAg or antiserum (anti-p, -y2, -a, -6) coupled to SRBC with chromic chloride was used as the plaque target-cell in the plaquing part of the combination rosetteplaque assay. A stock solution of 0.04 i’U chromic chloride in distilled water was stored at 4 C; for use, an aliquot was diluted 1: 20 in saline. WWAg or each rabbit antiglobulin was diluted in saline to give a concentration of 5 mg of protein/ml of antigen or antiglobulin. One-tenth milliliter of protein solution in saline was added to 0.1 ml of chromic chloride solution and 0.1 ml of packed SRBC. Fifteen minutes later, the cells were washed three times and made up to a 6% suspension in saline.

IN

14i

ASCARIASIS

Rosette Formation The technique used was a modification of the suspension-centrifugation rosette test reported by Jonas et al. (1965) and McConnell et al. (1969). A suspension (0.1 ml) of antigen-SRBC containing 1 x lo* cells/ ml was added to 1 x lo6 lymphocytes in 0.1 ml of MEM. The suspension was spun (500g) at 4 C for 2 min. Lymphocytes showing four or more adherent antigencoated SRBC were considered as rosettes (Fig. I). Ten ahquots of 1000 lymphocytes each were assessed for rosette formation for each preparation, and the percentage of rosette-forming cells (RFC) was calculated after the subtraction of background rosettes (i.e., those resulting after the centrifugation of normal SRBC with lymphocytes from infected animals plus rosettes with antigencoated SRBC and lymphocytes from control animals) from each assay. Results were recorded by calculating the mean and the standard error of each rosette-formation assay. Rosette-Formation

Inhibition

One-tenth milliliter of each rabbit antiserum (anti-gp, -p, -y2, -01,or -6) or 0.1 ml of WWAg was added to the lymphocyte suspension (1 x 10” cells) and the mixture was agitated for 15 min and was centrifuged at 500g for 2-3 min. Lymphocytes were washed once with saline and once with MEM and resuspended to 0.1 ml in MEM and were then rosetted. Percentage of rosette inhibition was calculated as follows:

(mean percentage of rosc+tcs formed _ (mean pcrccntagc of roscttcs formed in presence of ant’igcn or antiserum) in sbscnce of antiserum or WWAg) x 100. (mean percentage of rosettes formed in absence of ant,iserum or WWAg) The difference between the rosetting rcsponsc of lymphocytes in the presence of antiserum or antigen and in their ab-

sence was analyzed using Student’s t test. A probability (P) value of 0.05 or lower was considered to constitute a statistically

148

XHOURY

AND

SOULSBY

Fre. 1. A lymphoid cell expressing rosette formation. This cell type represents binding

receptored

cell.

-

significant difference between the responses obtained at each rosette-formation assay. Combination

Rosette-Plaque

an antigen-

-

Assay

The technique as developed by Bankert and Wolf (1973) with several modifications was used (Khoury 1973). The rosetting part of the technique was the same as described above. For the plaquing part of the technique, an aliquot of the suspension containing the rosetting mixture was resuspended with 0.05 ml of MEM, 0.05 ml of diluted (1: 15) guinea pig complement (gpc’), and 0.05 ml of a 6% suspension of SRBC coupled to antigen (antigen-SRBC) or to rabbit antiglobulin (anti-p, -E, -yz, or -a) (anti-Ig-SRBC), with chromic chloride. The suspension was then plaqued with an agar-free technique (Cunningham 1965). Thirty minutes later, slides were assessed for rosette-plaques (Fig. 2) and plaques (Fig. 3). For indirect (facilitated) plaquing, an aliquot of the suspension containing the rosctting mixture was suspended with 0.05 ml of anti-e, -y2, or -Q instead of MEM.

Both for direct and facilitated plaquing, l( aliquots of 1000 lymphocytes were assessec and the percentage of rosette-plaques OI plaques in a lymphoid cell population wa: quantified. Controls for the rosetting part of thi technique were the same as mentioned fo: the rosette formation. Controls for the plaquing part of the technique included th replacement of antigen-SRBC or anti-Ig SRBC with normal SRBC in the presenct of gpC’ and the addition of antigen-SRBC or anti-Ig-SRBC in the absence of corn. plement. The rosette-plaquing response wa: determined by calculating the mean ati the standard error of each rosette-plaquing assay. RESULTS

In Vitro Antigen-Induced LymphocyteTransformation Responses Lymphocyte transformation induced 1): WWAg is expressed as the ratio of the diz integrations per minute of *H per 16

LYMPHOID

FIG. cell.

2. A rosette-plaque. This cell type represents a receptored antibody-producing

lymphocytes from antigen-stimulated cultures to lymphocytes from control nonstimulated cultures. A value of 1.0 indicates no transformation; values greater than this

lymphoid

indicate lymphocyte transformation in response to antigen. There was no antigen-induced tramsformation of the lymphocytes of the ITies-

1;~. 3. A plaque. This cell type represents a nonreceptored

cell.

149

CELL RESPONSES IN ASCARIASIS

antibody-producing

lymphoid

KHOURY

AND SOULSBY

enteric, hepatic, or mediastinal nodes, of the spleen, or of the Peyer’s patches of guinea pigs not infected with Ascaris suum (Table I, Group IV). However, in orally infected animals (Group I), lymphocytes from the mesenteric, hepatic, and mediastinal nodes showed peak responses at Day$ 1, 2, and 7 after infection, respectively, while the responses of the spleen and the Peyer’s patches remained at a uniformly low level throughout the period of infection, In Group II animals which received 2L via the mesenteric vein, there were no lympho-’ cyte transformation responses in the mesenteric nodes, but peak responses occurred in the hepatic (at Day 2) and mediastinal (at Day 7) nodes. In animals (Group III) that received 3L via the saphenous vein, only the mediastinal nodes showed peak antigeninduced Iymphocyte transformation responses (at Day 2) (Table I). Rosette-Formation Responses The rosette-formation responses of the mesenteric, hepatic, and mediastinal nodes, of the spleen, and of the Peyer’s patches of Groups I, II, and III are presented in Figs. 4-6. These were similar to those of antigeninduced lymphocyte transformation in that peak responses occurred in the mesenteric, hepatic, and mediastinal nodes in Group I (Fig. 4) ; in the hepatic and mediastinal nodes in Group II (Fig. 5); and in the mediastinal nodes in Group III (Fig. 6). However, the peak responses of the meseni teric and hepatic nodes of Group I and of the hepatic nodes of Group II occurred later than those of antigen-induced lympho: cyte-transformation responses (Table I). For example, in Group I peak rosetteformation responses occurred in the mesenteric nodes at Day 2 (1 day after the peak lymphocyte-transformation response) and in the hepatic nodes at Day 5 (3 days after the peak lymphocyte-transformation response). The rosette-formation responses of the spleen in Groups I, II, and III were at a

LYMPHOID

CELL

RESPONSES

IN

ASCARIASIS

151

FIG. 4. Rosette-fomlation responses of the lymphoid organs of normal guinea pigs orally infected with 10,000 infective eggs of Ascaris swum (Group I).

low level at the early stages of the infection but increased uniformly as the infection progressed. However, the responses of the

Peyer’s patches in these three groups approached background levels (Figs. 4-6). No increased rosette formation was evi-

l’rc. 5. Rosette-formation responses of the lymphoid organs of normal guinea pigs which received 10,000 second-stage larvae of Ascaris suurn via the mesentcric vein (Croup II).

152

KHOURY

AND SOULSBY

FIG. 6. Rosette-formation responses of the lymphoid organs of normal guinea pigs which received 1500 third-stage larvae of Asc~ris suum via the saphenous vein (Group III).

dent with lymphocytes from uninfected animals (Group IV) at any time during the period of study. Inhibition

of Rosette Formation

Inhibition of rosette formation with WWAg measured quantitatively the percentage of RFC that expressed antigenspecific receptors on their surfaces. Inhibition with anti-gp, -JL,-6, -(Y,or -y2 measured those RFC that expressed antigen-specific Igs, IgM, IgA, IgE, or IgG, receptors on their surfaces, respectively. TABLE

II

Inhibition of Roset,te-Formation Responses of the Lymphoid Organs of Guinea Pigs that Received per OS10,000 Eggs of Ascaris .suurn (Group I) Lymphoid organ

Mesenteric node Hepstic node Mediastinal node Spleen d N.S. = inhibit& (P > 0.05).

Percentage of inhibition with anti-p 68.4-71.4 75.3-79.8 46.4-48.2 95.4-98.9

Percentage of inhibition with anti-6

15.6-16.6 6.7- 8.8 31.7~32.4 5.6- 6.2 (N.S.)-not sta.tistirnlly cignificant

The rosette-formation responses of the mesenteric, hepatic, and mediastinal nodes and of the spleen were inhibited to background levels (comparable to 0.06-0.15~ RFC in Group IV) throughout the period of the infection, with WWAg or with antigp in Groups I, II, and III. Statistically significant inhibition of rosette-formation responses occurred with anti-p (which was most marked with spleen cells) and anti-6 (which was most marked with mediastinal node cells), but not with anti-y, or anti-a in the mesenteric, hepatic, and mediastinal nodes of Group I (Table II). Similar results were obtained for animals in Group II with ceIIs from the hepatic and mediastinal nodes and the spleen and those in Group III with cells from the mediastinal nodes and spleen. Distribution of Rosetting, Rosette-Plaquing, and Plaquing Lymphocytes in Various Lymph Nodes

Data obtained from the inhibition of RFC with specific anti-heavy chain sera were combined with data obtained from direct and facilitated plaque assays of rosetting and nonrosetting cells. Conse-

LYMPHOID

CELL IiESPONSES IN ASCARLASIS

A

I

2

3

4

5

Oayr Illll

6

7

8

9

IO

I1

12

lllCcllOn

FIG. 7. The IgM antigen-specific responses of the lymphocytes of the (A) mesenteric, (B ) hepatic, and (C) mediastinal nodes of nomlal guinea pigs orally infected with 10,000 infective eggs of Ascaris suum (Group I). Ordinate: percentage of rosettes (-), rosette-plaques (-.-* ), or plaques (----).

quently, it was possible to rank reactivities of lymphocytes as follows: ( 1) lymphocytes with antigen-specific Ig receptors, not secreting antibody (rosetting cells); (2) lymphocytes with antigen-specific Ig receptors and also secreting specific antibody of various classes (rosette-plaquing cells) ; (3) lymphocytes without antigen-specific Ig receptors but secreting antibody of various Ig classes ( plaquing cells) ; (4) lymphocytes without antigen-specific Ig receptors, secreting Igs not necessarily specific for A. suum and detected by anti-heavy chain scra attached to plaque target-cells. The percentage of lymphocytes from the

mesenteric, hepatic, and mediastinal nodes of Group I animals which demonstrated IgM or IgE antigen-specific rosettes, rosette-plaques, and plaques ( reactivities 1, 2, and 3, respectively) are presented in Figs. 7 and 8. The major response in each lymph node was attributable to IgM, followed by IgE. Responses attributable to IgGz and IgA were minimal and barely above background levels. A sequentia1 appearance of rosetting cells, rosette-plaquing cells, and plaquing cells was observed in the various lymph nodes during infection. This sequence differed from one lymph node to another, but

154

KHOURY

AND

SOULSBY

r

FXG. 8. The IgE antigen-specific responses of the lymphocytes of the (A) mesenteric, (B) hepatic, and (C) mediastinal nodes of normal guinea pigs orally infected with 10,000 infective eggs of Ascuris suum (Group I). Ordinate: percentage of rosettes (-), rosette plaques (-*-a), or plaques (----).

did not differ with the same node for the different Igs. Thus, the peak IgM and IgE antigen-specific rosetting responses, peak rosette-plaquing responses, and peak plaquing responses occurred in the mesenteric nodes at Days 2, 5, and 7 after infection, respectively (Figs. 7A and SA); in the hepatic nodes at Days 5, 7, and 9 after infection, respectively (Figs. 7B and 8B); and in the mediastinal nodes at Days 7, 9, and 12 after infection, respectively (Figs. 7C and 8C ) . The highest IgM response was exhibited by the hepatic nodes (Fig. 7B),

whereas the highest IgE response was exhibited by the mediastinal nodes (Fig. SC). The responses of the spleen were uniquely IgM in character and they increased gradually as the infection progressed without showing detectable peak responses. Thus, significant IgM antigenspecific rosetting responses occurred on Days 5 (2.02%), 7 (2.86%)), 9 (3.04%)) and 12 (5.40%) after infection; whereas antigen-specific roestte-plaques IgM (2.05% ) or plaques (0.65% ) occurred at Day 12 after infection.

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153

CELL RESPONSES IN ASCARIASIS

The lymphocytes of the Peyer’s patches did not exhibit antigen-specific rosetting, rosette-plaquing, or plaquing responses, Throughout the period of the infection a certain population of the lymphocytes of the mesenteric, hepatic, and mediastinal nodes and of the spleen was secreting Igs that were not specific for the A. suum antigen. For example, during the first 5 days of infection, the lymphocytes of the above lymphoid organs did not secrete antigenspecific Igs but secreted nonspecific IgM, and IgA (Table III). These I@, W2, responses were generally higher than those of noninfected control animals and especially so in the case of IgE responses which were appreciably higher than those of control animals. After the fifth day of infection lymphocytes from these lymphoid organs secreted antigen-specific IgM and IgE (but not IgGZ or IgA) antibodies. Of the cells of the mesenteric, hepatic, and mediastinal nodes that were secreting IgM, approximately 68, 79 and 69% were secreting IgM antigenspecific antibodies, respectively; whereas, of the cells that were secreting IgE, approximately 50, 47, and 70% wcrc secreting IgE antigen-specific antibodies, respectively. In animals of Group II, the rosetting, and plaquing responses rosette-plaquing, and the Ig class of the hepatic and mediastinal nodes responsible for them were similar to those of Group I both in the type and the pattern of the responses and in the time of their sequential appearance. In animals of Group III, although the reTABLE

sponses of the mediastinal nodes were similar to those of Group I and II in their type (predominantly IgM and IgE) and their sequential pattern, they differed in the time of their appearance (Fig. 9). Thus, peak IgM and IgE antigen-specific rosettes, rosettc-plaqucs, and plaques occurred in the mediastinal nodes at Days 2, 5, and 7 after infection, respectively, (Fig. 9) compared with Days 7, 9, and 12 after infection in Groups I and II. DISCUSSION

In the present study lymphoid cell responsiveness to primary Ascaris suum infections in guinea pigs was characterized by the use of in vitro antigen-induced lymphocyte-transformation, rosette-formation, and rosette-plaquing techniques. The response of the lymphoid tissues draining parasitized organs was followed as the infection progressed in the host animal. Following an oral infection with infective eggs of A. suum, infective larvae released from the egg penetrate into the intestinal wall and migrate to the liver via the hepatic portal system. At this time, i.e., Day 1 after infection, cells harvested from the mesenteric lymph nodes exhibited marked antigen-specific transformation in culture. At this time also lymphocytes in these nodes were expressing antigen-binding receptors (predominantly IgM and IgE) on their surfaces and peak responses were reached 1 day later, i.e., Day 2 after infection. Secretion of specific Igs (IgM and IgE) as deIII

PlaqlCng liesponses Not Related to Ascaris suwn Antigen of the Lymphoid Organs of Orally Infected Guinea Pigs (Grollp I) during the First 5 Days of Infection Lymphoid

Percentage

organ

hlesenteric node Hepatic node 1Iediastinal node Spleen

of plaqlling

rells

IgXI plaques

IgE: plaques

IgcTz plaques

IgA plaques

1.12-1.68 I .28-1.34 1.42-2.23 2.644..!4

1.31-1.62 0.45-0.60 X45-3.98 0.21-0.34

1.30-1.61 2.12-2.32 2.62-3.63 2.56-3.82

1.22-l .98 0.11-0.16 2.00-3.21 0.01~0.02

KHOURY

AND

SOULSBY

FXC. 9. The (A) IgM and (B) IgE antigen-specific responses of the lymphocytes of the mediastinal nodes of normal guinea pigs which received 1500 third-stage larvae of Asca& suum via the saphenous vein (Group III). Ordinate: percentage of rosettes (-), rosette plaques (-e-e), or plaques (----).

tected by rosette-plaques and plaques occurred at Days 5 and 7 after infection, respectively, by which times the parasite has migrated to the liver or to the lungs ( Douvres and Tromba 1971). In the liver, second-stage larvae molt to third-stage larvae on Day 2 and these can be found in the liver up to the fifth day after infection (D ouvres and Tromba 1971). Peak antigen-induced lymphocytetransformation responses occurred in the hepatic nodes at Day 2 after infection, and peak IgM and IgE antigen-specific rosetting responses occurred approximately 3 days later at Day 5 after infection. Antibody-secreting cells were evident in the hepatic node 7 days after infection, i.e., when the majority of the larvae were in the lungs. Third-stage larvae are found to be in maximum numbers in the lungs at Day 7 after infection (Khoury 1973). Maximum antigen-induced lymphocyte-transformation responses and antigen-specific rosetting re-

sponses occurred in the mediastinal nodes at 7 days after infection. At Day 9 after infection, some 3L are still present in the lungs (Douvres and Tromba 1971) and, at this time also, lymphocytes of the mediastinal nodes were also secreting IgM and IgE antigen-specific antibodies. During the migration period of the infection, the spleen showed somewhat variable, but low, antigen-induced lymphocyte transformation, and marked transformation was not evident until 12 days after infection. Rosette formation by spleen cells was low, but increased progressively while antibodysecreting cells were not detected in significant numbers until the 12th day after infection. Collectively, the results obtained indicate that the lymph nodes draining an organ respond only when that organ is invaded by larvae. This is further exemplified by the results obtained with abbreviated infections, Thus, in animals infected by mesenteric vein injection of infective larvae

LYMPHOID

CELL RESPONSES IN ASCARIASIS

(Group II), and in which the intestinal phase of infection is eliminated, the mesenteric node responses were at a minimal level. When both the intestinal and hepatic phases were eliminated by the injection of 3L into the saphenous vein (Group III), only the mediastinal node responded. However, in this case (Group III) the mediastinal node responded promptly (by Day 2) since 3L would be carried directly to the lungs following saphenous vein injection. The lack of a distinctive response by cells of the spleen until the end of the migration of larvae would suggest that there is no major systemic distribution of antigen in the early stages of infection. Since, in the guinea pig 3L of A. ~uum generally die in the lungs and do not migrate to the intestine (Douvres and Tromba 1971), the antigenic stimulus to spleen cells may be provided by antigens released by degenerating larvae. Each lymphoid organ responded in an individual manner producing cell populations with a distinctive distribution of cells with Ig receptors. This distribution remained the same when infections were abbreviated (Groups II and III ) . This is illustrated in the response of the mediastinal nodes to saphenous vein-injected 3L (Group III) and to 3L arriving in the lungs after an oral infection with infective eggs (Group I). In both cases, the mediastinal node produced a distinctive distribution of IgM and IgE antigen-specific rosettes, rosette-plaques, and plaques. The present study does not provide enough evidence to decide whether the Ig responses of a lymph node are a function of the reactivity of the node or a function of the developmental stage which invades the organ drained by the node. Subsequent studies of secondary responses in previously sensitized animals (to be published) suggest that the Ig response is largely a function of the individual lymph node. In general, the lymphocyte responses of each of the draining lymphoid organs progressed from an antigen-sensitive state to

157

an Ig-receptored state, then to an Ig-reccptored state capable of antibody production, and finally to a nonreceptored antibodyproducing state. Antigen-sensitive lymphocytes which undergo transformation in vitro have been considered as thymus-derived (T) cells (Mills 1966; Oppenheim 1968; Green et al. 1968) or bone marrow-derived (B) cells (Benezra et al. 1969; Shevach et al. 1972; Elfenbein et al. 1972; Elfenbein and Rosenberg 1973). Thus, in the present study B- and T-cells may constitute the antigen-sensitive populations of the draining lymphoid organs. Most workers (Coombs et al. 1970; Rabellino et al. 1970; Raff et al. 1970; Unanue 1971; Pernis et al. 1972) consider Ig-receptorcd cells as B-cells clue to the presence of Igs on their surfaces that bind specifically to antigen. However, the presence of T-cells among these Igreceptored lymphocytes cannot be ruled out since Greaves ( 1970), Hogg and Greaves ( 1972), Dwyer et al. ( 1972), and Marchalonis and Cone (1973) have shown that L chains and heavy chains of the p specificity might be present on T-cells. In addition, Goldschneider and Cogen (1973) have suggested that surface Ig molecules exist in a masked form in “resting” T-cells, but become unmasked after stimulation with antigen and that such unmasked molecules are more readily detectable by anti-Ig sera. Whether such Ig molecules bind specifically to antigen is still debatable. Therefore, it would seem that the IgM and IgE receptored lymphocytes antigen-specific that were present in the draining nodes during the period of infection were predominantly B-cells although some of them could be T-cells. The Ig-receptored antibody-producing (IgM and IgE) lymphocytes, because they secrete antibody must be regarded as equivalent to B-cells. Consequently, in a primary infection with A. suum the immune response manifest by the draining lymphoid tissue of parasitized organs is primarily one which results in Igbearing and -secreting cells, the majority of which show specificity for the infection.

158

KHOURY AND SOULSBY

In vitro correlates of cell-mediated immunity (CMI) have been demonstrated during the migration phase of A. suum infections ( Soulsby and Muncey 1970). However, the present results would suggest that responses ascribable to B-cells predominate during the infection but may, together with CMI, constitute the immune response involved in a primary infection of A. suum. An interesting observation was the fact that lymphocytes from the mesenteric, hepatic, and mediastinal lymph nodes and spleen produced Igs that were not necessarily specific for the A. suum antigen. Such Igs could consist of antibodies to concomitant bacterial or viral infections to damaged tissue or possibly immunoconglutinins. In the case of IgE antibodies which did not show specificity to the A. suum antigen, it is possible that the A. suum infection facilitated the production of such antibodies to unrelated antigens that might be present at the time of parasitism. The ability of helminth infection to facilitate IgE production to unrelated antigen is well recognized; an example of this is the work of Orr and Blair (1969) who showed that Nippostrongylus brasiliensis in rats facilitates the production of IgE antibodies to egg albumin, ACKNOWLEDGMENTS We thank Dr. Sheelagh Lloyd for her advice on, and help in, the purification of the immunoglobulins. We also thank Mrs. Jane McDay, Mr. Derek Muncey, Miss Dragica Borojevic. Mrs. Rosetta GOSS, and Miss Norma Molina for their excellent technical assistance. This work was supported by USPHS Grant AI-06262.

REFERENCES BANKERT, R. B., AND WOLF, B. 1973. Simultaneous expression of hapten binding and antibody secretion by NIP-primed lymph node cells early in the immune response. Journal of Immunology 111, 1790-1799. BENEZRA, D., GREY, I., AND DASHES, A. M. 1969. The relationship between lymphocyte transformation and immune responses, II. Correlations between transformation and humoral and

cellular responses. Clinical and Experimental Immunology 5, 155-163. COOMBS, R. R. A., GURNER, B. W., JANEWAY, D. A., WILSON, A. B., CELL, P. G. H., AND KELUS, A. S. 1970. Immunoglobulin determinants on the lymphocytes of normal rabbits. Immunology 18,

417431. COVENTRY, F. A. 1929. Hypersensitiveness to helminth proteins. II. cutaneous and precipitin tests with Ascaris extracts in infected and immunized animals. Journal of Preventive Medicine 3, 43-62. CUNNINGHAM, A. J. 1965. A method of increased sensitivity for detecting single antibody forming cells. Nature (London) 207, 1106-1107. DOBSON, C., MORSETH, D. J., AND SOULSBY, E. J. L. 1971. Immunoglobulin E-type antibodies induced by AxarC suum infections in guinea pigs. 106, 128-133. Journal of Immunology DOBSON, D., AND SOULSBY, E. J. L. 1974. Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus cohLbriformis: Tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions and in vitro lymphocyte transformations during primary infections. Experimental Pamsitology 35, 16-34. DOUVRES, F. W., AND TROMBA, F. G. 1971. Comparative development of Ascaris .suum in rabbits, guinea pigs, mice and swine in 11 days. Proceedings of the Helminthological Society of Wa.rhington 38, 246-252. DWYER, J. M., WARNER, N. L., AND MACKAY, I. R. 1972. Specificity and nature of the antigencombining sites on fetal and mature thymus 108, 1439lymphocytes. Journal of Immunology 1446. ELFENBEIN, G. J., AXD ROSENBERG, G. L. 1973. In vitro proliferation of rabbit bone marrow-derived 8 and thymus-derived lymphocytes in response to vaccinia virus. Cellular Immunology 7, 516-521. ELFENBEIN, G. J., SHEVACH, E. M., AND GREEN, I. 1972. Proliferation of bone marrow-derived lymphocytes in response to antigenic stimulation in VitTO. Journal of immunology 109, 876-874. ESKENAZY, M., AND PETRUNOV, B. 1971. Sensitization of erythrocytes with lipopolysaccharides using glutaraldehyde as a coupling agent. Immunology 21,311313. FUDENBERG, H. H. 1967. Purification of antibody In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. M. Chase, eds.), pp. 307385. Academic Press, New York/London. GOLDSCHNEIDER, I., AND COCEN, R. B. 1973. Immunoglobulin molecules on the surface of activated T lymphocytes in the rat. Journal of Experimental Medicine 138, 163-175. GREAVES, M. F. 1970. Biological effects of antiimmunoglobulins: Evidence for immunoglobulin

LYMPHOID

CELL RESPONSES IN ASCARIASIS

receptors on T and B lymphocytes. Transplantation Reviews

5, 45-75.

GREEN, I., PAUL, W. E., AND BENACERRAF, B. 1968. Hapten carrier relationships in the DNPPll foreign albumin complex system: Induction of tolerance and stimulation of cells in vitro. Journal of Experimental Medicine 127, 43-53. Hocc, N. M., AND GREAVES, M. F. 1972. Antigenbinding thymus-derived lymphocytes. II. Nature of the immunoglobulin determinants. Immunology 22, 967-980. JASKOSKI, B. J., AND COLLUCCI, A. V. 1964. In vitro hatching of Ascaris suum eggs. Transactions of the American

Microscopical

Society

83, 29P

300. JEZIORANSKA, J., AND DOMBROWOLSKA, D. 1956. Odczyny immuologiczne przy askaridozie. Wiadomosci Parazytologiczne 2, 117-l 18. JONAS, W. E., GUIWER, B. W., NELSON, D. S., AXD COONBS, R. R. A. 1965. Passive sensitisation of

tissue cells. I. Passive sensitisation of macrophages by guinea pig cytophilic antibody. International Archives of Allergy 28, 86-104. KHOURY, P. B. 1973. “Local Lymphoid Cell Responses to Ascaris suum Infections in Guinea Pigs.” Ph.D. thesis, University of Pennsylvania, Philadelphia. MARCHALONIS, J .J., AND CONE, R. E. 1973. Biochemical and biological characteristics of lymphocyte surface immunoglobulin. Transplantation Reviews 14, 3-49. MCCONNELL, I., MUNRO, A., GURNER, B. W., AND COOMBS, R. R. A. 1969. Studies on actively allergised cells. International Archives of Allergy 35, 209-227. MILLS, J. A. 1966. The immunologic significance of antigen induced lymphocyte transformation in vitro. Journal of Immunology 97, 239-247. MORSETH, D. J., AND SOULSBY, E. J. L. 1969. Fine structure of leukocytes adhering to the cuticle of ASCUTLSsuum larvae. I. Pyroninophils. Journal of Parasitology 55, 22-31. OLIVER-GONZALEZ, J. 1946. Functional antigens of helminths. Journal of Infectious Diseases 78, 232-237. ORR, T. S. C., AND BLAIR, A. M. J. N. 1969. Potentiated reagin response to egg albumin and conalbumen in Nippostrongylus brasiliensis infected rats. Life Sciences 8, 1073-1077. ONKELINX, E., MELJLBERMANS, W., JONIAU, M., AND LONTIE, R. 1969. Glutaraldehyde as a coupling

reagent in passive hemagglutinination. ogy 16, 35-42. OPPENHEIM, J. J. 1968. Relationship

159 Immunol-

in in &TO lymphocyte transformation to in vitro delayed hypersensitivity in guinea pigs and man. Federation Proceeding.9 27, 21-28. PERNIS, B., FOIWI, L., ASD AIVIANTE, L. 1972. Immunoglobulin spots on the surface of rabbit lymphocytes. Journal of Experimental Medicine 132, 1001-1018. RABELL~O, E., COLON, S., GREY, H. M., AND UNANLJE, E. R. 1970. Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation. Journal of Experimental Medicine 133, 156167. RAFF, M. C., STERNBERG, M., AND TAYLOR, R. B. 1970. Immunoglobulin determinants on the surface of mouse lymphoid cells. Nature (London) 225, 553-554. SHEVACH, E., GREEN, I., ELLL~AN, L., AND MAILLARD, J. 1972. Heterologous antiserum to thymus-derived cells in the guinea pig. Nature (London) 235, 19-21. SOULSBY, E. J. L. 1972. Cell mediated immunity responses in parasitic infections. In “Immunity to Animal Parasites” (E. J. L. Soulsby, ed. ), pp. 57-95. Academic Press, New York/London. SOLJLSBY, E. J, L., AND MUE~‘CEY, D. W. 1970. Lymphoid cell reactions to tissue nematodes. In “Isotopes and Radiation in Parasitology II,” pp. 105-117. International Atomic Energy Agency, Vienna. STAVITSKY, A. B., AND INGRAHAM, J. S. 1964. and haemagglutination-inHaemagglutination hibition reactions with tannic acid- and bisdiazotized benzidine-protein-conjugated erythrocytes. In “Immunological Methods” (J. F. A. Achroyd, ed.), pp. 363-396. Blackwell Scientific, Oxford. STELOS, P. 1967. Salt fractionation. In “Handbook of Experimental Immunology” (D. M. Weir, ed.), pp. 3-9. Blackwell Scientific, Oxford. TAFFS, L. F. 1958. “Immunological Reactions of the Host to the Nematode ASCUT~~suum. Ph.D. thesis, University of Cambridge, Cambridge, England. UNANUE, E. R. 1971. Antigen-binding cells. I. Their identification and role in the immune response. Journal of Immunology 107, 1168-1174. WILLIAMS, J. F., AND SOULSBY, E. J. L. 1970. Antigenic analysis of developmental stages of Ascaris suum. I. Comparison of eggs, larvae and adults. Experimental Parasitology 27, 150-162.