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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I 75-26,685 WHITACRE, Caroline Clement, 1949- TRANSPLANTATION STUDIES IN A CANINE MODEL: CHARACTERIZATION OF A CANINE ANTI-LYMPHOCYTE SERUM.

Hie Ohio State University, Ph.D., 1975 Health Sciences, imnunology

Xerox University Microfilms,Ann Arbor, Michigan 48106

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. TRANSPLANTATION STUDIES IN A CANINE MODEL: CHARACTERIZATION

OF A CANINE ANTI-LYMPHOCYTE SERUM

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Caroline C. Whitacre, B.A.

********

The Ohio State University 1975

Reading Committee: Raymond W. Lang, Ph.D. Vincent V. Hamparian, Ph.D. Abramo Ottolenghi, Ph.D. Advisor/ Norman L. Somerson, Ph.D. Department cS$r Medica Microbiology ACKNOWLEDGMENTS

My respect and sincere thanks go to my advisor, Dr.

Raymond Lang, who was always available for consultation.

His dedication to his field provides an example for any beginning investigator. Many thanks also go to the members of my graduate advisory committee for their time and con­ structive criticism: Dr. Norman L. Somerson, Dr. Vincent

V. Hamparian, and Dr. Abramo C. Ottolenghi.

I would also like to acknowledge Dr. James A. Madura who was responsible for all coberman immunizations and bleedings.

I wish to express my thanks to my mother, Mrs. Rosalyn

Whitacre, for her understanding and encouragement toward graduate education. To my future husband, Michael Para, goes much gratitude for his unending moral support. VITA

November 4, 1949 Born - Cincinnati, Ohio

1971 B.A./ The Ohio State University, Columbus Ohio

1971-1975 Graduate Research Assoc­ iate, Department of Medical Microbiology, The Ohio State Univer­ sity, Columbus, Ohio

Publications

Whitacre, C., Wilson, G.P., Madura, J.A., St. Pierre, R. L., and Lang, R.W., "Specificity of DEAE Fractions of Canine Anti-Lymphocyte Serum." Abstract in Tissue Antigens, 3 (1973), 167.

Whitacre, C., and Lang, R.W., "Method for Isolation of Canine Lymphocytes." Abstract in Am. Soc. Microbiol., (1973),98.

Whitacre, C., and Lang, R.W., "Characterization of IgG- Containing DEAE Fractions of Canine Serum." Transplanta­ tion Proceedings, (1975), in press.

Whitacre, C., and Lang, R.W., "A Technique for Separation of Canine Lymphocytes and Their Use in the Lymphocytotoxic, Blastogenic, and Rosette Assays." Transfusion (1975), in press.

Fields of Study

Major Field: Medical Microbiology

Major Area of Study: Immunochemistry

iii TABLE OP CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iii

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

INTRODUCTION ...... 1

PART I. CHARACTERIZATION OF IgG- CONTAINING DEAE FRACTIONS OF CANINE SERUM ...... 10

Introduction ...... 11

Materials and Methods ...... 12

Results ...... 14

D i s c u s s i o n ...... 23

PART II. DEAE CHROMATOGRAPHIC CHARACTERIZA­ TION OF CANINE SERUM AND ISOLATION OF AN IgG SUBCLASS ...... 27

Introduction ...... 28

Materials and Methods ...... 29

Results ...... 32

Discussion ...... 47

PART III. A TECHNIQUE FOR SEPARATION OF CANINE LYMPHOCYTES AND THEIR USE IN THE LYMPHOCYTOTOXIC, BLASTOGENIC, AND ROSETTE ASSAYS ...... 50.

Introduction ...... 51

Materials and Methods ...... 52

Results ...... 55

iv Page D i s c u s s i o n ...... 62

PART IV. CHARACTERIZATION OF A DL-B TISSUE TYPING ANTISERUM ...... 64

BIBLIOGRAPHY ...... 73

V LIST OF TABLES

Table Page

1. Localization of cytotoxic activity in DEAE column fractions ...... 24

2. Analysis of leukocyte preparations ...... 56

3. Analysis of lymphocyte preparations ..... 57

4. PHA and PWM stimulation of lympho­ cytes following separation from canine blood ...... 59

5. E rosette formation of lymphocytes following separation from canine b l o o d ...... i ...... 60

6. Comparison of cytotoxic titers with lymphocyte preparations ...... 51

vi LIST OP FIGURES

Figure Page

1. The family history of the "coberman" dogs .... 13

2. DE-52 chromatograph of canine anti-lympho­ cyte serum SI4113 and chromatograph of pooled and concentrated pre-hemoglobin fractions...... 15

3. Immunoelectrophoresis of DE-52 fractions 1, 2, 3, 4, 5, commercial canine IgG, and commercial canine transferrin ...... 17

4. Osserman Immunoelectrophoresis test for identification of DE-52 column frac­ tions ...... 20

5. Analytical DE-52 chromatographs of canine serum and serum fractions :

A. Reconstituted lyophilized commercial dog serum ...... 33

B. Pooled normal mongrel serum ...... 34

C. Normal coberman serum ...... 35

D. Coberman anti-lymphocyte serum ...... 36 .

E. Commercial canine Cohn fraction II ... 37

F. Human serum, canine serum, human s e r u m ...... 39

6. Direct immunoelectrophoresis of fraction 1, fraction 2, and commercial canine IgG..... 41

7. Osserman immunoelectrophoretic technique for identification of fraction 3, and 2 components ...... 43

8. Polyacrylamide gel electrophoresis of fractions 1-5, whole serum S14113 and canine IgG ...... 44 vii Figure Page 9. Characterization of anti-fraction 1 and anti-fraction 2 antisera ...... 46

10. The immunizing dose and cytotoxic anti­ body response of dog 7 1 ...... 67

11. Percentage of dogs from populations react­ ing with whole ALS and the IgM frac­ tion of whole ALS and the level of response ...... 69

viii INTRODUCTION

Studies concerning the immunoglobulins of the canine were begun in the early 19 60's by Patterson and co-workers

at Northwestern University. The immunologic response of dogs was investigated with the finding that dogs were poor producers of precipitating antibodies in response to solu­ ble antigens (Patterson et ad., 1963a). However, the incor­ poration of Freund's adjuvant with the antigen resulted in high antibody levels as detected by tanned red blood cell hemagglutination and precipitation reactions. The demon­ stration of both canine precipitating and non-precipitating antisera against bovine serum albumin prompted further study into the canine immune response (Patterson et al., 1964a;

Patterson et al., 1964b). The precipitating antisera showed incomplete precipitation of antigen in the region of anti­ body excess as measured by coprecipitation and quantitative isotope precipitation. The non-precipitating antisera con­ tained only one active component, a $ 2 globulin, while the precipitating antisera demonstrated a $ 2 as well as a y globulin, the combined effect supposedly producing the characteristic canine precipitin curve.

The first completely comprehensive study of dog immuno­ globulins was carried cut by Jphnson and Vaughan (1967a) when

1 they described six immunoglobulin classes: y2a, y2b, y2c,

7Sy^, intermediate Sy^, and yM. y2a and y2b were found to

be inseparable on agar gel electrophoresis, ultracentrifuga­

tion both sedimenting at 6.7 S, starch zone electrophoresis,

and DEAE cellulose chromatography. y2c was designated as a

spur from the y2a, y2b line and an antiserum prepared against

a y2c myeloma was only observed to cross react with y2a, y2b

through antigenic determinants on Fab. The relationship of

the y2c globulin to the other IgG globulins has not been settled yet. Two immunoglobulins, 7Sy-^, and Int. Sy^, were

found in higher concentrations in colostrum than serum,

7Sy^ in 4 fold greater concentration and Int. Sy^ 80 fold.

Int. Sy^ was also found in saliva, bronchial secretions and in low concentrations in serum. The yM appeared analogous to the yM of other species by ultracentrifugation, electro­ phoresis and chromatography. In an accompanying paper,

Johnson and Vaughan (196 7b) demonstrated antibody activity in all six immunoglobulin classes. Antisera to hemocyanin,

BSA, and rabbit thyroglobulin consisted of y2a, y2b, and

7Sy^ globulins while anti-A isohemagglutinins exhibited reactions with all six anti-immunoglobulin sera. Serum from a dog with lupus erythematosus showed y2a, y2b, 7Sy^, and yM anti-nuclear antibody. Since Johnson and Vaughan's preliminary study, some of the immunoglobulin designations have undergone a change in nomenclature. The current desig­ nations for the six classes now are: IgGa, IgGb, IgGc, IgGd, 3 IgA, IgM.

In all reports to date, IgGa and IgGb have been consid­

ered together as IgGab. Patterson et a]L. (19 68) confirmed

the presence of IgGab in the first peak of a DEAE cellulose

column loaded with a 33.3% saturated ammonium sulfate (SAS)

fraction of whole serum. An antiserum to this first peak

after absorption with light chains recognized the major

IgGab line and a spur from that line, supposedly IgGb.

Immunochemical studies by Vaerman and Heremans (1968) local­

ize IgGa and IgGb in the second peak from Sephadex G-200,

throughout most of the effluent volume from DEAE cellulose, and in the most cathodal fractions from Pevikon block zonal electrophoresis. The presence of IgGab in both fecal ex­ tracts and colostrum has been documented (Reynolds and John­ son, 1970a; Reynolds and Johnson, 1970b). In addition, canine IgG globulin has been demonstrated to elicit a pas­ sive cutaneous anaphylaxis reaction in the guinea pig

(Rockey and Schwartzman, 19 67). Immunochemically and bio­ logically, canine IgGab seems analogous to the IgG of other species.

Very few studies were conducted on the Y2c immuno­ globulin class. Its relationship to the other IgG immuno­ globulin subclasses was demonstrated only by light chain cross reaction. Its immunochemical analysis (Vaerman and

Heremans, 1968) showed identical patterns as IgGab on

Sephadex and sucrose density gradients but a slower elution from DEAE cellulose and a faster migration pattern on Pevikon electrophoresis than IgGab. Its presence has been documented in canine colostrum and fecal extracts

(Reynolds and Johnson, 1970a; Reynolds and Johnson, 1970b).

An IgGc myeloma was described (Johnson and Vaughan, 1967a) which was used for preparation of an antiserum.

The 7Sy^ immunoglobulin originally described by

Johnson and Vaughan (1967a) was postulated to be canine IgA by Patterson et al. (1968). However, carboxy-terminal amino acid sequences of the octadecapeptides from IgGab and 7Sy^ showed considerable homology between the two classes with only 3 amino acid differences (Grant et al., 1972). Compar­ ison of both sequences with a human IgA amino acid sequence showed 8 of 10 amino acid differences. Therefore, this constitutes evidence of 7Sy-^ being a subclass of IgG globulin (i.e., IgGd). In addition, canine IgGab and IgGd were observed to cross react with equine IgG globulins

(IgG, IgB, and IgG (T)) (Allen and Johnson, 1972). The canine IgG globulins shared antigenic determinants in common with equine IgG and IgB but not with IgG (T). A procedure was outlined for the separation of this immunoglobulin class from colostrum (Reynolds and Johnson, 1970a). Following a

Sephadex G-200 chromatogram of clarified colostrum, peak 2 was found to contain IgGab, IgGc, IgGd, globulins. It was possible to separate IgGd from other components on a 5% acrylamide gel. IgG globulin bands from origin to Rf 0.13 while the IgGd Rf was 0.43. A canine non-precipitating

antisera to BSA was shown by radioimmunoelectrophoresis to

be IgGd (Johnson et al., 1967b). Also an IgGd myeloma

protein has been reported (Grant et aj., 1972).

The immunochemical characterization of IgM was carried

out by Vaerman and Heremans (1968). The macroglobulin was

localized in the first peak on Sephadex and sucrose density

gradients and the descending side of the last peak on DEAE

cellulose chromatography. In this same study, it was shown

that antisera specific for human IgM reacted with canine IgM

by immunoelectrophoresis and gel diffusion. Canine MgM has

been found in low concentrations in both colostrum and fecal

extracts (Reynolds and Johnson, 1970b; Vaerman and Heremans,

1969). Two reports of a canine IgM myeloma have appeared

(Capra and Hurvitz, 1970; Hurvitz et al^. , 1971). Character­

ization of one myeloma protein was carried out (Capra and

Hurvitz, 1970) and the molecular weight was found to be

880,000 with an S value of 18.8.

Johnson and Vaughan (1967a) initially suggested Int.

Sy^ as being the analogue to IgA in the human. However

Patterson et al. (1968) reported the 7Sy^ globulin as being

the canine analogue to human IgA. Their evidence was that

1) specific antisera directed against the canine non­ precipitating antibody (7Sy^) did not react with canine

IgG or IgM, 2) the antibody migrated as a y^ globulin and was found to be a 7S protein, 3) broad distribution on Sephadex G-200 suggested a polymeric form, 4) solubility

in 33.3% SAS, and 5) no other immunoglobulin was found in

canine sera analogous to human IgA. A number of other inves

tigators found Int. Sy^ was canine IgA (Reynolds and Johnson

1970a; Reynolds and Johnson, 1970b; Ricks et al., 1970;

Vaerman and Heremans, 1968; Vaerman and Heremans, 1969).

Vereman and Heremans (1968) showed that a protein present

in dog's serum and milk having the physical properties of

Int. Sy^, as described by Johnson and Vaughan (1967a)

(behavior in the ultracentrifuge, gel filtration, fast

electrophoretic mobility and excretion in milk, saliva, and

bronchial mucus) does cross-react with some antisera spec­

ific for human IgA. Vaerman and Heremans in an accompany­

ing paper (19 69) found canine IgA to be the major immuno­

globulin in milk, saliva, tears, hepatic bile, and intes­

tinal fluid. Canine serum IgA (10S) was observed to sedi­ ment in the ultra-centrifuge at a faster rate than human

serum IgA (7S). The molecular size of canine secretory IgA was found to be similar to or slightly larger than 11S human

secretory IgA. Treatment with mercaptoethanol was observed to reduce both canine serum and secretory IgA to 7S sub­ units. The colostrum to serum ratio of IgA was the highest of any of the immunoglobulins being 14 on day 1 and decreas­ ing to 4 by day 30 following parturition. A technique for the isolation of secretory IgA from colostrum was outlined by Reynolds and Johnson (19 70a). The technique utilized a combination of Sephadex and Sepharose columns in which IgA

enriched fractions were concentrated and filtered on Seph­

adex G-200 where peak 1 containing IgM and IgA and peak 2 containing IgA resulted. Refiltration of the IgA peak yielded 1 peak on Sephadex G-200. This isolated colostral

IgA exhibited reactions of identity with its counterpart in canine serum, and ileal secretions. Canine IgA was also found in fecal extracts (Reynolds and Johnson, 1970b) in a concentration four to eight times greater than that found in serum. A procedure for isolation of fecal IgA is re­ ported which used DEAE cellulose chromatography followed by two Sephadex filtrations. The purified IgA showed reac­ tions of identity with serum IgA and IgA from other canine secretions. Fecal and colostral IgA appear to be the same molecular species because of similarities in their sedimen­ tation coefficients, appearance on acrylamide gels, and molecular weights. A study of canine secretory IgA was carried out by Ricks et aJ. (1970) who prepared an anti­ serum against colostral IgA and, following absorption to remove anti-L chain antibodies, found that the antisera recognized one component in serum and the same plus one additional component in colostrum. When this antiserum was used to examine Sephadex column fractions of colostrum, two distinct locations of activity were found: one cor­ responding to the component only present in colostrum postulated to be secretory piece. Two canine IgA myelomas have been reported (Hurvitz et al., 1971; Rockey and

Schwartzman, 1967).

A dog with spontaneous pollen hypersensitivity was described (Patterson, 19 60) and subsequent studies were performed with this particular dog. Patterson and Sparks

(1962) showed that pollen hypersensitivity could be trans­ ferred passively to normal dogs by serum, but that the serum factor capable of transferring sensitivity was heat labile.

It was subsequently shown (Patterson et a_l. , 1963b) that canine antiserum against ragweed antigen was capable of trans­ ferring skin, respiratory, and anaphylactic sensitivity to normal animals. The active component of the serum was found to be precipitated with 50% SAS but soluble with 33% SAS and migrating as a B'globulin, probably 7S. The canine antibody is found to be very similar to human IgE antibody. Patter­ son et aJL. (1965) describe the immunization of a normal dog with ragweed antigen and the subsequent production of a non­ precipitating, heat stable antiserum which did not sensitize normal dog skin and migrates as a y^, globulin. This anti­ serum has "blocking" activity as it inhibits the reactions of dogs passively sensitized with canine reagin to challenge with ragweed antigen. Evidence for canine reaginic antibody as a separate class of immunoglobulin (yE) is stated by

Patterson et ad. (1968) as mainly a lack of inclusion in any other antibody class and its immunoelectrophoretic localiza­ tion similar to human IgE. More substantial evidence for canine reaginic antibody being the canine analogue to human

IgE was shown by Halliwell et a_l. (19 72) with the absorption of canine anti-ragweed antiserum with anti-human IgE which resulted in a reduction of PK titer to ragweed. Also anti­ human IgE was also effective in eliciting reversed cutaneous anaphylaxis in the dog.

Reynolds and Johnson (1970c) reported the concentra­ tions of canine immunoglobulins in serum, colostrum, and fecal extracts for mongrel and pure bred dogs. Serum values were as follows:

serum a Immunoglobulin Class_____ Pure bred Mongrel j^mg/ml IgM 1.56 (37) 1.45 (35)

IgA 0.83 (37) 0.79 (33)

IgGab 5.12 (37) 7.71 (35)

IgGd 3.00 (37) 5.62 (28)

IgGc 1.13 (21) 1.12 (20)

cl foxhounds and collies ■ number of animals tested PART I

CHARACTERIZATION OF IgG-CONTAINING DEAE

FRACTIONS OF CANINE SERUM

Caroline C. Whitacre and R. W. Lang

Accepted for publication

Transplantation Proceedings 1975 Due to the increasing use of the canine as a labora­ tory transplant model, several workers have investigated the immune response of this species to various antigens

(Johnson and Vaughan, 1967a; Johnson et al., 1967b; Patter- son et al., 1963). Six globulin classes with demonstrated antibody activity were classified according to electrophor­ etic mobility: IgGa, IgGb, IgGc, 7S gamma 1 (IgGd), inter­ mediate S gamma 1 (IgA), and IgM (Johnson and Vaughan, 1967a

Johnson et al., 1967b). Analysis of the four IgG globulins

(IgGa-d) revealed that IgGa and IgGb were inseparable by agar gel electrophoresis, ultracentrifugation, starch gel electrophoresis, ion exchange chromatography, and gel fil­ tration (Johnson and Vaughan, 1967a); therefore, they were collectively designated as IgGab (Reynolds and Johnson,

1970c; Vaerman and Heremans, 1968).

A canine tissue typing antiserum (S14113 Columbus USA,

International Workshop on Canine Immunogenetics (Vriesen- dorp et al., 1973), produced by intrafamily leukocyte immun­ ization, was examined for its immunoglobulin content. The antiserum exhibited high anti-lymphocyte cytotoxic titers and demonstrated a specificity different from known DL-A. antigens (Vriesendorp et al., 19 73). This report describes the column chromatographic fractionation of -that tissue typing antiserum with the separation of the IgGa and IgGb components and localization of cytotoxic activity. 12

MATERIALS AND METHODS

Antiserum for Chromatography

Canine tissue typing antisera were produced within a

family of "coberman" dogs, a cross between a collie and

doberman (Figure 1). Peripheral leukocytes from the F 2 gen­

eration dogs were injected biweekly into generation

cobermans yielding antisera with high anti-lymphocyte cyto­

toxic titers. One of these antisera (S14113 Columbus USA,

First International Workshop on Canine Immunogenetics)

showed segregation patterns different from the known DL-A 1 antigens (Vriesendorp et al., 1973). Also chromatographed were unimmunized F^ coberman serum, commercial dog serum

(Pentex), Cohn Fraction II of pooled dog serum (Pentex),

and fresh pooled serum from three unconditioned mongrel dogs.

Column Chromatography

Sera were chromatographed on DEAE (Whatman DE-52)

anion exchanger using a modification of the method of Fahey

et al. (1958). Of a Tris-phosphate continuous gradient

(0.01 M phosphate pH 8.4 to 0.3 M phosphate pH 4.5) for

elution of serum proteins. Each sample was dialyzed for 18

hours against 2 changes of 0.01 M phosphate buffer and

centrifuged prior to application to the column. The optical

density at 280 nm and 413 nm, pH, and conductivity were

determined on all effluent fractions. Kale □ ' Doberman Kale Collie Female

Female o -

*1

6 (CD

Immunisation

C, D, CD: Blood Croups o—Donor *o Recipient Evolution of Inbred strain of "Coberman" dogs.

Figure Is The family history of the "coberman" dogs showing leukocyte immunization scheme. Numbers refer to individual dogs. The designation S14113 was given to serum from dog 71. 14 Serological Techniques

Immunoelectrophoresis was performed as described by

Scheiddegger (1955) using 1% Noble agar and barbital acetate buffer, pH 8.6. Antisera for the development of immuno- electrophoretic patterns were produced by biweekly intra— dermal injections of rabbits with equal volumes of coberman serum and complete Freund's adjuvant (Difco).

The Osserman technique for immunoelectrophoresis

(Osserman, 1960) was utilized to define antigenic relations among column fractions, and to identify individual compon­ ents using known proteins: canine IgG (Pentex) and canine transferrin (Pentex).

The micro-lymphocytotoxicity assay of Terasaki and

McClelland (1964) was used to examine whole serum and column fractions for cytotoxic activity. Lymphocytes were obtained from canine peripheral blood by a three—step separation tech­ nique employing phagocytosis of iron particles, differential sedimentation, and gradient centrifugation (Whitacre and

Lang, 1975b). Rabbit serum absorbed with pooled canine leukocytes was utilized as a complement source and eosin Y dye exclusion was used to assess cell viability.

RESULTS

Typically, a 20 ml. sample of coberman serum S14113 was chromatographed on a Pharmacia K16 column as described above. The elution pattern showed eight protein peaks

(Fig. 2). Hemoglobin, assayed by optical density at 413 nm, 15

cE O CO CVJ o o INITIAL CHROMATOGRAPH

1.0-

To W To To TO e’o W Tbo % EFFLUENT VOLUME

Figure 2: DE->-52 chromatograph of canine anti-lymphocyte serum S14113 and (in the insert) chromatograph of pooled and concentrated pre-hemoglobin frac­ tions. Numbers 1-8 on the abscissa refer to pooled fractions. 16 was localized in peak 6 and found to elute routinely at 6 3-

70% of the effluent volume. All column fractions prior to peak 6 were pooled, concentrated to 10 ml., dialyzed against

0.01 M phosphate buffer, and rechromatographed under similar conditions. The protein scan of the rechromatographed sample closely resembled that of the initial chromatograph (insert

Fig. 2). Effluent fractions were pooled as represented by the numbers on the abscissa and the resulting 8 fractions were concentrated to 2 ml. each. The gamma globulin portion of the chromatogram, represented by fractions 1 through 5, was assayed by immunoelectrophoresis and cytotoxicity.

The elution patterns obtained with unimmunized cober- man serum, commercial dog serum, and fresh pooled dog serum appeared similar to the initial chromatograph of Figure 2.

In each case prehemoglobin peaks were observed at approx­ imately the same per cent effluent volume and corresponding to similar molarities of phosphate. Cohn fraction II eluted as a small unbound peak corresponding to peak 1 and a large bound peak encompassing the regions occupied by peaks 2 and

3. The immunoelectrophoretic patterns of DE-52 fractions

1 through 5 were developed with rabbit anti—canine antiserum

(Fig. 3). Fraction 1 shows only one major arc in the gamma two region. Fraction 2 exhibits 3 lines, a major line in the gamma 2 region (line 1) and an equally intense line crossing line 1 (line 2). Line 2 produces a "gull line" indicating two electrophoretically different arcs that 17

fr 1 3

traps: q

Figure 3: Immunoelectrophoresis of DE-52 fractions 1, 2, 3, 4, 5, commercial canine IgG (Pentex) and commercial canine transferrin (Pentex), Rabbit anti-coberman whole serum was placed in the trough. Cathode is on the right. 18

coalesced indicating immunological identity. Line 3 is a minor component in the gamma 1 region (not visible on photo­

graphic reproduction). Fraction 3 shows two equally prom­

inent lines, one migrating in the gamma 2 region (line 1) and one migrating in the gamma 1 region (line 2). Frac­ tions 4 and 5 yield similar immunoelectrophoretic patterns to fraction 3.

By the Osserman technique, the lines in fractions 1 through 5 were identified using commercial canine IgG and commercial canine transferrin as known proteins. Canine IgG in direct Immunoelectrophoresis (Fig. 3) showed 4 compon­ ents: a prominent line with 2 spurs and a separate line in the gamma 1 region which proved to be transferrin contamina­ tion. Early workers described a similar pattern for canine

IgG and found both IgGa and IgGb occurring in the prominent line, IgGc in the more cathodal spur and IgGd in the more anodal spur (Johnson and Vaughan, 19 67a). Transferrin

(Fig. 3) contains 3 components: a prominent line in the gamma 1 region and two distinct lines in the beta region which proved to be beta globulin contaminants.

When fractions 1 through 5 were electrophoresed and developed with rabbit anti-canine antiserum (lower trough) and canine IgG (upper trough) in the usual Osserman pro­ cedure, line 1 in each fraction (the most cathodal arc), showed identity with the major IgG component (Osserman line), indicating that line 1 of all 5 fractions contained 19 one or more canine IgG subclasses (not shown). Each frac­ tion was also assayed for transferrin using the Osserman technique. No transferrin was observed in fraction 1 and only a slight amount corresponding to a weak line (line 3) was seen in fraction 2. However, the major arc in the gamma

1 region (line 2) of fractions 3, 4, and 5 (Fig. 3) con­ nected with the transferrin Osserman line (Fig. 4f) indicat­ ing substantial amounts of transferrin in these fractions.

When IgG was electrophoresed and developed with frac­ tion 1 (Fig. 4a), the single Osserman line of fraction 1 coalesced with only the lower segment of the primary IgG line, the upper segment forming a spur. This reaction served to confirm the observations of Johnson and Vaughan

(1967a) that there were indeed two components in the major arc of IgG, only one of which was present in fraction 1.

There was no reaction with the other components spurring from the major IgG line.

A study of fraction 2 showed that lines 1 and 2 (Fig.

3) were both IgG components. When fraction 2 was electro— phoresed and developed with IgG (Fig. 4b), both arcs showed identity with the major IgG Osserman line. Moreover, when

IgG was electrophoresed and developed with fraction 2 (Fig.

4c), the single major Osserman line was observed to connect completely with the major IgG arc, while the minor Osserman line proved to be transferrin. The major Osserman line on the slide (Fig. 4c) was shown, in a separate experiment, to 20

Figure 4: Osserman immunoelectrophoresis test for iden­ tification of DE-52 column fractions. Anti­ gens were electrophoresed from the wells as indicated in slides a through f. Cathode is on the right. Rabbit anti-coberman whole serum was placed in the lower trough. Osser­ man reagents were placed in the upper troughs as indicated on the right margin of the figure.

22 contain the two major components of fraction 2 (lines 1 and

2). When fraction 2 was electrophoresed and developed with

fraction 2, the single major Osserman line coalesced with

lines 1 and 2 (not shown).

The relationship of the one IgG component of fraction

1 to the two IgG components of fraction 2 was clarified by

the Osserman procedure in which fraction 2 was electrophor­

esed and developed with fraction 1 (Fig. 4d). The single

Osserman line of fraction 1 connected with only line of

fraction 2, indicating that of the two populations of IgG present in fraction 2, only one is present in fraction 1. I By comparison of electrophoretic mobilities (Fig. 3), line

1 in both fractions appears very similar. The "gull" line of fraction 2 can be interpreted as electrophoretically slow and fast populations of the same immunoglobulin subclass.

The relationship of the two IgG components of frac­ tion 2 to the one IgG component of fractions 3, 4 and 5 was shown by electrophoresing fraction 3 and developing with fraction 2 (Fig. 4e). The major Osserman line was observed to connect completely with line 1 of fraction 3 proving the presence of both fraction 2 IgG components in that pre­ cipitin arc. The same reaction was observed when fractions

4 and 5 were electrophoresed and developed with fraction 2. * The location and level of cytotoxic activity was followed throughout the fractionation procedure. The cytotoxic titer of whole serum (S14113) was 2048 prior to its application to the DE-52 column. After chromatography. 23 each fraction was assayed for cytotoxic activity (Table 1) resulting in titers as follows: fraction 1, undiluted; frac­

tion 2, 256; fraction 3, 256; fraction 4, 256; fraction 5,

256; fraction 6, 16; fraction 7, 8; and fraction 8,0. Cyto­

toxic activity indicates the distribution of immunoglobulins

in the fractions.

DISCUSSION

Evidence indicates that DE-52 fraction 1 of canine serum S14113 contains a single subclass of IgG. Direct Im­ munoelectrophoresis of fraction 1 reveals a single arc ap­ pearing as a typical IgG line in the gamma 2 region. How­ ever, this arc is not the total IgG arc typically seen with whole serum, as indicated by the spur formation with com­ mercial IgG when fraction 1 is used in the Osserman pro­

cedure (Fig. 4a). Although not indicated on direct immuno- electrophoresis, the major arc of commercial IgG consists of

two superimposed lines, only one of which is identical with

fraction 1. The two major arcs can best be seen in the im— munoelectrophoretic pattern of fraction 2(Fig. 3). Here

again fraction 1 coalesces with only one component of frac­

tion 2, the slower migrating arc (Fig. 4d). Since the two major components of fraction 2 appear as a single Osserman

line, the complete fusion of this line with the major IgG arc without spur formation (Fig. 4c) confirms that both com­

ponents are present in the IgG arc. The presence of two IgG

subclasses appearing as a single line is also indicated in 24

Table 1: DISTRIBUTION OF CYTOTOXIC ACTIVITY IN DEAE COLUMN FRACTIONS

Fraction # IgG Subclasses Cytotoxic Titer

1 IgGa 1

2 IgGa, IgGb 256

3 IgGa,IgGb 256

4 IgGa, IgGb 256

5 IgGa, IgGb 256

6 IgGa, IgGb 16

7 IgGa, IgGb 8

8 0 25 fractions 3, 4 and 5, where line 1 of each fraction coalesces with both IgG lines in fraction 2 and with the major commer­ cial IgG line.

Johnson and Vaughan (1967a) describe canine IgG sub­ classes gamma 2a and gamma 2b as electrophoretically insepar­ able on agar gel. However, an effective separation of one component has been achieved in the high pH, low molarity ef­ fluent of a DE-52 cellulose column. The single component present in fraction 1 can be provisionally identified as

IgGa using the criteria established previously (Johnson and

Vaughan, 1967a) of the most cathodal of the serum proteins.

IgGa and IgGb appear as electrophoretically distinct compon­ ents in fraction 2. In fractions 3, 4 and 5, both components appear as a single arc. In contrast to earlier reports

(Johnson and Vaughan, 1967a; Vereman and Heremans, 1968) describing IgGa and IgGb as eluting together from DEAE cel­ lulose with initial buffer of high pH (8.0) and low molarity

(0.01 M phosphate), our results indicate IgGa precedes the elution of other immunoglobulins in a pH 8.4, low molarity

(0.01 M phosphate) buffer.

The cytotoxic activity can be localized to the IgG globulins and more specifically appears to be associated with the gamma 2b subclass. Cytotoxicity is associated with the first seven fractions from DE-52 cellulose which cor­ responds to the elution region of canine gamma globulin.

However, the greatest cytotoxic activity (256) is observed 26 with fractions 2, 3, 4 and 5, in which IgGb is present in higher concentrations. Fraction 1 is not observed to con­ tain any IgGb and the corresponding cytotoxicity is min­ imal. It therefore appears that the lymphocyte antigens elicited primarily an IgGb response. PART II

DEAE CHROMATOGRAPHIC CHARACTERIZATION OF

CANINE SERUM AND ISOLATION OF AN IgG SUBCLASS

Caroline C. Whitacre and R. W. Lang

Prepared for submittal to the

Journal of Immunology

27 28

Interest in studying canine immunoglobulins stems pri­ marily from similarities of human and canine gammopathies

including multiple myeloma (Hurvitz, et al., 1971; Rockey

and Schwartzman, 1967), systemic lupus erythematosus (Lewis et al., 1965), and spontaneous ragweed pollen hypersensitiv­

ity (Patterson, 1960). In a comprehensive study on dog immunoglobulins, Johnson and Vaughan (1967a) described six

antigenically distinct populations (y2a, y2b, y2c, 7Sy^,

Int Sy^, and yM) all of which possessed antibody activity

(Johnson et al., 1967b). The y2a and y2b globulins, analogous to IgG globulins of other species, have similar properties: sedimentation in the ultracentrifuge as a single peak (S° on 6.7), identical electrophoretic 6U / w mobility in agar gel, and simultaneous elution on anion exchange chromatography (Johnson and Vaughan, 1967a). Be­ cause of their virtually identical physicochemical proper­ ties, these two subclasses of canine IgG are often referred to as IgGab (Reynolds and Johnson, 1970c; Vaerman and

Heremans, 1969). The canine 7Sy^ class described by John­ son and Vaughan (1967a) was classified as an IgG subclass

(IgGd) based on amino acid sequence homology with other mammalian yG immunoglobulins (Grant et ajL., 1972). While

y2c is assumed to be an IgG subclass also (Reynolds and

Johnson, 1970c), conclusive evidence is lacking. Vaerman and Heremans (1968) identified the Int Sy^, and yM classes as canine IgA and IgM, respectively. 29

The present work concerns a study of various prepara­

tions of canine whole serum and serum fractions using anion

exchange chromatography. As described in a previous report

(Whitacre and Lang, 1975a), this technique facilitated the

separation of IgGa from other canine immunoglobulins. A par­

tial characterization of IgGa is presented.

MATERIALS AND METHODS

Sources of canine sera and serum fractions

The following canine sera and serum fractions were frac­

tionated by anion exchange chromatography: reconstituted

lyophilized dog serum (Miles Laboratories, Kankakee, Illinois),

canine Cohn Fraction II (Miles Laboratories), fresh pooled sera from three mongrel dogs, unimmunized F^ coberman serum

(Dog 31) and a canine tissue typing antiserum. The anti­ serum was produced in a family of "coberman" dogs (cross be­ tween a collie and doberman) by biweekly injections of F 2 generation peripheral leukocytes into F-^ generation cober- mans (Figure 1). One high titered anti-lymphocyte cyto­ toxic serum (Dog 71, final bleed) was shown to have high specificity for non-DL-A antigen(s) and was designated

S14113 Columbus USA (Vriesendorp et a]^., 1973) .

Anion exchange column chromatography

The procedure of Fahey et al. (1958) was followed for

DEAE cellulose chromatography except that Whatman DE-52 30 cellulose was used and the pH of initial buffer was revised to 8.4. A continuous non-linear phosphate gradient (0.01 M phosphate pH 8.4 to 0.3 M phosphate pH 4.5) was produced by the Varigrad mixing device (Buchler Instruments). Before application to the column, the sample was dialyzed overnight against two changes of 0.01 M phosphate buffer and centri­ fuged to remove any precipitate. One ml analytical and 20 ml preparative samples were chromatographed on Pharmacia

K-9 and K-16 columns, respectively. All effluent fractions were assayed for pH , conductivity and optical density at

2 80 nm and 413 nm. Column results were plotted as OD at

280 nm versus % effluent volume in order to facilitate com­ parison of analytical and preparative columns. Protein determinations of fractions were made according to the method of Lowry et al. (1951).

Immunoelectrophoresis

Immunoelectrophoresis was performed according to the micromethod of Scheiddegger (1955) using barbital acetate buffer (pH 8.6, ionicity 0.05). Rabbit anti-dog serum

(Miles Laboratories, Kankakee, Illinois) was used to develop the immunoelectrophoretic pattern.

The Osserman technique for immunoelectrophoresis

(Osserman, 1960) was employed in the identification of pre­ cipitin arcs using known proteins: canine IgG preparation

(Miles Laboratories) and canine transferrin preparation

(Miles Laboratories). 31

Polyacrylamide Gel Electrophoresis (PAGE)

Samples were electrophoresed according to the method of Davis (1964) using a 7% running gel, and 2.5% stacking and sample gels in a Buchler apparatus. Electrophoresis was carried out at 3 ma per tube with a .38M Tris-glycine buffer pH 8.3 until the bromphenol blue tracking dye reached the end of the 5 cm. gel columns. Gels were stained with 1% amido schwartz in 7% acetic acid. Sample volumes ranged from

5 to 50 ul depending upon the protein concentration.

Cytotoxicity analysis

Cytotoxic antibody activity in whole serum and column fractions was assessed by the micro-lymphocytotoxicity test

(Terasaki and McClelland, 1964). Lymphocytes were obtained from canine peripheral blood by a three-step separation procedure (Whitacre and Lang, 1975b). Absorbed rabbit serum ' served as a complement source and eosin Y dye exclusion was used to monitor cell viability. Serum end-point titers were the reciprocal of the antibody dilution giving 50% cell death.

Antisera

Rabbits were immunized intradermally over the back with an emulsion containing equal volumes of antigen and complete Freund's adjuvant (Difco). 32

RESULTS

Anion exchange chromatography of canine sera

To obtain a representative canine serum chromatogram,

1 ml samples of reconstituted lyophilized commercial dog serum, pooled normal mongrel serum, normal coberman dog 31 serum and coberman dog S14113 antiserum were fractionated on DE-52 cellulose (Fig.5A-D). A continuous gradient of de­ creasing pH and increasing phosphate molarity was used for all column separations and is represented in Figure 5D.

Similar elution patterns were obtained with all four sera yielding 7 to 8 peaks. Starting at 8-10% of the effluent volume, IgG is seen to elute first, followed by transferrin beginning at approximately 22—25%. Hemoglobin occurs at

50-68% and is followed by a major peak seen in Fig. lb and lc which contains °= - 2 macroglobulin (Vaerman and Heremans,

1968). Albumin is the primary component of the last peak, and IgM elutes at 90-100%. The appearance of four peaks in the first 50% of the column effluent was consistent while sometimes the 3 peaks appearing in the last 50% of the column effluent resolved into 4 peaks (Fig. 5A) depending upon the slope of the molarity gradient. Hemoglobin was monitored at 413 nm and eluted between 50% and 68% of the effluent volume (Fig. 5D). DE-52 chromatography of Cohn fraction II (Fig. 5E) yielded a small initial peak (0-15%) and one large peak eluting at 35-50% of the effluent volume. iue : nltcl . l) E5 fatoain o cnn serum. canine of (.1 fractionations DE-52 ml.) Analytical 5: *Figure

OD 280 nm. .5' ( A) reconstituted lyophilized commercial dog serum. dog commercial lyophilized reconstituted (A) 20 30 EFUN VOLUME EFFLUENT % 40 50 60 70 090 80 iue5 Aayia ( l) E5 fatoain o aie serum. canine of fractionations DE-52 (1ml.) Analytical 5: Figure

OD 280 nm. . 2 - B poe omlmnrl serum. mongrel normal pooled (B) 20 30 EFUN VOLUME EFFLUENT % 40 070 50 60 90 io Figure 5: Analytical (1 ml.) DE-52 fractionations of canine serum. canine of fractionations DE-52 (1ml.) Analytical 5: Figure

OD 280 nm. .5- . 2 - C Dg 1 nra oemn serum. coberman) (normal 31 (C) Dog 20 30 EFUN VOLUME EFFLUENT % 0 60 5 0 40 807 0 90 CO /l C iue : nltcl 1m. D-2 rcintoso aie serum. canine of fractionations DE-52 (1ml.) Analytical 5: Figure

OD 280 nm. 413 nm .- D Cbra nilmhct eu (S14113). serum anti-lymphocyte (D) Coberman 20 30 EFUN VOLUME EFFLUENT % 70 80 9040 60 50 Q. — I 6 O' U3 iue5 Aayia ( l) E5 fatoain f aie serum. canine of fractionations DE-52 (1ml.) Analytical 5: Figure

OD 280 nm. (E) Commercial canine Cohn fraction II. fraction Cohn canine (E) Commercial 20 EFUN VOLUME EFFLUENT % 0 50 40 60 0 8 9030 70 Id 38

These chromatograms of canine serum differed markedly

from the chromatograms of human serum reported by Fahey

et al., 1958. To verify this apparent dissimilarity, human

serum and canine serum were chromatographed over the same

DE-52 column under similar conditions. After one ml of human serum was chromatographed, the DE-52 cellulose was washed with final buffer until eluate OD was zero and then

equilibrated with initial buffer before 1 ml of canine serum was chromatographed. In the same way the cellulose was

again treated with final and initial buffers before a second

1 ml sample of human serum was chromatographed. The pH

gradients of the three fractionations were similar and the conductivity plots were superimposable. The final human serum chromatogram, which differed slightly from the initial sample, is shown in Figure 5F with the canine serum chrom­ atogram. With human serum, three major peaks were observed corresponding to gamma globulins, beta globulins, (includ­ ing transferrin), and albumin respectively (Fahey et al.,

1958). The dog chromatogram differs from the human prin­ cipally in the size of the first peak and the presence of

1 or 2 additional canine peaks eluting before human trans­

ferrin. A 20 ml sample of coberman antiserum S14113 was chromatographed on a preparative K-16 column (Fig. 2) and the pre-hemoglobin portion was pooled, concentrated to 10 ml and rechromatographed as described previously (Whitacre

100 HUMAN- - CANINE 9080 60 50 40 % % EFFLUENT VOLUME 20 (F) (F) Humanserum, canine serum, and human serum sequentially was passedover the same column.

IUUQ82 0 0 Figure 5: Analytical ml.) (1 DE-52 fractionations of canine serum. 40 and Lang, 1975a). Individual samples were pooled and con­ centrated producing eight fractions, the first five of which contained the gamma globulins (and some transferrin). Frac­ tions 1-5 were studied by immunoelectrophoresis and all arcs were identified by means of the Osserman procedure (Whitacre and Lang, 1975a).

Immunoelectrophoretic study of serum and serum fractions

Immunoelectrophoresis of fraction 1, when developed with rabbit anti-dog whole serum, showed a single precipita­ tion arc of Y2 mobility (Fig. 6A). Fraction 2 showed 3 precipitation arcs: one occurring in the Y2 region, another occurring as a "gull" line in the Y^ region, and a third weak Y^ arc (not visible on photographic reproduction) which proved to be transferrin (Fig. 6B). By means of the Osser­ man test, using a commercial canine IgG preparation, the precipitation arc in fraction 1 and the two major arcs in fraction 2 were identified as canine IgG.

Immunoelectrophoresis of canine IgG showed a major precipitation arc with 2 minor spurs (Fig. 6C) (the separate minor arc is due to transferrin contamination), Early workers (Johnson and Vaughan, 1967a) had established that the major arc contains IgGab, the more cathodal spur rep­ resents IgGc, and the more anodal spur is IgGd. Rabbit anti-dog whole serum (lower trough) was used to develop the pattern of electrophoresed IgG and to produce an Osserman Figure 6: Immunoelectrophoresis of A. fraction 1 from DE-52 fractionation of serum S14113. B. fraction 2. C. commercial canine IgG. Anti­ body troughs contain rabbit anti-dog whole serum. Cathode is on the right. 42 line with fraction 1 diffusing from the upper trough (Fig.

7A). The fraction 1 Osserman line fused with only the lower portion of the major IgG arc, thus forming a spur with the upper portion. This reaction suggested that fraction 1 contained only one of the two components present in the major IgG arc, that is the more cathodal IgGa. When IgG was electrophoresed and fraction 2 was placed in the upper trough (Fig. 7B), the major Osserman line fused completely with the primary IgG arc, indicating the presence of IgGa and IgGb in fraction 2. Thus the 2 electrophoretically distinct arcs seen in fraction 2 (Fig. 6B) corresponded to

IgGa and IgGb. Fractions 3, 4 and 5 were seen to contain transferrin and IgGa, IgGb occurring together as a single precipitation arc (Whitacre and Lang, 1975a).

PAGE Analysis

Polyacrylamide gel electrophoresis was performed on

DE-52 column fractions, canine IgG and unfractionated serum

S14113 (Fig. 8). Fraction 1 exhibited a dark staining region at the top of the gel Rf .01 tO .21 corresponding to gamma globulin and a faint band at Rf .38. Fraction 2 demonstrated eight components: gamma globulin occurring at Rf .13 to .19, transferrin at Rf ,40, and other uniden­ tified components occurring at Rf .32, .36, .47, .59, .64,

.68. Figure 7: Osserman immunoelectrophoretic technique for identification of fraction 1 and 2 components. Canine IgG is electrophoresed, lower troughs contain rabbit anti-dog whole serum and upper troughs contain a. fraction 1 and b. fraction 2. From the fusion of precipitation lines with IgG, fraction 1 appears to contain only IgGa and fraction 2 appears to contain IgGa, IgGb, and some transferrin. Cathode is on the right. Figure 3: Polyacrylamide gel electrophoresis of a. fraction 1, b. fraction 2, c. fraction 3, d. fraction 4, e. fraction 5, f. whole serum S14113, g. canine IgG. 45

Cytotoxic Antibody Activity

Anti-lymphocyte serum S14113 exhibited a high cyto­

toxic titer (204 8) with reactive lymphocytes and therefore

it was possible to assess cytotoxic activity in individual

fractions (Table 1). Fraction 1 demonstrated a titer of undiluted; fraction 2, 256; fraction 3, 256; fraction 4,

256; fraction 5, 256; fraction 6, 16; fraction 7, 8; and

fraction 8, 0 (Whitacre and Lang, 1975a). The protein con­ centration of fractions 1 and 2 were not markedly different

(.044 and .069 mg/ml. respectively).

Analysis of Anti-Immunoglobulin Sera

Fraction 1 and fraction 2 preparations were each used to immunize rabbits. Sera Rflb from a rabbit injected with

fraction 1 (IgGa) formed a single arc with electrophoresed

IgG which coalesced completely with an Osserman line formed with fraction 1 (Fig 9a). The absence of a cathodal spur as seen in Fig. 7a indicated that antibodies to the IgGb subclass were not present. Sera obtained early from rabbit

Rf2c, which had been injected with fraction 2 (IgGa, IgGb) showed reactivity only to the IgGb component (Fig. 9b).

Through absorption of this early serum with fraction 1, anti­ bodies to L chains and to common Y chain determinants were removed making the antiserum specific for the IgGb subclass.

Sera taken later in the immunization schedule of rabbit Rf2c demonstrated antibody activity against both IgGa and IgGb

(Fig. 9c). Figure 9: Characterization of anti-fraction 1 (Rflb) and anti-fraction 2 (Rf2c) antisera. a. IgG is electrophoresed, the upper trough contains fraction 1 and the lower trough contains antisera Rflb. The develop­ ment of a single precipitation arc with no spurs indicates the ab­ sence of antibodies specific for other IgG subclasses (IgGb, IgGc, IgGd). b. and c. Fraction 2 is electrophoresed, the lower troughs contain rabbit anti-dog whole serum (Rad)t and the upper troughs con­ tain Rf2c obtained 9 days after immunization (b) and 85 days after immunization (c). Early antisera appears to detect only IgGb while later antisera detects both IgGa and IgGb. 47

DISCUSSION

The DE-52 chromatograms of various whole canine sera

(Fig. 5A, B, C, D, F) appear similar. Although the height of the individual protein peaks may vary, their number (7-8) and location in the column effluent are similar. The point of elution for a given peak did vary from column to column, and therefore peaks are designated by percentage ranges of eluate. The chromatograms illustrated appear to be represen' tative of canine serum, regardless of breed of dog.

A comparison of the human and canine serum chromato­ grams (Fig. 5F ) reveals that the two sera differ markedly in charge characteristics. Human IgG is observed to elute primarily in the large unbound protein peak while canine

IgG is seen in the first four peaks. It would seem to indi­ cate that the subclasses of human IgG are inseparable on

DEAE cellulose while it has been shown here that canine IgGa can be obtained from DEAE free of other immunoglobulins.

In order to separate canine IgGa, it was necessary to rechromatograph the concentrated pre-hemoglobin DEAE frac­ tions using initial buffer at pH 8.4 (conductivity 1<0) instead of pH 8.0 as outlined in the classical procedure of

Fahey et al. (1958). The distribution of IgGa throughout much of the DEAE effluent volume suggests that it is a heterogeneous protein. The IgGa in fraction 1 therefore represents only molecules with a low charge density at pH

8.4. Acrylamide gel analysis showed fraction 1 to consist 48 of slowly migrating gamma globulins when compared to the entire distribution of IgG (Fig, 8g).

Electrophoresis of fraction 1 and subsequent develop­ ment with anti-whole serum revealed one arc of y2 mobility providing evidence for the isolation of a single immuno­ globulin. More conclusive evidence was provided by the

Osserman procedure in which IgG was electrophoresed and fraction 1 was used as the Osserman reagent. Fusion of the

Osserman line with only the more cathodal portion of the major IgG arc indicated the sole presence of IgGa in frac­ tion 1 (Fig. 7a). In addition/ when fraction 1 was used to immunize rabbits, the resultant antisera demonstrated reac­ tivity with IgGa but not with IgGb subclass specific deter­ minants (Fig. 9a). However, the antisera did react with other immunoglobulins, presumably through antibodies to L chains or gamma H chain common antigen(s). Had IgGb, IgGc, or IgGd paraproteins been available for absorption, the cross-reacting antibodies could possibly have been removed.

Sera Rf2c, prepared against fraction 2, recognized two components, IgGa and IgGb. However, polyacrylamide gel analysis of fraction 2 demonstrated eight separately stain­ ing components, many of which appeared to be non-antigenic since only 2 arcs were observed by IEP, This antiserum was rendered specific for the IgGb subclass through absorp­ tion with fraction 1.

The cytotoxic activity of antiserum S14113 was local­ ized principally in fractions 2, 3, 4, and 5, which 49 corresponded to the elution profile of IgGb (Whitacre and

Lang, 1975a). It was postulated from a previous study

CWhitacre and Lang, 1975a) that the coberman non-DL-A lymph­ ocyte antigen(s) had elicited primarily an IgGb response.

This speculation was supported by the fact that fraction 1 had no significant cytotoxicity, while fraction 2 of ap­ proximately equal protein concentration and containing both

IgGa and IgGb had a high titer of cytotoxic antibody. The possibility exists that IgGa is a non-complement fixing antibody, as is human IgG^ (Natvig and Kunkel, 1973) or that the nature of antigen or its distribution on the lymphocyte surface prohibits bound IgGa antibody from leading to com­ plement lysis.

The preparation of canine IgGa by DEAE chromatography was attempted only with antiserum S14113. Since the DE-52 chromatograph of normal dog serum (Fig. 5A) is very similar to that of antiserum S14I13 (Fig. 5D), isolation of IgGa is not likely to be dependent upon the fractionation of immune serum especially since it appeared that IgGa did not have antibody activity.

The isolation of canine IgGa allows, for the first time, a characterization of this IgG subclass. PART III

A TECHNIQUE FOR SEPARATION OF CANINE LYMPHOCYTES

AND THEIR USE IN THE LYMPHOCYTOTOXIC, BLASTOGENIC

AND ROSETTE ASSAYS

Caroline C. Whitacre and R. W. Lang

Accepted for publication, Transfusion 1975

50 51

The randomly bred canine has been used widely as a

laboratory model for transplantation research. Several

groups have shown a correlation between graft survival and

histocompatibility between donor and recipient dogs (Daus-

set, et all, 1971; Epstein et al., 1968; Rapaport, et al.,

1970; Storb et al., 1970). The existence of a major histo­

compatibility locus in the dog (DL-A), analogous to the

HL-A locus of the human, has been documented by graft sur­

vival, serologic studies (Boyd, et al., 19 71; Westbroek

et al., 19 71), and by mixed lymphocyte cultures (MLC)

(Templeton and Thomas, 1971). The lymphocytotoxicity and

MLC assays have been increasingly valuable in selection of

compatible donor-recipient pairs for organ transplantation.

However, both assays require a relatively pure target

lymphocyte preparation (Johnson et al., 1971; Walford et al.,

1965). Historically, the separation of lymphocytes from

whole blood has involved the application of differential

sedimentation (Chaplin et al., 1959), gradient centrifuga­

tion (Perper et al., 1968), phagocytosis of iron particles

(Levine, 1956) or selective adsorption (Greenwalt et al_. ,

1962). A number of techniques have been applied to the

canine system ( (Cassen et al., 1958; Epstein £t al., 1968;

Miller et al., 1971) but with the similarity in size of canine lymphocytes and polymorphonuclear neutrophils (Kay et al. , 1973), the purity of the lymphocyte preparations was low and variable. When a combination of these procedures was used with canine blood, cell preparations consisting of

52-74% lymphocytes were obtained (Kay et al. , 1973; Miller et al., 1971).

This report describes a method involving in sequence, phagocytosis, plasmagel sedimentation, and gradient centri­ fugation, yielding a preparation with greater than 90% canine lymphocytes. In spite of this somewhat rigorous pro­ cedure, the lymphocytes are shown to be functional by

1) stimulation with phytohemagglutinin (PHA), and pokeweed mitogen (PWM), 2) E rosette formation with human erythro­ cytes and 3) antibody-mediated lympho cytotoxicity.

MATERIALS AND METHODS

Ten milliliters of venous blood is drawn and trans­ ferred to a 25 ml Erlenmeyer flask containing about 10, 3-mm glass beads for defibrination. The flask is swirled for 10 minutes, then the glass beads and fibrin clot are removed.

Defibrinated blood is transferred to a 15 ml tube contain­ ing 200 mg SF special carbonyl iron powder (GAF Corporation,

New York, N.Y.) and mixed thoroughly. The blood-iron mix­ ture is incubated at 37C and the tube inverted at 5 minute intervals for mixing. After 30 minutes, the mixture is divided into two 5 ml aliquots and 1 ml of Plasmagel (HTI 53

Corporation/ Buffalo, N.Y.) is added to each tube, mixed, and

held at room temperature for 30 minutes. The lymphocyte-

rich supernates are aspirated, pooled and subjected to den­

sity gradient centrifugation in the following manner. The

supernatant fluid is transferred to a 50 ml conical centri­

fuge tube and diluted with three volumes of Seligmann's

Balanced Salt Solution (SBSS). A Ficoll-Hypaque mixture

[10 parts 34% w/v sodium diatrizoate (Winthrop Laboratories,

New York, N.Y.) with 24 parts 9% w/v Ficoll (Pharmacia,

Uppsala, Sweden)] is layered beneath the diluted sample

using a pipette fitted with a plasma aspirating needle.

Ficoll-Hypaque (specific gravity 1.072) is added at the

ratio of 0.25 ml/1 ml of diluted sample. Centrifugation is

then performed at 400 g for 25 minutes in a swinging bucket

type rotor. The lymphocyte layer located just above the

serum-gradient interface is removed into a separate tube

and sedimented by centrifugation at 600 g for 15 minutes.

This supernate is discarded leaving one drop for resuspen­

sion of the cells. In order to lyse remaining erythrocytes,

3 ml distilled water is added to the resuspended cell pellet

followed in 30 seconds by 1 ml of 4X SBSS. Then 0.4 ml whole autologous serum is added followed by centrifugation

at 260 g for 10 minutes. The supernate is removed, again

leaving one drop for resuspension of the pellet and at this time a sample is obtained for differential leukocyte count­

ing based on 200 cells. The cell number is adjusted to the 54

desired concentration by dilution with Hank's Balanced Salt

Solution (HBSS) containing 25% autologous serum and counted

with a Coulter counter. Viability studies were performed on

the final cell suspension at various time intervals using

the supravital stain Eosin Y. Leukocyte suspensions, pre­

pared by a Ficoll-Hypaque separation of defibrinated blood

followed by lysis of remaining erythrocytes, were included

for comparison with lymphocyte suspensions.

PHA and PWM stimulations were carried out using a

modified microtechnique of Strong et al. (1973) in Micro­

test II Tissue Culture Trays (Falcon Plastics). The medium

used was Eagle's Minimum Essential Medium (MEM) and was

supplemented with 25% fetal calf serum, 0.5% antibiotics

(penicillin 200 units, streptomycin 200 ug/ml) and 0.5% glutamine (200mM). Lymphocytes at a concentration of 5 5.0 x 10 cells/well were stimulated with 20 ul/ml of PHA

(M) (General Biochemicals, Chagrin Falls, Ohio) and 50 ug/ml of PWM (Grand Island Biological Company, Grand Island,

N.Y.). Stimulation was measured by uptake of tritiated thymidine (New England Nuclear, Boston, Mass..) after 72 hours of culture.

The rosette assay was carried out following the method of Kelly et al. (1971) in which 0.25 ml of washed human red Q cells (1 x 10 /ml) were mixed with 0.2 ml of a canine lymphocyte suspension (15 x 10^/ml). The cell suspension 55

was diluted with 0.55 ml HBSS and immediately centrifuged

at 200 g for 5 minutes. The pellet was gently resuspended

and the % rosette-forming lymphocytes (RFL) determined. A

RFL was defined as a lymphocyte with three or more adherent

red cells.

The antibody-mediated lymphocytotoxicity assay (Tera-

saki and McClelland, 1964) employed lymphocytes as target

cells, high-titered canine anti-dog lymphocyte sera for

cytotoxic antibody, and rabbit serum absorbed with canine

leukocytes as a complement source. Results were reported

as the reciprocal of the serum dilution giving 50% cell

death when measured by the Eosin Y dye exclusion technique.

RESULTS

The differential cell counts of five lymphocyte pre­

parations obtained by the one-step Ficoll-Hypaque gradient

centrifugation procedure, are compared with the mean of cell

counts of untreated whole blood representing samples from

eleven different dogs (Table 2). Whole blood had an average of 23% lymphocytes, while gradient preparations averaged

30.6% lymphocytes, with a neutrophil contamination of 63.2%.

Blood from the same five dogs was processed according to the proposed three-step procedure, and the differential counts are shown in Table 3. The mean percentage of lympho­ cytes increased to 91.4%, while the neutrophil contamination Table 2: ANALYSIS OF LEUKOCYTE PREPARATIONS

Peripheral Blood v Differential count (%)

■Dog # White cell Polymorphonuclear

.Whole blood^ 23 7 63 7

.Gradient c Preparations

1 18.4 38 0 61.5 0.5 99

2 49.7 32 2.5 60 5.5 99

3 36.6 29.5 2 64.5 4 99

4 37.4 19 1 70.5 9.5 99 5 32.1 34.5 2 59.5 4 99

mean 34.8 30.6 1.5 63.2 4.7 99

a Yield from 10 ml of defibrinated whole blood,

k Represents average counts on freshly drawn blood of eleven dogs.

M Whole blood was treated by the one-step Ficoll-Hypaque (sp. gr. 1.072) centrifugation procedure including

<7 > lysis of erythrocytes and resuspension of cells in media containing 25% fetal calf sera. Table 3: ANALYSIS OF LYMPHOCYTE PREPARATIONS

.Peripheral Blood

Differential count (%)

White cell Polymorphonuclear (%) Dog # yield (xlO^) Lymphocyte» Monocyte Neutrophil Others Viability

Lymphocyte ^ Preparations

1 13.2 94 2.5 0 3.5 99

2 22.4 91 0 1 8 99

3 16.0 93.5 0 0.5 6 99

4 15.1 85 0 3 12 99

5 18.0 93.5 0 0.5 7 99

mean 16.9 91.4 0.5 1.0 7.3 99

Yield from 10 ml of defibrinated whole blood.

^ Prepared by three-step procedure (see Materials and Methods) including iron phagocytosis, Plasmagel

sedimentation, Ficoll-Hypaque gradient centrifugation.

Ul “ •vj 58 decreased to 1%. An average 30% recovery of lymphocytes was calculated based on standard values for canine whole blood 3 3 (using 7.5 x 10 leukocytes/mm ). By dye exclusion, 95% of the lymphocytes were shown to be viable for at least 21 hours when maintained in the 25% serum medium.

In order to evaluate the preparations obtained by the three-step procedure, blast transformation studies using PHA and PWM were performed and the results are depicted in

Table 4. All five lymphocyte preparations, treated with PHA or PWM, incorporated tritiated thymidine at a greater level than control cultures containing lymphocytes and media alone as evidenced by the stimulation ratios. For a given mitogen, incorporation varied among the dogs. Two cell preparations that exhibited high cpm values with PHA stimulation were also high with PWM stimulation and one of these had control values twice that of the other preparations.

E rosette formation with human erythrocytes was also used to test the function of five lymphocyte preparations

(Gupta et al., 1974). The per cent rosette-forming lympho­ cytes (RFL) ranged from 1.5 - 8.6% (Table 5).

The standard canine antilymphocyte serum was used to assess the reproducibility of titers in the lymphocyto- toxicity test using several lymphocyte preparations from a number- of dogs. Lymphocyte suspensions prepared from bleed­ ings of five different mongrel dogs showed that for each dog, the titer was reproducible within one tube dilution (Table 6). Table 4: PHA AND PWM STIMULATION OF LYMPHOCYTES FOLLOWING SEPARATION FROM CANINE BLOOD

Counts per minute (CPM)

PHA treated Stimulation PWM treated Stimulation Control: Lymphocytes Dog it lymphocytes ratio0 lymphocytes ratio and media

+ 1 4481 ±•30b 118 3725 + 69 98 38 2 + + 2 13,097 t 1835 117 11,921 788 106 ' 112 17 + + 3 8820 t 294 170 10,707 566 206 52 1 + + 4 4356t 196 81 2627 48 49 54 11 + + 5 3170 t 75 50 4956 160 77 64 18

3 ^ Eight hour pulse of 1 uC H-thymidine/well containing 0.5 x 10 cells. b Numbers represent mean 1 standard error of triplicate cultures. c Stimulation ratio = test cpm control cpm

U l VO Table 5: E ROSETTE FORMATION OF LYMPHOCYTES FOLLOWING SEPARATION FROM CANINE BLOOD

Dog # Lymphocyte Preparations %RFL

+ b 1 1.5 I .2

2 5.9 t .6 3 • 6.6 t -2 4 2.2 t .5

5 8.6 t 1.4

mean 5.0 i 1.3

a %RFL = number of rosette-forming lymphocytes total number of lymphocytes

k Numbers represent %RFL t. standard error of two determinations : 6: COMPARISON OF CYTOTOXIC TITERS3 WITH LYMPHOCYTE PREPARAT!

LYMPHOCYTE PREPARATIONS

Dog Bleeding 1 2 3

• •

1 512 512

n JL 1024 512

3 512 1024 • 1024

4 2048 2048 1024

5 512 1024 512

The titer of this antiserum varied when tested against

lymphocyte preparations from different dogs due to their

histocompatibility differences. 62

DISCUSSION

The three-step method for separation of canine lympho­ cytes was used successfully in this laboratory with over 100 dogs. The number of lymphocytes recovered from blood sam­ ples using’ this procedure was remarkably constant for the dogs studied (Table 3).

Previously reported methods have resulted in eryth­ rocyte, granulocyte, or platelet contamination of lymphocyte preparations (Cassen et al^., 1958; Epstein et al. , 1968? Kay et al., 1973; Miller et al., 1971). The present technique removes erythrocytes by Plasmagel sedimentation and hypo­ tonic lysis, platelets by defibrination (Thierfelder, 1964) and granulocytes by iron ingestion followed by sedimenta­ tion of iron containing cells on a Ficoll-Hypaque gradient while simultaneously banding lymphocytes. A recent report

(Kay et al., 1973) also suggests removal of granulocytes by iron ingestion, but here iron laden cells were removed by repeated magnetic extraction. Their preparations contained red cells (15-71%) and presumably platelets, since anticoag­ ulants were used and no specific procedure for platelet extraction was described.

In the only published report to date on canine E rosettes, Kelly et al. (19 71) stated that a "small percentage" of dog lymphocytes formed rosettes with human erythrocytes.

Our finding of a mean 5% RFL in canine peripheral blood confirms the observation of the earlier report. The results of the blastogenic and lymphocytotoxic assays may be altered by the presence of neutrophils, plate­ lets, erythrocytes and other cell contaminants. Neutrophils may interfere in the blastogenic response by released en­ zymes degrading tritiated thymidine to thymine and dihydro­ thymine which inhibits DNA synthesis (Elves, 1972). In the lymphocytotoxic assay, granulocytes give a questionable reaction upon supravital staining making a quantitative judg­ ment of the per cent cell death difficult. Reading of the test is hindered, too, by cellular debris which shows up as a diffuse background. The presence of neutrophils and macro­ phages could be responsible for the non-immune killing of lymphocytes due to release of hydrolytic enzymes. The pres­ ence of platelets and other leukocytes containing histo­ compatibility antigens could effectively reduce the titer of antisera by an absorption effect. With our lymphocyte preparations, positive and negative reactions were easily and clearly determined, and estimations of 50% endpoint titers can be made in repeat titrations which agree within one tube dilution.

The present technique yields a viable cell preparation containing greater than 90% lymphocytes which are reactive in blastogenic, rosette and lymphocytotoxic assays. PART IV

CHARACTERIZATION OF A DL-B TISSUE TYPING ANTISERUM

Caroline C. Whitacre, R. W. Lang, James A. Madura

Prepared for submittal as a

Brief Communication to

Transplantation 65

CHARACTERIZATION OF A DL-B TISSUE TYPING ANTISERUM

The use of the outbred mongrel dog as a model for.organ

transplantation research has necessitated a study of canine

histocompatibility antigens. The existence of a major histo­

compatibility locus in the dog (DL-A) /analogous to the human

HL-A locus, has been demonstrated by graft survival using

serologic studies (Blumenstock et erl., 1971; Boyd et al. ,

1971; Epstein et al. , 1971; Westbrock et a]L., 1971) and the j mixed lymphocyte reaction (MLR) (Dausset et al., 1971).

DL-A typing antisera were produced (Epstein et al., 1968;

Mollen et al., 1968; Saison, 1972) by cross-immunization of

related dogs, and these sera were valuable in experimentally

matching or mis-matching donor recipient pairs for trans­

plantation studies. Non-DL-A typing antisera (DL-B (Second

International Symposium on Canine Immunogenetics, 1974))

recognizing minor histocompatibility loci have also been

reported (Vriesendorp et al., 1973). In this paper, a whole

DL-B antiserum and DEAE fractions of that antiserum are

examined for reactivity against a random panel of dogs.

The antiserum was produced within a family of coberman

dogs (Figure 1) (Whitacre et al^. , 1973) . Immunizations were

carried out from the F 2 to the F^ generation in 3 cases and within the F 2 generation in one case. Leukocytes obtained 66 by density gradient centrifugation (Perper et: al., 1968) were injected biweekly by the intraderxnal route. Immuni­ zations were made between dogs of identical blood groups

(blood types determined by Doctor Robert S. Bull, Michigan

State University). The sera chosen for study were obtained from bleedings of dog 71 which produced high titers of cyto­ toxic antibody.

Cytotoxicity titers of various bleedings of dog 71 were obtained using the micro-lymphocytotoxicity assay of

Terasaki and McClelland (1964). One ul of antisera and 1 ul of lymphocytes (4 x 10^/ml) were incubated in a micro­ test tissue culture tray (Falcon Plastics) followed one hour later by 4 ul of rabbit complement. Five ul Eosin Y and 4 ul formalin were added after an additional hour of incubation, and the number of cells staining determined t under an inverted phase microscope.

Lymphocytes for use in the cytotoxic assay were ob­ tained using a three-step technique (Whitacre and Lang,

1975b) . Defibrinated whole blood was incubated with car­ bonyl iron particles followed by Plasmagel sedimentation of erythrocytes. The final step of Ficoll-Hypaque density gradient centrifugation resulted in preparations containing

90-100% lymphocytes which were 99% viable.

The immunizing dose and cytotoxic antibody response of dog 71 measured against donor lymphocytes are shown in

Figure 10. Near the end of the two year immunization CANINE ANTIBODY RESPONSE TO

a n

M

•4 U

14

o X oe • S .

OATS OATS

Figure 10: The immunizing dose and cytotoxic antibody response of dog 71. The nuirber of leukocytes injected is represented by vertical bars at the bottom of the figure. Following day 500 of the immunization schedule, leukocyte inocula were no longer quantitated and are represented by arrows on the abscissa. Dog 71 received an average of 125 x 10^ leukocytes at first biweekly, then weekly, and then biweekly.- Cyto­ toxic titers were determined using lymphocytes from the immunizing donor, and represent the mean + standard error of at least two determinations performed on different days. 68 schedule# the titer was boosted significantly by an injec­ tion of spleen cells from the donor. Representative sera# obtained early, intermediate, and late in the immuniza- • tion schedule were assayed in the cytotoxic test against a panel of 92 dogs: 13 collies, 11 dobermans, 61 mongrels, and 7 family cobermans. Sera obtained at various times during the immunization schedule gave comparable results and the averages are presented in Figure 11. The antisera reacted with the cells from 100% of the family dogs tested and titered at approximately 256. Forty-four per cent of mongrels and 82% of dobermans reacted with the cytotoxic sera titering at 256, but 100% of collies showed cyto­ toxicity at a level of only 4. A clear distinction could be made between reactive and non-reactive family, mongrel, and doberman dogs, the positive dogs having high levels of cytotoxicity while others were completely negative.

Serum from dog 71 was analyzed at a workshop where eighteen families with a total of 101 pups were available for testing. This serum exhibited a gene frequency of

0.1125 and showed segregation patterns different from known DL haplotypes. The antiserum was given the work­ shop designation S14113 (Columbus, U.S.A.) and classified as a lymphocytotoxin determining factors other than DL-A

(Vriesendorp et al., 1973). At the second International

Workshop on Canine Immunogenetics (1974), further family studies confirmed the non-DL-A nature and high specificity PERCENTAGE of DOGS FROM POPULATIONS REACTING with WHOLE ALS and

the IgM FRACTION of WHOLE ALS and the LEVEL of RESPONSE 256 n 64- E S 3 lg M Cytotoxicity 16- COLLIE 4

DOBERMAN

Ui 256

MONGREL

256-

FAMILY COBERMANS

20 30 40 50 60 70 80 100 PERCENTAGE

Figure 11: The percentage and level of activity of whole anti-lymphocyte serum (S14113) and the IgM fraction of S14113 with family cobermans, and a random panel of mongrels, dobermans, and collies. 70

of these antisera.

In an attempt to determine the immunoglobulin class

exhibiting cytotoxic activity, antiserum 71 was fraction­

ated on Whatman DE-52 cellulose using a continuous non­

linear gradient (0.01 M phosphate pH 8.0 to 0.3 M phos­ phate pH 4.5) (Fahey et al., 1958). Individual fractions were measured for pH, conductivity, and optical density

(280 nm) and protein peaks were pooled and concentrated

for testing. By immunoelectrophoresis, IgG was localized to the first five effluent peaks and IgM was found only in the last peak. These fractions were subsequently tested for cytotoxic activity. All five IgG fractions were cytotoxic for doberman, mongrel, and coberman cells while the IgM fraction showed low level cytotoxicity for 18% of dobermans and 29% of cobermans (Figure 11). The IgM fraction was not cytotoxic for lymphocytes from any of the mongrels tested. Collies, however, exhibited cytotoxicity consistently with only one IgG fraction and 5 of 13 dogs

(39%) reacted with the IgM fraction. Collie results were difficult to determine as the titers were low.

Although the particular antigens recognized by this antiserum do not occur with the same frequency in the family, doberman, and mongrel dog populations, there were indica­ tions that the reacting antigens are the same in these three species. The whole antiserum was observed to be cytotoxic for lymphocytes from dobermans, cobermans, and mongrels 71 at the same mean titer of 256. Furthermore, all three lym- .

phocyte populations reacted consistently with the five IgG

fractions. The cytotoxic reaction with collie lymphocytes,

however, appeared markedly lower than that of the other

three dog populations and this activity was localized to

only one IgG fraction and IgM. Apparently the typing serum

contains at least two antibody specificities, only one of which is cytotoxic for collie lymphocytes, whereas the high

titer antibody reactivity is against cells from the other

three breeds.

The recognition of DL-B antigens by serum S14113 can be attributed to the possible DL-A identity of donor and

recipient cobermans. Another antiserum has been reported which also recognizes DL-B antigens, and indeed when donor

and recipient were typed, they appeared to be DL-A iden­

tical (Vriesendorp et al., 1973). In the case of serum

S14113, the donor-recipient pair were unavailable for typing.

It has been suggested that the length of the immunization

schedule could have favored the development of antibodies to minor histocompatibility loci (Vriesendorp et al., 1973).

Westbroek et al. (1972) have shown that short term immun­

ization, in the form of a renal transplant, did not elicit the production of any allolymphocytotoxins in the DL-A identical dogs.

In summary, an antiserum recognizing DL-B factors was produced within a family of coberman dogs. The whole 72

antiserum demonstrated high cytotoxicity titers when tested

against a panel of family, doberman, and mongrel cells, but

low titers when tested against collie cells. The cytotoxic

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