Biochhnica et Biophysica A cta, 415 (1975) 253-271 .~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 85149

IMMUNOGLOBULINS LYMPHOID

AND

ALLOANTIGENS

ON THE SURFACE

OF

CELLS

ELLEN S. VITETTA and J O N A T H A N W. UHR Department of Microbiology, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, Texas 75235 (U.S.A.) (Received February 10th, 1975)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

253

il.

Immunoglobulins on B lymphocytes . . . . . . . . . . . . . . . . . . . . . .

255

Ill.

Binding of immunoglobulins to plasma membrane . . . . . . . . . . . . . . . . .

258

IV.

Sites of synthesis and intracellular transport . . . . . . . . . . . . . . . . . . . .

260

V.

Turnover of immunoglobulins on the plasma membrane . . . . . . . . . . . . . .

262

VI.

Biochemistry of alloantigens . . . . . . . . . . . . . . . . . . . . A. H-2 alloantigen . . . . . . . . . . . . . . . . . . . . . . . . B. TL alloantigen . . . . . . . . . . . . . . . . . . . . . . . . C. GIX alloantigen . . . . . . . . . . . . . . . . . . . . . . . D. la alloantigen . . . . . . . . . . . . . . . . . . . . . . . . . E. Thy-I alloantigen . . . . . . . . . . . . . . . . . . . . . . .

264 264 264 265 266 266

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VII. Site of synthesis and turnover of H-2 antigen on the plasma membrane . . . . . . . .

267

VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269

1. INTRODUCTION L y m p h o c y t e s are the c e l l u l a r r e p o s i t o r i e s for i m m u n e responsiveness.

Current

c o n c e p t s based o n c o n s i d e r a b l e e v i d e n c e i n d i c a t e t h a t there are a large n u m b e r ( p r o b a b l y e x c e e d i n g I0000) o f distinct c l o n e s o f l y m p h o c y t e s e a c h o f w h i c h is c o m m i t t e d to s y n t h e s i z e a specific i m m u n o g l o b u l i n m o l e c u l e [1-5]. D u r i n g its p r i o r d i f f e r e n t i a t i o n , e a c h cell in the c l o n e has s y n t h e s i z e d an i m m u n o g l o b u l i n o f a p a r t i c u l a r specificity a n d has t r a n s p o r t e d it to the cell surface.

In general, these cells are

m e t a b o l i c a l l y q u i e s c e n t , h a v i n g little c y t o p l a s m a n d a p a u c i t y o f o r g a n e l l e s f o r m a n u f a c t u r i n g p r o t e i n s [6]. T h e p r o g e n y o f a p a r t i c u l a r c l o n e are t h e r e f o r e r e a d y to i n t e r a c t w i t h a n t i g e n s w h i c h are c o m p l i m e n t a r y t o t h e r e c e p t o r o n t h e i r surface.

254 Under appropriate conditions of immunization, the interaction between lymphocyte receptors and antigen leads to proliferation and differentiation into antibody-secreting plasma cells [7-9]. In contrast to lymphocytes, plasma cells have the machinery for highly efficient synthesis and secretion of immunoglobulios i.e. they possess large numbers of polyribosomes, a well developed endoplasmic reticulum and a prominent Golgi complex [10]. They secrete immunoglobulin molecules of the same specificity as the receptor immunoglobulin on their ancestral clone of lymphocytes.. Another pathway of lymphocyte differentiation following interaction with antigen is the development of an increased number of precursor cells [9,11,12]. These cells are responsible for immunologic memory i.e. a more effective response when an animal encounters antigen for the second time. Tile role of antigen, therefore, is to interact with clones whose receptors bind to the immunogen witll sufficient energy so that they are selected for preferential stimulation and replication. The result of these two events, replication and differentiation, is an enormously amplified response to the initial immunogenic signal. In the last decade, it has become apparent that there are two major populations of lymphocytes and that stimulation of both types of cells is needed for antibody formation to the majority of antigens [13-15]. Thus, there are bone-marrow derived ceils which differentiate into immunocompetent cells (B cells) in the Bursa of Fabricius in the chicken and in the mammal in unknown anatomical sites. These cells, which are precursors of plasma cells, are characterized by a high concentration of immunoglobulin molecules on their surface as discussed above. In addition, there is another major population of cells which differentiate in the mammalian thymus into immunocompetent lymphocytes (T cells). Although these cells also appear to exhibit specificity for antigen, the nature of the antigen specific receptor on their surface is as yet unknown. For stimulation of B lymphocytes to the majority of antigens, it is essential that T cells be stimulated simultaneously by the same antigen [16-21]. With hapten-protein conjugates, receptors on T lymphocytes appear to recognize the protein (carrier) portion of the conjugate whereas those on B lymphocytes recognize the haptenic portion [22-27]. Thus. the B lymphocyte may have a receptor for a T cell signal (pharmacologic) as well as its antigen-specific receptor and both receptors may have to be occupied at the same time for stimulation of the B cell to occur. Another significant feature of T-B cell collaboration is that surface alloantigens which are under the control of genes in the major histocompatibility locus, appear to play a major role in cell cooperation. For example, there is evidence that T and B cells must display the same H-2K and/or la antigens on their cell surface in order for cooperation to occur in certain defined experimental systems [29-30]. Finally, there is accumulating evidence that macrophages are essential for induction of an immune response. They probably are concerned with presenting antigen to T and B cells [31]. Macrophage-T cell cooperation may also require identity of H-2 and/or la surface antigens [32]. Much of the behavior therefore of these subpopulations of lymphocytes depends upon interactions that take place on the cell surface via membrane-bound receptors.

255 To understand induction of an immune response at the molecular level, the relevant macromolecules must first be characterized biochemically. These include the antigen specific receptors on T and B cells, the molecules involved in cell cooperation (e.g. Ia, H-2 antigens) and probably other plasma membrane molecules concerned with transducing the surface stimulus into a cytoplasmic signal for stimulation (perhaps adenyl cyclase, ionic pumps, etc.). Later steps would include purification of such molecules, reconstitution of artificial membranes with them, elucidation of their interactions within the membrane and testing of the proposed transducing mechanism in immunocompetent cells. The studies to be discussed in this review represent a beginning in the biochemical characterization of surface molecules including immunoglobulin and a number of alloantigens on lymphocytes. Prior to our studies, a good deal of information was available on surface H-2 antigens primarily from Nathenson and his co-workers who obtained the antigens from cells by treatment with papain and purified them by conventional chromatographic techniques [33-34]. However, surface immunoglobulin and all other alloantigens had been identified almost exclusively by nonbiochemical techniques i.e. immunofluorescence, cytotoxic assays, etc. Our own studies began in 1971 with the first description of the biochemistry of a surface immunoglobulin molecule. Extending the observation of Phillips and Morrison [35] that the surface proteins of intact erythrocytes could be enzymatically radioiodinated, we similarly labeled surface proteins on intact lymphocytes, lysed the ceils in nonionic detergent and "isolated" the radiolabeled molecule in question by precipitating it with specific antibody [36]. We than were able to perform conventional biochemical techniques on the labeled molecule.

II. IMMUNOGLOBULINS ON B LYMPHOCYTES One of the major problems in studying immunoglobulin receptors on B lymphocytes is that these cells also have a receptor for the Fc portion of IgG and probably other classes of immunoglobulin [20,37-39]. Hence, these B lymphocytes can acquire exogenous homologous immunoglobulin from the serum or in vitro from treatment with heterologous antibodies directed against unrelated antigens. Therefore, if lgG is detected on the cell surface, it is necessary to prove that it was synthesized by the cells on which it resides. This can be accomplished by exposing cells to proteolytic enzymes or anti-immunoglobulin in order to denude the surface of existing immunoglubulin molecules, followed by in vitro incubation in order to allow resynthesis to occur. Of course, this method will only be effective in the absence of lgG-secreting plasma cells, which could theoretically secrete sufficient new immunoglobulin to saturate Fc receptors on surrounding lymphocytes. In addition, the phenomenon of allelic exiusion in immunoglobulin synthesis can be used to show that in a heterozygote lor an immunoglobulin-genetic marker, a single cell shows surface immunoglobulin of one parental type only, Thus, allelic exclusion can be

256 employed to ascertain whether the surface immunoglobulin is adsorbed or synthesized by the cell in question. Our studies of normal young mice indicate that lgM is lhe predominant immunoglobulin on splenocytes of such animals [40]. Occasionally trace amounts of cell surface lgG were detected on these cells. Presumably, the extensive washing and/or iodination procedure had removed cytophillic immunoglobulin. The identity of the lgM was established by immunoprecipitation with antiserum monospeciIic to /~ chain and by mobility on acrylamide gel electrophoresis of the ,u and L chains from the reduced and alkylated immunoprecipitate. Acrylamide gel electrophoresis of the unreduced IgM indicated that it was predominantly in its monomeric form (p2L2) and had a molecular weight of approximately 190000. Such findings were confirmed by Marchalonis and his colleagues, using similar techniques [41]. The finding that lgM is in its monomeric form on the cell surface was unexpected, since serum IgM in mammals is usually pentameric (bt~oLto; S constant is 19-S) [42]. To insure that a breakdown of 19-S immunoglobulin had not occurred during the iodination and extraction procedure, iodination experiments were carried out with pentameric lgM. These experiments revealed no depolymerization of pentameric IgM by the methods used. Recent findings indicate that there is another major cell surface immunoglobulin on murine lymphocytes which had not been previously detected [43-44]. The new class of immunoglobulin has properties similar to that described for human IgD. We have therefore tentatively called it "lgD-like". For simplicity, we will refer to it as IgD in this review, but will keep in mind the reservation that definitive classification awaits demonstration of antigenic cross reactivity or structural homology with human lgD. Mouse lgD had escaped detection because it is not present on the splenocytes of young animals (less than 2 weeks of age); it is very rapidly degraded presumably by ploteolytic enzymes released during lysis of cells, and on 5 ~ acrylamide gel electrophoresis in sodium dodecyl sulfate, it migrates with lgM both under reducing and non-reducing conditions. The co-migration is due to the facts that the 6 polypeptide chain is smaller than/~ chain (probably the size of a y chain) but 6 has a higher carbohydrate content than ,u chain. Therefore the reduced mobility from the "extra" carbohydrate counteracts the increased mobility of the small 6 polypeptide chain. At higher concentrations of acrylamide, the contribution of the carbohydrate portion of the molecule to mobility is decreased and the chains are readily separated (Fig. !). Further studies indicate that lgD is probably acquired late in the differentiation of virgin B lymphocytes i.e. lymphocytes which are immunocompetent but have not as yet encountered antigen. The evidence is that: (1) IgM is detectable on spleen cells of newborn mice whereas lgD is first detected at about 2 weeks of age [44] (Fig. 2); (2) bone marrow cells which contain stem cells for the B cell series have cell surface lgM but not cell surface IgD (44); (3) in lethally irradiated, bone-marrow reconstituted mice, surface lgM appears prior to surface lgD [45]; (4) lgD appears on spleen ceils of germ free mice and conventionally raised mice at the same time [44].

257

!0 • Anti-/~

0Anti-I(] 8 o

6

x

E

~4

1

10

i

20

1

30

4~) 50 Fractic~s

60

70

Fig. I. Cell surface lgM and IgD on splenocytes of 3 week old mice. Separate aliquots of the lysates from radioiodinated cells were immunoprecipitated with either anti-# or anti-immunoglobulin. The precipitates were dissolved, reduced, and electrophoresed on dodecyl sulfate-polyacrylamide gels [44].

I

12 ,~

4 doys

FJ

'or

v

8 -

;>5 doys !

~Anti-p. -Anti-Iq

t

1

i i

t

L

42 doys

i

__

,=

I

_ _

85 doys

:i

't

1o

20

30

40

50

60

70 t0 Fractions

20

30

40

50

60

70

Fig. 2. Age related appearance of IgD on the surface of BALB/c splencMcytes. (see Fig. 1) [44].

258 Tile acquisition of lgD appears to be thymus independent, since lgD appears in conventional amounts in athymic (nu/nu) mice [44]. Studies in the human of normal and neoplastic lymphocytes suggest that lgM and IgD can be synthesized by and be on the surface of the same cell. Thus, Rowe et al. [46] have demonstrated that lgM and IgD can be independently capped on individual cord blood lymphocytes. After "stripping" the cells with trypsin, lgD reappears. Fu et al. [47] have shown that ill a human chronic lymphocytic leukemia with macroglobulinemia, idiotypic antibody to the serum macroglobulin, interacts with IgD and IgM on the same cell. This finding suggests that igM and lgD on the same cell in humans, probably have the same variable ( V~ sequences.* Similar conclusions have also been reached by Pernis et al. [48]. The question arises as to whether the acquisition of lgD represents a switch in synthesis from cell surface lgM or whether both classes of immunoglobulin continue to be synthesized by the same cell. There is not a definitive answer to this question, but the data in the human [47,48] suggests a large population of cells bearing both classes of immunoglobulin. This observation suggests that both immunoglobulins may be synthesized by the same cell, at the same time. If this preliminary conclusion is verified, additional mechanisms at the genetic or RNA level will have to be invoked to explain simultaneous synthesis of the products of I V g e n e a n d 2 Cgenes.

111. BINDING OF IMMUNOGLOBULIN TO PLASMA MEMBRANE The biochemistry of the immunoglobulin molecule in the plasma membrane is basic to the problem of trtggering of lymphocytes by antigen. Thus, binding of receptor immunoglobulin by ligand is necessary but insufficient for stimulation of B lymphocytes by the majority of antigens [49]. As mentioned previously, a second signal imparted by activated T cells is obligatory for stimulation by most antigens. This signal could be received by the immunoglobulin receptor or another membrane molecule such as la (to be discussed later). A simple possibility is that both antigen and the T cell signal interact with surface immunoglobulin and change its conformation in the membrane. The complex then could interact with other membrane proteins resulting in transduction of the external signals into cytoplasmic ones. As will become evident in the subsequent discussion, studies of cell surface immunoglobulin have not as yet answered many of the fundamental biochemical questions ~oncerning this molecule. One of the first questions of interest is what part of the immunoglobulin mole" Both L and H chains have an N-terminal sequence of amino acids (approximately 110 amino acids) which vary among different immunoglobulins of the same class ( V regions). These V regions are responsible for the antigen binding specificity of the immunoglobulin. The remainder of the L and H chains are relatively invariant within a class of H chain (or type of L chain). These constant regions of the H chain (C regions) determine the class of immunoglobulin, i.e. lgM, IgD, lgG, etc. There are two types of L chains, 2 and =..:,and both are present in each class of immunoglobulin.

259 cule is bound to the plasma membrane of lymphocytes? A priori, it would be predicted that the Fab portions of the molecule (which contain the antigen binding sites) would be exposed to the exterior and that the Fc portion of the molecule would be bound to the plasma membrane. Earlier studies of the antigenicity of cell surface immunoglobulin are consistent with this idea [50-51] in that certain antibodies to the Fc portion were not able to stain immunoglobulin on cells. Additional support for this concept was obtained by comparing the capacity of rabbit antibody to L chain and # chain to bind to radioiodinated cell surface immunoglobulin on splenocytes from young mice [52,53]. The binding step was measured by lysing the washed ceils after treatment and by treating the lysate with antibody to rabbit immunoglobulin to precipitate the cell surface mouse immunoglobulin that had been bound to the rabbit anti-mouse immunoglobulin. The results show that antibody to L chain (anti-~ in the mouse) was able to bind approximately 90 ~ of radioiodinated cell surface immunoglobulin on murine splenocytes whereas antibody directed to/~ chain could bind approximately 50 ~ of such molecules. Either a portion of/z chain is buried in the plasma membrane or its binding to the membrane made the determinants on the/~ chain inaccessible to the anti-/~ molecule. In either case, the evidence indicates that immunoglobulin is probably attached by its Fc fragment to the cell surface. More elegant evidence favoring this conclusion was recently reported by Fu and Kunkel [54]. They demonstrated that certain antisera to human lgM were incapable of binding to human leukemia cells with surface IgM. Their data indicates that these particular antisera are directed towards antigenic determinants in the last 50 amino acids of the carboxylterminal end of the/~ chain suggesting that this portion is either buried in or hidden by the plasma membrane. What is the nature of the chemical bond between the Fc fragment and the plasma membrane? There are two major possibilities that we are considering: (Fig. 3) (I) hydrophobic binding to the hydrocarbon portions of the lipid bilayer (2) interaction with an integral membrane protein. We exclude iomc bonding because cell surface lgM is not readily removed by treatment with 3M KCI [55]. There is considerable information about the structure of secreted IgM and it has a bearing

A

/3

Fig. 3. Models for the attachment of immunoglobulin to the plasma membrane. In (A) hydrophobic portions of the Fc interact directly with the lipid bilayer. In (B) immunoglobulin interacts with an integral protein in the membrane.

260 on the possibility of hydrophobic binding. Thus, lgM is a relatively soluble molecule, with 5 distinct carbohydrate moieties attached to each/~ chain (see ref. 42 for review). These moieties restrict the portions of the ,tt chain that could be buried in the plasma membrane. The complete amino acid sequence of a secreted lgM is known [56,57] and there is no sequence of hydrophobic amino acids analogous to that found in glycophorin [58]. It would be necessary therefore to postulate a hydrophobic loop lbrmed from several non-sequential portions of the molecule. For example, this could be accomplished in the fifth domain (the carboxyl terminal end) of the molecule in which there is only I carbohydrate moiety [56,57]. It would not be necessary to postulate that the loop penetrates completely through the bilayer. In fact, it is more likely from the considerations mentioned above and the shedding phenomena (to be discussed) that the molecule may only be imbedded part way. It is also possible that surface immunoglobulin is bound to a membrane protein and is not imbedded in the bilayer. Thus, the molecule could be bound to the Fc receptor [59} or a similar type of protein which does have a hydrophobic sequence and thereby extends deeply into the bilayer. The binding would presumably be noncovalent since the bond appears exquisitely susceptible to detergent. At the present time. there is no information which excludes either possibility. Regardless of the type of chemical bond between surface immunoglobulin and plasma membrane, the question arises as to how some immunoglobulin molecules are secreted whereas others of the same isotype are bound to the cell surface. The simplest hypothesis is that there is a structural difference between surface and secreted H chain of the isotype and that this difference is ~he signal for binding of immunoglobulin to plasma membrane. For example, surface lgM could have an extra peptide at the carboxy-terminal end of the t~ chain which anchors the molecule to ~he plasma membrane. Another possibility is that there are differences in the carbohydrate moieties between cell surface and secreted immunoglobulin. Either underor over-glycosylation (e.g. extra sialic acids residues) could lead to conformational changes favoring binding to membrane as discussed above. Reported electrophoretic differences between secreted and cell surface/~ chain [60] (which preceded the discovery of murine b chain) do not appear to be completely accounted for by 6 chain and, therefore, may provide a clue to the nature of this proposed structural difference.

IV. SITES OI- SYNTHESIS AND INTRACELLULAR TRANSPORT Extensive studies of murine myeloma cells has led to considerable insight into the assembly and transport of the immunoglobulin molecule destined for secretion. Thus, it has been shown (Fig. 4j that I_ zmd H chains are synthesized on polyribosomes of different classes, that L chains are released into an intracellular pool of excess L chains in the cisternae of the endoplasmic reticulum [61-63], that assembly of the L-H dimer occurs on the H chain polyribosome [61], that the bridge sugar (usually glucosamine in its n-acetyl form) in incorporated at the level of polyribosomes

261 Fc')t Y~IR~,SOMF

_ CHAIN" PL)LYRIBOSO~ [

II

I \ r 555 t