Processing and Presentation of Antigens

295 Pages·1988·5.55 MB·English

by  Benvenuto Pernis (Eds.)

PROCESSING AND PRESENTATION
OF ANTIGENS
Edited by
Benvenuto Pernis
Samuel C. Silverstein
Henry J. Vogel
College of Physicians and Surgeons
Columbia University
New York, New York
Academic Press, Inc.
Harcourt Brace Jovanovich, Publishers
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Library of Congress Cataloging-in-Publication Data
Processing and presentation of antigens.
Includes index.
1. Antigen-antibody reactions. 2. Antigens—Receptors.
3. T.cells. I. Pernis, Benvenuto. II. Silverstein,
Samuel C. III. Vogel, Henry J. (Henry James), Date
[DNLM: 1. Antigen-Presenting Cells—immunology.
2. Antigens—immunology. QW 573 P963]
QR187.A1P76 1988 616.07'92 87-26971
ISBN 0-12-551855-2 (alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA
88 89 90 91 9 8 7 6 5 4 3 2 1
Preface
Antigen processing and presentation have been much discussed in im-
munology since the time when "instructive" theories of antibody formation
postulated that antibodies are molded on the surface of antigens. Since
the bulk of antigen administered to an organism ends up in macrophages
and related cells, it was assumed that macrophages are the cellular sites
of antibody production. This is not the case, and the nature of the early
events taking place after the introduction of antigens has been a matter
of uncertainty and controversy for a long time. Recently, however, intense
interest has been focused, from a new perspective, on the fate of antigens
at the very beginning of the immune response. It is now clear that Τ cells
generally do not respond to intact protein antigens and that antigens must
be processed and presented by antigen-processing cells. The best studied
among such cells are macrophages and dendritic cells, but the concept is
emerging that any cell, e.g., a virus-infected fibroblast, can actually process
protein antigens and present them. Antigen processing appears to consist
essentially of limited intracellular proteolysis followed by recycling of the
relevant peptides, in association with histocompatibility antigens, to the
cell membrane. Macrophages, dendritic cells, and antigen-binding Β lym-
phocytes all have the capacity to present antigens. The presentation of
immunogenic peptides involves an interaction with histocompatibility
molecules, and the structural basis for this interaction is beginning to be
understood.
This volume brings together the views of outstanding immunologists,
biochemists, cell biologists, and virologists who have a common interest
in the early phases of the immune response. Antigen processing and pre-
sentation comprise a series of events that are much more complex and
sophisticated than had been thought for a long time and that constitute
an essential part of the biology of the immune response to T-dependent
protein antigens.
xiii
xiv PREFACE
It is a pleasure to acknowledge the advice and help of Dr. Baruj
Benacerraf, Dr. Charles R. Cantor, Dr. Franco Celada, Dr. Leonard
Chess, Dr. Harold S. Ginsberg, Dr. Hugh O. McDevitt, Dr. David
Sabatini, and Dr. Jack L. Strominger in the development of this volume.
We are grateful for the fine support of the College of Physicians and
Surgeons (P&S) of Columbia University without which this volume would
not have reached fruition. This volume was developed from a P&S
Biomedical Sciences Symposium held at Arden House, on the Harriman
Campus of Columbia University, from May 30 through June 1, 1986.
Benvenuto Pernis
Samuel C. Silverstein
Henry J. Vogel
1
Processing of Native Antigen by Accessory Cells
and Presentation of Membrane-Bound
MHC-Associated Antigen to Specific Τ Cells1
BARUJ BENACERRAF, LOUIS D. FALO, JR., AND
KENNETH L. ROCK
Department of Pathology
Harvard Medical School
Boston, Massachusetts 02115
INTRODUCTION
The understanding of the numerous and complex steps which, starting
with the introduction of a protein antigen, its uptake, processing by ac-
cessory cells, and suitable presentation to clonally specific Τ cells in as-
sociation with class II MHC molecules, results ultimately in specific cel-
lular and humoral responses, has been one of the major achievements of
twentieth century biology.
Immunologists concerned with the phenomenology of immune responses
and with the fate of the antigens soon realized that the clonal selection
theory which postulated the selective activation of specific lymphocytes
by antigen bound to their antibody receptors was unable to explain the
fundamental requirement demonstrated by Landsteiner (1) that a carrier
protein must be immunogenic to elicit antibody responses to the haptenic
determinants it bears. The protein carrier requirement indicated that im-
munogenecity is indeed distinct from specificity and was only understood
when it was established that Τ cells are required for an antibody response
(2,3) and recognize antigen only on the surface of live cells as a complex
with MHC molecules (4,5). These discoveries defined one of the major
problems in immunology: Immunogenecity requires the transformation of
'Based on the Opening Address delivered at the symposium "Processing and Presentation
of Antigens," held at Arden House on the Harriman Campus of Columbia University, May
30 through June 1, 1986.
PROCESSING AND PRESENTATION 3 Copyright © 1988 by Academic Press, Inc.
OF ANTIGENS All rights of reproduction in any form reserved.
4 BARUJ BENACERRAF et al.
a water soluble native protein into a membrane-associated protein by cells
specialized for this process.
As is the case for major discoveries, Nature grudgingly revealed the
secrets that permitted immunologists to assemble appropriately the various
pieces of this puzzle. Moreover, it is only recently that one of the most
important steps in this process, antigen processing and presentation by
accessory cells to Τ lymphocytes, has become sufficiently clarified to be
the topic of this treatise in which immunologists, cell biologists, and phys-
ical chemists will discuss their findings. We are beginning, finally, to ap-
preciate the problems involved in transforming a water soluble hydrophilic
native protein antigen into a membrane-associated protein, or fragment
thereof, capable of reacting specifically with both the membrane-bound
la molecules on antigen-presenting cells and the Τ cell receptor.
I propose first to discuss the major advances and insights that have
brought us where we are today, and then some recent results from our
laboratory concerning the interaction of antigen with class II MHC mol-
ecules and the precise mechanism involved in antigen processing, the single
major issue left unresolved to date.
HISTORICAL
The first insights into the complexity of antigen processing were as fol-
lows: (a) The realization by Unanue and associates (6-7) and us (8) that
the critical requirement for accessory cells, originally macrophages, was
to present T-dependent antigens and to initiate immune responses. Unanue
and Askonsas made the important and unexpected observation that mac-
rophages, after degrading most of the ingested antigen, showed "persis-
tence of immunogenicity associated with a small percentage of antigen
retained by the cells in a form which was protected from rapid breakdown
and elimination." (b) The demonstration by Gell and Benacerraf that the
specificity of cellular immune reactions, which reflects the immunity of
Τ lymphocytes, was directed to denatured unfolded sequential determi-
nants, whereas the epitopes recognized by humoral antibodies were in
most cases determined by the tertiary structure of the intact antigen (9).
This unexpected finding was confirmed in other systems by several lab-
oratories (10,11).
The study of the cells besides macrophages with antigen-presenting
properties, generally referred to as accessory cells, revealed that several
cell types, closely related to macrophages, function as highly effective
antigen-presenting cells; among these are the dendritic cells of the spleen
1. ANTIGEN PROCESSING AND PRESENTATION 5
(12) and the Langerhans cells (13) of the skin. More recently it was realized,
as will be documented later in this volume, that class II MHC-bearing Β
cells are also very effective antigen-presenting cells, particularly for an-
tigens previously bound to their immunoglobulin receptors (14,15).
The seminal contributions of Rosenthal and Shevach (4) and of Yano
et al. (16), alluded to earlier, then established that the in vitro pulsing of
live macrophages with antigen enabled these cells to present antigen to
specific Τ lymphocytes in the MHC-restricted fashion required for their
activation.
ANTIGEN PROCESSING
The efforts of several laboratories became concerned with an analysis
of the steps involved in antigen processing by macrophages to present
membrane-bound antigens to specific Τ cells, and of the changes imparted
to the antigens to make them immunogenic for Τ cells. Ziegler and Unanue
(17) showed that inhibition of macrophage catabolism by ammonia or
chloroquine is associated with impaired antigen presentation to Τ cells.
Similar results were reported by Lee et al. (18) and by Berzofsky and
associates (19). Moreover, it was determined that the essential step in the
processing of antigens, blocked by these inhibitors of lysosomal enzymes,
is indeed the denaturation or unfolding of the antigens. Accordingly, the
processing requirements are bypassed when the antigen is denatured in
vitro previous to pulsing, as shown for ovalbumin (20), Listeria, hen egg
lysozyme (21,22), and sperm whale myoglobin (19,23).
Dissociating effectively the antigen processing from the presenting step,
Grey and associates (20) demonstrated that dead, glutaraldehyde-fixed
cells, which could effectively present previously pulsed and processed
ovalbumin, could not present the native antigen; however, such glutar-
aldehyde-fixed cells were capable of presenting in vitro, denatured and
proteolytically cleaved ovalbumin. An analysis by several groups (19-24)
of the changes effected in the antigen by denaturation which permitted
antigen presentation by fixed cells revealed the unmasking and exposing
of hydrophobic sequences. Moreover, DeLisi and Berzofsky (24) pointed
out the presence of amphipathic structures in several of the immunogenic
peptides they studied which were capable of bypassing processing by live
accessory cells.
The general conclusion from these experiments was that antigen is nor-
mally ingested by accessory cells and metabolized in lysozomes to unfolded
chains or fragments capable, in many cases, of associating by themselves
6 BARUJ BENACERRAF et al.
with the cell membrane to permit presentation to Τ cells, in association
with la molecules. The demonstration by several laboratories that antigen,
with membrane-binding properties, could be effectively presented by iso-
lated la-bearing membranes (25) or liposomes (26,27), and activate Τ cells,
was in agreement with this interpretation.
However, in spite of these considerable advances, the process by which
a denatured antigen fragment is stably associated with the membrane of
the antigen-presenting cell remains largely undefined.
To explain the specificity of Ir gene function and the definitive role
played by la molecules in determinant selection for Τ cells (28), we pro-
posed in 1978 that processed antigens and class II MHC molecules are
capable of some degree of specific association, previous to and independent
of the interaction of these two components with the Τ cell receptor. The
accumulating evidence in favor of this hypothesis will be detailed in another
section. As part of this discussion, however, we want to emphasize that
the affinity of the binding between antigen and la molecules reported re-
cently by Unanue's laboratory (29) is much too weak to account for the
stable association of pulsed processed antigen with the macrophage's
membrane, observed over days in culture. Moreover, it would seem un-
likely that nature would not have evolved one or more specific mechanisms
to ensure the integration of processed antigens and their transport to the
membrane of the antigen-processing cell.
The recent analysis of the membrane anchoring of several immunolog-
ically relevant integral membrane proteins, such as Thy-1 (30) and decay
accelerating factor (DAF) (31), had identified phosphatidylinositol as the
membrane-anchoring domain of these molecules, which share with pro-
cessed antigens the remarkable requirement for considerable mobility on
the plane of the membrane. This is not to imply that there is any evidence
other than analogy that processed antigen might be anchored by phos-
phatidylinositol or a similar covalently bound lipid soluble moiety, but
rather that the search for such a possible binding may prove informative
and has indeed been undertaken in our laboratory (32).
To approach this problem, antigen-pulsed cells were treated with various
enzymes and immediately fixed with paraformaldehyde to prevent antigen
reexpression. These cells were then added to specific Τ cell hybrids to
determine whether the membrane-bound processed antigen was effectively
released. As a control, the integrity of class II MHC molecules as detected
on the same cells by an alloreactive Τ cell following the enzymatic treat-
ment was tested in parallel. It has been already established that treatment
with proteolytic enzymes such as trypsin or pronase (under conditions
that spare la molecules) do not interfere with antigen-presenting properties
of antigen pulsed, fixed cells (21,33). Our own experiments were in com-
1. ANTIGEN PROCESSING AND PRESENTATION 7
plete agreement with these conclusions. However, we observed that, in
contrast to proteases, both phospholipase A, purified from Naja naja ven-
om, and phospholipase C, purified from Clostridium perfringens, were
able to abolish totally the antigen-presenting capacity of antigen-pulsed
fixed cells to antigen-specific Τ cells in several antigen systems, while
leaving unaffected the capacity of these same cells to stimulate alloreactive
hybrid Τ cell clones (32). Since these experiments were conducted with
broadly active phospholipases capable of hydrolyzing many types of
phosphatidyl bonds, they do not identify the critical structure or structures
to which processed antigen may be anchored on the membrane of the
antigen-presenting cell. Nevertheless, this can be considered the first evi-
dence that such specific bonds may exist and in many cases might be the
objective of the antigen processing step. In addition, such a reaction might
provide a general mechanism by which an antigen-presenting cell could
stably associate otherwise water soluble proteins with their plasma mem-
brane. The issue remains, however, whether denatured antigen with hy-
drophobic sequences of amphipathic structure, as referred to earlier, might
not be capable of binding stably to the membrane of the antigen-presenting
cells by themselves, without the need for an intervening lipid. A com-
parative study of the reversibility of binding of such material to fixed
membranes as compared to physiologically processed antigen should
answer these critical questions. In addition, the analysis for covalent-
ly bound lipids of lipase released labeled antigen should also prove very
informative.
SPECIFIC INTERACTION OF ANTIGEN WITH CLASS II
MHC MOLECULES
The specificity of Ir gene phenomena resulting from MHC polymorphism
and the demonstrated role of Ia molecules in determinant selection for Τ
cell responses led us to propose together with Rosenthal that processed
antigen is capable of specific interaction with class II MHC molecules
(28,34). However, the small number of Ia molecules, even in a hetero-
zygous individual, was considered a major objection to this hypothesis.
We felt, notwithstanding, that the increasing realization that the Τ cell
has a single receptor capable of binding both processed antigen and class
II MHC molecules required some degree of intimate interaction between
these two entities. Furthermore, even if the alternative explanation
promoted by Klein and Nagy (35) to explain Ir gene phenomena, i.e.,
Τ cell-repertoire defects caused by tolerance to autologous molecules
8 BARUJ BENACERRAF et al.
in the context of self-la, were a valid mechanism, it also presupposes a
similar specific interaction between antigen and la molecules at the stage
of induction of tolerance.
The first evidence that antigens and la molecules could associate re-
versibly came from functional studies of Τ cell activation involving com-
petition phenomena in antigen-presenting cells. Werdelin was the first to
demonstrate that the response of primed Τ cells in culture to antigen-
pulsed macrophages could be inhibited by closely related nonimmunogenic
molecules in the guinea pig (36). We investigated this phenomenon in the
mouse using Τ cell hybrid clones, antigen-presenting Β cell lines, and in-
itially, copolymer antigens under Ir gene control. We confirmed and ex-
tended Werdelin's observation and were able to demonstrate that non-
immunogenic GT copolymer at 0.1 mg/ml competed effectively with
comparable pulsing concentrations of immunogenic GAT, at the level of
the H-2d antigen-presenting cell, and rendered such a cell unable to activate
a GAT-specific, H-2d restricted Τ cell hybrid. Moreover, this phenomenon
was I-Ad restricted, as it was not observed with H-2 b-presenting cells, and
quantitatively reversible by increasing the concentration of GAT. In ad-
dition, a H-2(dxbF) accessory cell pulsed with GAT and GT could be com-
1
peted for by presentation to H-2 d- but not to H-2 b-restricted lines (37).
This MHC-restricted competition phenomenon at the level of the antigen-
presenting cell was confirmed with other copolymer systems, GLphe,
GLleu, restricted to I-E (38), and more recently with a native antigen,
bovine insulin. In this last system we showed that the presentation of
processed bovine insulin, which for H-2 b Τ cells involves a determinant
contributed by both alpha and beta chains, is inhibited by the nonim-
munogenic alpha chain (39). Similar results involving Ir gene-associated
antigen competition for the la molecule in antigen-presenting cells have
been recently reported by Unanue and associates (40).
Using a different approach to document the interaction of selected pro-
cessed antigens with class II MHC molecules, we reasoned that such an
interaction should cover or allosterically alter unique epitopes on la mol-
ecules normally recognized by selected alloreactive cells. Considering that
H-2b cells, in contrast to H-2d cells, were not suitable to demonstrate GAT/
GT competition, we prepared a series of H-2 b anti-H-2d hybrid clones
specific for I-Ad and explored whether any of these alloreactive clones
could be inhibited if the H-2 d stimulator cell had been pulsed with GAT
or GT. Approximately 5% of such alloreactive cells could be selectively
inhibited if GAT or GT, but not other unrelated antigens, had been used
to pulse the stimulator cells (41). Moreover, when we compared the ef-
fective concentrations of GAT required to demonstrate these inhibitory