I m m u n o l o g y Today, Vol. 10. No.

9, 1989 :;' ~ -

The most critical property of the immune

system is its ability to discriminate self im i ogenic'tym signals i , :2,3 ... and i
from nonself. Failure to respond to non-
se!f can lead to overwhelming infection,
while failure in the ability not to respond
by~arle$ Janeway
to self, or self tolerance, leads to auto-
immunity. A meeting held in Steamboat one nonself molecule from another, self-nonself discrimination. While
Springs* focused on this iscue under the since responses to self are difficult to the nature of the ligand is becoming
title of 'lmmunogenicity'. This brief sum- elicit. This focus on those highly dis- clear, its generation in cells remains
mary focuses on one of the key issues criminant forms of specificity has led to be characterized. Studies of M.
to a very precise picture of inter° Geffer (lmmunologics) and E. Sercarz
considered at this conference - the sig- (UCLA) show that processing of a
actions between the antigen-specific
nals involved in the induction of lympho- receptor and its ligand that generate protein in cells is not random, and
cyte activation. In addition, an hypothesis signal one. By contrast, the analysis that smalt immunogenic peptides
of immune system function that appears of second signals has lagged, prob- may not be recognizable when pre-
to follow from these findings is de- ably for two reasons. First, signals sented as part of an intact protein
scribed. that lack antigen specificity are in- molecule. While there is general
herently more difficult to character- agreement that proteins entering a
The basic premise ize and second, because the accurate cell via endocytic or phagocytic
The premise on which the confer- characterization of signal two re- routes are degraded to peptides that
ence was based is that specific T-cell quires that signal one delivery be are presented by MHC class II mol-
activation is the critical event in self- fully characterized and fixed in an ecules, while peptides degraded
nonself discrimination. All sustained experimental system. within the ER or cytoplasm of a cell
immune responses involve the acti- are presented by MHC class I mol-
vation of specific T lymphocytes. Signal one: detailed analysis of receptor- ecules (M. Bevan, Scripps; J. Yew-
Perhaps the most important mes- ligand interaction dell, NIH), the cell biology of these
sage of the conference was that the Progress towards understanding separate pathways is unclear. Do
initiation of T-cell activation requires the interaction between the TCR and MHC classll moleculesrecyclevia endo-
two signals: one delivered via the its complex ligand of peptide bound somes in APCs (B. Pernis, Columbia),
T-cell receptor (TCR)-major histo- to the cleft of an MHC molecule has or is recycling a minor behavior of
compatibility complex (MHC) inter- been very rapid. The first half of the such molecules (P. Cresswell, Duke)?
action, commonly referred to as sig- symposium was devoted to updating Are MHC class I molecules actually
nal one, and the other derived from information in this area, which is folded arour'd peptides during their
antigen-presenting cells (APCs) and becoming a matter of fine molecular assembly in the endoplasmic reticulum
delivered nonspecifically (signal detail. Six major questions were essentially preventing them from
two). Immunogenicity requires that addressed: the precise nature of the further peptide binding (P. Parham,
these two signals are delivered ligand, its generation in cells, its Stanford)? Where are proteins nor-
appropriately. By analogy, one may recognition by the TCR, the roie mally degraded, by what enzymes,
also speak of antigenicity in the of 'accessory molecules', the and how are they brought to the cell
T-cell system. While two signals are transduction of signals and the surface? These and many other
required for the induction of T-cell .development of the TCR repertoire. questions require detailed analysis. A
activation, it appears that only the It is now clear that the ligand for monoclonal antibody reported by
specific signal, or signal one, is the TCR must closely resemble the D. Murphy (Albany) that appears to
required to trigger an activated T cell models published by Wiley's group detect the complex of a self peptide
to mediate its effector function. last year. R. Germain (NIH) presented presented by an MHC class It mol-
Thus, APCs are distinguished from data showing that mutations in amino ecule may allow the kind of cell
other cells, not so much by their acids pointing into the putative biological studies required to unravel
ability to generate and present the antigen binding cleft of an MHC such complex issues.
antigen-MHC iigand for the TCR, as ~,~oiecui~ affect peptide binding to Fhe recognition of .L~,,~
....
~+~'4-',,"~-
by their ability to generate signal MHC, while mutations of amino MHC complex ligand by the TCR and
two. acids on the top of one of the major its associated structures is of central
Specificity in immune responses helices of the MHC molecule affect importance to understanding im-
has long been the major focus of MHC restriction. An interesting mune recognition. The key here is to
immunological research, as this is property of the ligand can be de- move from response assays to bind-
their most striking characteristic. duced from mutations in the anti- ing assays, and from there to three-
However, this specificity is usually genic peptide sequence (A. Sette, La dimensional structure, as has now
described not in terms of the ability Jolla): many changes in peptide been achieved for antibodies (P. Col-
to discriminate self from nonself - structure allow binding to MHC mol- man, Melbourne). To do so requires
the most important biological conse- ecules, but few ailow T-cell recog- a soluble form of the TCR. M. Davis
quence of specificity - but rather in nition. Thus, as would appear to be (Stanford) has attached the 3' end of
terms of the ability to discriminate necessary to allow a small number of the phosphoinositol (PI)linked cell
MHC molecules to present all anti- surface protein decay accelerating
gens, peptide binding is degenerate, factor to the 5' domains of the c~and
*The UCLASymposiumon Immunogenicitywas 13chains of the TCR. Such molecules
held at SteamboatSprings,Coloradoon 23-29 while T-cell recognition is extremely form stable dimers that can be 283
January1989. precise, a requirement for accurate
(~) 1989, ElsevierSciencePublishersLtd. UK 0167-4919/89/$03,50

cleaved in soluble form from the cell
surface with a PI-specific phospho-
lipase. To date, no specific bi.lding
has been observed. This could reflect
low affinity, a failure to retain con-
formation in the soluble receptor, or
a requirement for other proteins in
MHC binding. The most likely candi-
dates here are CD4 and CD8, which
bind to MHC class II and class I
molecules respectively, and clearly
form a physical component of the
TCR (C. Janeway, Yale; S. Burakoff,
Harvard). These molecules also par-
ticipate in signal transduction, being
associated with a tyrosine kin3se,
p56 ~ck, that can phosphorylate the
chain of the TCR (A. Veillette, NIH
and Montreal). Additional possible
components in ligand-receptor in-
teractions are the proteins that make
up the minor lymphocyte stimulating
(MIs) locus products. C. Janeway
(Yale) provided evidence for the
binding of similar molecules to MHC
class II and to V~ on the TCR, bring-
ing these two molecules together• J.
Kappler (Denver) showed that many
such effects exist, and play a major
,ole in shaping the TCR repertoire.
The nature and mechanism of action
of these molecules is unknown, and
this forms a major goal for future
research. Janeway favors the hy-
pothesis that such molecules func-
tion as intact proteins binding to the
outside of the TCR-MHC complex,
but this is not a concensus view. An
alternative hypothesis is that these
molecules are 'superantigens', a
term needing more accurate mol-
ecular definition. Further work is
needed to clarify this important area.
Once receptor and ligand come
together, how is signal one ~rans-
duced to the cell? it is clear that in
addition to the commonly recog-
nized signals triggered via phos-
phoinositol breakdown, tyrosine
phosphorylation (R. Klausner, NIH),
tyrosine phosphatase activity in
CD45 (J. Ledbetter, Seattle), and
transmembrane calcium channels (R.
Lewis, UC Irvine) play a role in such
signalling. This is clearly going to be
a highly complex area, especially as
each class of T ceil may have a
distinct signal transduction mechan-
ism related to its unique functional
activity.
The TCR repertoire plays a major
role in what an immune system can
and cannot recognize. Despite major
differences in the genes encoding
284 this repertoire in different mouse
strains, it has been difficult to dem-
onstrate effects of the gene pool on
specificity. By contrast, it is clear that
selective events in the thymus have
major effects on the TCR repertoire.
Negative selection can remove auto-
reactive clones of T cells, whether
they recognize class-II-associated
proteins like MIs (Davis, Kappler) or
minor or major H antigens (yon
Boehmer, Basel). It is clear that posi-
tive selection of T cells whose recep-
tors allow recognition of foreign pro-
teins presented by self MHC is a
critical process in T-cell development
as well (H. yon Boehmer, Davis). This
precise recognition of MHC requires
a complete TCR, while deletion by
MIs and related proteins requires
only the TCR 13chain (Davis).
In summary, most proteins are
potentially immunogenic, in most
strains and species, by generating at
least one peptide that can bind to
MHC and be recognized by a TCR in
that individual. Work is ongoing to
define the rules behind this im-
munogenic potential, and to refine
the molecular details of antigen-
MHC-TCR interactions. The com-
pletion of this phdse seems possible
at present; however, ~t is abunua,,~,y
• " ,, ,.4
clear that many issues remain, par-
ticularly in the area of cell biology.
Nevertheless, it seems fair to state
that signal one is now quite well
characterized, and can be substi-
tuted for in many systems with anti-
TCR antibody or with pharmaco-
logical agents such as ionomycin
and phorbol myristate acetate
(PMA). Signal two remains to be
understood.
Signal two: the key to immunogenicity?
One of the main purposes of this
conference was to focus on the role
of cells in the delivery of signal two
to T cells. In this area, it is becoming
clear that resting T cells require not
only signal one, delivered via the
TCR, but also signal two in order to
expand clonaily and differentiate
into effector cells. Indeed, for some
cell types, signal one alone is actually
inhibitory to further clonal expan-
sion. R. Schwartz and D. Mueller
(NIH) have developed such a system
using cloned CD4 T-cell lines that
respond to antigen and APC by pro-
ducing interleukin 2 (IL-2); if the APCs
are chemically modified, the T cells
produce effector lymphokines, such
as gamma-interferon (IFN-~) but not
IL-2. More important, they cannot be
Imm~,'nology Today, Vol. 70, No. 9, 1989
restimulated by fresh antigen and
APC. This behavior is not observed
with IL-4-producing cloned T-cell
lines; however, these latter cells do
not expand clonally after receptor
cross-linking unless IL-1 is present•
Thus, both IL-2 and IL-4 producing
CD4 + T cells require both signal one
and signal two for clonal expansion.
J. Sprent (Scripps, La Jolla) has
shown similar requirements for both
signal one and signal two in CD8 ÷
T-cell activation.
Several models for signal two re-
quirements have been reported. For
instance, D. Lo (Penn) showed that
transgenic mice whose pancreatic 13
cells express MHC class II molecules
present antigen to cloned IL-2 pro-
ducing T cells in a form that induces
inhibition of IL-2 production. T cells
from mice with such transgenes are
tolerant to the MHC class II allele
expressed on the 13 cell, and this
appears to be due to peripheral T-
cell inactivation rather than clonal
deletion (L. Burkley and R. Flavell,
pers. commun.). Thus, the require-
ment for signal two appears to be
important in vivo, and the absence
of signal two appears to be a
,,.,+i,,
mechanism by which T cells can be
inactivated to tissue antigens in the
periphery; this has been a problem
in self-nonself discrimination for a
long time. Another interesting set of
findings in this area is that different
APCs appear to play a role in priming
different CD4 ÷ T-cell subsets. Immu-
nization with soluble antigens
primes he!per T cells but not IL-2-
producing T cells in B-cell deprived
mice, and A. Abbas (Harvard) has
shown that antigen-specific B cells
are required to present antigen
directly to IL-2-producing CD4 T cells
for in-vivo priming. By contrast,
helperTcells cannot be primed in mice
(D. M,,:his, Strassbourg; R. Flavell,
Yaie) or chickens (P. Matzinger, Basel)
if antigen is not presented by macro-
phages. This finding is in keeping
with the requirement for IL-1 in the
clonal expansion of IL-4-producing
CD4 T cells which seem to mediate
most B-cell activation. Why B cells
are required for presentation of sol-
uble antigens, why macrophages are
required for priming of helper T cells,
why dendritic cells are the dominant
stimulator of mixed lymphocyte re-
sponse (R. Steinman, Rockefeller),
and what dendritic cells contribute
to responses to foreign antigens all
need to be better understood.
 Immunology Today, Vol. 10, No. 9, 1989
.................................................................................................................................................................................... C ~ ?) G, :~* ~ ~ ~,~'~~ < ............

Signal thr~e: T-cell induction of si§nals three could also be important in the initial responding cell produces a sig-
one and two development of autoimmune states: nal that in turn activates an effector
It is becoming clear that certain a local release of IFN-'y could induce cell. These defence mechanisms lack
cytokines can act on cells to render increased MHC levels and co- discrimination between ligands, but
them immunogenic. Much work has stimulatory activity on tissue cells, may have evolved under heavy selec-
focused on the induction of MHC leading to immunization of tissue- tive pressure to recognize constitu-
class II molecules on cells. IFN--y is specific T cells. The issue of co- ents unique to microorganisms that
known to induce MHC class II ex- stimulatory factor expression on tis- are not found in the primitive host.
pression on cells llormally lacking sue cells will probably be a critical However, as the host became more
these molecules. However, exper- one for understanding self-nonself complex, so too did the microorgan-
iments of K. Onoue and colleagues discrimination and the generation of isms besetting it. This necessitated a
(Kumamoto, Japan) have also shown tolerance and autoimmunity. more flexible and mere accurate
that IFN-~/induces co-stimulatory ac- means of self-nonself discrimi-
tivity in cells independent of MHC Signal O: what normally inducessecond nation, and the rearranging receptor
induction, and N. Sarvetnik (Genen- signal expression? systems evolved. However, the dif-
tech) has shown that mice express- These studie~ lead one to ask a ferent classes of lymphocytes bear-
ing IFN-x under the control of the different question: if the production ing these receptors appear to have
insulin promoter develop auto- of signal three requires prior ex- retained the response to cell-derived
immune diabetes, whereas mice that pression of signals one and two, factors as a requirement for acti-
express class II under the same pro- what is the normal pathway by vation. It is easier to believe that
moter do not. These experiments which signal two expression is in- non-specific responses evolved before
appear to dissociate two effects of duced? There appear to be two antigen-specific ones, and that the
IFN-~/, the induction of co- possible answers: specialized APCs requirement for second signals
stimulatory activity and the induction constitutively express signal two ac- reflects the retention of a tested and
of ligand. Of the two, the former tivity; this may be the case for den- highly successful response mechan-
activity appears to be more critical dritic cells, referred to by Steinman ism rather than a system that
for a response. E. Unanue (St Louis) as 'nature's adjuvant'. However, it evolved after the evolution of
and colleagues have shown that does not appear to be the case for the exquisitely specific self-nonself
tumor necrosis factor (TNF) present either macrophages or B cells, which discriminator represented by the
in T-cell supernatants will induce IL-1 normally fail to elicit primary mixed rearranging antigen receptor gene
production, and that a T-cell- lymphocyte responses, yet which can families. Viewed this way, we may
associated activity different from TNF be shown to be important for immu- eventually renumber signals, so that
may have a similar effect. Mueller nization of proliferating and helper APCs deliver signal one and the TCR
(NIH) showed that T cells that are CD4 T cells in responses to protein signal two.
activated with ionomycin and PMA, antigens. My personal belief is that
or certain cloned T-cell lines, also the normal inducers of signal two Is ~ the first specificrecognition system?
r:_ __11..
express co-stimuiatory activity. are microbial in origin, and . . . . . . . . rlfldlly, i[ifUi rridi.ion t.JI~:~ClII.EU"I clI.4+
Most of these activities derive main function of bacterial adjuvants this meeting may point to the ~/~
from activated T cells and require would be the effective generation of T-ceil receptors as the most primitive
signal one and signal two for their signal two activity in APCs. This of the recognitive systems, First, the
production; here such activities are approach leads to a different view of 3to receptors found in skin (J. Allison,
termed signal three. This designation self-nonself discrimination and of Berkeley) and gut epithelium (A.
makes it clear that signal three can- the evolution of the immune system Hayday, Yale) in the mouse have very
not normally function in the in- that I will briefly outline below. restricted variability. Assuming such
itiation of an immune response. Mv the~is is that setf-nonself dis- cells play a role in host defence, they
However, signal three activity is crimination arose long before the appear unlikely to make fine dis-
probably critical in sustaining im- development of specific recognition, criminations between different
mune effector responses as antigen and that even today, many immune organisms. Second, W. Born and R.
concentrations drop. The effect of defence mechanisms are based on O'Brien (Denver) presented evidence
IL-4 on B-cell MHC class II expression, such r,on-antigen specific responses. that ~/~ T cells derived from young
fol instance, may be critical in main- There are many examples of non- thymocytes recognize heat shock
taining T-cell-B-cell collaboration. A antigen specific effector mechanisms proteins of bacterial origin; such
possible example of this corrles from that are closely related to effector proteins are known to be highly con-
MHC class II transgenic mice that can responses that show antigen served in evolution, and are also
make specific antibody although the specificity, such as the alternative expressed by cells in conditions of
relevant MHC class II is expressed on pathway of complement activation, stress. Thus, this recognition system
macrophages and not B cells. Such phagocytosis, T-independent macro- could allow a relatively invariant
mice are found to express MHC class pr:age activation by lipopolysacchar- receptor to recognize a change in
II on B cells after immunization with ide, natural killer (NK) cells, and IFN- cellular physiology induced by infec-
antigen. In this case, it appears that e~ and IFN-[} production. A second tion. Such a system might function
T-cell priming occurs on macro- phase of responses has also been wel! in epithelia, where cells are
phages, leading to IL-4 production defined, such as the activation of NK rapidly regenerating, but would
and the expression of MHC class II on cells by interferons or the production perhaps be too likely to kill critical
B cells, which then allows T-cell-B- of acute phase proteins by the liver cells within the body. Thus, the
in response to IL-6, in which the subsequent development of c~13 T 285
cell collaboration to proceed. Signal

................ !i •
cells to monitor intracellular infec-
tion and of B cells to monitor the
extracellular spaces may have been
the most recent evolutionary ad-
vance in the immune system.
Summary
The most interesting conclusion
drawn from this conference is the
critical nature of second signals in
the functioning of the immune sys-
tem. Indeed, I would argue that
second signals really come first, at
least in evolution and perhaps also in
the response. If the construction out-
lined above is correct, this meeting
also could initiate an exciting phase
of integration in immunology, in
which the usually separate dis-
ciFlines of innate immunity and
specific T-cell induction are brought
together into aq integrated response
system, giving "lew insights into pro-
tective immunity, adjuvants, auto-
The fourth international conference* on
monoclonal antibody immunoconjugates
for cancer heard of great progress in the
area of rad;oimmunoconjugates, and of
continued research into the potential
of immunotoxins, chemoimmunoconju- from an audience that included
gates and anti-immunoglobulin therapy m a n z _ ° f :_ tbe~_ authori_tie_s _fr°m
=c n ~ f ~ n f ; ~ l r ~ n t - ~ r f h ~ r ~ r ~ i ~ e
gd l,,l~ll.~..I , I . l U l l~.Ul l~b.qi.I
The format of this conference dif-
fered somewhat from its predeces-
sors since there were no panel dis-
cussions and each subject area
began with an overview, followed by
a small series of selected presen-
tations. The overviews were gener-
ally excellent. Two additional fea-
tures were workshops which were
led by R.A. Reisfeld (Scripps, La Jolla)
and S.E. Halpern (VA Medical Cen-
ter, San Diego), and an elaborate
buffet of posters. The organizers, I.
Royston (Univ. California, San Diego)
and R.O. Dillman (Scripps Clinic,La
Jolla) are to be congratulated for
providing an excellent and compre-
hens. ~. selection of topics, pre-
senters and content in the brief
period of two and a half days, which
also gave stimulating discussion
*TheFourthInternationalConferenceof M0noclonal
AntibodyImmunoconjugatesfor Cancerwasheldin
286 SanDiego,Californiafrom30March- I April1989.
LI I 1 ~ , 1 C . i ~ l l ~ . 2 .
immunity and the evolution of the
immune system. The molecular
analysis of second signals, and of the
response systems that induce their
expression on APCs, should be a
most fruitful avenue for new
research, both in basic science and in
the control of the immune response.
This progress is made possible by the
maturation of our understanding of
signal one in T cells: the generation
of ligands, the recognition of
ligands, and the transduction of
signals across the membrane. Finally,
the analysis of innate and specific
immunity also rationalizes effector
mechanisms, since both innate and
specific responses appear to use the
same nonspecific effector mechan-
isms. Apart from the impossibility of
recreating evolution, the real missing
links in our understanding of signal
two induction may be found by care-
ful analysis of existing d~ta about
Targeted cancertreatment
dCdU~lllld, IrlOU:~l.ry dnO governmenT.
The most impressive aspect of the
conference was the evidence that
steady progress was being made in
the field of radioimmunoconjugates.
It was shown that simple and accu-
rate methods of detecting small can-
cer sites with Tc-99m-labeled and
In-Ill-labeled antibodies are now
available. It was also apparent that
emphasis on radioimmuno,:onju-
gates is progressing to radioimmuno-
therapy (RAIT). where the focus of
this conference was on clinical
results. In contrast, for immuno-
toxins and chemoimmunoconjugates
the emphasis is still on the design of
new agents, solving pharmacokinetic
and low targeting problems, animal
model results, and host responses to
both the antibody and the toxic
chemical.
G.L. DeNardo (Univ. California,
Davis) gave an overview of radio-
immunoconjugates, concentrating
on the current status of RAIT. Many
different antibodies have entered
clinical trials: 1311is the most preva-
1989, ElsevierScience PublishersLtd, UK. 0167-4919/89/503.50
Immunology Today, VoL 10, No. 9, 1989
innate immunity systems. A critical
examination of primitive immune
systems should perhaps focus more
on pathogen recognition and less on
tissue grafting and antigen speci-
fioty, if it is to lead us closer to an
understanding of how our system
recognizes and responds to antigen,
and how it discriminates self from
nonself.
The author wishes to thank the UCLA
Symposia and ICl Pharmaceuticals for
sponsoring the symposium, Jon Sprent
and Eli Sercarz, his co-organizers, and
Kim Bottomly for pointing out the im-
portance of signal 0 in understanding
immune responses.
CharlesJanewayis at the Sectionof Immuno-
biology, Howard Hughes Medical Institute,
Yale University School of Medicine, New
Haven, CT06510, USA.
fromDavidM. Goldenberg
lent radionuclide, and some early
experienceo~vith 13 emitters, such as
-'"Y ano "~uKe, was a l s o repor~eo.
DeNardo focused on the good
responses of lymphomas to RAIl,
beginning with the work of S.E.
Order (Johns Hopkins Univ., Baltimore)
using polyclonal anti-ferritin anti-
bodies labeled with 1311and, more
recently, 9oy in Hodgkin's disease.
The DeNardos showed the exciting
potential of RAIT when a radio-
sensitive tumor is chosen. Their
therapy trial of 20 patients with lym-
phoma, using an 1311-labeled B-cell
monoclonal at~tibody (mAb), re-
sulted in a 10% complete remission
rate, an 85% partial remission rate
and progression of disease in the
remaining 5%; only a 15% human
anti-mouse antibody (HAMA) re-
sponse rate was reported. These very
promising findings indicate that even
with the current limitations of low
mAb uptake in tumors, the radiation
doses achieved are adequate for in-
ducing responses in drug-refractive,
radiosensitive neoplasms. I his work
needs to be extended and con-
firmed, especially in terms of the