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DiscussionGenetic regulation of the immune response.

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DISCUSSION: Genetic Regulation of the Immune Response
Uhr: Although there is considerable information available concerning the immunological abnormalities of
SLE in the human and in the SLE-like syndromes of
mice, it remains unclear as to which subset of lymphoreticular cells are responsible for initiating the
immunologic dysfunctions. The most likely candidate
according to current concepts would be suppressor T
cells. Thus, it could be postulated that T-cell suppression that is of major importance in controlling B cell
function is inadequate, thereby allowing autoantibody formation to a wide variety of antigens. Other
major possibilities for a primary cellular defect include: 1) B cells resistant to either the induction of or
maintenance of immunologic tolerance; and 2) macrophages that do not metabolize macromolecules
such as nucleic acids normally or are unable to present antigen in an immunogenic fashion to particular
subsets of T cells which are needed for immunoregulation. This latter possibility is underscored by
the rapidly accumulating evidence that Ir genes can be
expressed at the level of the macrophage. Therefore,
one major problem for future research is to determine
which subsets of lymphoreticular cells are responsible
for initiating autoantibody formation in SLE. In this
regard it should be emphasized that there could be
more than one pathogenetic mechanism in SLE leading to a common effector pathway. Thus, it is possible
that there are different forms of SLE representing
abnormalities of different subsets of cells.
The relationship of virus infection to SLE also
remains unclear. There are several different ways that
viruses could cause the observed immunological abnormalities.
The possible immunologic and etiologic complexity of SLE has implications for genetic considerations. There may well be a number of genetic factors that can affect the development of SLE. Hence,
different patients with the disease might show different genetic predispositions. For example, some patients with SLE might have an HLA linked gene that
plays an important role in susceptibility, whereas others might not. For these reasons, it is desirable not to
restrict our interests in the genetic factors because of
overly simplistic hypotheses.
We should move now to consider factors inArthritis and Rheumatism, Vol. 21. No. 5 Supplement (June 1978)
volved in the regulation of the immune response. Dr.
Klein, why don’t we begin with you.
Klein: There are a large number of MHC genes because
a large number of genes are needed to carry out the
MHC function. If the function of the MHC is, indeed,
to assist in antigen recognition, then perhaps a large
number of genes are needed because there are a large
number of antigens and different types of antigen
require assistance of different classes of MHC genes.
On the other hand, one has to emphasize that we
really d o not know for sure that the number of MHC
genes is exceedingly large. When the gene counting is
over, there might not be more than a dozen genes per
MHC-that is, far less than some people have predicted.
The force that keeps the genes apart is probably the need for different genes to assist in the recognition of different antigen types. But here again, the
genes are probably not kept completely in isolation. I
would not be surprised to find-once we learn more
about the population genetics of the MHC-the
MHC stability to be a dynamic one with genes, at the
species level, constantly being added to the complex
and deleted from it.
Kunkel: I’d like to ask Dr. Klein why he didn’t mention
at all the primary B-cell expression in the I system as
compared to the presence of H-2K and D determinants on most cells, and whether this does not account for many of the differences that you see. At
least, how do you bring the differences in the cellular
reactivity for this system into your concept of similarities and differences?
Klein: The difference in the expression of class I and
class I1 antigens probably has something to do with
the specific way the two classes of antigens function. I
believe that the function of both classes is to assist in
antigen recognition and that the principle of the recognition is the same for both classes-namely that of the
Zinkernagel-Doherty phenomenon.
But the specific way the recognition occurs is
probably different. Class I molecules assist primarily
in the recognition of virally infected cells, and since
viruses can infect a variety of cells, the MHC mole-
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DISCUSSION
cules need to be ubiquitously expressed. Class I1
molecules, on the other hand, assist primarily in the
recognition of antigens to which antibodies are
formed. And since the main actor in antibody production is the B cell, there is a need for class I1 molecules
to be present primarily on B cells.
However, this speculation does not answer the
question of why recognition of different antigens is
assisted by different M H C molecules. To answer this
question one would have t o know something about
the way the T-cell receptor diversity is generated. If
one would postulate that the generation of diversity is
driven by M H C molecules in the thymus, then one
can go a step further and say that different MHC
molecules drive the differentiation of different T cells,
which then assist in the recognition of different antigens.
Benacerraf: There are indeed some cases, as you’ve
shown, where Ir genes appear to map in the K region.
It is not a unique phenomenon. Most Ir genes map in
the I region of the H-2 complex.
Klein: The asymmetry in the distribution of Ir genes
with respect to the MHC regions could be illusory.
Most Ir genes have been mapped into the I region
because the antigens that have been studied are all of
a similar kind. Dr. Benacerraf, you yourself pointed
out some time ago that the antigens share certain
common characteristics. So, one can say that they are
all of the same class. To find more Ir genes that would
map in the K or D region, perhaps one should begin
to study the immune response to different classes of
antigens. For instance, it may turn out that the response to most membrane alloantigens is controlled
by the K-D rather than the I region.
Gershon: In the cross reactive killers you demonstrated
with anti-I region immunization in a CML, did you
see any cross-killers against K and D regions? I
couldn’t see all your combinations. Have you mapped
it to show that there are only I, or could there also be
K and D?
Klein: No, we did not find any evidence for cross reactivity of killers directed against the K- and D-region
antigens. We are in the process of mapping the Iregion cross reactivity. It might turn out that the cross
reactivity will be actually directed against antigens
controlled by genes closely linked to either K or D.
Gershon: A possible explanation for your results is suggested by the observations of Dr. Schwartz and his
colleagues (Nature 263:699, 1976) that cultured T
cells can become “promiscuous killers” and that the
generation of these promiscuous killers is inhibited by
the induction of a mixed lymphocyte reaction (MLR).
One could speculate that the allo-K and - D region
antigens on MLR stimulator cells cause the inhibition
of promiscuous killers. This possibility is supported
by an observation made by Dr. Harvey Cantor and
me that allo-K and -D region antigens preferentially
activate Ly 23 suppressor cells whereas allo-I region
antigens d o not (Development of Host Defenses. Edited by M D Cooper and D H Dayton. New York,
Raven Press, pp. 155-161). Thus, the absence of an
allo-K and -D region on the stimulator cells could
have been responsible for the generation of promiscuous killers rather than the allo-I region difference
producing “cross reactive killers.”
Klein: That is a possibility worthy of testing.
Agnello: Dr. Klein, could you tell us your thoughts as to
why complement genes are linked to histocompatibility genes?
Klein: In a very general way one can say that the reason
for the linkage is the functional relatedness of complement genes with the M H C genes, since both systems are clearly involved in immune functions. What
kind of speciJc relatedness exists between complement and M H C genes is hard to tell at this moment. One possibility is that the K and D molecules
are directly involved in cell killing in a manner similar
to that exerted by some of the complement components. One could speculate that the genes for some
of the complement components are derived from ancestral class I genes. The main step in the derivation
could have been freeing of the active molecules from
the cell membrane. Amino acid sequencing should
soon show whether there is any merit t o such speculations.
Paul: Dr. Klein, the explanation you offer is that there is
a common progenitor gene for the various genes that
are encoded in histocompatibility antigens. But I’m
sure there are many instances in which genes with
common progenitors become dispersed in the course
of evolution. Wouldn’t one require some type of
coordinate regulation of the entire complex in order
to explain the maintenance of linkage? That is, should
not one postulate that it is important for these genes
to be expressed together, because they are in some
way co-regulated, or because their coordinate expression has survival value? I don’t know that anyone
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has offered an entirely satisfactory explanation for
why genes for complement and genes for histocompatibility antigens-even if they prove to have a common progenitor, or even if they both prove to be
involved in cytotoxicity in the broadest sense-would
nonetheless act more efficiently if coded for together
than if coded for separately.
Klein: Yes, you are right. A common origin is not sufficient reason to keep genes together. One still needs
something besides evolutionary relatedness. A common regulatory mechanism is as good an explanation
as any. Unfortunately, we know so preciously little
about the ways the MHC genes are regulated that it is
very difficult to come up with some concrete suggestion of how regulation might keep complement genes
associated with the MHC genes.
Uhr: With regard to regulation, I find it difficult t o
speculate fruitfully. An example of regulation is the
synthesis of a molecule with two dissimilar subunits.
Presumably, it is desirable to regulate the synthesis of
these subunits so that they are synthesized simultaneously and in similar amounts. Invariably, however,
the genes for such subunits are on different chromosomes, for example immunoglobulin, hemoglobin,
and so forth.
I want to ask Dr. Benacerraf a question related
to the spontaneous release of the T-cell factor in the
contact hypersensitivity system. On the one hand, the
notion has arisen from a significant body of evidence
that in the T-cell system, cell interactions are important; two examples are antigen-pulsed macrophages
stimulating various subsets of T cells and killer T cells
contacting target cells. On the other hand, in the last
few years, from your own work and that of others, it
appears that T-cell factors are extraordinarily effective and at great dilution. You shove a little factor
into an animal and it acts like serum antibody and
does its job. How does one reconcile these two notions?
Benacerraf: The factors and cellular phenomenon are
not mutually exclusive. The contact specific suppressor factor appears to act on other cells. As shown by
Asherson and by Gershon in this particular system,
and we confirm their observations, the suppressor
factor is taken up by peritoneal exudate cells very
well. These cells, after having absorbed the factor, can
then mediate specific suppression of contact reactivity. It is quite possible, therefore, that the target cell
for the suppressor factor in contact reactions is the
DISCUSSION
macrophage, which may then mediate its suppression
of a helper function.
Waldmann: In trying to clarify similarities or differences,
Dr. Tada sees an H-2 restriction in his system which
you do not see in your system. Do you feel this may be
related to the fact that Dr. Tada’s system is predominantly a secondary immune response, whereas
yours is a primary one? Or do you have other explanations for this?
Benacerraf: I think that the absence of H-2 restriction in
our experiments with suppressor factor is the only
major difference between the data we have obtained
and the data from Tada’s laboratory. We find restriction in certain strains and not in others; these restrictions d o not concern the H-2 region. Moreover, since
our experiments have been concerned with only suppression of the primary response, it is possible that the
differences between our laboratories could be, as you
state, that Tada studied the effects of specific suppressor factor on the secondary response.
So my assumption is that it is basically a difference in primary and secondary responses.
Waldmann: I know that AJ mice cannot make a factor
but can respond to it. Have you determined whether
SJL mice make a factor? Do they lack a receptor for
such a factor?
Benacerraf: This has not been tested in the GAT system.
The SJL mice were not tested but the A S W strain,
which is another strain bearing the H-2s haplotype,
has been tested for suppressor factor production in
the GAT system. A.SW mice produce GAT specific
suppressor factor and accept the factor from DBA 1,
H-2* mice.
Waldmann: A final point-just to compare this with
Asherson’s system. This system obviously was antigen specific in its generation and required antigen to
be present in its final suppression. But there was an
element of nonspecificity in it, because if one immunizes with two antigens, A and B, and introduces a
suppressor to A and then tests with B, no suppression
to B is found. But if one assays the response to A and
B administered simultaneously, the response to B is
suppressed.
Benacerraf: There is another difference in the way Asherson and Zembala’s experiments were done and in
the way we carried out our experiments. Both immunization procedures and the mode of obtaining the
factor were the same, but Asherson and Zembala
tested their factor on the transfer of contact hyper-
DISCUSSION
sensitivity, and in their hands the factors inhibit the
capacity of sensitized cells to transfer contact reactions and therefore block the reaction at the effector
level. We used the same type of suppressor factor
material, and we have shown that it inhibits the development of contact sensitivity in our experiments.
Warner: I am intrigued as to how the factor generates
further suppressor cells.
Benacerraf: So am I.
Warner: Have you passed that factor over an anti-TNP
column to remove any potential complexes that might
be there? In other words, it may be the antigen that is
actually triggering the cell. If so, would this infer that
the antigen is far more efficient in generating suppressors when in combination with the I-J molecule? This
in turn might suggest that the I-J is in polymeric form
or the single I-J chain has multiple binding sites.
Benacerraf: As usual, with your good acumen you have
put your finger on a critical point in both systems. In
both the polymer systems and the T N P systems, the
activity in the crude extracts may be absorbed specifically by an immunoabsorbent. Thus in the GAT system the activity can be removed specifically with a
GAT-immunoabsorbent and also with an anti-GATimmunoabsorbent; in the TNP contact system the
activity can be removed by a T N P specific immunoabsorbent and by an anti-TNP-immunoabsorbent.
Both immunoabsorbents removed the suppressive activities from the crude extracts. This implies that the
active fraction is indeed a complex of an I region
product specific for antigen with a fragment of antigen present in very small amount, which by itself is
unable to produce suppression.
Ziff: Dr. Benacerraf, would you like to discuss the nonspecific immunosuppressor substance that has been
generated by Con A? It is of some pertinence because
the NIH group has reported the suppression of NZB
disease by just two or three injections a week given
early in life. Dr. Uhr asked about the amounts involved, and you replied that there has to be a generation of cells which takes over or adopts the suppressor
activity. Would you suppose that the nonspecific suppressor substance, since it’s working in the whole
animal, will have to do the same thing?
Benacerraf: First, I should make it clear that I bear no
prejudice against the nonspecific suppressor factor. It
was discovered in our laboratory by Carl Pierce and
Bob Rich who worked on the Con A suppressor fac-
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tor at my urging. We ourselves haven’t worked with
the nonspecific suppressor factor because it was their
problem. My interpretation is that both suppressor
materials, specific and nonspecific, function in the
same manner; however, the nonspecific suppressor
factor is present in much larger amounts and therefore does not need to be focused specifically by binding with antigen.
Talal: Dr. Benacerraf, A/Jax mice are another strain
that spontaneously develops autoimmunity. They
were studied some years ago by Drs. Teague and
Friou who, even prior to the discovery of suppressor
cells, presented evidence that autoantibodies in this
strain could be suppressed by thymus cells.
DNA is an antigen that may be under active
suppression. The only way that one can experimentally induce an immune response to DNA is to denature it and conjugate it to methylated albumen, a
procedure similar to the one you use.
I wonder if you have any evidence from your
own experiments, or if you would care to speculate
about, controlling mechanisms that might be involved
in the response to nucleic acids, in NZB mice, in/AJ
mice, or in lupus patients.
Benacerraf: We have no knowlege at present of the cell
type on which suppressor factor acts, and therefore it
is inappropriate to speculate about the situation in the
NZB mice.
Williams: Just to follow up on Dr. Warner’s question,
I’m having a bit of trouble visualizing exactly what is
happening in your suppressor substance. You think
that there is a part of the antibody combining site,
combined to a portion of H-2, and that it in turn has
been combined with the tiny amount of antigen.
- Just
from a molecular standpoint, how much of the specificity of the putative combining site is involved in this
material?
Benacerraf: There are regulatory molecules produced by
T-cells which are specific for antigens; these molecules
are controlled by the I region of the H-2 complex.
These factors have been shown to regulate the immune response either in the positive (helper) or negative (suppressor) sense. The suppressor molecules
bear I-J determinants; the helper molecules bear I-A
determinants. Both suppressor
and helper factors are
._
antigen specific and are removed by the appropriate
antigen immunoabsorbents. Moreover, they do not
bear determinants of the constant regions of immunoglobulins.
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Williams: Would it be possible to absorb the material
with peritoneal exudate cells, to put your material on
an immunoabsorbent column, and thereby purify
suppressor cells?
BenacerraC: Suppressor cells have been purified by specific antigen columns by Tada very efficiently; I believe that these investigators obtained a purification of
maybe 100 fold by this technique.
Dixon: I wonder how one can put in proper perspective
the suppressor substances in something as complicated as murine lupus. If one has immunologic suppressor specificity, as you demonstrate, then just to
show in a model system that a mouse does or does not
have immunosuppressor materials doesn’t necessarily
translate to unresponsiveness to an antigen, which in
real life is quite different. I presume that you can’t
generalize.
On the other hand, if one stimulates the system
with Con A and gets what one might consider nonphysiological levels of suppressors that are obviously
effective in suppressing all responses, is that really
philosophically different from using cyclophosphamide, which also suppresses? Anything that would
suppress the entire immune response would heighten
the disease. How does one make this transition?
Benacerraf: Of course the models that we have been
working with are designed t o look at the intimate
mechanism of the regulation of immune response in
model systems. In the course of these experiments we
have also studied the general properties of suppressor
T cells. One of the properties of suppressor T cells is
their dependence on the presence of an adult thymus.
Furthermore the suppressor T cells are exquisitely
sensitive, as compared to other types of T-regulatory
cells, to radiation or cyclophosphamide. One of the
many ways in which we can demonstrate a suppressor
phenomenon is to treat mice with small doses of cyclophosphamide and observe that we have abolished
suppressor effects. Five milligrams of cyclophosphamide per kilogram are sufficient t o d o away with
suppressor type response in the polypeptide systems.
This dose of cyclophosphamide is also capable of
turning a nonresponder into a responder mouse.
Uhr: I’d like to ask Peter Doherty if he has used antibody against particular influenza type A hemagglutinins as a possible inhibitor of the cell-mediated cytotoxicity in order t o dissect out further fine specificity
in this system.
DISCUSSION
Doherty : Yes, we’ve done some experiments like that.
Inhibiting these virus immune cytotoxic T-cell responses with antibody has been notoriously difficult.
Some people report that they can do it. The group
that works with Vaccinia in Germany says that they
can inhibit with an antiviral antibody. The group in
Canberra with Bob Blanden that is using an almost
identical virus, the ectromelia virus, says that they
can’t inhibit with antiviral antibody. Our experience
to date with the various viruses we’ve used has been
that the T-cell response cannot be readily inhibited
with antiviral antibody. This has been one of the
problems in thinking about T-cell specificity and the
sort of recognition unit that the T cell is using. If you
could get direct inhibition, it would be more likely
that it would be using exactly the same receptor.
Uhr: What is your interpretation of the inability to
inhibit with excess antibody presumably of high affinity?
Doherty: In this particular situation, it may be that the T
cell is not seeing hemagglutinin at all. That’s one
possibility. The second revolves around the matrix,
We’ve done only one inhibition assay so far, and
adding antibody actually increased the level of killing.
What this might reflect is that we are seeing
antibody-dependent cell-mediated cytotoxicity as a
result of having small amounts of antibody in the
system, and any effect on T-cell blocking is being
masked. So we are currently separating the types of
cells that would cause that sort of cytotoxicity to see if
we d o get an inhibition.
The other possibility, of course, is that the T
cell is recognizing some sort of neoantigen and not the
virus antigen.
Schwartz: There is some precedent for an internal viral
protein, a matrix protein, being represented on the
surface of the cell it infects. I’m speaking in particular
of the GS antigen of murine leukemia viruses which
does appear on the cell surface of tumor cells infected
by the virus; these cells can be killed by antibodies
against this antigenic component in the presence of
complement.
Doherty: Yes, this is the work from Shellam, Knight,
and Mitchison using the Gross virus-rat system. Initially the antigen they recognized was thought to be
an internal component. Later it was shown by
Nowinsky and Watson that a glycosylated analog,
and not the antigen itself, is actually present on the
cell surface.
DISCUSSION
Schwartz: One other thing. In your altered self-neoantigen single site hypothesis, the virus would either
have to bud at the area of H-2 representation on the
cell surface or insert a protein at the H-2 site, or
contiguous with the site. With respect to vesicular
stomatitis virus, there is recent evidence that the virus
does actually bud at the H-2 site in the membrane.
But do you know of other evidence where viruses
either bud or insert their proteins at H-2 sites or
modify these sites?
Doherty: For a start, I’m not particulary emotionally
attached to the idea that the virus and the H-2 are
together. In fact, we’ve rather been going away from
that sort of system. Rolf Zinkernagel and I argued the
alternative case in an attempt to find alternatives to
our earlier model. However, the experiments with
influenza and internal budding may reflect that the
cross reactive T cells are recognizing some sort of
“altered self-component.”
The case with vesicular stomatitis virus: vesicular stomatitis virus is a rhabdo virus; it’s an enormous
virus. I t rips out the cell membrane as it goes, to be
rather dramatic about it, but it’s one of these things
that puts an enormous stalk out on the cell. It was
shown some years back, I think by Hecht and Sumners, that the vesicular stomatitis virus is actually
carrying some H-2 antigen in it. I don’t know about
the current status of this phenomenon.
With other viruses, for instance, Aoki showed
that the murine leukemia virus is actually budding
away from the H-2 sites and that the murine leukemia
virus tends to displace the H-2 as it buds out. Of
course, if it’s a nonviral protein or a protein that
one would normally not expect to see on the surface,
the researchers would have missed that.
The other thing concerns the model that the
Woodruffs have been studying, the Coxsackie model.
Coxsackie is an enterovirus that is not thought to put
antigens on the cell surface. However, there are
changes in t h e morphology of the cell-if you have
ever seen a cell infected with an enterovirus, it goes
from a nice big fat healthy cell to a little squidged up,
horrible cell.
So it could be that it is producing some
stoichiometrical or morphological change on the cell
that is not even associated with virus. Or perhaps it’s
putting on a nonviral antigen that we don’t expect to
see. Virus budding is not essential for T-cell recognition. Ectromelia is not normally a budding virus.
Ziff: This is a comment rather than a question. The
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rheumatologist is interested in mechanisms of chronic
inflammation. And in your last slide you really were
developing what might be a mechanism for chronic
inflammation if one had a chronic virus infection. For
example, if the virus were in the target cell and it put
an antigen or a neoantigen on the surface and then T
cells came out of the blood vessels in response to this
antigen and attracted macrophages, a chronic inflammatory reaction would occur.
Doherty: I tend to think of T cells as being a trigger for
inflammation. Even in persistent infections-for instance, the lymphocytic choriomeningitis modeleven if the virus does persist, the T-cell response does
seem to be regulated. Cytotoxic T-cell activity goes up
and then it comes down, and it doesn’t necessarily
correlate with antigen persistence. In subacute sclerosing encephalitis the virus is not controlled, and one
might t h i n k it’s not controlled in the brain because the
T-cell response is in some way defective. This argument was put many years ago by Sir MacFarlane
Burnett.
The result is that there is localization of numerous antibody-producing cells that produce lots of
antibody against the measles-type virus but don’t control the infection, because the virus goes readily from
one cell to another.
It could be that many chronic inflammatory
processes represent a failure of the T-cell response.
Whether it was involved in triggering initially I don’t
know. We don’t know a lot about what triggers the
inflammatory process. We know that immune complexes, or complement, can be involved in some situations. We know that T cells are involved in others. But
there are a lot of other situations we don’t understand. For instance, we don’t understand what the
message is to get the T cell in there.
Ziff: But, if T cells were chronically attracted and then
they were followed by macrophages, chronic inflammation would exist.
Doherty: We’re trying to look at that at the moment. We
can certainly show T cells localizing in inflammatory
exudates. The populations are very potent. Whether
they would persist there I don’t know.
Talal: Is there any evidence that either a dual recognition or altered self-mechanism might be involved in
recognition of endogenous viruses? For example, the
type C viruses that we heard so much about yesterday.
Doherty: You mean, the H-2 restriction-type phenomenon.
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Talal: Right. Such a phenomenon would have the advantage, for lupus models, of requiring neither a
unique H-2 or Ir gene nor a unique virus, but rather a
unique relationship between some component of
MHC and perhaps any one of a number of viruses. In
particular, there has been work on the EL4 tumor
which suggests that gp70 and H-2 can co-cap. I’ve
also heard some criticism of that work based on specificity of reagents. Do you know of any other evidence
that type C viruses show the H-2 restriction?
Doherty: I think that you’re summing up the situation
just about as it stands at present. There is some evidence of co-capping with the gp70 and H-2 from
Schrader and Edelmann. Schrader has also been
working recently with inactivated Sendai virus that he
puts into the cell membrane. The virus goes directly
into the cell membrane; indeed that is the basis of its
ability to cause cell fusion.
Schrader says that he also has an association
between that virus and the H-2 antigens. On.the other
hand, Zinkernagel and Oldstone have looked at cocapping with the lymphocytic choriomeningitis virus
and H-2 and have not found convincing results. The
whole thing is really up in the air.
The H-2 restriction, at least in some laboratories, does apply to the murine oncornaviruses. The
only doubt at present is t o a certain extent from
Herberman’s laboratory; he shows at least a syngeneic
preference.
Benacerraf: To what extent can one demonstrate in your
system cross reactivity of killing on allogenic targets
that have been infected with the virus?
Doherty: With the LCM model you’ve seen the data and
you’ve seen that some of the data are not all that
clear. With the influenza model, we’ve been recently
interested in a phenomenon that is a little bit analogous to that described by Burakoff and yourself; that
is, the CBA, immune lung exudate cell is killing p8 15,
which is an H-2d virus-infected-target cell at about
50% of the level that we would see in the syngeneic
situation.
The level of killing is variable, sometimes 50%
sometimes 20%. The line we followed is to see whether
the killer cell survives nylon wool passage. Since it
doesn’t, the killing is probably not due to T cells. We
haven’t done any further analysis yet. However, the
problem is that sometimes we get a lot of killing,
sometimes we don’t. The particular experiments
where we haven’t seen any enriching through nylon
DISCUSSION
wool were in situations where we’ve had a rather low
level of cross reactivity.
Jacobs: I would like to comment along the same lines as
Dr. Benacerraf and remind people that the LCM
cytotoxicity assay was initially described with actively
immunized mice and LCM virus-infected L cells. In
fact, if these data are compared with some of the
Australian data, the effector cellharget cell ratio is
simply much higher. Jerry Cole has shown that killing
can still be demonstrated across H-2, thus I think that
we’re talking about degree; H-2 is certainly not an
absolute phenomenon-that is, this is not an absolute
restriction.
Doherty: We disagree strongly with Dr. Cole on that, as
far as the LCM system is concerned. It was originally
described by two groups: one was the Danish group
with Marker and Volkert, who used a syngeneic system to get a very strong killing. I don’t know why this
discrepancy arose. We speculated about it early on.
We tried very hard to check it out. We just didn’t
basically find the same phenomenon.
Ziff: You’ve implied that the killer cell is in fact the cell
that is resulting in the pathology that we see in some
of the viral systems we’ve discussed, specifically LCM
and Ectromelia. The electronmicrographs that you
showed in your model for the influenza virus system
are very much like the electronmicrographs that are
seen in some acute LCM lesions, as Mongan has
shown, in which macrophages are present. You don’t
see cell lysis as you’d expect. Do you think the killer
cell is doing any damage? Or is in fact the killer cell a
coincidental marker and are we dealing with a more
typical in vivo cell-mediated immune response?
Doherty: As far as we’re concerned, the cell that transfers effector function in LCM is subjected to the same
constraint for H-2 compatibility as the cytotoxic T
cell. It maps in H-2k or it maps in H-2D and, in H-2
mutant mice, which Dr. Klein mentioned briefly, discrimination is associated with what are probably
fairly minor differences in structure. We also see the
same discrimination in vivo.
Of course, as far as the T cell goes, it’s difficult
to sort out the exact mechanism occurring in a pathological focus, and one is left with speculation. We do
know that in the LCM model there are very potent
cytotoxic T-cell populations in the lesions. We also
know the same about inflammation in influenza.
There are very potent cytotoxic T-cell populations in
DISCUSSION
the lung. Whether o r not a direct killing phenomenon
operates in vivo is rather difficult to sort out, but there
is one experiment that would tend to indicate that it
may do so. The LCM-transfer model results in the
production of disease in a cyclophosphamide suppressed recipient-a procedure that may be augmented by cortisone treatment to knock out the host
macrophages.
If this immunosuppressive regime is used in the
Listeriu system, which depends on T-cell macrophage
interaction to eliminate the bacteria, the mice die. The
T-cells are not sufficient to eliminate the bacteria by
themselves. They have to have macrophages there as
well.
In the LCM system cyclophosphamide and
cortisone treatment make absolutely no difference,
and that would tend toward the viewpoint that macrophages are not required in the LCM system. But I
think it would be foolish to be dogmatic about it.
Frank: Dr. Warner, do you know the antigens on the
red cell surface to which the antibodies are directed?
Warner: Edgington at Scripps Clinic has extensively
studied the autoantigens on red cells. The major point
to emphasize is that there is not one antigen but at least
several distinct antigens. There is an external membrane antigen and an internal red cell antigen. Other
groups have indicated even further red cell specificities, so what is detected by a Coombs’ test could
clearly be a composite of antibodies with several different specificities.
Frank: Could part of the genetic control in this system
relate to the expression of an alloantigen on the red
cell surface which influences the development of the
autoimmunity?
Warner: I very much doubt it, because early studies by
Long and Burnet showed that the eluted Coombs’
positive antibody showed absolutely no preference for
red cells of any different mouse strain studied.
Paul: Dr. Warner, in referring to the fact that the New
Zealand chocolate mice made only a modest in vitro
response to sheep erythrocytes, you pointed out that
that defect could be partially corrected by adding 2mercaptoethanol to the system. You concluded that
this was evidence for a macrophage defect. Although
that’s perhaps the most logical possibility, it is also
conceivable that those experiments indicate a defect
among B cells and that some B-cell responses are
relatively more dependent on 2ME or macrophages
S125
than others. In fact, I think that there are instances in
which that’s likely to be the case. I wonder if you have
some more information supporting the macrophage
defect idea.
Warner: I think your point is well taken and needs
further assessment. However, the deficiency in the in
vitro response of N Z C applies only to anti-erythrocyte
antibody responses and not to anti-flagellin responses.
This would imply that only one B-cell subset was involved, or rather that a single defect existed in an
accessory cell. The defect does not affect in vitro CML
generation.
Gershon: Dr. Warner, you showed that the NZB X
NZC F, generation was unique in the fact that the
autoimmunity was dominant. Then you mentioned
that in NZB bred with a number of strains the ability
to produce tolerance became dominant so that in
these cases the male NZB parent was contributing
something that was dominant. But you didn’t mention the NZB X NZC in terms of the tolerance phenomenon.
Warner: As far as the tolerance work has gone, the NZC
is perfectly normal in the induction and development
of immunological tolerance. We have also shown that
in the inbred NZC, this tolerance is associated with
induction of specific suppressor cells.
Gershon: But has that become dominant when you
make an F, between an NZB and an NZC?
Warner: That particular combination and backcrosses
of that type have not been studied yet.
Gershon: That particular cross would be the important
one if one is thinking that autoimmunity and tolerance are etiologically related.
Warner: Yes. In the same sort of study we’re attempting
to look at the F, generation to see if the same mouse
that develops immunological tolerance would then go
on to develop autoimmunity.
Shulman: In regard to the question of antinuclear antibodies in NZB/W mice, Dr. Schwartz showed that in
his NZBs 100% had anti-DNA antibodies, if I remember correctly. In the study that Dr. Hahn and I did in
older animals, we found about the same frequency of
antinuclear antibodies in NZB/W mice. It was about
20-30%.
Warner: Yes. I think that, if you go through the litera-
S126
ture, there is considerable variability in the percentage
of mice that have anti-DNA antibodies, particularly
when one reviews the genetic analyses in detail. This is
obviously a complicated question and the results of
any study depend on the exact age of the animals as
well as the methodology of the assay, particularly
with reference to the particular nucleic acid substrate
used.
Hahn: I’d like to remind every one of Braverman’s data
on the genetic passage of the autoimmune abnormalities in New Zealand Blacks and their hybrids. He also
showed that antierythrocyte and antinuclear antibodies are transmitted through backcrosses and hybrids as a mendelian dominant. Nephritis, however,
occurred in close to 100% of all the backcrosses and
hybrids. A polygenic basis for the nephritis was inferred, but it is also possible that the nephritis is not
genetically controlled.
Warner: Yes, the Braverman work is one of the more
extensive earlier studies on genetics in this field. I
think it raised some questions that are still unanswered; for example, not only the dissociation in incidence of nephritis verus serologic findings, but even
in the cases of hemolytic anemia he stressed a dissociation between the presence of the antierythrocyte
antibody and a full picture of hemolytic anemia.
Clearly, further genetic analysis of these aspects is still
necessary.
However, the main point a t the moment that
we are stressing is that there is a modifying regulatory
gene contributed by the non-NZB parent, and Braverman’s work on the genetics of the ANA is very much
in accord with bringing out that dominant gene that
we observed in the hemolytic anemia.
Uhr: Dr. Warner, can we get you to discuss the role of
histamine in modifying the expression of the autoimmunity genes?
Warner: Clearly one of the directions we wish to take is
to determine the gene product produced by this dominant gene and to determine how it is working.
Histamine offered a tantalizing possibility.
One could build a composite picture that suppressor
cells appeared to be particulary susceptible to inactivation by histamine, since suppressor cell activation in’ some experiments involving tolerance was negated by the presence of histamine. On the basis of
this finding and the observation of Burnet that NZB
thymuses appeared to be full of mast cells, we attempted to give NZB mice antihistamines and to give
DISCUSSION
normal mice histamine to see if anything unusual
could happen in regard to tolerance or autoimmunity.
So far the results are negative. I now think that sex
hormone regulation may be another, and perhaps
more likely example, of a modifying factor that could
be under genetic control. We will determine serum
levels of various androgens and hormones in the
serum of the new congenic NZB and NZB Chocolate
mice as another approach t o this problem of gene
level of action.
Gershon: Would you elaborate on the role of histamine
in preventing tolerance induction?
Warner: These were unpublished data from an in vitro
tolerance model system and I am not sure if the paper
was subsequently published.
Gershon: Just for the record, I can say that we’ve been
studying the effect of histamine on the generation of
suppressor T cells in vitro, and it seems to act synergistically with antigen. The presence of histamine increases the generation of suppressor T cells two- to
threefold. Thus, it does seem that the H2 receptor on
the T cell can be functionally stimulated by the agonist.
Uhr: Dr. Gershon, does histamine affect the function of
suppressor cells after they have been generated?
Gershon: The original data were obtained in vitro but
have recently been confirmed in in vivo studies on the
regulation of DTH.
Ziff: I would like to ask Dr. Schwartz a question. You
have worked with a xenotropic virus. Is it possible
from a lot of the evidence that’s been given at this
meeting that it’s not the xenotropic virus that we
should be interested in but rather an ecotropic virus
that might be involved in stimulating the immunity?
Schwartz: We have no evidence that NZB mice produce
ecotropic viruses. Moreover, in a cross like AKR/
NZB F1, which does produce ecotropic virus, there is
no evidence that this agent contributes to the disease.
Ziff: Does it follow that the amount of virus that you
find in these combinations is necessarily related to the
immune response to the virus or the stimulation of the
immune response that the virus might give? Gross
amounts might not necessarily be important.
Schwartz: We have found no correlation whatsoever
between the presence, absence, or titer of virus, and
either positive Coombs’ tests, anti-DNA antibodies
DISCUSSION
whether denatured or native, and the presence or
absence of nephritis.
Viola: Since your criteria for the presence of virus is the
ability to score for foci on a cat cell line transformed
by murine sarcoma virus (CCC-MSV), is it possible in
your SWR or NZB hybrid mice that you are actually
producing a xenotrope with an altered host range that
won’t score on CCC-MSV but will on another type of
cell from a different species?
Schwartz: Yes, that’s a good point. However, we have
also assayed xenotropic virus on mink cells with an
immunofluorescent technique, and the results are the
same as in the focus forming test. Naturally, we cannot exclude an undetectable virus.
Dixon: The fact that one doesn’t get viral isolates
doesn’t mean that there are no viral antigens, since
these viral antigens can be expressed in the absence of
virions or out of proportion to a few virions.
Schwartz: Right, and Dr. Dixon is going to check it for
us. We have the necessary material for that.
Barnett: Regarding your statement about the lack of
correlation among DNA antibodies of any type,
Coombs’ positivity, or hemolytic anemia and renal
disease, does that hold true for NZB F, and F,? And
for the high responders in the F,s and F,s?
Schwartz: The central point to emphasize is that the two
phenotypes-virus
expression and autoimmunityare segregating independently. All the F,s are viruspositive, and their virus content is for all practical
purposes identical to the NZB. As you saw in the case
of the Coombs’ test, 96% of the F, mice were negative.
The same pattern also holds true for the backcross
and the F,. However, we don’t have enough animals
t o contrast the occurrence of differing degrees of
nephritis according to varying levels of antibody.
Aaronson: Do the SWR X NZB develop nephritis?
Schwartz: Yes, they can develop nephritis that morphologically resembles typical NZB/W nephritis.
Aaronson: I ask because the SWR strain possesses an
ecotropic endogenous virus, B-tropic in host range,
along with information for xenotropic virus which is
expressed as antigen in the absence of virus release.
The NZW strain contains inducible ecotropic and
xenotropic viruses, along with noninducible xenotropic virus.
You mention the possibility that viremia or
S127
release of virus may be related to the immunologic
abnormality. I think we’ve ruled that out in crosses
involving NZB and the NIH Swiss mouse. The latter
contains the same xenotropic virus as NZB but expresses its antigens in the absence of viral release. In
the genetics of virus expression using embryo cells in
culture, where immunologic factors play no role,
there is segregation of a single codominant gene that
determines the level of virus expression.
Schwartz: First, in our own laboratory we’ve never been
able to isolate an ecotropic virus from SWR mice.
They may express a protein related to these viruses,
but we haven’t tested that.
Aaronson: The isolation of the SWR ecotropic virus is
well documented in the literature.
Schwartz: I stress again that all that we’ve been looking
at in these studies is expression of xenotropic virus.
In these experiments we’re not concerned with the
obvious point of partial expression of viral genome. It
very well may be ultimately that there is some cosegregation with some viral polypeptide or glycoprotein. We have no data on this. As for your comment about ecotropic viral genes in NZW and SWR
mice, the point is that these genes are not expressed
by the animal. Your studies dealt with chemical induction of viral genes in vitro and are therefore
irrelevant to the question of spontaneous expression
in vivo.
Mellors: Dr. Schwartz, I’ve enjoyed your paper greatly
and I think it is important to make it clear that one
must look at viral antigen expression-subviral protein antigen expression. I have been studying an inbred strain of NIH Swiss mice, now at 15 generations
of inbreeding. The F, hybrid cross of the inbred NIH
Swiss with the NZB would, I think, be somewhat
analogous t o your SWR cross. Some of the NZB/
NIH F, mice do develop nephritis, and when you
examine their glomeruli you find deposits of the gp70
antigen.
Schwartz: I have absolutely no doubt that this is correct.
In fact we would predict from our data that this kind
of cross would be uniformly virus-positive and some
of the animals will develop nephritis. That’s not the
issue here. The issue is whether the expression of
xenotropic virus particles initates the autoimmune
disease, or is the prime mover-or the cause or whatever other word you’d like to use-for this condition.
That’s been paramount in everybody’s think-
S128
ing about NZB. Is it or isn’t it the etiology of the
disease? We know that immune deposit nephritis with
viral antigens occurs in many strains of mice. AKR,
for example, is a very good system in which there are
viral antigens and a very high incidence of immune
deposit nephritis.
But I would interpret that as a secondary phenomenon. In other words, in the cross that you mentioned or in our SWR crosses, which are very similar,
the virus is supplying an antigen that can be deposited
in the glomeruli. As Dr. Dixon pointed out yesterday,
viral antigen is present, but in amounts that constitute
about only 10 or 15% of the total complex in the
kidney.
DISCUSSION
Ziff:Have you looked a t the kidneys for viral antigen?
Schwartz: I don’t have the answer to that yet, but it is a
current project.
Ziff: If it is an endogenous virus, how do you explain the
fact that the animal develops antibody to the virus,
which is a “self’ component?
Schwartz: It’s an autoimmune disease!!
Ziff: Thank you! But which is the chicken and which is
the egg? Presumably the response to the virus is the
cause of the autoimmunity.
Schwartz: I thought that I had shown which is which!
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