S118 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- S119 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 s120 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- s121 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. s122 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 S123 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. S124 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!