Mechanisms of recovery from a generalized viral infection: mousepox. I. The effects of anti-thymocyte serum.

Agglutination and immunofluorescence tests in vitro showed that the ATS used in these experiments cross-reacted with macrophages and RBC. However, ATS was not toxic in vivo, and small doses given subcutaneously depleted thymus-dependent areas of lymphoid tissues and selectively depressed blood lymphocyte counts without affecting other cell types in the blood. Furthermore, the function of littoral macrophages as indicated by the clearance of blood-borne virus and its subsequent behavior over a 48 hr period in the liver and spleen was not changed by ATS. Thus, the innate resistance of these vital target organs was not depressed. A similar regimen of subcutaneous ATS caused a highly significant increase in mortality from mousepox with an associated failure to control virus growth in the liver and spleen which was manifest by 6 days after infection. The interferon and neutralizing antibody responses were not impaired in ATS-treated mice, but the cell-mediated immune response was significantly suppressed. This evidence, and consideration of the timing of these host responses during the course of infection in relation to the control of virus growth in the liver and spleen, led to the conclusion that cell-mediated immunity probably contributed an essential acquired recovery mechanism. However, no evidence was obtained concerning the nature of this antiviral mechanism.

Mechanisms of recovery from a generalized viral infection: mousepox. II. Passive transfer of recovery mechanisms with immune lymphoid cells.

The following passive transfer experiments evaluated the contributions of the various host responses in recovery from mousepox. (a) Immune spleen cells transferred highly efficient antiviral activity, but preinfected recipients of these cells made no detectable splenic interferon or antibody in the 24 hr interval after cell transfer. (b) Passively administered interferon was ineffective. (c) Recipients of hyperimmune serum had much more antibody than recipients of immune spleen cells but significantly less antiviral activity. (d) Immune spleen cell populations with antiviral activity contained mediators of CMI to virus antigens. (e) The antiviral activity of immune spleen cells was specific; it was inhibited by in vitro treatment with ATS, anti-light chain serum, and anti-theta ascitic fluid, but not by removal of mononuclear phagocytes from the immune population. These results are interpreted to mean that recovery mechanisms conferred by immune spleen cells were triggered by specifically sensitized, thymus-derived lymphocytes, and that antibody and interferon responses were of less importance. A radiosensitive recipient component was necessary for the full expression of the antiviral activity of both immune cells and immune serum. It seemed likely that this component was the blood monocyte.

Mechanisms of recovery from a generalized viral infection: mousepox. 3. Regression infectious foci.

Histological and immunofluorescence techniques showed that mononuclear cells invaded virus-infected foci in the livers of passively immunized mice within 10 hr of the receipt of immune spleen cells or hyperimmune serum; by 24 hr, marked destruction of virus antigens had occurred in these lesions. Immune cell transfer promoted denser packing of mononuclear cells in the foci and more efficient destruction of infectious material than immune serum. Similar liver lesions developed by the 6th day after sublethal, primary, subcutaneous infection in normal mice. In contrast, in mice with GVHR which were immunosuppressed but possessed hyperactive macrophages and unimpaired splenic interferon response, mononuclear cells did not invade liver lesions and the animals died. These results, together with data reported previously, indicated that mononuclear cell invasion of infected liver foci, triggered by CMI, was of key importance in recovery from primary mousepox. The roles of specifically sensitized lymphocytes and macrophages within lesions were not directly evaluated, but indirect evidence suggested that lymphocytes could cause no more than a halt in virus multiplication, and that macrophages were required for the inactivation of preformed virions. Possible augmentation of the efficiency of macrophages by locally-produced lymphocyte interferon, neutralizing antibody, or stimulation of their phagocytic and intracellular digestive capacity cannot be excluded.

Regulation of the T-cell response to ectromelia virus infection. I. Feedback suppression by effector T cells.

Spleen cells and serum from mice immunized with ectromelia virus suppressed the immune response to infectious virus when transferred intravenously into recipient mice given an immunizing virus dose. The suppression was reflected in decreased cytotoxic T-cell activity directed against H-2 compatible virus-infected target cells in the spleens of recipients. Suppression was observed when immune cells or serum were transferred 1-2 h or 1 day after immunization of recipients, but not 2 days after, and was maximal when 6-day immune spleen cells were used as suppressor cells. H-2 compatibility between donor and recipient mice was necessary for suppression to be expressed. Use of recombinant mice showed that I-region compatibility was neither sufficient nor necessary, and that D-region compatibility was sufficient. Specificity of suppression was suggested by the finding that cells and serum from mice immunized with Listeria monocytogenes, a bacterium, had no suppressive activity on the antiviral response. Anti-theta treatment eliminated the ability of immune cells to suppress, and the suppressive effect was not markedly dose-dependent with respect to both cell dose and virus dose under the conditions employed. Virus levels in the spleens of recipients were significantly reduced after injection of immune cells. Adult thymectomy had no effect on the primary immune response to ectromelia virus infection, thus indicating no role for T1 cells in the suppressive mechanism. The results obtained therefore suggested that suppression in this system was due to effector T cells which triggered clearance of virus (and thus, of virus-induced antigens) necessary for the induction of precursors of effector T cells, and that this simple feed-back mechanism normally plays an important role in the regulation of the primary immune response to ectromelia infection at the level of precursor induction. The existence of other postinduction regulatory mechanisms, however, is unknown and under investigation.

A single genetic element in H-2K affects mouse T-cell antiviral function in poxvirus infection.

Cell transfer experiments using mice with recombinant H-2 haplotypes were used to map the H-2 regions which must be shared by ectromelia-immune T-cell donors and virus-infected recipients for transfer of virus clearance mechanisms in the spleen. K- or D-region genes were necessary and sufficient; I-region genes were not involved. The remainder of the mouse genome could be varied widely without impairing the efficacy of T-cell antiviral function, provided either a K or a D region was shared in the donor-receipient combination. A mutation in a single genetic element of the K region of the H-2 complex abolished the antiviral effect of immune T-cell transfer in a donor-recipient combination which shared the K end.

H-2-linked control of cytotoxic T-cell responsiveness to alphavirus infection. Presence of H-2Dk during differentiation and stimulation converts stem cells of low responder genotype to T cells of responder phenotype.

Secondary Tc cells generated against Sindbis virus (SIN) are restricted to Dk. All other H-2K or D regions tested show low specific responsiveness. F1 hybrids between low and high responders show dominance of responsiveness but lack complementation. When BALB/c (KdIdDd) low responder fetal liver stem cells were allowed to mature in irradiated high responder recipients C3H.OH (KdIdDk) a response to Dk plus SIN could be generated with Tc cells of BALB/c origin. This result, together with the failure of complementation in the F1 hybrids, implies that the lesion of low responsiveness is in the inability of viral antigen to stimulate a Tc-cell response in association with any self H-2K or H-2D molecule (of those tested) other than H-2Dk. Hypotheses compatible with these data are discussed.

Cytotoxic T-cell responses show more restricted specificity for self than for non-self H-2D-coded antigens.

The specificity of recognition of H-2 antigens by various subsets of Tc cells was investigated with respect to the two separate molecules known to be coded in the H-2D(d) region (a) D which carries the private specificity H-2.4 and (b) D'which carries the public specificity H-2.28. BALB/c.H-2(db) mutant mice express D but not D' on their cell surfaces, whereas wild-type BALB/c mice express both D and D'. H-2 restricted Tc cells specific for viral-plus- H-2D(d)-coded antigens on infected self cells, or minor H-plus-H-2D(d)-coded antigens on H-2-compatible cells apparently recognize D, but do not detectably recognize D. In contrast, BALB/c-H-2(db) anti-BALB/c Tc cell responses do recognize D' (the only known antigen which is not shared by mutant and wild-type); furthermore, D' is also detectably recognized by a significant proportion of the Tc cells that respond in MLR to H-2D(d)-coded antigens. In these latter responses, D' was recognized separately from D, i.e., the response was not "H-2 restricted". These results indicate that H-2 restricted Tc cell responses to modified-self cells are more specific for self H-2D(d)-coded antigens then are allogeneic Tc cell responses directed at the same antigens, in that haplotype-unique (private) specificity recognition (of the D molecule) exclusively occurs only in the former, not the latter case. The implications of this specificity of H-2 restricted responses for possible processes of somatic selection of anti-self recognition structures on progenitor Tc cells are briefly discussed.

Interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin 2 infection.

Athymic nude mice recover from an infection with recombinant vaccinia virus (VV) encoding murine interleukin 2 (IL-2), but treatment with a mAb to IL-2 accentuated infection. Administration of a mAb against interferon gamma (IFN-gamma) to mice infected with the IL-2-encoding virus completely prevented the IL-2-induced mechanisms of recovery. Both asialo-GM1+ (NK) and asialo-GM1- (non-NK) cells were participants in the IFN-gamma-mediated recovery of nude mice from infection with the IL-2-encoding VV recombinant. Depletion of asialo-GM1+ cells exacerbated infection, though not as much as anti-IFN-gamma mAb. In vitro, both asialo-GM1+ and asialo-GM1- nude mouse splenocytes produced IFN-gamma in response to IL-2.

Primary anti-viral cytotoxic T-cell responses in semiallogeneic chimeras are not absolutely restricted to host H-2 type.

Chimeras produced by reconstitution of 950 rads irradiated type A or type B host mice with (AXB)F1 fetal liver stem cells were examined in primary (in vivo) and secondary (in vitro) Tc-cell responses to ectromelia virus infection. Of 33 individual chimeras which gave primary responses, 26 produced significant specific lysis of infected targets of both A and B type, though host type targets were invariably lysed more efficiently (host bias). The other 7 chimeras gave lysis of infected host type targets only (absolute restriction). 12 individual chimeras were used in secondary responses. Nine showed host bias, and three showed absolute restriction. Whether an individual chimera showed host bias or absolute restriction seemed to be unrelated to whether the response was primary or secondary, to the time after reconstitution (ranging from 4 to 22 wk), to strain of mouse, or to the batch of fetal liver stem cells used.

Quantitative differences in the expression of parentally-derived H-2 antigens in F1 hybrid mice affect T-cell responses.

Quantitative absorption with specific anti-H-2 sera has shown that the H-2Kb and H-2Dd antigens coded by the B10.A(5R) haplotype are expressed in about fourfold lower amount on the spleen cells of [B10.A(5R) X B10.A(2R)]F1 hybrids than on parental B10.A(5R) cells. In contrast, the H-2Kk and H-2Db antigens of B10.A(2R) are expressed equally on parental and F1 cells. These quantitative differences are reflected in cytotoxic T-cell (Tc-cell) function. Macrophage target cells from F1 mice are killed less efficiently than B10.A(5R) targets by alloreactive or H-2 restricted Tc cells specific for H-2Kb or H-2Dd, and spleen cells of F1 mice are less efficient stimulators of alloreactive Tc cells specific for B10.A(5R) H-2 antigens, whereas F1 and B10.A(2R) cells are equal as targets and stimulators for Tc cells recognizing B10.A(2R) H-2 antigens.

Different D end-dependent antigenic determinants are recognized by H-2-restricted cytotoxic T cells specific for influenza and Bebaru viruses.

Two different BALB/c anti-CBA (H-2k)monoclonal antibodies that bind to Kk and Dk antigens blocked Tc cell-mediated lysis of L929 (Kk, Dk) target cells, but with quite different specificity. One antibody (30R3) powerfully blocked Kk-specific lysis mediated by alloreactive or Kk-restricted Tc cells immune to ectromelia, Sendai, or influenza viruses. The other antibody (27R9) blocked these anti-Kk Tc cells much less than 30R3, but in contrast, 27R9 blocked anti-Dk lysis much more than 30R3. Most importantly, 27R9 strongly blocked Dk-restricted anti-influenza Tc cells, but did not significantly block Dk-restricted anti-Bebaru (BEB) lysis. This result indicated that different H-2 determinants coded in the D end of H-2k were recognized by influenza-and BEB-immune Tc cells. These determinants may be carried on two different molecules coded by the H-2D and H-2L loci, but other possibilities are not yet excluded.

Mechanisms of suppression of cytotoxic T-cell responses in murine lymphocytic choriomeningitis virus infection.

The cytotoxic T-cell response to lymphocytic choriomeningitis (LCM) virus infection was suppressed either in vitro or in vivo by addition of a high level of syngeneic virus-infected cells or syngeneic cells from congenital LCM virus carriers to the environment of the responding cells. This effect was not duplicated by formaldehyde-fixed carrier cells, nor could it be accounted for by 'cold' target competition by carrier cells at the level of the cytotoxicity assay. Conversely, suppression was produced in vivo by water-lysed, ultrasonically treated carrier cell suspensions, or by a large dose of LCM virus equivalent to that contained in the carrier cells. Thus a high level of infectious virus was a common factor in all observed examples of suppression. Based upon this, the following hypothesis, a form of 'forbidden clone deletion,' was proposed to account for virus-specific cytotoxic T-cell tolerance in LCM virus congenital carriers, or in high dose suppression. A high level of virus in lymphoid tissues, while not cytopathic per se, may result in infection of all or most T cells; this then may lead to deletion either via 'suicide' of individual, infected, cytotoxic T cells with receptors specific for virus-induced antigenic patterns on their own surface membranes, or by mutual lysis of two adjacent T cells.

Specific adsorption of H-2-restricted cytotoxi T cells to macrophage monolayers.

These experiments tested whether Tc cells specific for foreign antigen (X) plus self H-2 adsorbed to macrophage monolayers displaying (a) X with allogeneic H-2; (B) self H-2 alone; (c) X plus self H-2. Specific adsorption occurred only in case (c), a result compatible with altered self and requiring further operational assumptions in dual recognition models.

Neonatal tolerance of major histocompatibility complex antigens alters Ir gene control of the cytotoxic T cell response to vaccinia virus.

The K region of H-2 controls the Tc cell response to vaccinia-Db. The Kb, Kd, and Kq alleles allow good Tc cell responses against vaccinia-Db. In contrast, the presence of Kk in H-2 recombinants 2R (Kk,Db) and 4R (Kk,Db) or in F1 hybrids greatly reduces the anti-vaccinia-Db response. The defect does not lie in antigen presentation, as infected 4R cells can stimulate anti-vaccinia-Db Tc cells in vitro. Furthermore, nonresponder animals possess Tc cell precursors for vaccinia-Db, as transfer of F1 nonresponder spleen cells into infected, lethally irradiated responder recipients allowed generation of anti-vaccinia-Db effector Tc cells. Secondary responses to vaccinia-Db can also be obtained in vitro from T cells of 4R animals. Feedback inhibition was excluded in experiments with mixed chimeras in which Kk and Db were expressed on separate cell populations. Neonatal tolerance of B10 animals to Kk suppressed the anti-vaccinia-Db response but did not affect anti-vaccinia-Kb, anti-lymphocytic choriomeningitis virus, or anti-H-2d responses. In cold target competition experiments, H-2k competitors inhibited vaccinia-Db-specific target cell lysis by Tc cells, which suggests that anti-vaccinia-Db and anti-H-2Kk Tc cells may cross-react. Therefore, we propose that the suppressive influence of Kk on anti-vaccinia-Db Tc cell responses is a consequence of self-tolerance and that suppression of anti-Kk Tc cells results in cross-reactive suppression of anti-vaccinia-Db Tc cells.

The host response to Calmette-Guérin bacillus infection in mice.

Heterologous organisms (L. monocytogenes and S. typhimurium) were used to study the rate of development, magnitude, and persistence of the antimicrobial resistance engendered in mice by vaccination with BCG. These same methods were used to investigate the influence of prior vaccination on the host response to reinfection. The rate of onset and magnitude of the resistance produced by BCG varied with the vaccinating dose. Increased resistance was detected within 48 hr of injecting large numbers of BCG (approximately 10(8) viable units), but concurrent treatment with isoniazid interrupted its further development. An equal number of heat-killed organisms failed to influence host resistance significantly. The development of tuberculin sensitivity was also dependent upon the continued survival of the immunizing population of BCG. When vaccinated mice were reinfected with BCG, host resistance in spleen and liver was rapidly augmented to the accompaniment of striking changes in the morphology and microbicidal activity of the peritoneal macrophages. These changes occurred most rapidly in mice with a high level of delayed hypersensitivity at the time of reinfection.

Host-parasite relations in mouse typhoid.

The development of acquired resistance to Salmonella typhimurium has been studied in mice infected intravenously with small numbers of streptomycin-sensitive or streptomycin-resistant organisms. By the 14th day of a primary infection the mouse develops a mechanism capable of destroying completely a super infecting dose of organisms, but is unable to eliminate organisms of the primary infection. The latter are constantly returned to the circulation from necrotic foci at the sites of implantation. Passive transfer of serum from actively infected or vaccinated animals, and immunization with heat-killed organisms, increase the capacity of the host to clear organisms from the blood, but do not interfere to any significant extent with their subsequent multiplication in the tissues. It is concluded that the resistance of actively infected animals depends on a nonhumoral mechanism capable of destroying organisms from endogenous or exogenous sources.

Infection-immunity in experimental salmonellosis.

Salmonella enteritidis is highly virulent for the mouse causing an infection resembling mouse typhoid. Survivors of the infection are completely resistant to reinfection and eliminate a large challenge dose of virulent organisms within 72 hr. The antigenically related Salmonella gallinarum was almost avirulent for the mouse but animals vaccinated with this organism were equally capable of eliminating a lethal dose of virulent S. enteritidis. Living Salmonella pullorum, on the other hand, was quickly eliminated from the tissues of normal mice. Vaccination with this organism failed to evoke an effective bactericidal mechanism. Alcohol-killed vaccines of these three Salmonellae all produced an increase in blood clearance rate, but gave only marginal protection against S. enteritidis. Liver and spleen counts on these mice revealed a 1 to 2 day delay before any net increase in the total bacterial population could be observed. Immunization of mice with increasing doses of living Salmonella montevideo resulted in progressively greater killing of a challenge dose of S. enteritidis despite the absence of common somatic antigens between the two strains. The degree of protection varied with the size of the residual population of S. montevideo in the vaccinated mice. The significance of these findings in assessing the importance of various factors involved in the development of acquired resistance to Salmonella infections is discussed.

Mechanisms of acquired resistance in mouse typhoid.

Experiments in vitro comparing normal mouse peritoneal macrophages with cells from Salmonella typhimurium-infected mice have shown that the "immune" macrophages have conspicuously enhanced microbicidal properties. Whereas normal macrophages could inactivate only 50 to 60% of intracellular S. typhimurium pretreated with immune serum, cells from infected animals killed virtually all ingested organisms and did so at an accelerated rate. Macrophages from Listeria monocytogenes-infected mice were shown to possess similarly enhanced microbicidal activity against S. typhimurium. Furthermore, the growth of S. typhimurium in the liver and spleen was more effectively restricted in Listeria-infected mice than in animals vaccinated with heat-killed S. typhimurium, even though the Listeria-infected animals possessed no demonstrable cross-reacting antibody to S. typhimurium. The lack of resistance in the mice vaccinated with heat-killed organisms could not be attributed to any deficiency of humoral factors, since the serum from these animals was as effective at promoting phagocytosis and killing by macrophages as serum from actively infected (and demonstrably resistant) mice. Conversely, Salmonella-infected mice were totally resistant to intravenous challenge with L. monocytogenes. The level of resistance in individual animals was related to the numbers of residual Salmonellae remaining in the tissues; mice with heavier residual infections being the more resistant. Specific antiserum from mice vaccinated with heat-killed S. typhimurium was found to be significantly protective only when the intraperitoneal route of challenge was employed. The foregoing studies have been interpreted to mean that enhancement of the microbicidal ability of macrophages is the mechanism of major importance in acquired resistance to S. typhimurium infection in mice.

Epithelium of mouse yolk sac placenta lacks H-2 complex alloantigens.

The only fetal cell membrane exposed to the mother in the mouse yolk sac placenta is the apical membrane of the endodermal epithelial cells. In yolk sac preparations in vitro, this apical membrane was exposed to reagents or cells in the incubation medium. By using several techniques we were not able to detect fetal major histocompatibility complex (MHC) antigens in this membrane. Immunoferritin labeling with and without prefixation and after neurominidase and trypsin digestion indicated that the apical membrane could contain no more than approximately 1% of the H-2 complex antigens that were present on peritoneal macrophages. Incubation of yolk sac preparations in anti-H-2 complex antiserum and complement had no cytotoxic effect on the endodermal epithelium, nor did incubation in an excess of alloreactive lymphocytes. Dissociated preparations of prefixed yolk sac contained endodermal epithelial cells and vascular endothelial cells whose entire surface membranes were exposed to the medium. H-2-complex antigens were not detected by immunoferritin labeling in either the apical or the laterobasal membrane of the yolk sac endoderm, but they were present in low density on the vascular endothelium. Also, incubation of unfixed, dissociated cells in anti-H-2-complex serum and complement had no detectable cytotoxic effect on endodermal epithelial cells. These observations indicate that H-2 antigens are sparse or absent in both the apical and laterobasal membranes of endodermal epithelial cells. The deficiency of MHC antigens in the apical membrane may account for the failure of sensitized females to reject the yolk sac, whereas the composition of the laterobasal membrane is probably less important to maternal-fetal relations. The present observations are consistent with labeling studies of adult-lining epithelial cells, which indicate that self-marker MHC molecules are absent from the apical membranes oriented toward the outside world and variably expressed in the laterobasal self-side membranes. It is suggested that the corresponding exclusion of fetal self-marker molecules from the apical membranes of some kinds of placental epithelia would deprive the mother of target sites for an alloimmune reaction at the maternal-fetal interface.

H-2 compatibility requirement for virus-specific T-cell-mediated cytolysis. Evaluation of the role of H-2I region and non-H-2 genes in regulating immune response.

Lymphocytic choriomeningitis virus (LCMV) and ectromelia virus-specific T-cell-mediated cytotoxicity was assayed in various strain combinations using as targets peritoneal macrophages which have been shown to express Ia antigens. Virus-specific cytotoxicity was found only in H-2K- or D-region compatible combinations. I-region compatibility was not necessary nor alone sufficient for lysis. Six different I-region specificities had no obvious effect on the capacity to generate in vivo specific cytotoxicity (expressed in vitro) associated with Dd. Low LCMV-specific cytotoxic activity generated in DBA/2 mice was caused by the non-H-2 genetic background. This trait was inversely related to the infectious virus dose and recessive. Non-H-2 genes, possibly involved in controlling initial spread and multiplication of virus, seem to be, at least in the examples tested, more important in determining virus-specific cytotoxic T-cell activity in spleens than are Ir genes coded in H-2.