Relationship among immunodominance of single CD8+ T cell epitopes, virus load, and kinetics of primary antiviral CTL response.

The primary CTL response of BALB/c mice infected with the lymphocytic choriomeningitis (LCM) virus strain WE is directed exclusively against one major epitope, n118, whereas a viral variant, ESC, that does not express n118 induces CTL against minor epitopes. We identified one minor epitope, g283, that induces primary lytic activity in ESC-infected mice. Infections of mice with WE and ESC were used to study the hierarchical control of a T cell response. Presentation of minor epitopes is not reduced in WE-infected cells. Generation of CTL against n118 does not suppress the generation of minor epitope-specific CTL systemically, as mice coinfected with WE and ESC developed CTL against n118 and g283. However, elimination of ESC and development of minor epitope-specific CTL in ESC infection were slower than elimination of WE and development of CTL against n118. CD8+ T cells against the minor epitope were activated in ESC and WE infection, but did not expand in the latter to show lytic activity in a primary response. We explain the absence of minor epitope-specific lytic activity in WE infection by the fast reduction of virus load due to the early developing n118-specific CTL. Immunodominance of CTL epitopes in primary virus infections thus can be explained as a kinetic phenomenon composed of 1) expansion of CD8+ T cells specific for individual epitopes, 2) stimulatory effect of virus load, and 3) negative feedback control on virus load by the fastest CTL population.

Protective immunity in BALB/c mice against the simian virus 40-induced mKSA tumor resulting from injection of recombinant large T antigen. Requirement of CD8+ T lymphocytes.

BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg-expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg-expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes.

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.

Emergence of virus escape mutants after immunization with epitope vaccine.

BALB/c and C57BL/6J mice were immunized with recombinant vaccines consisting of lymphocytic choriomeningitis virus CD8+ T-lymphocyte epitopes and a carrier protein. During challenge infection with WE strain lymphocytic choriomeningitis virus, mutants with alterations in distinct amino acid residues of the epitopic nonapeptides appeared and multiplied. Splenocytes from WE-infected BALB/c mice lysed cells coated with the WE-type epitope; lysis was considerably less effective when the epitopic nonapeptide with which the syngeneic cells had been sensitized was the mutated form. Neither target was lysed by splenocytes from BALB/c mice infected with the variant virus. Mutants were not detected in F1 hybrid mice immunized with two viral epitopes that were restricted by class I molecules of both parents.

Granzyme A-deficient mice retain potent cell-mediated cytotoxicity.

Granzyme A, a granule-associated serine proteinase of activated cytotoxic T cells and natural killer cells, has been reported to play a critical role in DNA fragmentation of target cells. To address the question of the biological role of granzyme A, we have now generated a granzyme A-deficient mouse mutant by homologous recombination. Western blot analysis, enzyme assays and reverse transcription-PCR confirmed the absence of granzyme A in activated T cells. In addition, deletion of granzyme A does not alter the expression patterns of other granule components, such as granzymes B-G and perforin. Granzyme A-deficient mice are healthy and show normal hematopoietic development. Most notably, their in vitro- and ex vivo-derived cytotoxic T cells and natural killer cells are indistinguishable from those of normal mice in causing membrane disruption, apoptosis and DNA fragmentation in target cells. Furthermore, granzyme A-deficient mice readily recover from both lymphocytic choriomeningitis virus and Listeria monocytogenes infections and eradicate syngeneic tumors with kinetics similar to the wild-type strain. These results demonstrate that granzyme A does not play a primary role in cell-mediated cytotoxicity, as has been assumed previously.

Murine hepatitis caused by lymphocytic choriomeningitis virus. II. Cells involved in pathogenesis.

BACKGROUND: Human virus hepatitides are often assumed to result from pathogenic immune responses rather than from direct viral cytopathic effects, but the details are largely unknown. Hepatitis of the mouse undergoing infection with lymphocytic choriomeningitis (LCM) virus is an immunopathologic phenomenon, and its analysis may help us to understand some of the events leading to the human illnesses. EXPERIMENTAL DESIGN: Mice were infected with LCM virus and were depleted of T cells or their subsets by inoculation of monoclonal antibodies; other infected mice lacked all T lymphocytes or the CD8+ subset because of genetic defects. Also, mice were infected and transfused with unsorted or CD(4+)-enriched LCM-immune spleen cells. Subsequently, the infectious titers were determined, the cytolytic activities of mononuclear cells isolated from the livers were measured, and the disease process was studied. RESULTS: In LCM virus-infected mice devoid of all T lymphocytes, pathologic alterations remained undetectable. In contrast, immunocompetent animals responded with a severe hepatitis, at the height of which the liver contained large numbers of cytolytic mononuclear leukocytes. Experiments with mice depleted of subset T lymphocytes revealed a predominantly CD8+ T lymphocyte-mediated phase, which was characterized by panlobular inflammation, whereas later there was a periportal inflammatory reaction, in which mainly CD4+ T lymphocytes were involved. Infusion of syngeneic immune spleen cells from immunocompetent donor mice into infected thymus-less mice resulted in virus elimination and damage to liver cells. With a similar protocol and the use of congenic mice, CD8+ T lymphocytes were observed to rapidly enter the recipients' livers, where they were present at the time liver cell injury was apparent. In mice genetically deficient in CD8+ T lymphocytes due to disruption of the gene for beta 2-microglobulin, a somewhat different type of LCM hepatitis developed that was largely dependent on CD4+ T lymphocytes. Liver cells were also damaged in infected nude mice that had been infused with positively selected CD4+ spleen cells from infected +/+ mice. CONCLUSIONS: Our findings published previously (Löhler J, Gossmann J, Kratzberg T, Lehmann-Grube F. Lab Invest 1994; 70:263-78) and related here suggest that the hepatitis in mice undergoing infection with LCM virus consists of three consecutive phases, which are mediated predominantly by NK cells, CD8+ T lymphocytes, and CD4+ T lymphocytes, respectively. Presumably, other elements of the immune system, such as mononuclear phagocytes and B lymphocytes, contribute to the pathogenesis.

Antiviral protective immunity induced by major histocompatibility complex class I molecule-restricted viral T-lymphocyte epitopes inserted in various positions in immunologically self and nonself proteins.

Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein.

T lymphocyte-mediated antiviral immune responses in mice are diminished by treatment with monoclonal antibody directed against the interleukin-2 receptor.

Blocking the interleukin-2 receptor's alpha-chain in lymphocytic choriomeningitis virus-infected mice by treatment with monoclonal antibodies diminished the increase of numbers of CD8+ T lymphocytes in spleens and prevented CD8+ T lymphocyte-mediated virus clearance from organs as well as generation of virus-specific cytotoxic T lymphocytes. Also, the CD8+ T cell-mediated early phase of the delayed-type hypersensitivity footpad swelling reaction was decreased. The same treatment had no effect on the number of CD4+ spleen T lymphocytes, which, however, did not enlarge during infection, but these cells' heightened DNA synthesis and cytokine production were reduced by antibody treatment; yet the generation of antiviral antibodies remained unaffected, and the CD4+ T lymphocyte-mediated second part of the footpad reaction was somewhat augmented. We conclude that blocking of the interleukin-2 receptor by antibody in lymphocytic choriomeningitis virus-infected mice diminishes both CD8+ and CD4+ T cell-mediated antiviral immune responses, the former more than the latter.

CD8+ T lymphocyte-mediated antiviral immunity in mice as a result of injection of recombinant viral proteins.

A major portion of the nucleoprotein (amino acids 67 through 300) and the glycoprotein-2 of lymphocytic choriomeningitis (LCM) virus were synthesized by using recombinant technology and were injected together with SDS twice in portions of 5 micrograms into BALB/c mice. As evidenced by diminished replication of LCM challenge virus, both proteins induced antiviral immunity, which was comparable in extent with the immunity caused by infection with LCM vaccinia recombinant viruses. Primed LCM-viral CTLs could not be demonstrated in these mice by culturing splenocytes in the presence of LCM virus, and Abs appeared slowly and in low quantities; but, after injection of large infectious doses, CTLs appeared faster and in higher numbers than in mice not previously treated with viral proteins. Depletion of CD8+ cells, but not of CD4+ cells, by treatment of mice with mAb abolished the antiviral immunity, demonstrating that protection was mediated by CD8+ T lymphocytes. Absence of CD4+ T lymphocytes before and during the period of immunization did not measurably affect the animals' antiviral immune status, indicating that activation of the CD8+ T lymphocytes was not dependent on help by CD4+ cells.

MHC class I molecule-restricted presentation of viral antigen in beta 2-microglobulin-deficient mice.

Normally, Ag is presented to CD8+ T lymphocytes as a tripartite complex consisting of peptide epitope, MHC-encoded class I heavy (alpha) chain, and beta 2-microglobulin (beta 2-m) light chain. Although there is agreement about the function of both peptide and alpha-chain, the role of beta 2-m has remained uncertain. In particular, can Ag be presented without its participation? We have sought to obtain an answer by using mice in which the gene for beta 2-m had been disrupted by homologous recombination. As a consequence, no light chains are synthesized and, furthermore, few if any CD8+ T lymphocytes are formed. Elimination of lymphocytic choriomeningitis (LCM) virus from the tissues of acutely infected mice is mediated solely by CD8+ T lymphocytes; hence, in the beta 2-m-lacking mutants the infection cannot be terminated. Here it is shown that infusion of immune spleen cells from syngeneic beta 2-m+/+ mice and from mice compatible in K or D of the MHC resulted in virus clearance. Approximately five times more cells were required to achieve antiviral effects in beta 2-m-deficient than in wild-type mice but attempts to improve elimination by treatment of the former with IFN-gamma or beta 2-m have failed. Depleting the immune splenocytes of CD8+ T lymphocytes but not of CD4+ T lymphocytes abolished the antiviral potential. We conclude that LCM virus-infected murine cells can present viral Ag to CD8+ effector T lymphocytes together with class I MHC molecules K and D, despite the total absence of beta 2-m.

Murine hepatitis caused by lymphocytic choriomeningitis virus. I. The hepatic lesions.

BACKGROUND: The hepatitis that occurs after adult mice are infected with lymphocytic choriomeningitis (LCM) virus is immune mediated, although the details of the pathogenetic mechanisms are largely unknown. To better understand the sequence of events leading to alterations typical for hepatitides with immunopathogenesis, livers of immunocompetent mice infected with LCM virus were examined. EXPERIMENTAL DESIGN: Virus replication and histopathology in the livers and concentrations of liver enzymes in the sera of C57BL/6 mice were followed from day 3 through day 14 after intraperitoneal infection with 10(6) mouse infectious units of LCM virus. Histologic, histochemical, immunohistologic, and in situ hybridization methods were used to determine the cells involved in the inflammatory process. RESULTS: Infectious virus rose to 10(9) mouse infectious units/g of liver by day 7 and declined thereafter. Viral RNA and antigen were localized in foci of hepatocytes and in Kupffer and endothelial cells of the sinusoids. Disseminated spotty necroses, steatosis, a marked sinusoidal reaction, and lobular and (later) periportal mononuclear infiltrates were observed. In the infiltrates, T cells predominated followed by macrophages and NK cells; the number of B and plasma cells rose moderately. Among T lymphocytes the CD8+ cells increased preferentially, and the CD4/CD8 ratio changed from 1.7 to 0.3. Other features were major histocompatibility complex antigen expression on hepatocytes, enhanced immunocytochemical evidence of fibronectin and ICAM-1 in sinusoids, and deposition of immunoglobulin, complement, and fibrinoid. Changed activities of liver enzymes and bilirubin levels paralleled the pathologic alterations. CONCLUSIONS: Although CD8+ T cells seem to be central in the pathogenesis of LCM hepatitis, probably more than one immunopathologic mechanism is operative.

Antiviral immune responses of lymphocytic choriomeningitis virus-infected mice lacking CD8+ T lymphocytes because of disruption of the beta 2-microglobulin gene.

Mice infected intracerebrally with lymphocytic choriomeningitis virus (LCM virus) develop a characteristic central nervous system disease and usually die. If the intravenous or intraperitoneal route is used, the infection leads to less severe clinical signs and the virus is eliminated. Illness and virus clearance are immunological phenomena, which are assumed to be caused exclusively by CD8+ T lymphocytes. In contrast, of the two phases of a delayed-type hypersensitivity reaction caused by inoculation of the virus into the mouse's foot, only the first is mediated by CD8+ cells, whereas the second is mediated by CD4+ cells. We have examined LCM virus-specific immune responses in mice devoid of CD8+ T lymphocytes as a result of disruption of the beta 2-microglobulin gene. As expected, the virus persisted but footpad swelling did not occur, although intracerebral infection resulted in CD4+ T-lymphocyte-mediated illness and antiviral antibodies were produced. Different results had been obtained by Fung-Leung et al. (W.-P. Fung-Leung, T. M. Kündig, R. M. Zinkernagel, and T. W. Mak, J. Exp. Med. 174:1425-1429, 1991), who, is essentially identical experiments but with mice lacking CD8+ T lymphocytes as a result of disruption of the Lyt-2-encoding gene, recorded control of the infection and development of a local delayed-type hypersensitivity reaction. We consider these differences important, because they provide us with clues that may help to understand the mode of action of the CD8+ T cells in cell-mediated antiviral immunity.

Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope.

For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CD8+ CTL are essential. In the infected BALB/c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I MHC-encoded molecule L; K- or D-restricted antiviral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant C-H-2dm2 should not be capable of eliminating the virus. The experimental evidence proves the contrary, which is explained by K- and D-restricted CTL that this mouse generates. Why such cells remain undetectable in BALB/c mice is currently unexplained, because there is no lack of precursors and the corresponding virus Ag is presented. Despite the absence of lytic activity in vitro, other than the one associated with L, transfusion of day 8-immune spleen cells from BALB/c into infected C-H-2dm2 (L-deficient) mice results in accelerated virus elimination from the organs of the latter, which was manifest as soon as 8 h after cell transfer. Furthermore, lytic activity did not attain measurable levels in the recipients' spleens. Obviously, this infection can be terminated by CD8+ T lymphocytes even when these cells' lytic activity is below detectability.

Antiviral immune responses of fully allogeneic irradiation bone marrow mouse chimeras.

In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.

Entry of antivirally active T lymphocytes into the thymus of virus-infected mice.

The thymus has long been regarded as an immunologically "privileged site" by being shielded against the entry of exogenous Ag as well as protective elements of the immune system. After i.p. infection of mice, the lymphocytic choriomeningitis virus multiplied in this organ. Viral Ag was found predominantly in the epithelial-reticular cells of the medulla and to a lesser extent in such cells of the cortex. Beginning on day 7 after infection, the virus disappeared, a process that could be blocked by depleting the mice of peripheral T lymphocytes or of CD8+ cytotoxic T lymphocytes. Viral clearance was accelerated by i.v. injection of splenocytes from mice, which themselves were just eliminating the virus. CD8+ cells from CD8-congenic donor mice, most with blast morphology, were detected immunocytochemically in the thymus, predominantly in the medulla. A few CD8+ T lymphocytes from donors not previously infected were also demonstrated in the thymic tissue of infected mice. Our findings indicate that the lymphocytic choriomeningitis virus has access to the thymus and suggest that it is cleared by the antiviral activity of circulating cytotoxic T lymphocytes. Thus, just as other organs, the thymus appears to be subject to immune surveillance by mature T lymphocytes that have their origin in secondary lymphoid tissues.

Mode of replication of lymphocytic choriomeningitis virus in persistently infected cultivated mouse L cells.

During persistent infection of mouse L cells with strain Armstrong lymphocytic choriomeningitis virus, the latter undergoes characteristic changes, including loss of mouse pathogenicity and failure to form plaques on cultivated cells. We call this virus L(Arm) and have analyzed transcription and translation of its S-RNA, which codes for the viral nucleoprotein (NP) and the glycoprotein precursor (GP-C). In L(Arm) virus-infected L cells, S-RNA and genomic-sized viral complementary S-RNA (VC-S-RNA) were detected and, in addition, considerable quantities of shortened molecules of either species. The cells' content of NP was high, but they contained little GP-C; instead, a viral glycoprotein with MW 65,000 was present. We propose a hypothesis in which it is assumed that along the VC-S-RNA there is more than one recognition site for the viral RNA-dependent RNA polymerase, which leads to the generation of truncated forms of S-RNA, VC-S-RNA, and mRNA for GP-C; this, in turn, results in relative overproduction of NP and relative underproduction of GP-C as well as the emergence of a new form of viral glycoprotein.

IFN-gamma production in tissues of mice during acute infection with lymphocytic choriomeningitis virus.

As shown by a single-cell solid-phase ELISA, splenocytes as well as liver lymphoid cells from unmanipulated specific-pathogen free mice synthesized and released IFN-gamma. Synthesis of this lymphokine could not be demonstrated either on the transcriptional level by Northern blotting or immunocytochemically. Thus, IFN-gamma is constitutively produced in mice, although in low quantities. During acute infection with lymphocytic choriomeningitis virus, IFN-gamma mRNA became detectable in spleen and brain but not in the liver. In spleens and livers of these mice, the numbers of cells synthesizing the lymphokine were increased and many were seen in foot tissue undergoing a delayed-type hypersensitivity reaction after intraplantar inoculation of the virus. In contrast, few IFN-gamma-producing cells were found in the inflammatory infiltrates of leptomeninges and choroid plexus after intracerebral infection.

Virus-specific delayed-type hypersensitivity (DTH). Cells mediating lymphocytic choriomeningitis virus-specific DTH reaction in mice.

We had previously shown that the local lymphocytic choriomeningitis virus-induced delayed-type hypersensitivity (DTH) reaction in mice consists of two well delineated phases that are mediated by CD8+ and CD4+ T lymphocytes, respectively. These findings have been confirmed and extended by showing that the first CD8+ cell-dependent part of the response was enhanced by either the presence of CD4+ cells or systemic treatment with IL-2 and that it developed in the absence of detectable numbers of mononuclear phagocytes, whereas the later CD4+ cell part required monocytes or related elements. Furthermore, in the DTH reaction that was elicited with noninfectious viral Ag in mice previously immunized by infection, only the CD4+ cells participated. Thus, the two phases of the lymphocytic choriomeningitis-viral DTH reaction are principally different, which has to be taken into account when trying to assess the relevance of DTH during this virus infection.

Mechanism of recovery from acute virus infection. IX. Clearance of lymphocytic choriomeningitis (LCM) virus from the feet of mice undergoing LCM virus-specific delayed-type hypersensitivity reaction.

As shown previously, after inoculation into the footpad of a mouse the lymphocytic choriomeningitis (LMC) virus multiplies locally. Beginning on day 6 or 7 after infection, the foot undergoes a delayed-type hypersensitivity (DTH) reaction which consists of two distinct phases that are mediated by CD8+ cells and CD4+ cells, respectively, and at about the same time the virus is eliminated. In general, for terminating infection of the mouse with LCM virus the CD8+ cytotoxic/suppressive T lymphocyte (CTL) is essential; we have now determined the cells that mediate control of the virus in a tissue undergoing a specific DTH reaction. Depletion, in infected mice, of all T lymphocytes by treatment with anti-Thy-1 monoclonal antibody prevented virus elimination from the foot, and the same was true when the CD8+ CTLs were removed. Depletion of the CD4+ helper/suppressor subset only marginally impaired the ability of the mice to rid themselves of the virus. The conclusion that here too the principal antiviral element is the CD8+ CTL was confirmed by experiments in which footpad-infected mice were adoptively immunized with virus-immune splenocytes from syngeneic mice selected for subclasses of T lymphocytes, or from mice differing in defined regions of the major histocompatibility complex (MHC), and also by experiments in which monocytes were virtually absent. However, CD8+ CTL alone or cells from MHC recombinant mice with identity in class I loci were never as antivirally active as unseparated splenocytes from syngeneic donor mice. Since the CD8+ cells' performance could be optimized by interleukin-2, we assume that the CD4+ T lymphocytes function as accessory cells; the same probably applies to monocytes.

Mechanism of recovery from acute virus infection. VIII. Treatment of lymphocytic choriomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic T lymphocytes and virus elimination.

In acutely infected mice the lymphocytic choriomeningitis (LCM) virus multiplies to high titers in essentially all tissues. Around day 6, virus clearance sets in, which has previously been shown to be mediated by CD8+ cytotoxic T lymphocytes (CTL), probably by releasing (or inducing other cells to release) anti-viral cytokines. To ascertain whether interferon-gamma plays a role, infected mice were injected once i.v. with monoclonal antibody known to neutralize this lymphokine, and the effect this had on both termination of the infection and development of LCM virus-specific CTL was determined. Administration 1 day after infection blocked virus elimination from spleen and liver and decreased the generation of CTL; also, limiting dilution analysis revealed absence of activation of CTL precursors. In contrast, when the antibody was given 3 days after or 1 day before the virus, neither clearance nor generation of CTL was measurably affected. Furthermore, the antiviral effect of immune spleen cells after their transfer into infected recipients was not altered by treatment of the latter with monoclonal antibody. We conclude that in the generation of LCM virus-specific CTL an early event is dependent on constitutively produced interferon-gamma; when its activity is blocked, CTL do not mature, resulting in the mouse's inability to terminate the infection.