Membrane phenotype of murine effector and suppressor T cells involved in delayed hypersensitivity and protective immunity to herpes simplex virus

The membrane phenotype of T cells involved in delayed hypersensitivity (DH), protective immunity, and suppression of delayed hypersensitivity to herpes simplex virus (HSV) has been determined. T cells from immune lymph nodes transferring DH and antiviral immunity to normal recipients were characterized as Lyt 1+2-. There appeared to be no detectable antiviral role for Lyt 1-2+ cells in the transferred cell suspension. Splenic T cells suppressing the induction of DH to HSV were characterized as being both Lyt 1+2- and Lyt 1-2+ 4 weeks after their induction. At earlier times, i.e., after 7 days, the suppression was mediated solely by the Lyt 1+2- population. Thereafter, a progressive increase in the contribution of the Lyt 1-2+ suppressor was observed. Both the early and later phases of suppression were due to I-J positive cells. The nature of the two suppressor cell types is discussed in relation to suppressor cell "cascades" and to the pathogenesis of herpes simplex virus infection.     

Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology.

Herpetic stromal keratitis (SK), a frequent cause of visual impairment, is considered to represent an immune-mediated inflammatory response to persistent herpes simplex virus virions or subcomponents within the corneal stroma. The experimental disease in mice involves the essential participation of T lymphocytes, but the role of T-lymphocyte subsets in either mediating or controlling the disease is uncertain. In this report, rat monoclonal antibodies were used to selectively deplete mice in vivo of CD4+ (helper-inducer) and CD8+ (cytotoxic-suppressor) T-cell populations and the effect on herpetic SK was evaluated. As measured by flow cytometry, mice treated with anti-CD4 monoclonal antibody (GK 1.5) were greater than 95% depleted of CD4+ T lymphocytes and mice treated with anti-CD8 monoclonal antibody (2.43) were 90% depleted of CD8+ T lymphocytes. Depleted and nonspecific mouse ascites-treated control mice were infected topically on the corneas with herpes simplex virus type 1, and the induction of various immune parameters during the acute infection was evaluated. CD4+-depleted mice failed to produce either a significant antiviral antibody or delayed-type hypersensitivity response but were capable of producing normal cytotoxic T-lymphocyte responses. In contrast, CD8+-depleted mice produced antiviral antibody and delayed-type hypersensitivity responses comparable with those in control animals, but cytotoxic T-lymphocyte responses were markedly reduced. Clinical observations of the corneas revealed that SK in CD4+-depleted mice was significantly reduced, whereas in CD8+-depleted mice SK developed more rapidly, was more severe, and involved a greater percentage of mice. These observations implicate the CD4+ T-lymphocyte subset as the principal mediators of SK and CD8+ T lymphocytes as possible regulators that control the severity of SK.     

Immunity to herpes simplex virus type 2. Suppression of virus-induced immune responses in ultraviolet B-irradiated mice

Ultraviolet B irradiation (280 to 320 nm) of mice at the site of intradermal infection with herpes simplex virus type 2 increased the severity of the herpes simplex virus type 2 disease and decreased delayed-type hypersensitivity (DTH) responses to viral antigen. Decrease in DTH resulted from the induction of suppressor T cells, as evidenced by the ability of spleen cells from UV-irradiated mice to inhibit DTH and proliferative responses after adoptive transfer. Lymph node cells from UV-irradiated animals did not transfer suppression. DTH was suppressed at the induction but not the expression phase. Suppressor T cells were Lyt-1+, L3T4+, and their activity was antigen-specific. However, after in vitro culture of spleen cells from UV-irradiated mice with herpes simplex virus type 2 antigen, suppressor activity was mediated by Lyt-2+ cells. Culture supernatants contained soluble nonantigen-specific suppressive factors.     

Augmentation of immunity to herpes simplex virus by in vivo administration of interleukin 2

The immune mechanisms responsible for recovery from herpesvirus infections are multiple and include a principle role for aspects of T cell immunity. Our investigations add further support for this notion. We show that the ability of immune lymphocytes from animals infected i.p. 6 wk previously with herpes simplex virus type one (HSV-1) clear virus more effectively when interleukin 2 (IL 2) is injected into recipients of the adoptive transfers. Mice were treated on two consecutive days with cyclophosphamide and infected in the pinnae with 4 X 10(6) plaque-forming units of HSV-1. Three hours post-infection lymphocyte populations were injected i.v., and after a further 3 days the pinnae were removed, homogenized, and the content of infectious virus assayed. Purified IL 2 obtained from EL-4 cells either was given i.v. 2 hr before and 24 and 48 hr after cell injection or was given subcutaneously 2 hr before and 3, 24, and 48 hr after cell injection. The latter three injections were given i.p. and suspended in 15% gelatin. The immune lymphocyte cell populations were splenocytes and were either injected immediately after preparation of cell suspensions or after 5 days in vitro secondary stimulation with HSV-1. This latter cell population showed greater viral clearance activity, a function shown previously to be a property of Lyt-2+ cells. The clearance activity of cells was markedly enhanced in animals given IL 2 but only with a regimen that included injections in gelatin, a procedure that enhances in vivo circulation time of IL 2. The cell involved in clearance was a T cell and principally the Lyt-2+ subset. Treatment of recipient mice with anti-asialo GM-1 did not affect the clearance efficiency, indicating that NK cells were not responsible for the observed effect. Our experiments indicate that IL 2 may provide an important regulator of immune function in vivo and may warrant its investigation as a therapeutic agent to enhance antiviral immunity in certain circumstances.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

Anti-glycoprotein D monoclonal antibody protects against herpes simplex virus type 1-induced diseases in mice functionally depleted of selected T-cell subsets or asialo GM1+ cells.

Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for the functional loss of one but not two T-lymphocyte subpopulations.     

Virus-lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo.

The lifelong persistence of lymphocytic choriomeningitis virus (LCMV) in neonatally or congenitally infected mice is accompanied by a suppression of virus-specific T-cell responses. In this study, we identified the subset of T cells infected with LCMV during persistent infection in vivo. Using specific monoclonal antibodies to separate the different lymphocyte cell populations and employing both an infectious center assay and immunofluorescence to detect the virus, we found that infection is confined primarily to T cells of the helper subset (L3T4+ Lyt2-), with minimal involvement of cytotoxic T cells (Lyt2+ L3T4-) and mature B cells. About 0.54 to 1.1% of L3T4+ T cells were producing the virus, as determined by the infectious center assay. In contrast, 9.1 to 12.2% of these L3T4+ T cells contained viral antigen, as shown by immunofluorescence studies. This finding suggested that, at any given time, a substantial number of infected T cells were not producing infectious virus. This infection of T helper cells may be involved in the suppression of LCMV-specific T-cell responses observed in persistently infected mice.     

Thy1+ NK [corrected] cells from vaccinia virus-primed mice confer protection against vaccinia virus challenge in the absence of adaptive lymphocytes

While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+) subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+) NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.     

Thy1+ Nk Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1⁺ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1⁺ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.     

Cell-mediated immunity in herpes simplex virus-infected mice: H-2 mapping of the delayed-type hypersensitivity response and the antiviral T cell response

An adoptive transfer system was used to investigate the H-2 restriction of delayed-type hypersensitivity (DTH) to herpes simplex virus. A successful DTH transfer was achieved when donor and recipient were compatible at the I-A region, with K and D region compatibility unnecessary. However, the rapid clearance of infectious virus from the inoculation site was found only when the donor and recipients were compatible at H-2K (and presumably D) and I-A regions.     

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.     

Human CD4+ CD25 high cells suppress proliferative memory lymphocyte responses to herpes simplex virus type 2

Lymphocytes with the regulatory CD4+ CD25+ phenotype frequently suppress memory T-cell responses. Murine herpes simplex virus type 1 (HSV-1) models have shown that CD4+ CD25+ cells can limit immunity-mediated corneal damage but slow viral clearance. We investigated the effect of CD4+ CD25+ cells from healthy HSV-2-infected humans on recall proliferative (CD4) responses to HSV-2. Depletion and reconstitution experiments were consistent with a suppressive effect of autologous blood-derived CD4+ CD25+ cells for whole HSV-2 antigen. Regulatory T cells may modulate human CD4 memory responses to HSV-2 and influence their antiviral and inflammatory functions.     

Transgenic Expression of IFN-γ in the Eye as a Model for Studying Ocular Inflammatory Disease

To study the effects of IFN-γ on intraocular inflammation we used mice with transgenic expression of IFN-γ in the retina of the eye. These transgenic mice (rhoγ) were challenged intraocularly with soluble and cellular antigens and then studied for development of uveitis and delayed-type hypersensitivity reaction after a second intracutaneous challenge with the same antigen. Further experiments tested the influence of IFN-γ on infection with herpes simplex (HSV) by intraocular virus inoculation in rhoγ mice and rhoγ mice crossed with MHC class I or class II deficient mice. Pathology was studied by morphology immunocytochemistry and electron microscopy. Growth of HSV was assessed by plaque assay on Vero cells. Rhoγ transgenic mice produced IFN-γ in the photoreceptors of the retina and secreted the cytokine into the vitreous of the eye. These animals suffered from cellular infiltration of the eyes, cataracts, and degeneration of the photoreceptors. In contrast to control mice, transgenic mice had increased uveal inflammation and developed delayed-type hypersensitivity after intraocular challenge with alloantigens such as allogeneic splenocytes and inactivated virus. Rhoγ mice also experienced protection from infection with herpes simplex virus-1 (HSV-1) and HSV-2, suffering a less severe course of disease than control mice. Cytokine-induced protection was not based on viral replication block and did not require simultaneous expression of MHC class I and class II. The rhoγ transgenic mouse provided a model suitable for testing mechanisms of IFN-γ action in the eyein vivo.The surprising results offer new arguments in the evaluation of cytokine effects.     

Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells

The lifelong chronic lymphocytic choriomeningitis virus (LCMV) infection established in neonatally or congenitally infected mice can be eliminated by adoptive transfer of lymphoid cells from LCMV-immune mice. In this study, we have identified the effector cells mediating the clearance of persistent and disseminated LCMV infection. Using mice that are recombinant in the H-2 region and by selective depletion of lymphocyte subpopulations, we show that viral clearance was mediated by LCMV-specific Lyt2+ L3T4- T cells that are restricted to the class I genes of the major histocompatibility complex. In addition, our results show a requirement for host-derived bone marrow cells for the effective elimination of virus from the liver. These studies emphasize the importance of virus-specific T cells and an intact bone marrow function in viral clearance.     

Identification of suppressor T cells in virgin non-responder spleen cells responsible for primary unresponsiveness to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

T cell subsets that regulate antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in mice that are Ir gene non-responders have been further characterized. We previously defined several T cell subsets in GAT-primed non-responder mice. The Lyt-2+ suppressor-effector T cells suppress responses to GAT and GAT complexed to methylated BSA (GAT-MBSA). The Lyt-1+ cell population is complex and can be separated into I-J- Th cells, which support responses to GAT and GAT-MBSA. After priming, the Lyt-1+, I-J+ cell population contains suppressor-inducer cells that activate precursors of suppressor-effector cells to suppress responses to GAT and GAT-MBSA as well as Ts cells that directly inhibit responses to GAT but not GAT-MBSA. By contrast, the Lyt-1+ cells from virgin mice contain only cells that directly suppress responses to GAT but not GAT-MBSA. The major question addressed in the present studies was whether the Lyt-1+, I-J+ Ts cells in virgin and primed mice and the suppressor-inducer cells in GAT-primed mice were functionally and serologically distinct subsets. The studies used mAb and panning procedures to separate cell populations and inhibition of PFC cell responses to functionally define the activity of the cell populations. We used the following two mAb that were raised by immunizing rats with GAT-specific suppressor factors: 1248A4.10 (known to react with suppressor-inducer cells) and 1248A4.3, another reagent from the same fusion. Lyt-1+ cells from virgin spleens contained Ts cells that were A4.10-, A4.3+ and no suppressor-inducer T cells, whereas Lyt-1+ cells from GAT-primed spleens contained Ts cells that were A4.10-, A4.3+ as well as A4.10+, A4.3- suppressor-inducer cells. Thus, the Lyt1+, I-J+ cell subset can be divided into two functionally and serologically distinct subsets, direct Ts cells (1248A4.3+), which suppress responses to GAT but not GAT-MBSA, and GAT-primed suppressor-inducer T cells (1248A4.10+).     

In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus.

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

Immune T cells can protect or induce fatal neurological disease in murine lymphocytic choriomeningitis

Adoptively transferred immune spleen cells induce fatal neurological disease in cyclophosphamide-suppressed recipients injected intracerebrally (ic) with a large, but not small, dose of neurotropic lymphocytic choriomeningitis (LCM) virus. The elimination of virus from brain in the latter group, which survives without developing symptoms, depends upon the presence of Lyt 2+ lymphocytes. However removal of Lyt 2+ subset which is cytotoxic in vitro does not diminish the severity of the inflammatory process in vivo, though the onset of clinical disease is delayed in mice given Lyt 2-depleted populations and a larger ic dose of virus. The present findings are consistent with the idea that fatal LCM results from acute, synchronous damage to key functional cells in the central nervous system by virus-immune Lyt 2+, lymphocytes. Even so, if the number of virus-infected CNS cells is still relatively small at the time of T cell invasion, neurological symptoms are not recognized and the mice survive.