Significant frequency of cytotoxic T lymphocyte precursor cells specific for TNP-modified allogeneic cells in normal lymphocytes

It was tested whether the cytotoxic T-lymphocyte precursor (CLP) repertoire in normal mice is biased toward recognizing foreign antigen in association with self H-2 as opposed to allogeneic H-2. The frequencies of CLPs in normal mice (H-2b,k,d) specific for TNP-modified syngeneic and TNP-modified allogeneic cells have been compared by limiting dilution analysis. Normal spleen cells were cultured at a limiting dilution with TNP-modified (TNP-self) or TNP-modified allogeneic (TNP-allo) stimulator cells. Cultures were split into four aliquots and assayed against TNP-self, TNP-allo, unmodified syngeneic, and unmodified allogeneic Concanavalin A blast targets and classified for cytotoxic activity directed against TNP-self, TNP-allo, and allo H-2 determinants. In disagreement with our expectations from the literature, the frequencies of CLPs in H-2b and H-2d responder cells recognizing TNP-modified H-2k were higher than the frequencies of CLPs recognizing TNP-self. There was no clear preference for TNP-self in the case of H-2b responder and H-2d allogeneic cells, nor vice versa. Only in the case of H-2k responder cells was there a distinct preference for TNP-self. The significance of a considerable number of TNP-specific, allo H-2-restricted CLPs in normal lymphocytes is discussed.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Detection of shared H-2Kk and H-2Dk antigens that function as self determinants for cell-mediated lympholysis to trinitrophenyl-self

Spleen cells from C3H/He mice immunized in vivo to trinitrophenyl (TNP)-self were sensitized in vitro to TNP-self. These spleen cells displayed strong lysis on TNP-modified H-2D end-matched (Kd-Dk) targets as well as enhanced cytotoxicity against H-2 matched (Kk-Dk) or H-2K end-matched (Kk-Dd) target cells. Cold target-blocking studies showed that the lysis of TNP-Kd-Dk targets could be blocked by the addition of TNP-modified Kk-Dk, Kk-Dd Kk-Db, or Kd-Dk, but not by TNP-modified Kd-Dd, Kb-Db and Kq-Kq spleen cells. These results demonstrate that the lysis of TNP-Kd-Dk targets is not due to cross-reactive clones against TNP-Kd-Dd, Kb-Db or Kq-Dq antigens. Inhibition of the TNP-Kd-Dk target lysis by TNP-Kk-matched (Kk-Dd or Kk-Db) as well as TNP-Dk-matched (Kd-Dk) blockers also reveals that this target is lysed by clones directed against shared antigens between Kk-TNP and Dk-TNP, indicating that no cytotoxic response restricted for Dk-TNP only could be detected even after in vivo priming.     

H2Kk can influence whether cytotoxic T lymphocytes recognize trinitrophenyl in association with H2Dk unique or H-2Kk and H-2Dk shared self determinants

The present study investigates the effect of trinitrophenyl- (TNP) modified H-2Kk (TNP-Kk) antigens on the generation of anti-TNP-Dk restricted cytotoxic T lymphocyte (CTL) responses. C3H.OH mice were primed to TNP-self by skin-painting with trinitrochlorobenzene, and spleen cells from these primed mice were subsequently stimulated in vitro with TNP-self. The effector cells generated exhibited appreciable lysis of TNP-modified C3H.OH blast target cells. Cold target inhibition studies demonstrated the generation of two effector cell populations: one that recognizes TNP in association with unique Dk self determinants, and one that recognizes TNP in association with self determinants shared between TNP-Kk and TNP-Dk. This was in contrast to primed C3H/He spleen cells, which did not generate CTL that recognized TNP in association with unique Dk self determinants. When spleen cells from (C3H/He x C3H.OH)F1 mice primed to TNP were stimulated in vitro with TNP-C3H.OH cells, unique Dk self determinants were recognized in association with TNP. However, in vitro stimulation of the same F1 responding cells with TNP-C3H/He or TNP-F1 cells failed to elicit CTL that utilized these Dk-unique self determinants. The findings of this study demonstrate that unique or shared H-2Dk determinants can be differentially utilized by CTL populations, depending on the H-2 alleles expressed by the stimulator cells.     

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.     

Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.     

Heterogeneity of the lymphokine-activated killer cell phenotype.

Lymphokine-activated killer cells (LAK) are functionally defined by their ability to mediate the MHC-unrestricted lysis of a range of tumor targets, while sparing normal cells. They can also lyse TNP-modified normal syngeneic lymphoblasts. We show here that lysis of TNP-modified targets is mediated by CD8+ LAK in a self-MHC-restricted manner, whereas lysis of tumor targets is largely by CD8- LAK and is MHC-unrestricted. LAK generated from the immune-deficient strains Balb/c nude and C.B-17 scid lyse tumor targets as effectively as LAK from normal mice but do not lyse TNP-modified normal targets. Further, lysis of TNP-modified targets, but not tumors, can be inhibited by antibody to the T cell receptor complex. These experiments strongly suggest that recognition of TNP-modified targets is not accomplished by the same mechanism as that of tumors. Rather, they are consistent with recognition of TNP-modified targets by CD8+ LAK cells being mediated via recognition through the T cell receptor.     

Quantitative studies on the precursors of cytotoxic lymphocytes. IV. Specificity and cross-reactivity of cytotoxic clones.

Murine cytotoxic lymphocyte precursor cells (CLP) appear to be specificity restricted, yet 2 to 4% of the total pool appears to be activated in a response against a single foreign H-2 alloantigen. The simplest model that provides enough CLP for all possible reactions is that H-2 antigens have cross-reacting determinants. Experimentally, we observed that activation of RNC-nu/+ (H-2k) LN cells with H-2b spleen cells activated 1200 to 1800 CLP per 10(6) LN cells capable of lysing H-2b target cells. Of these, 3 to 4% could also lyse H-2d target cells. In addition, a small number of clones (approximately 25/10(6) LN cells) were activated which could lyse H-2d but not H-2b target cells. However, the total number of such apparent nonspecifically activated clones is small since greater than 80% of all the CLP activated on culturing with H-2b alloantigens will lyse H-2b targets. The results are discussed in terms of specificity restriction and possible subpopulations of CLP.     

Helper cells in the autologous mixed lymphocyte reaction. I. Generation of cytotoxic T cells recognizing modified self H-2

The autologous mixed lymphocyte reaction (AMLR) in mice measures the proliferative response of T cells to determinants on syngeneic non-T spleen cells. Normally, cytotoxic T lymphocytes (CTL) are not generated in this reaction. However, the addition of trinitrophenyl-modified mitomycin C-treated syngeneic T cells (TNP-Tm) to the AMLR results in the generation of TNP-specific CTL but does not alter the proliferative response. Significant cytotoxic activity is not detectable against TNP in association with Ia unless TNP is present on cells bearing those determinants. Thus, if unselected spleen cells are TNP-modified and used as stimulators in the AMLR, the proliferative response is enhanced and CTL are generated that recognize TNP in association with K, D, and I region-encoded determinants. The CTL generated in the AMLR are H-2 restricted and dependent on the presence of adherent cells in the sensitization cultures. The experiments presented here suggest that the AMLR can provide the help necessary for generating cytotoxic T cells in vitro.     

Tolerance to Mls-disparate cells induces suppressor T cells that act at the helper level to prevent in vivo generation of cytolytic lymphocytes to hapten-altered self

We have been examining the mechanisms that control in vivo development and down regulation of cytolytic T lymphocytes (CTL) to trinitrophenyl (TNP)-altered self antigens. In vivo generation of hapten-specific CTL requires an auxiliary antigenic stimulus, which can be provided by H-2 compatible but Mls-disparate cells. These experiments were designed to study the effect of tolerization with such Mls-disparate cells on CTL development. C3H/HeN (H-2k, Mlsc ) mice sensitized in the footpads with C3H-TNP spleen cells plus CBA/J (H-2k, Mlsd ) spleen cells develop CTL in the draining lymph nodes that will lyse 51Cr-labeled TNP-modified C3H targets. However, we have found that if C3H/HeN mice are given tolerizing doses of CBA/J spleen cells 5 to 7 days before sensitization, a splenic suppressor T cell (Ts) appears. This Ts will suppress CTL development in its tolerant host, and can be transferred adoptively to function in naive mice. Ts and its precursor are cyclophosphamide insensitive and therefore different from the naturally existing suppressor cell present in mice. When triggered by cells with Mlsd , the Ts produces a factor (TsF) that hinders helper factors from functioning in an in vitro CTL assay. Furthermore, TsF acts to prevent utilization of IL 2 by an IL 2-dependent cell line. Thus, evidence has been provided that the in vivo generation of CTL toward hapten-altered self can be down regulated at the level of helper signals by a Ts. The latter is inducible by the Mls-disparate cells that are needed at a different site to trigger the helper factors in this CTL system.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Respiratory syncytial virus infection in C57BL/6 mice: clearance of virus from the lungs with virus-specific cytotoxic T cells.

We describe respiratory syncytial virus (RSV)-specific cytotoxic T-cell (CTL) lines and clones developed from the spleens of C57BL/6 and BALB/c mice. Line 7 and clones derived from it were H-2Kb restricted, whereas line 12 had both Kb and Db components. Both lines, and all the clones except one, could lyse targets infected with either strain A or strain B RSV. Line 7 or 7-11E1 cells (8 x 10(6) to 10 x 10(6) given intravenously cleared RSV from the lungs of infected mice. There was no morbidity or mortality in any of the infected mice whether or not they received T cells. The C57BL/6 mouse is a useful model system in which to study the role of the CTL response in protective immunity to RSV. CTL lines and clones can mediate clearance of RSV from the lungs of normal mice without producing any associated morbidity.     

Specificities of killing by T lymphocytes generated against syngeneic SV40 transformants: studies employing recombinants within the H-2 complex.

T lymphocyte effectors to syngeneic SV40-transformed cells, generated by secondary in vitro sensitization of immune spleen cells, lyse SV40 transformed targets that are syngeneic at the H-2 locus. In this study we have employed recombinants within the H-2 region to examine in detail this H-2 specificity. H-2b effectors were found to lyse SV40-transformed targets from recombinants bearing either H-2Kb or H-2Db.H-2k effectors recognized only SV40-transformed H-2Kk, and not H-2Dk target cells. By using the same protocol for sensitization, no effector cells could be detected in H-2d mice. Effectors generated in H-2 recombinant mice showed that the response capacity resides with K and D. For example, HTG, which is H-2d except at the D locus (H-2Db), produced effector cells specific for SV40-transformed H-2Db targets. Thus, the secondary in vitro response to SV40 transformants was found to depend only on the K and D alleles and not to be modified by the I region to any measurable extent.     

Specific T-cell-mediated killing of autologous lung tumour cells

The phenomenon that strong syngeneic T-cell-mediated cytotoxicity is observed if killer, stimulator, and target cells share H-2 histocompatibility antigens is called H-2 restriction. Here a syngeneic model system making use of hapten-coupled stimulator and target cells is used to explore whether H-2 restriction is absolute or not. Using TNP-coupled spleen or tumor cells as stimulator or target cells in syngeneic and allogeneic situations, it is shown that neither the induction step nor the effector step of TNP-dependent killing is H-2 restricted. By varying the experimental assay conditions more or less H-2-restricted, TNP-dependent killing can be observed. For instance, suboptimal coupling of TNP to targets may result in H-2-restricted killing. Similarly, the use of spleen cell targets as opposed to spleen blast cells or tumor cells may result in H-2-restricted lysis. In contrast optimal coupling of TNP to sensitive target cells and coupling of TNP to cells with certain H-2 haplotypes may lead to significant TNP-dependent killing which is not H-2 restricted. Since hapten-coupled cells lacking H-2 are neither stimulators nor targets these results suggest that the T-cell receptor recognizes TNP-modified H-2 antigens simply as nonself-H-2. Thus hapten coupling of syngeneic cells appears to lead to a histocompatibility antigen change similar to the situation in an allogeneic cytotoxic reaction. Experiments are presented which support this view showing that TNP-coupled and uncoupled syngeneic or allogeneic stimulator and target cells cross-react. For instance allogeneic sensitization may lead to killing on TNP-coupled targets syngeneic to the effector cells and TNP-coupled stimulator cells syngeneic to the effector cells may induce killing on uncoupled syngeneic targets. TNP-dependent cytotoxicity can therefore be envisaged as a kind of allogeneic reactivity due to modification of H-2 antigens by the TNP coupling. This conclusion may have bearing on other model systems in which syngeneic killing appears to be H-2 restricted. In support of this possibility it is shown that allogeneic sensitization may lead to priming of memory cells able to respond to minor histocompatibility antigens.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

T cell clones specific for IgG2a of the a allotype: direct evidence for presentation of endogenous antigen

T cell clones specific for IgG2a of the alpha allotype have been isolated from C57BL/6J mice. Antigenic determinants recognized by these clones were localized by using a panel of hybrid IgG2b-IgG2a myeloma proteins. These experiments provide evidence for two distinct antigenic sites, one located in a segment encompassing the hinge region and most of the CH2 domain, and the other in a segment spanning the CH3 domain and the C-terminal eight residues of the CH2 domain. As judged by their failure to respond in the presence of B6.C-H-2bm12 spleen cells, all the clones recognize determinants created, in part, by the I-A beta chain. A strong proliferative response was observed in the presence of spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP, and BALB.B. Experiments testing reactivity directed toward spleen cells from appropriate allotype-congenic mouse strains demonstrated that this response was controlled by Igh-linked genes. These results clearly indicated 1) that Igh-1a-specific T cell clones are stimulated by endogenously synthesized IgG2a, and 2) these T cells recognize shared determinants expressed on IgG2a molecules of various strains. These experiments thus provide strong evidence for presentation of self antigens under normal physiological conditions.     

Primary in vitro cytotoxic response of F1 T lymphocytes against parental antigens.

Spleen cells from adult female (AKR/J x BALB/(c)F1 mice can respond to mitomycin C-treated spleen from AKR/J mice and can generate effector CTL in a 5-day primary in vitro culture. The response is comparable in magnitude to the response to allogeneic H-2K or H-2D antigens. The response is T cell mediated and is directed to antigen(s) present only on the parental cells. The target cell must be homozygous at H-2Kk to be lysed and H-2Dk antigens do not serve as a target in this response. Spleen cells from (B10.BR x B10.D2) hybrids that have been stimulated with AKR/J lyse B10.Br as well as AKR/J target cells. Similar H-2k/d hybrid F1 anti-H-2k parent responses are seen in certain other strain combinations. A number of possible interpretations of these responses are discussed.     

The products of transferred H-2 genes express determinants that restrict hapten-specific cytotoxic T cells

Cell lines into which cloned H-2 genes had been introduced (i.e., transformants) were used to correlate the genes and their products that are capable of functioning as H-2 restriction elements for hapten-self-(AED and TNP) specific cytotoxic T cells (CTL). These transformants provided a unique system in which major histocompatibility restricted (MHC) T cell recognition could be examined by using cells that express only H-2Ld or only H-2Dd gene products. BALB/c (H-2d) anti AED-self CTL lysed both the H-2Ld and Dd transformants, but not parental, i.e., untransformed, cells. The AED-self lysis of the Ld and Dd transformants was shown to be specifically inhibited by anti-H-2Ld and anti H-2Dd monoclonal antibody, respectively. In contrast to these results, BALB/c anti TNP-self CTL were found to lyse readily the Dd but not Ld transformed lines, supporting reports indicating that H-2Ld-restricted TNP-self CTL could not be detected. The results of this study thus demonstrate that the cell surface products encoded by these transferred MHC class I genes contain self determinants recognized by CTL.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)