Heterogeneity of immunologic memory T cells specific for H-2 antigens and detection of their receptors

The in-vivo-induced memory T cells (MC) of mice, specific to H-2 antigens, are assayed by the generation of the secondary cytotoxic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) activated by heat-killed stimulator cells. The MC are shown to adhere selectively to the corresponding target monolayer that gives rise to both the loss of MC activity in the population of non-adherent lymphocytes and gain in MC activity in the population adherent and eluted from the same monolayer. In addition to the revealing of MC H-2 antigen-binding receptors, the absorption-elution technique allows the separation of the MC into two categories: secondary CTL precursors bearing these receptors, and secondary amplifier cells non-adherent to the monolayer and assayed by promotion of the CTL generation from the primary precursors activated in MLC by heated stimulators. The difference in the receptor properties between the primary and secondary CTL precursors raises the possibility that the MC are generated not only in the amplifier cell population but also in the independent CTL precursor population.     

The main difference between the repertoire of receptors of effector T-killers and their secondary precursors (memory cells)

Receptor repertoire analysis of in vivo induced secondary cytotoxic T lymphocyte precursors (pCTL-2) was performed, using the technique of their specific adherence to macrophage monolayers with subsequent elution of the adherent pCTL-2 and their activation by heat-killed donor stimulator cells. The capacity of anti-H-2Kb pCTL-2 receptors to contact H-2Kbm has been revealed only in a minor pCTL-2 component whose progenitors were able to lyse mutant bm1 target cells (TC). Unlike poor cross-reactivity of CTL descended from anti-Kb, pCTL-2 which were eluted from the donor monolayer, CTL-progenitors of anti-Kb pCTL-2 eluted from bm 1 or B10. A (4R) third-party monolayers lyse in equal quantities the donor TC and those third-party TC from which they have been eluted, but fail to lyse other TC. Enrichment of pCTL-2 eluted from bm 1 or B10. A (4R) monolayers is 6- or 12-fold lower, respectively, than after their elution from the donor monolayer. The findings indicate that anti-class I MHC pCTL-2 are separated into fractions, with their receptors being strongly specific (with high affinity) to the particular fragment of the same complex epitope without cross-reactivity to other fragments. These data differentiate pCTL-2 receptors from effector CTL ones which are homogenous in specificity to a whole (single) complex epitope with variable degree of complementarity. A cardinal distinction of receptor repertoire between CTL, pCTL-2 and suppressor T cells specific to the same class I MHC molecule and alteration of the active site during pCTL-2 differentiation have been suggested.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Allogeneic lymphocyte induction in vivo of highly active T-killers specific for the molecule of the H-2 class-I complex and the detection of their receptors in vitro

The optimal conditions are found for in vivo irradiated lymphocyte induction of high cytotoxic T lymphocytes (CTL) specific to the H-2Kb molecule with a subsequent monoculture differentiation. B10.D2(R101) CTL have a pronounced excess as compared to B10.A (4R) CTL, with respect to lysis intensity of the same target cells (TC), and requires a lower term of the monoculture incubation in spite of their specificity to the same H-2Kb molecule. As the susceptibility of TC for CTL lysis is higher (M phi as compared to EL-4 thymoma cells), CTL are much more inactivated with the monoclonal antibodies to Lyt-2 and Lyt-3 antigens without complement. Anti-H-2Kb CTL differentiated in the monoculture cross react to TC bearing third-party H-2 molecules (Kk, Dq, Dk). Unlike a stable CTL adherence to the donor M phi monolayer, nonspecific CTL adherence to the syngeneic M phi monolayer declines in the presence of EGTA, and as a result of repeated detachment of lymphocytes. The findings give rise to study receptor affinity expressed on the in vivo induced CTL surface, the CTL receptor monoclonal antibody and the CTL differentiation factor.     

T helper cell lines that augment in vivo cytotoxic T-cell responses to minor alloantigens

We have investigated the ability of long-term cultured T helper (Th) cell lines to help an in vivo cytotoxic T lymphocyte (CTL) response to non-H-2 alloantigens (minor antigens). Th cell lines specific for various single or undefined minor antigens were selected by regular restimulation with antigen in vitro. They were antigen specific and H-2 restricted in proliferation assays and were found to be able to help primary CTL responses to multiple minor antigens and secondary CTL responses to single minor antigens. Although the Th were antigen specific they did not determine the specificity of the CTL. Th cells were both necessary and limiting for an effective CTL response indicating that "helper-independent" CTL are not in themselves sufficient to generate a strong in vivo response. Under conditions where a CTL response was clearly H-2 restricted, Th cells were not. Thus, the Th cells appeared to be activated by reprocessed antigen rather than antigen on the surface of the injected antigenic cells even though the CTL themselves reacted directly to the injected antigen.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

Antigen-independent activation of memory cytotoxic T cells by interleukin 2

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.     

Beta (1-3) glucans as a probe for T cell specific immune adjuvants. II. Enhanced in vitro generation of cytotoxic T lymphocytes.

The capacity of five antitumor polysaccharides (PS) of the β (1–3) glucan type, lentinan, pachyman, pachymaran, carboxymethylpachymaran and hydroxyethylpachyman to augment the in vitro generation of alloreactive cytotoxic T lymphocytes (CTL) in a primary murine mixed lymphocyte culture was tested in detail. All of the PS tested, each at its own optimal concentration, induced enhanced alloreactive CTL responses. Lentinan, pachymaran and hydroxyethylpachyman increased the magnitude of the in vitro CTL responses up to 28 fold. The PS tested appeared to enhance the responsiveness of alloreactive prekiller T cells rather than the immunogenicity of the stimulator cells. Kinetic experiments indicated that in vitro CTL responses could be augmented when PS were added as late as 72 hr after initiation of the cultures, suggesting that PS influences the differentiation of antigenically triggered CTL precursors into cytotoxic effector cells. PS also augmented in vitro the generation of H-2 restricted hapten-specific CTL. The results indicate that the PS tested are effective immune adjuvants of both alloreactive CTL responses and H-2 restricted CTL responses specific to foreign antigens.     

T cell-mediated cytotoxicity against dengue-infected target cells

A cytotoxic T lymphocyte (CTL) response to dengue virus-infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H-2k haplotype compared to H-2d, and H-2 compatibility was required between CTL and target cells. CTL generated showed some cross-reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.     

The cellular mechanism of maintenance of neonatally induced tolerance to H-2 class I antigens

We used limiting dilution analysis protocols to investigate the mechanism by which in vitro cytotoxic T lymphocyte (CTL) hyporeactivity is maintained in adult mice that had been neonatally tolerized to major histocompatibility complex-encoded antigens. Class I molecules, presented on donor cells having an H-2 K or D region haplotype difference from recipients, readily induce tolerogen-specific CTL hyporeactivity. All attempts to identify any in vitro effects of active suppressive cells operative in the maintenance of this hyporeactivity have been unsuccessful. We conclude that this cytotoxic deficiency is the consequence of in vivo mediated clonal inactivation of the precursors of tolerogen-specific CTL. A presentation and evaluation of the assumptions inherent in this conclusion are made. In contrast to class I molecules, class II molecules, presented on donor cells having an H-2 I region haplotype difference from recipients, are unable to induce tolerogen-specific CTL hyporeactivity, even when injected neonatally at high doses. This inability of class II molecules to induce CTL tolerance parallels the considerable difficulty of inducing helper T lymphocyte tolerance to class II molecules.     

Recognition of the beta-2 microglobulin-B molecule by a CTL clone

In this report we offer evidence that the beta-2 microglobulin-B (beta 2M-B) molecule is recognized by a cytotoxic T lymphocyte (CTL). Production of GA5, a CTL clone reactive against a membrane antigen with the same strain distribution pattern as beta 2M-B, is described. This clone lysed syngeneic target cells in which native beta 2M-A molecules had been exchanged with beta 2M-B molecules by incubating the cells in serum from beta 2M-B-positive mouse strains. Conversely, the CTL clone GA5 failed to lyse its specific target when beta 2M-B molecules had been exchanged with beta 2M-A molecules by incubating the cells in serum from beta 2M-A-positive, beta 2M-B-negative mouse strains. Strain combinations were chosen so as to limit reactivity to beta 2M and to preclude reactivity to H-3 antigens, thus indicating CTL clone GA5 to be reacting specifically with beta 2M-B.     

The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes.

Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.     

Class I-restricted presentation occurs without internalization or processing of exogenous antigenic peptides

Previous studies have shown that glutaraldehyde-fixed cells can present fragmented, but not native, Ag to class II-restricted T cells. This presumably occurs via direct binding of peptides to class II molecules at the cell surface. More recently, it has been shown that viable target cells can present peptides and endogenous, but not exogenous, protein Ag in association with class I MHC molecules to CTL. We have derived CTL specific for a chicken OVA peptide (OVA258-276) recognized in association with H-2Kb. These CTL recognize target cells that endogenously synthesize OVA and cells "loaded" with native OVA but fail to recognize target cells in the presence of exogenous native OVA. Thus, OVA must be intracellularly located to be processed and presented for CTL recognition. It remains unclear, however, whether exogenous peptides require internalization and further processing by target cells or are able to associate directly with class I molecules at the cell surface for CTL recognition. We provide evidence that glutaraldehyde-fixed cells can present synthetic peptides to H-2Kb- and H-2Db-restricted CTL and that such presentation does not require internalization or processing. The peptides used range in size from 16 to 48 amino acids in length. In contrast, glutaraldehyde-fixed cells are incapable of presenting Ag to CTL specific for influenza nucleoprotein and OVA if the cells are fixed within 1 h of viral influenza infection or loading with OVA. Thus, CTL recognition of antigenic peptides appears to occur via direct binding of peptides to class I molecules at the cell surface and does not require any intracellular processing events.     

Predominant utilization of V beta 8+ T cell receptor genes in the H-2Ld-restricted cytotoxic T cell response against the immediate-early protein pp89 of the murine cytomegalovirus

Cytotoxic T cell responses to the murine Cytomegalovirus (MCMV) were elicited in BALB/c mice (H-2d) by infectious virus. Eight days after infection, MCMV-primed local lymph node T cells were either depleted for T cells expressing a V beta 8+ TCR or separated into V beta 8+ and V beta 8- subpopulations by a cell sorter using the mAb F23.1. T cells were then expanded in vitro under limiting dilution conditions in the presence of IL-2 and in the absence of viral Ag to avoid selection by Ag in vitro. Frequencies of CTL precursors specific for the Immediate-Early-Ag 1 of MCMV and restricted to H-2Ld were determined. L cells of the endogenous haplotype H-2k cotransfected with the genes for MCMV-IE 1 and H-2Ld were used as target cells. Detection of a CTL response required previous priming of the animals by infection in vivo (less than 1/10(6) for nonimmunized animals). In primed animals CTL precursors of this specificity and restriction were three to fivefold more frequent in the V beta 8+ population (1/9.900 to 1/22.300) than in the V beta 8- population (1/57.000 to 1/87.200). Control experiments showed that frequencies were not influenced by the treatment with the anti-V beta 8-antibody and the fluorescein-labeled anti-Ig itself. V beta 8+ and V beta 8- T cells did not reveal any frequency differences when several other responses were determined (TNP-specific self-restricted CTL precursor; Th cells specific for keyhole limpet hemocyanin or Listeria monocytogenes).     

Effects of antigen-feeding on intestinal and systemic immune responses. I. priming of precursor cytotoxic T cells by antigen feeding.

The ability of antigens administered by the intestinal route to alter the cytotoxic T lymphocyte (CTL) population in intestinal and extraintestinal sites was examined by feeding mice tumor cells bearing H-2 or non-H-2 gene-coded cell surface alloantigenic differences. Peyer's patches from mice fed tumor cells differing at H-2 demonstrated a 12-fold increase in specific CTL activity over controls after stimulation of culture but the precursor CTL population in Peyer's patches was not expanded by feeding mice cells bearing minor H gene coded alloantigenic differences. In contrast, the precursor CTL pool in spleen was increased both in mice fed cells bearing alloantigenic differences coded for by H-2 and non-H-2 gene loci. The precursor CTL population in mesenteric lymph nodes (MN) was not expanded significantly by tumor cell feeding. Feeding was nearly as effective as i.p. priming at expanding the Peyer's patch precursor CTL population when tumor cells bearing H-2 differences were used, whereas i.p. priming was more effective than feeding in expanding the precursor CTL population in spleen.     

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation of memory cell-mediated immune responses after secondary infection of mice with pichinde virus

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.     

Estimates of the precursor frequency of cytotoxic T lymhocytes against antigens controlled by defined regions of the H-2 gene complex: comparison of the effect of H-2 differences due to intra-H-2 recombination vs mutation.

Lymph node cells were sensitized in a limiting dilution assay against B10.D2 (H-2d) and the frequency of cytotoxic T lymphocyte (CTL.P) precursors was determined. A mean CTL.P frequency of 0.047% was observed when responding strains differed from the stimulators at the entire H-2 gene complex. When intra-H-2 recombinant strains were sensitized against B10.D2, lower frequencies of CTL.P were observed. Responding strains that differed from the stimulators at the H-2K-end only had 2- to 6-fold more CTL.P compared to strains sensitized against the D-end only. In order to study the CTL.P frequency against minor antigenic differences, the B10.D2 (M504-H-2da) mutant strain, which carries a mutation with an antigenic gain-loss in the D-region of H-2d, was examined. This mutant showed an identical CTL.P frequency against H-2d as H-2D-end recombinant strains. Therefore, this H-2 mutant (M504) has either undergone extensive mutation or the qualitative nature of the antigenic loss in this strain results in a high CTL.P frequency against the strain of origin.