Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes

A polypeptide fragment obtained by CNBr cleavage of the hemagglutinin from A/JAPAN/305/57 influenza virus has been purified by using high performance liquid chromatography. The first five N-terminal amino acids as determined by sequential Edman degradations have localized this peptide to the HA2 subunit of the hemagglutinin between residues 103 and 123. This peptide, denoted HA2(103-23), can generate both proliferative and cytolytic responses from spleen cells of BALB/c mice previously immunized with A/JAPAN/305/57. These results demonstrate that a single nonglycosylated fragment of the influenza hemagglutinin as small as 21 amino acid residues is capable of being recognized as an antigenic determinant to generate influenza CTL from primed precursors.     

The recognition of a viral antigenic moiety by class I MHC-restricted cytolytic T lymphocytes is limited by the availability of the endogenously processed antigen.

The transmembrane hydrophobic domain of the type A influenza A/JAPAN/305/57 (H2N2) hemagglutinin (HA) contains an immunodominant site encompassing amino acids 523-545 (J523-545) recognized by class I MHC-restricted cytolytic T lymphocytes (CTL). Class I CTL of two fine specificity subsets map to this transmembrane (TM) site. One of these CTL subpopulations is subtype specific. These T lymphocytes recognize the site generated during infection of target cells with A/JAPAN/305/57 virus (H2N2) but not target cells expressing the comparable TM site of the influenza A/PR/8/34 virus (H1N1) hemagglutinin (P527-549) after infection with this virus. The other CTL subpopulation is cross-reactive and recognizes the TM site of the A/JAPAN/305/57 HA and the A/PR/8/34 HA with similar efficiency. Analyses of the critical amino acids in the TM site necessary for CTL recognition with the use of synthetic peptides unexpectedly revealed reactivity for the A/PR/8 HA TM site by subtype-specific CTL. This reactivity was only observed with truncated peptides corresponding to a limited portion of the A/PR/8 HA TM site but also required peptide concentrations greater than 10(-7) M. These results suggested either that the endogenously processed A/PR/8 HA TM site generated during infection was larger than the site defined by the truncated cross-reactive peptides or that the concentration of endogenously processed TM site produced during infection was limiting. To distinguish between these possibilities, we expressed in target cells synthetic minigenes encoding only the portion of the A/PR/8 HA transmembrane sites defined by the synthetic peptides. Unlike the peptides, the "preprocessed" endogenous minigene products were not recognized by subtype-specific CTL. These data suggest that the level of available endogenously processed Ag rather than selectivity in the site of fragmentation of newly synthesized Ag may play a critical role in determining whether the complex of the antigenic moiety and class I MHC is efficiently presented to and recognized by class I CTL.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

HA2 subunit of influenza A H1 and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response

Influenza H1 subtype-specific CTL can be induced by secondary stimulation of a hybrid protein of the first 81 amino acids of the viral NS1 non-structural protein and the HA2 subunit of A/Puerto Rico/8/34(H1N1) hemagglutinin. In addition, a derivative of this protein with 65 amino acids deleted from the N-terminal end of HA2 can also generate H1 subtype-specific CTL in bulk cultures. CTL clones established by stimulation with the derivative protein demonstrated cross-reactive lysis of target cells infected with virus strains of the H1 and H2 subtypes. Cold target competition experiments with CTL clones as effectors demonstrated that the Ag specificity between these two hybrid proteins is identical. Adoptive transfer of the CTL clone significantly reduced virus titers in the lungs of mice infected with the virus strains of the H1 or H2 subtype but not those infected with the H3 subtype virus in vivo, which reflects the in vitro CTL clone activity. These experiments demonstrate that an epitope on the hemagglutinin that is conserved on virus strains of the H1 and H2 subtypes induces a protective CTL response. These results suggest an alternative approach for developing influenza vaccines by using conserved antigenic sites on the hemagglutinin HA2 subunit to avoid the problem of frequent antigenic mutations of the HA1 subunit antibody binding sites.     

In vitro antibody response to influenza virus. II. Specificity of helper T cell recognizing hemagglutinin

Intraperitoneal immunization of mice with liver influenza virus was shown to induce helper T (TH) cells with specificity for the hemagglutinin (HA). The interaction of virus-primed TH cells with purified HA was studied independently of B cell reactivity to the same antigen by using the generation of nonspecific help as an index of activation of HA-specific TH cells. TH cells from mice primed with any of the H3 viruses A/Aichi/68 X A/Bel/42 (H3N1), A/Memphis/102/72 X A/Bel/42 (H3N1) or A/Port Chalmers/73 (H3N2) were strongly cross-reactive towards HA of other strains within the H3 subtype. In addition, several examples of cross-reactivity towards HA of a different subtype were observed, usually of a lower magnitude. TH cells from mice primed to any of the H3 viruses above or to A/Bel/42 (H1N1) virus cross-reacted with the HA of A/Japan/305/57 (H2); furthermore, priming with A/Bel/42 or with A/Jap/305/57 X A/Bel/42 (h2N1) virus yielded TH cells that cross-reacted with certain of the H3 HA preparations. The cross-reactivity observed between subtypes was not due to the common chicken host carbohydrate component of HA, since no response to the purified type A HA preparations was obtained with T cells from mice primed with egg-grown influenza B/Hong-Kong/8/73 virus. The results indicate that HA of different subtypes may share cross-reactive antigenic determinants recognized by TH cells. Within a subtype, HA are highly cross-reactive with respect to tH cell recognition.     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

Impacts of avidity and specificity on the antiviral efficiency of HIV-1-specific CTL

Although CD8(+) CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.     

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

The cross-priming APC requires a Rel-dependent signal to induce CTL

Induction of OVA-specific CTL by cross-priming requires help from CD4 T cells, which use CD154 to signal CD40 on the APC. To further dissect the molecular pathways involved in cross-priming, we examined the role of Rel, an NF-kappaB family member. c-rel(-/-) mice failed to generate OVA-specific CTL by cross-priming, but could induce CTL to HSV-1. Using chimeric mice, Rel expression was shown to be required by the APC, but not by the T cells. Notably, the deficiency in Rel could be overcome by triggering CD40, implying that the APC required Rel before receipt of the CD40 signal. These data suggest that the cross-priming APC must receive two signals before it can stimulate CTL. The first signal is Rel dependent and is required before activation of CD4 helper T cells, which then deliver the second signal using CD154 to trigger CD40.     

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.     

Peptide affinity and concentration affect the sensitivity of M3-restricted CTLs induced in vitro.

In vitro stimulation of mouse splenocytes with hemagglutinin (HA) 173-190, a peptide derived from influenza virus hemagglutinin (A/JAP/305/57, H2N2), induces CTLs that are directed to the MHC class Ib molecule, H2-M3. M3 preferably binds peptides bearing an N-terminal formylmethionine. In this study, we show that several related nonformylated peptides can induce anti-HA CTLs in vitro: MLIIW (the minimal epitope), derived from HA186-190 at the C-terminal end of HA173-190; MLIIWG; MLIIWGV; and MLIIWGI, as well as formylated MLIIW. The heptamer peptides correspond to a polymorphism of HA192 in H2 strains of influenza; they have the highest relative affinities for M3 of the nonformylated peptides and higher affinities than some formylated mitochondrial peptides. Depending on the affinity of the peptide, a range of concentrations can be used to induce CTLs. One nanomolar of the high affinity f-MLIIW peptide can induce anti-HA CTLs, whereas 100-fold more of the lower affinity MLIIW peptide is needed. Lines induced with high concentrations (1 microM or greater) of f-MLIIW recognize Ag poorly, and the most efficient CTLs are induced with the lowest concentrations of peptide. Analysis with a panel of anti-TCRVbeta Abs shows that different T cells respond to high vs low peptide; the repertoire of cells responding to higher concentrations is more diverse, consistent with the expansion of more, but less efficient, clones. Thus, peptide affinity and concentration should be considered together for generating efficient antipeptide CTLs in vitro.     

Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

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.     

Host immune response to reovirus: CTL recognize the major neutralization domain of the viral hemagglutinin

Previous studies have demonstrated the importance of the hemagglutinin (HA) of reovirus in determining virus-specific immune responses. In this study, we have used anti-HA monoclonal antibodies and reoviruses with antigenically altered HA proteins to further analyze the CTL response against the HA. We showed that reovirus-specific CTL primarily recognize one distinct antigenic domain on the HA. These results emphasize the fine specificity of the T cell receptor that recognizes viral determinants. The CTL response is directed against the region of the HA that is also responsible for neurotropism and binding to neutralizing antibody.     

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.     

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.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

In vitro influenza virus-specific antibody production in man: antigen-specific and HLA-restricted induction of helper activity mediated by cloned human T lymphocytes

Cloned human T lymphocytes induced with influenza A virus (A/Texas/1/77) and maintained in continuous culture with T cell growth factor were assayed for helper function in the in vitro production of anti-influenza antibody. Helper function mediated by both cloned helper T cells and normal peripheral blood lymphocytes was highly antigen dose-dependent, requiring lower concentrations than that necessary to induce blastogenesis. Optimal help was observed with 1 X 10(2) cloned T cells per culture, whereas excess helper cells inhibited the response. After culture with influenza A virus-induced cloned helper T cells, the antibodies formed were directed against influenza A and not B virus. Furthermore, the cloned helper T cells despite being specific for matrix protein collaborated in the production of predominantly anti-hemagglutinin antibody, suggesting associative recognition of the two discrete antigens. Cellular interactions between cloned helper cells from an HLA-Dw1,3 DR1,3 individual and erythrocyte rosette-negative cells required HLA-Dw1; DR1 compatibility for the production of specific antibody. This was confirmed by using subclones. Finally, it was observed that supernatants of the cloned helper T cells contained functional activity capable of replacing the parent cells in the production of anti-influenza A virus antibody.     

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

Induction of cytotoxic T lymphocytes in mice against the principal neutralizing domain of HIV-1 by immunization with an engineered T-cytotoxic-T-helper synthetic peptide construct.

Peptide constructs were engineered by colinear synthesis of two short synthetic peptide determinants; a determinant recognized by T helper cells (TDh) and a determinant recognized by T cytotoxic cells (TDc). Three types of constructs were synthesized: TDc-TDh, TDh-TDc, and TDh-KK-TDc, where KK are two lysine residues. In vivo immunization with free construct induced cytolytic lymphocytes (CTL) only in the case of TDc-TDh. However, immunization with spleen cells to which these constructs had been internalized by hypertonic shock, induced CTL activity in all three cases. No CTL could be induced after immunization with free TDc in either protocol. These results indicate that cell internalization of the construct might be essential for CTL induction, and also, that "help" from the TDh seems to be required.