T-cell mediated immune responses in calves primary-infected or re-infected with Cooperia oncophora: similar effector cells but different timing

Cooperia oncophora is the most prevalent intestinal nematode of cattle occurring in Western Europe. Primary infection with 100000 third stage infective larvae (L3) induces acquired immunity in a high proportion of the animals but there is little information on immunity against re-infection. In the current experiment, the contribution of the T-cell mediated immunity in protection against re-infection with C. oncophora was investigated in detail. Priming elicited long-lasting protective immunity that was evidenced by a significantly decreased worm burden and egg excretion in primed animals compared to challenge control animals. Lymphocyte proliferation tests with excretory/secretory products (ESP) of C. oncophora and with three distinct ESP fractions indicated an enhanced reactivity in primed animals and suggested that by fractionating of ESP we selected for proteins involved in protective immunity against re-infection with C. oncophora. Phenotypic analysis of T cell subsets at diverse anatomical locations revealed that the enhanced reactivity of lymphocytes from peripheral blood and lymph nodes of the infected animals coincided with a significantly increased frequency of CD4(+) cells at these locations but a deceased frequency of CD4(+) cells in the lamina propria. These findings were independent of the immune status of the animals but more pronounced in the primed animals than in the challenge control animals. In addition we demonstrated that primary and secondary infections with C. oncophora were associated with two waves of eosinophils and that the kinetics of this cell population differed as a result of priming. Based on the observed correlations we propose that the early increase of eosinophils is T cell independent and merely a consequence of inflammation in the parasitised gut. In contrast, the second wave of eosinophils depends upon CD4(+) cells and correlations with parasitological parameters at this time point support a role of eosinophils as effector cells against adult stages of C. oncophora.     

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.     

Experimental investigations on the effectiveness of fenbendazole in parasitic helminths of the stomach, intestines and lung of cattle (author's transl)]

Helminth-free bulls were injected artificially with Ostertagia ostertagi (120 000 L3), Cooperia oncophora (240000 L3), Haemonchus spec. (1 000 L3) and/or Dictyocaulus viviparus (60 L3/kg) orally or intraruminally. The animals were treated with fenbendazole during the prepatent period or after having reached maturity. The effect of 5 mg fenbendazole/kg p.o. on 3-d, 10-d- and adult stages of Ostertagia ostertagi, Haemonchus spec. and Cooperia oncophora ranges between less than 94% and 100%. Particularly noticeable was the effect on 10-d-old Ostertagia ostertagi with less than 94%. 5 mg/kg fenbendazole orally removed nearly 100% of 6-d-, 13-d-, and adult stages of Dictyocaulus viviparus. Fenbendazole may be administered as a drench or as medicated feed. The safety of the compound permits the administration of excessively high doses without clinical signs.     

Delayed expansion and contraction of CD8+ T cell response during infection with virulent Salmonella typhimurium

Ag presentation to CD8(+) T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (approximately 7 days), resistant mice (129 x 1SvJ) harbor a chronic infection lasting approximately 60-90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8(+) T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62L(high)IL-7Ralpha(high)CD44(high)) CD8(+) T cells. However, by day 14-21, majority of the primed CD8(+) T cells display an effector phenotype (CD62L(low)IL-7Ralpha(low)CD44(high)). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62L(low)IL-7Ralpha(high)CD44(high)) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8(+) T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8(+) T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8(+) T cell recognition, conferring a survival advantage to the pathogen.     

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.     

Induction of autoimmunity by expansion of autoreactive CD4+CD62Llow cells in vivo

The prerequisites of peripheral activation of self-specific CD4(+) T cells that determine the development of autoimmunity are incompletely understood. SJL mice immunized with myelin proteolipid protein (PLP) 139-151 developed experimental autoimmune encephalomyelitis (EAE) when pertussis toxin (PT) was injected at the time of immunization but not when injected 6 days later, indicating that PT-induced alterations of the peripheral immune response lead to the development of autoimmunity. Further analysis using IA(s)/PLP(139-151) tetramers revealed that PT did not change effector T cell activation or regulatory T cell numbers but enhanced IFN-gamma production by self-specific CD4(+) T cells. In addition, PT promoted the generation of CD4(+)CD62L(low) effector T cells in vivo. Upon adoptive transfer, these cells were more potent than CD4(+)CD62L(high) cells in inducing autoimmunity in recipient mice. The generation of this population was paralleled by higher expression of the costimulatory molecules CD80, CD86, and B7-DC, but not B7-RP, PD-1, and B7-H1 on CD11c(+)CD4(+) dendritic cells whereas CD11c(+)CD8alpha(+) dendritic cells were not altered. Collectively, these data demonstrate the induction of autoimmunity by specific in vivo expansion of CD4(+)CD62L(low) cells and indicate that CD4(+)CD62L(low) effector T cells and CD11c(+)CD4(+) dendritic cells may be attractive targets for immune interventions to treat autoimmune diseases.     

Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of antitumor immunity in vivo.

Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

Immunological study of cellular components of Pseudomonas aeruginosa. IV. Fractionation of Pseudomonas aeruginosa mucus by diafiltration and biological properties of isolated fractions]

In fractionation of Pseudomonas aeruginosa mucus (strains No. 8 and 1463) by means of diafiltration on the system of membranes Diaflo XM-300, XM-100A, PM-30, and PM-10 there was obtained a successive series of fractions differing by the molecular weight and chemical composition. According to the results of gel chromatography fractions with the mol wt of 100000 dalton and over apparently represented protein-polysaccharide components of mucus in the form of complexes; fractions with the mol wt of 30000 dalton and lower contained a considerable amount of free protein along with the protein-polysaccharide complex. The fractions obtained differed by biological properties: fractions with the mol wt of 100000 dalton and over were toxic for mice and possessed weak antigenic properties in the precipitation in agar test and immunoelectrophoresis; fractions with the mol wt lower than 30000 dalton expressed in the mentioned test distinct antigenic properties and proved to be practically nontoxic for mice. Thus the use of diafiltration method permitted to separate the antigenic, weakly toxic component of Ps. aeruginosa mucus from the toxic factor with weak antigenic properties.     

Prevalence of Cooperia punctata, C. pectinata and C. oncophora infections in dairy calves in Brazil

Cooperia is the most prevalent helminth parasitizing calves in Brazil. Three species of this genus occur most often: C. punctata, C. pectinata and C. oncophora. Six calves from dairy farms in the south of the State of Minas Gerais aged six to 15 months were killed and necropsied each month over a period of two years. The Cooperia species were identified, counted, and the numbers related to the calves' age. The worm burdens due to the three species of Cooperia were statistically different. C. punctata was the most prevalent species and had a positive correlation with the age of the calves; C. pectinata appeared with lower intensity but was always present, and C. oncophora was not found in calves older than 11 months. These results show the existence of different degrees of resistance to Cooperia species among calves as the three species did not behave similarly. It seems to be an acquired resistance. C. punctata appears to be less immunogenic than C. pectinata and C. oncophora. As C. punctata and C. pectinata are more pathogenic than C. oncophora, it seems that this pathogenicity can be related to immunogenic aspects associated with the species.     

B7-H1 signaling is integrated during CD8+ T cell priming and restrains effector differentiation

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cells is integrated during priming of naïve CD8⁺ T cells and functions to limit the differentiation of effector T cell responses. CD8⁺ T cells primed by B7-H1-deficient dendritic cells exhibit increased production of IFN-γ, enhanced target cell killing, and improved anti-tumor activity. Additionally, enhanced memory populations arise from CD8⁺ T cells primed by B7-H1-deficient dendritic cells. Based on these findings, we suggest that early blockade of B7-H1 signaling should be investigated as a strategy to improve dendritic cell-based anti-tumor immunotherapy.     

Naive and memory T cells induce different types of graft-versus-host disease

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.     

CD8 T cell expansion and memory differentiation are facilitated by simultaneous and sustained exposure to antigenic and inflammatory milieu

Understanding the factors contributing to the generation of immune memory is important for rational vaccine design. In this study, we addressed the individual and combined roles of Ag and inflammation in sustaining the ability of primed CD8 T cells to clonally expand and differentiate into memory cells. We transferred CD8 T cells that were primed for a brief period into naive mice, mice infected with a pathogen not carrying the specific Ag (inflammation only), mice infected with a pathogen carrying the donor cell-specific Ag (inflammation plus Ag), or into mice exposed to soluble Ag (Ag only). We found that the donor CD8 T cells continued to proliferate in all the four conditions, but their ability to clonally expand and differentiate into memory cells was approximately 1000-fold higher when transferred into mice acutely infected with pathogen carrying the relevant Ag. Memory cells generated under conditions of sustained exposure to inflammation and Ag during the priming phase were superior in their ability to elicit recall responses on a per cell basis. Thus, simultaneous and sustained exposure of donor CD8 T cells to inflammatory and antigenic stimuli, following the initial priming phase, leads to the greatest expansion of CD8 T cells at the peak of the immune response and induces an optimal memory differentiation program. These results suggest that vaccination strategies should attempt to provide sustained exposure to Ag plus inflammation but not either alone following the initial priming.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.     

Cutting edge: L-selectin (CD62L) expression distinguishes small resting memory CD4+ T cells that preferentially respond to recall antigen

Naive CD4+ T cells use L-selectin (CD62L) expression to facilitate immune surveillance. However, the reasons for its expression on a subset of memory CD4+ T cells are unknown. We show that memory CD4+ T cells expressing CD62L were smaller, proliferated well in response to tetanus toxoid, had longer telomeres, and expressed genes and proteins consistent with immune surveillance function. Conversely, memory CD4+ T cells lacking CD62L expression were larger, proliferated poorly in response to tetanus toxoid, had shorter telomeres, and expressed genes and proteins consistent with effector function. These findings suggest that CD62L expression facilitates immune surveillance by programming CD4+ T cell blood and lymph node recirculation, irrespective of naive or memory CD4+ T cell phenotype.     

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.     

In vivo priming of helper T cells in the absence of B cell activation

This paper describes an adjuvant-free immunization regimen that results in the priming of T cells but not B cells. B10.A mice were primed s.c. with syngeneic spleen cells that had been pulsed with the peptide 81-104 derived from pigeon cytochrome c. The T cell response was measured by using a sensitive limiting dilution assay that measures lymphokine production. The precursor frequency of Ag-specific cells found in these mice was indistinguishable from the frequency found in mice primed in the footpads with 81-104 in CFA. A striking difference in antibody induction was found, however, when these two immunization regimens were compared. Mice primed with 81-104 in CFA developed significant serum antibody responses against the peptide, whereas mice primed with Ag-pulsed spleen cells produced no detectable anti-peptide antibodies. This lack of antibody did not result from detectable differences in the T cells that were primed: no differences were seen in IL-2 and IL-4 production or in the ability to provide help to B cells in vitro. In vitro stimulation with LPS suggested that the B cells were not primed by the Ag-pulsed spleen cells. The B cells were not tolerized, however, because boosting the mice with Ag in CFA resulted in the induction of an antibody response. The failure to induce an antibody response by priming with Ag-pulsed spleen cells was not caused by the site of immunization or the total amount of Ag used for priming. The critical variable may be the introduction of the Ag on the surface of an APC; in this form, B cell Ag recognition was apparently inefficient, whereas T cell Ag recognition was optimal.     

Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.