Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

A hybrid protein between IFN-gamma and IL-2

The complementary DNAs encoding human interferon-gamma (IFN-gamma) and human interleukin-2 (IL-2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E. coli to investigate the interactions of these two proteins at the molecular level. Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN-gamma and IL-2. The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN-gamma and the ability to support the growth of natural killer (NK) cells derived from IL-2. Comparing the enhancement of NK cell activity of this hybrid protein with IFN-gamma and IL-2, this hybrid protein appears to conserve each activity almost completely without diminishing the other.     

IL-2 and IFN-gamma are two necessary lymphokines in the development of cytolytic T cells

A filler cell-free limiting-dilution microculture system has been developed for the expansion and differentiation of a high proportion of single CD4-CD8+ T cells into cytolytic T cell (CTL) clones. The stimulus used was PMA together with the calcium ionophore ionomycin. The growth and differentiation factors were rIL-2, together with either a Con A-stimulated spleen cell supernatant (CAS) or rIFN-gamma. CTL activity was monitored by an autoradiographic 111In-release assay. With CAS and rIL-2 present, 50% of all potential precursors (CTL-p) produced cytolytic clones. Substitution of rIFN-gamma for CAS gave a similar efficiency with up to 42% of CTL-p producing cytolytic clones. rIL-2 alone allowed only a small proportion (6%) of CD4-CD8+ T cells to become cytolytic clones. Addition of rIL-2 and rIFN-gamma at various stages of the culture demonstrated that IL-2 was required throughout, but exogenous IFN-gamma was required only during the early stages. It is concluded that for at least 40% of all CTL-p, the lymphokines IL-2 and IFN-gamma are essential and act in synergy to induce proliferation and differentiation into CTL.     

Mycobacterium tuberculosis-specific CD4+, IFNgamma+, and TNFalpha+ multifunctional memory T cells coexpress GM-CSF

Multifunctional T cells expressing several cytokines in parallel are thought to play a crucial role in protection against different infections. To characterize T cell cytokine patterns associated with disease and protection in Mycobacterium tuberculosis infection we determined the expression of IFNγ, IL-2, TNFα, and GM-CSF in T cell subpopulations from children with tuberculosis (TB) and healthy latently M. tuberculosis-infected children (LTBI) after short-term in vitro restimulation. We identified CD4⁺ effector memory T cells (T_EM) as the major source of all measured cytokines after antigen-specific restimulation. T_EM from children with TB expressed higher proportions of IFNγ, TNFα, and IL-2 after Mtb restimulation while no differences were detected for GM-CSF between both study groups. GM-CSF secretion strongly depended on antigen-specific stimulation. Analyses of multiple cytokine patterns revealed that the majority of GM-CSF-positive M. tuberculosis-specific memory T cells coexpressed IFNγ and TNFα therefore showing a characteristic feature of multifunctional T cells. We conclude that children with active TB possess higher proportions of IFNγ-, TNFα-, and/or IL-2-positive T_EM than children with LTBI while GM-CSF coexpression reveals a novel subpopulation within CD4⁺ memory T cells not increased in children with active TB.     

Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells

Memory T cells (T(M)) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as T(M) undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 T(M) by comparing them to defective CD8 T(M) generated in the absence of CD4 T cells. Unhelped CD8 T(M) are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-gamma. Our data show that this defect in IL-2 and IFN-gamma production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-gamma loci and substantial histone acetylation at the IFN-gamma promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-gamma locus. Thus, CD4 T cell help influences epigenetic modification during CD8 T(M) differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 T(M).     

IL-2 absorption affects IFN-gamma and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays

Cytokine assays are gaining increasing importance for human immune monitoring because they reliably detect antigen-specific T cells in primary PBMC, even at low clonal sizes. Double color ELISPOT assays permit the simultaneous visualization of cells producing two different cytokines. Permitting the simultaneous assessment of type 1 and 2 immunity and due to the limited numbers of PBMC available from human study subjects, double color assays should be particularly attractive for clinical trials. Since the performance of double color assays has not yet been validated, we set out to compare them to single color measurements. Testing the recall antigen-induced cytokine response of PBMC, we found that double color assays regularly provided lower numbers of IFN-gamma and IL-5 spots than single color measurements when IL-2 detection was part of the double color assay. We showed that the inhibitory effect resulted from IL-2 absorption and could be overcome by either antibody free preactivation cultures or by inclusion of anti-CD28 antibody. In contrast, the simultaneous detection of IL-2 did not affect the numbers of IL-4 spots. Therefore, unlike IL-2/IL-4 and IFN-gamma/IL-5 assays, IL-2/IFN-gamma, and IL-2/IL-5 assays require compensation for the IL-2 capture to provide accurate numbers for the frequencies of cytokine producing memory T cells.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.     

Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs) are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

IL-2 receptor blockade inhibits late,but not early,IFN-gamma and CD40 ligand expression in human T cells: disruption of both IL-12-dependent and -independent pathways of IFN-gamma production

mAbs directed against the alpha-chain (Tac/CD25) of the IL-2R are an emerging therapy in both transplantation and autoimmune disease. However, the mechanisms underlying their therapeutic efficacy have not been fully elucidated. Therefore, we examined the effect of IL-2R blockade on Th1 and Th2 cytokine production from human PBMC. Addition of a humanized anti-Tac Ab (HAT) to activated PBMC cultures inhibited IFN-gamma production from CD4 and CD8 T cells by 80-90%. HAT partially inhibited production of TNF-alpha and completely inhibited production of IL-4, IL-5, and IL-10. Furthermore, IL-12, a central regulatory cytokine that induces IFN-gamma, was undetectable in treated cultures. As T cell-dependent induction of IL-12 is regulated via CD40/CD40 ligand (CD40L) interactions, we examined the effect of HAT on CD40L expression. We found CD40L expression to be biphasic with an early (6 h) peak that is CD28/IL-2-independent, but a later peak (48 h) being CD28/IL-2-dependent and inhibited by HAT. Similarly, IFN-gamma production at 6 h was CD28/IL-2-independent but CD28/IL-2-dependent and inhibited by HAT at 48 h. Nonetheless, addition of rCD40L or exogenous IL-12 to HAT-treated cultures could not restore IFN-gamma production. The IFN-gamma deficit in such cultures appears to be due to a direct inhibition by HAT of IL-12-independent IFN-gamma production from T cells rather than altered expression of either the IL-12Rbeta1 or IL-12Rbeta2 chains. These data demonstrate that IL-2 plays a critical role in the regulation of Th1 and Th2 responses and impacts both IL-12-dependent and -independent IFN-gamma production.     

Differential CD3/T cell antigen receptor-mediated IL-2 production in jurkat T cells. Dissociation of IL-2 response from total inositol phosphate and calcium responses

We used three anti-human anti-CD3 mAb each recognizing different surface CD3 epitopes to differentially perturb the CD3/TCR complex on the surface of Jurkat T cells. In the presence of phorbol ester, these anti-CD3 mAb triggered differential IL-2 production in Jurkat T cells, which could not be explained by differences in kinetics of IL-2 production, by differences in IL-2 adsorption caused by differential surface expression of p55 or p75 IL-2R, by effects on IL-2 secretion rather than actual synthesis, or by differential toxicities of the anti-CD3 mAb to Jurkat cells. In addition, this differential anti-CD3-induced IL-2 production could not be explained by differences in mAb isotype or in avidities of the anti-CD3 mAb for the Jurkat cells. Moreover, anti-CD3 mAb covalently immobilized onto beads also differentially induced IL-2 production in Jurkat cells, suggesting that the differential IL-2 response is not based on differential rates of anti-CD3-induced modulation of Jurkat cell surface CD3. Although differences among the anti-CD3 mAb in the initial rates of binding to Jurkat cell were observed, this was also believed unlikely to explain the differential IL-2 response. Regardless of the anti-CD3 mAb used, anti-CD3-induced total inositol phosphate (IP) production did not necessarily correlate with anti-CD3-induced IL-2 production. Nevertheless, despite the differences among the anti-CD3 mAb in inducing IL-2 production, the calcium responses were grossly similar. Taken together, these observations indicate that CD3/TCR-mediated IL-2 production in Jurkat cells can be dissociated from total IP generation, and the basis of differential CD3/TCR-mediated IL-2 production in these cells does not appear to be at the level of the initial activation-induced calcium response. These studies suggest that the nature of the CD3/TCR ligand (its physical form and/or the specific epitope it perturbs) can either directly influence intracellular events distal to the generation of IP and increase in intracellular free calcium leading to differential IL-2 production or can trigger IP-independent pathways that affect IL-2 production.     

Possible association between IL-2 receptors and class I HLA molecules on T cells

In the process of evaluating murine hybridomas for an antibody to the beta-subunit of the IL-2R (p70) we identified an antibody that immunoprecipitated a 55- to 57-kDa complex from cross-linked lysates. We demonstrate that this complex is composed of IL-2 (15.5 kDa) cross-linked to the H chain of HLA class I (40 to 42 kDa), suggesting a molecular interaction between HLA class I molecules and IL-2R. Although the exact role of this association remains to be determined, the specific cross-linking of IL-2 to HLA class I Ag is intriguing in view of published claims for a role of HLA class I in OKT3-induced lymphocyte proliferation and in NK cell lytic activity.     

IL-2, regulatory T cells, and tolerance

IL-2 is a potent T cell growth factor that for many years was assumed to amplify lymphocyte responses in vivo. Accordingly, IL-2 has been used clinically to enhance T cell immunity in patients with AIDS or cancer, and blocking Abs to the IL-2R are used to inhibit T cell responses against transplanted tissues. It was later shown in mice that, unexpectedly, disruption of the IL-2 pathway results in lymphoid hyperplasia and autoimmunity rather than immune deficiency, indicating that the major physiological function of IL-2 is to limit rather than enhance T cell responses. This apparent paradox has recently been resolved with the discovery that IL-2 is critical for the development and peripheral expansion of CD4(+)CD25(+) regulatory T cells, which promote self-tolerance by suppressing T cell responses in vivo. Our new understanding of IL-2 biology prompts a re-evaluation of how best to clinically manipulate this important immunoregulatory pathway.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.