Cytokines regulate the capacity of CD8alpha(+) and CD8alpha(-) dendritic cells to prime Th1/Th2 cells in vivo

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8alpha(-) DC induces a Th2-type response, whereas injection of CD8alpha(+) DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-gamma, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-gamma are required for Th1 priming by CD8alpha(+) DC, whereas IL-10 is required for optimal development of Th2 cells by CD8alpha(-) DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8alpha(+) DC. Conversely, incubation with IFN-gamma down-regulates the Th2-promoting capacities of CD8alpha(-) DC and increases the Th1-skewing properties of both subsets.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

CD8 T cells inhibit IgE via dendritic cell IL-12 induction that promotes Th1 T cell counter-regulation.

Th1 and Th2 cells are counterinhibitory; their balance determines allergic sensitization. We show here that CD8 T cell subsets break these rules as both T cytotoxic (Tc)1 and Tc2 cells promote Th1 over Th2 immunity. Using IL-12(-/-), IFN-gamma(-/-), and OVA(257-264)-specific Valpha2Vbeta5 TCR-transgenic mice, we have identified the key steps involved. OVA-specific IFN-gamma(-/-) CD8 T cells inhibited IgE responses equivalent to wild-type CD8 T cells (up to 98% suppression), indicating that CD8 T cell-derived IFN-gamma was not required. However, OVA-specific CD8 T cells could not inhibit IgE in IFN-gamma(-/-) recipients unless reconstituted with naive, wild-type CD4 T cells, suggesting that CD4 T cell-derived IFN-gamma did play a role. Transfer of either Tc1 or Tc2 Valpha2Vbeta5 TCR-transgenic CD8 T cells inhibited IgE and OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses, suggesting a potential role for a type 1 inducing cytokine such as IL-12. CD8 T cells were shown to induce IL-12 in OVA(257-264)-pulsed dendritic cells (DC) in vitro. Furthermore, CD8 T cells were unable to inhibit IgE responses in IL-12(-/-) recipients without the addition of naive, wild-type DC, thus demonstrating a pivotal role for IL-12 in this mechanism. These data reveal a mechanism of IgE regulation in which CD8 T cells induce DC IL-12 by an IFN-gamma-independent process that subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN-gamma is the final step that inhibits B cell IgE class switching. This demonstrates a novel regulatory network through which CD8 T cells inhibit allergic sensitization.     

Chlamydia muridarum infection subverts dendritic cell function to promote Th2 immunity and airways hyperreactivity

There is strong epidemiological evidence that Chlamydia infection can lead to exacerbation of asthma. However, the mechanism(s) whereby chlamydial infection, which normally elicits a strong Th type 1 (Th1) immune response, can exacerbate asthma, a disease characterized by dominant Th type 2 (Th2) immune responses, remains unclear. In the present study, we show that Chlamydia muridarum infection of murine bone marrow-derived dendritic cells (BMDC) modulates the phenotype, cytokine secretion profile, and Ag-presenting capability of these BMDC. Chlamydia-infected BMDC express lower levels of CD80 and increased CD86 compared with noninfected BMDC. When infected with Chlamydia, BMDC secrete increased TNF-alpha, IL-6, IL-10, IL-12, and IL-13. OVA peptide-pulsed infected BMDC induced significant proliferation of transgenic CD4(+) DO11.10 (D10) T cells, strongly inhibited IFN-gamma secretion by D10 cells, and promoted a Th2 phenotype. Intratracheal transfer of infected, but not control noninfected, OVA peptide-pulsed BMDC to naive BALB/c mice, which had been i.v. infused with naive D10 T cells, resulted in increased levels of IL-10 and IL-13 in bronchoalveolar lavage fluid. Recipients of these infected BMDC showed significant increases in airways resistance and decreased airways compliance compared with mice that had received noninfected BMDC, indicative of the development of airways hyperreactivity. Collectively, these data suggest that Chlamydia infection of DCs allows the pathogen to deviate the induced immune response from a protective Th1 to a nonprotective Th2 response that could permit ongoing chronic infection. In the setting of allergic airways inflammation, this infection may then contribute to exacerbation of the asthmatic phenotype.     

Dendritic cell-derived IL-12 is not required for the generation of cytotoxic, IFN-gamma-secreting, CD8(+) CTL in vivo.

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

Dendritic cell-derived IL-12 promotes B cell induction of Th2 differentiation: a feedback regulation of Th1 development.

B cells convert what are normally conditions for Th1 differentiation into an environment suitable for Th2 development. This capacity is dependent on CD40 as B cells from CD40-/- mice do not elicit Th2 differentiation. To elucidate the basis of this effect, we surveyed cytokine RNA made by naive B cells after activation with anti-Ig and anti-CD40. Resting B cells make TGF-beta message only, however, 4 days after activation, RNA encoding IL-6, IL-10, and TNF-alpha was found. The expression of these messages was accelerated by 2 days in the presence of IL-12. The relevance of these observations to T cell differentiation was investigated: addition of OVA peptide to splenic cells from DO.11.10 transgenic mice causes most T cells to make IFN-gamma. Coactivation of B cells in these cultures reduces the number of IFN-gamma-producing T cells and increases the number synthesizing IL-4. Abs to IL-6 and IL-10 block the IL-4 enhancement. Dissection of the component APC demonstrated that interaction of B cells with IL-12-producing dendritic cells is crucial for B cell-mediated IL-4 enhancement: Thus, B cells preactivated in the presence of dendritic cells from IL-12-/- mice show little IL-4-inducing activity when used to activate T cells. This immune regulation is initiated by IL-12 and therefore represents a feedback loop to temper its own dominant effect (IFN-gamma induction).     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

Characterization of tumor-infiltrating CD4+ T cells as Th1 cells based on lymphokine secretion and functional properties

Recent studies have subdivided the Th cells into mutually exclusive Th1 subset producing IL-2 and IFN-gamma and Th2 cells producing IL-4 and IL-5. The relative role played by these two subsets in the antitumor immunity is not clear. We earlier demonstrated that treatment of C57BL/6 mice bearing a syngeneic Ia- T cell lymphoma, LSA, with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) resulted in 90 to 100% survival of the mice. Furthermore, host's T cell responses were critical for successful BCNU-mediated cures. In our study we observed that immediately after BCNU treatment, there was a dramatic increase in the percentage of CD4+ T cells at the site of tumor growth in the peritoneal cavity. The percentage of CD4+ T cells increased from approximately 3 to 4% found in normal or tumor-bearing mice to approximately 41% in BCNU-treated tumor-bearing mice. The percentage of CD8+ T cells also increased although to a lesser degree. Also, these alterations were primarily restricted to the site of tumor-growth inasmuch as they were not seen in the thymus and were less pronounced in the spleen. The tumor-infiltrating CD4+ T cells obtained after BCNU-treatment, when further characterized, were found to secrete only IL-2 and IFN-gamma but not IL-4, after tumor-specific stimulation. Furthermore, the supernatants from LSA-activated CD4+ T cell cultures failed to provide help to the B cells but were able to activate the macrophages to inhibit the tumor cell proliferation. The CD4+ T cells when adoptively transferred could also protect the nude mice from LSA tumor challenge and induced tumor-specific delayed-type hypersensitivity reaction. Together our data suggest that in the LSA tumor model, the tumor-infiltrating CD4+ T cells have the properties of Th1 cells and these cells can mediate tumor-rejection independent of the CD8+ T cells by activating the macrophages.     

The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice

Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-gamma, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.     

IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.     

CD4+ subset regulation in viral infection. Preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice.

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL-2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Antigenic epitopes regulate the phenotype of CD8+ CTL primed by exogenous antigens

We previously reported that insulin-specific, MHC class I-restricted CTL precursors can be primed by injecting C57BL/6 mice with bovine insulin in CFA. These bovine insulin-primed CTL displayed a type 0 CTL phenotype, producing IL-4, IL-5, IL-10, low levels of IFN-gamma, but no TNF-alpha. By contrast, CTL generated from C57BL/6 mice primed with OVA in CFA produced IFN-gamma and TNF-alpha but no IL-4, IL-5, or IL-10 and therefore were classified as type 1 CTL. Although CD4+ T cell subsets have been compared extensively in the literature, CTL subsets are less well characterized. Here, the phenotype, function, and requirements for the in vivo activation of type 1 and type 0 CTL cells were studied. Although both types of CTL express many of the same cell-surface Ags, OVA-specific CTL but not bovine insulin-primed CTL expressed CT-1, a carbohydrate epitope of CD45, and bovine insulin-primed CTL but not OVA-specific CTL expressed Fas constitutively. Priming of CTL was abrogated by depletion of phagocytic cells but not CD4+ T cells, whereas depletion of CD4+ T cells but not phagocytic cells inhibited Ab responses in the same mice. Neither endogenous IL-4 nor the dose of priming Ag altered the CTL phenotypes, but the antigenic peptides of OVA and bovine insulin were key to determining the differentiation of either type 1 or type 0 CTL. To our knowledge, this is the first time that antigenic epitopes have been demonstrated to influence the phenotype of Ag-specific CTL responses. These results may be relevant to the development of peptide vaccines in which a particular type of CTL response is desired.