Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Inducible costimulator protein controls the protective T cell response against Listeria monocytogenes

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.     

Immune modulation in multiple sclerosis patients treated with the pregnancy hormone estriol

The protective effect of pregnancy on putative Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, is associated with a Th1 to Th2 immune shift during pregnancy. The hormone estriol increases during pregnancy and has been shown to ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis. In addition, estrogens induce cytokine changes consistent with a Th1 to Th2 shift when administered in vitro to human immune cells and in vivo to mice. In a pilot trial, oral estriol treatment of relapsing remitting multiple sclerosis patients caused significant decreases in enhancing lesions on brain magnetic resonance imaging. Here, the immunomodulatory effects of oral estriol therapy were assessed. PBMCs collected longitudinally during the trial were stimulated with mitogens, recall Ags, and glatiramer acetate. Cytokine profiles of stimulated PBMCs were determined by intracellular cytokine staining (IL-5, IL-10, IL-12 p40, TNF-alpha, and IFN-gamma) and cytometric bead array (IL-2, IL-4, IL-5, IL-10, TNF-alpha, and IFN-gamma). Significantly increased levels of IL-5 and IL-10 and decreased TNF-alpha were observed in stimulated PBMC isolated during estriol treatment. These changes in cytokines correlated with reductions of enhancing lesions on magnetic resonance imaging in relapsing remitting multiple sclerosis. The increase in IL-5 was primarily due to an increase in CD4(+) and CD8(+) T cells, the increase in IL-10 was primarily due to an increase in CD64(+) monocytes/macrophages with some effect in T cells, while the decrease in TNF-alpha was primarily due to a decrease in CD8(+) T cells. Further study of oral estriol therapy is warranted in Th1-mediated autoimmune diseases with known improvement during pregnancy.     

Transforming growth factor-β protein inversely regulates in vivo differentiation of interleukin-17 (IL-17)-producing CD4+ and CD8+ T cells

TGF-β is a pleiotropic cytokine that predominantly exerts inhibitory functions in the immune system. Unexpectedly, the in vitro differentiation of both Th17 and Tc17 cells requires TGF-β. However, animals that are impaired in TGF-β signaling (TGF-βRIIDN mice) display multiorgan autoimmune disorders. Here we show that CD4(+) T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation and, paradoxically, that CD8(+) T cells from these animals spontaneously acquire an IL-17-producing phenotype. Neutralization of IL-17 or depletion of CD8(+) T cells dramatically inhibited inflammation in TGF-βRIIDN mice. Therefore, the absence of TGF-β triggers spontaneous differentiation of IL-17-producing CD8(+) T cells, suggesting that the in vivo and in vitro conditions that promote the differentiation of IL-17-producing CD8(+) T cells are distinct.     

The attachment (G) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both Th1 and Th2 CD4+ T cell responses

BALB/c mice immunized with a vaccinia virus expressing the attachment (G) glycoprotein of respiratory syncytial virus (RSV) develop a virus-specific CD4(+) T cell response that consists of a mixture of Th1 and Th2 CD4(+) T cells following intranasal infection with live RSV. Recent work has shown that both Th1 and Th2 CD4(+) T cells are elicited to a single region comprising aa 183-197 of the G protein. To more precisely define the CD4(+) T cell epitope(s) contained within this region, we created a panel of amino- and carboxyl-terminal truncated as well as single alanine-substituted peptides spanning aa 183-197. These peptides were used to examine the ex vivo cytokine response of memory effector CD4(+) T cells infiltrating the lungs of G-primed RSV-infected mice. Analysis of lung-derived memory effector CD4(+) T cells using intracellular cytokine staining and/or ELISA of effector T cell culture supernatants revealed a single I-E(d)-restricted CD4(+) T cell epitope with a core sequence mapping to aa 185-193. In addition, we examined the T cell repertoire of the RSV G peptide-specific CD4(+) T cells and show that the CD4(+) T cells directed to this single immunodominant G epitope use a restricted range of TCR Vss genes and predominantly express Vss14 TCR.     

TGF-beta1-mediated control of central nervous system inflammation and autoimmunity through the inhibitory receptor CD26

The T cell marker CD26/dipeptidyl peptidase (DP) IV is associated with an effector phenotype and markedly elevated in the human CNS disorder multiple sclerosis. However, little is known about the in vivo role of CD26/DP IV in health and disease, and the underlying mechanism of its function in CNS inflammation. To directly address the role of CD26/DP IV in vivo, we examined Th1 immune responses and susceptibility to experimental autoimmune encephalomyelitis in CD26(-/-) mice. We show that gene deletion of CD26 in mice leads to deregulation of Th1 immune responses. Although production of IFN-gamma and TNF-alpha by pathogenic T cells in response to myelin Ag was enhanced in CD26(-/-) mice, production of the immunosuppressive cytokine TGF-beta1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-beta1 production, responsiveness to external TGF-beta1 was normal in T cells from CD26(-/-) mice, excluding alterations in TGF-beta1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-beta1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-beta1 production in T cells from CD26(-/-) mice. The striking functional deregulation of Th1 immunity was also seen in vivo. Thus, clinical experimental autoimmune encephalomyelitis scores were significantly increased in CD26(-/-) mice immunized with peptide from myelin oligodendrocyte glycoprotein. These results identify CD26/DP IV as a nonredundant inhibitory receptor controlling T cell activation and Th1-mediated autoimmunity, and may have important therapeutic implications for the treatment of autoimmune CNS disease.     

Cutting edge: anti-CD1 monoclonal antibody treatment reverses the production patterns of TGF-beta 2 and Th1 cytokines and ameliorates listeriosis in mice.

Protection against intracellular bacteria by T cells is regulated by Ag-presenting molecules, which comprise classical MHC class I molecules, MHC class II molecules, and nonclassical MHC class Ib molecules. The role of CD1 molecules, which are structurally similar to classical MHC class I gene products, but less polymorphic, is not understood so far. We show that CD1 surface expression increased on APC in Listeria-infected mice. The in vivo treatment with anti-CD1 mAb reduced TGF-beta 2 levels and concomitantly increased secretion of the proinflammatory cytokine TNF, the Th1 cell promoting cytokine IL-12, and the Th1 cell cytokine IFN-gamma at the onset of listerial infection. These findings point to a regulatory role of CD1-reactive cells in the immune response against listeriosis.     

Effector function of diabetogenic CD4 Th1 T cell clones: a central role for TNF-alpha

Effector function of T cells in autoimmune diabetes has been widely studied with mixed populations of lymphoid T cells stimulated ex vivo, but this approach does not permit evaluation of the contribution by a single T cell clone in the inflammatory site during pathogenesis. We have investigated cytokine production both in vitro and in vivo in a panel of diabetogenic CD4 Th1 T cell clones derived from the NOD mouse. SuperArray analysis showed a common pattern of mRNA expression for inflammatory cytokines and receptors in vitro after TCR stimulation. Ex vivo intracellular cytokine staining demonstrated that two important inflammatory cytokines, IFN-gamma and TNF-alpha, were being made by these T cells recovered from the pancreas 6 days following adoptive transfer. TNF-alpha produced in the pancreas by pathogenic T cell clones and recruited macrophages was not the membrane-bound form. Secreted TNF-alpha can lead to production of multiple inflammatory chemokines, as were observed in the pathogenic clones by intracellular cytokine staining. Our results not only define the nature of an inflammatory cytokine response critical to development of diabetes, but also suggest its role in the regulation of other events during pathogenesis induced by CD4 T cells. Similar analyses in other models demonstrated that disease induced by CD4 T cell clones closely resembles spontaneous autoimmune diabetes in which both CD4 and CD8 T cells are required. Thus, cloned T cells in effect amplify effector function of T cells which otherwise may be difficult to detect without ex vivo stimulation.     

Novel function of IFN-gamma: negative regulation of dendritic cell migration and T cell priming

IFN-gamma is considered to be a Th1 cytokine with immunomodulatory effects on a variety of immune cells. In this study, we determined whether dendritic cell (DC) function was aberrant in IFN-gamma knockout (GKO) mice. The results demonstrated that IFN-gamma deficiency did not interfere with bone marrow-derived DC development and maturation in vitro. However, functional analysis showed that bone marrow-derived DC from GKO mice had altered cytokine secretion, allostimulatory and Ag presentation capacity, chemokine receptor expression, and in vitro chemotaxis. LPS induced the recruitment of DC from different organs into the spleen; epicutaneously sensitized DC with hapten (FITC) accumulated in the draining lymph nodes and CD11c(+) DC levels in the draining lymph nodes from autoantigen (interphotoreceptor retinoid-binding protein) immunized mice were enhanced in GKO mice as compared with wild-type mice. After treatment of GKO mice with i.p. IFN-gamma injection restored IFN-gamma levels in vivo, DC migration decreased in response to LPS or FITC. IFN-gamma altered the adaptive immune responses in vivo, since T cell priming and IL-2 production were increased in interphotoreceptor retinoid-binding protein-immunized GKO mice. Furthermore, in IFN-gamma-treated GKO mice, experimental autoimmune uveitis score enhancement and T cell activation were eliminated. Taken together, IFN-gamma appears to play a negative regulatory role on in vivo DC function, resulting in suppression of Ag-specific T cell priming.     

Preferential Th1 immune response in invariant chain-deficient mice

MHC class II molecules associate with the invariant chain (Ii) molecule during biosynthesis. Ii facilitates the folding of class II molecules, interferes with their peptide association, and is involved in MHC class II transport. In this study, we have investigated the in vitro and in vivo immune response of Ii-deficient mice (Ii(-/-)). Our results have demonstrated that CD4(+) T cells from Ii(-/-) mice proliferate normally in vitro after in vivo immunization with protein Ags. However, cytokine secretion profiles of Ag-primed CD4(+) T cells from Ii(-/-) mice differ from CD4(+) T cells from wild-type mice. Whereas cells from wild-type mice secrete IFN-gamma and IL-4, cells from Ii(-/-) mice secrete mostly IFN-gamma. Moreover, Ii(-/-) mice exhibit a normal Th1 response in the delayed-type hypersensitivity and trinitrobenzene sulfonic acid colitis models; however, these mice lack an in vivo Th2 response, as demonstrated in the asthma model. Therefore, we suggest that defective Ag presentation in Ii(-/-) mice leads selectively to a Th1 effector response.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

Inhibition of Th2-mediated allergic airway inflammatory disease by CD137 costimulation

The engagement of CD137 (4-1BB), an inducible T cell costimulatory receptor and member of the TNF receptor superfamily, by agonistic Abs can promote strong tumor and viral immunity mediated by CD8(+) T cells and stimulate IFN-gamma production. However, its role in Th2-mediated immune responses has not been well defined. To address this issue, we studied the function of CD137 engagement using an allergic airway disease model in which the mice were sensitized with inactivated Schistosoma mansoni eggs followed by S. mansoni egg Ag challenge directly in the airways and Th1/2 cytokine production was monitored. Interestingly, treatment of C57BL/6 mice with agonistic anti-CD137 (2A) during sensitization completely prevents allergic airway inflammation, as shown by a clear inhibition of T cell and eosinophil infiltration into the lung tissue and airways, accompanied by diminished Th2 cytokine production and reduced serum IgE levels, as well as a reduction of airway hyperresponsiveness. At various time points after immunization, restimulated splenocytes from 2A-treated mice displayed reduced proliferation and Th2 cytokine production. In accordance with this, agonistic Ab to CD137 can directly coinhibit Th2 responses in vitro although it costimulates Th1 responses. CD137-mediated suppression of Th2 response is independent of IFN-gamma and T regulatory cells. Our study has identified a novel pathway to inhibit Th2 responses in a CD137-dependent fashion.     

Roles of vasoactive intestinal peptide (VIP) in the expression of different immune phenotypes by wild-type mice and T cell-targeted type II VIP receptor transgenic mice

Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1 and VPAC2, are quantitatively prominent and functionally critical in the immune system. Transgenic (T) mice constitutively expressing VPAC2 selectively in CD4 T cells, at levels higher than those found after maximal induction in CD4 T cells of wild-type (N) mice, have elevated blood concentrations of IgE, IgG1, and eosinophils; enhanced immediate-type hypersensitivity; and reduced delayed-type hypersensitivity. In contrast, VPAC2-null (K) mice manifest decreased immediate-type hypersensitivity and enhanced delayed-type hypersensitivity. The phenotypes are attributable to opposite skewing of the Th2/Th1 cytokine ratio, but no studies were conducted on the roles of T cell-derived VIP and altered expansion of the Th subsets. Dependence of the Th phenotype of T mice, but not of N or K mice, on T cell-derived VIP now is proven by showing that eliminating VIP from TCR-stimulated T cell cultures with VIPase IgG normalizes the elevated number of IL-4-secreting CD4 T cells, decreases the secretion of IL-4 and IL-10, and increases the secretion of IFN-gamma. Flexible responsiveness of CD4 T cells from N and K mice, but not T mice, to exogenous VIP in vitro and in vivo is shown by increased numbers of IL-4-secreting CD4 T cells, greater secretion of IL-4 and IL-10, and lesser secretion of IFN-gamma after TCR stimulation with VIP. The level of VIP recognized by CD4 T cells thus is a major determinant of the relative contributions of Th subsets to the immune effector phenotype.     

Human herpesvirus 6 infection of CD4+ T-cell subsets

The immune system includes CD4+ regulatory T (T reg) cells that play a role in self-tolerance and demonstrate functional variations that govern immune responses. HHV-6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T-cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV-6 to specific T-cell populations such as CD4+CD25 high, which includes the majority of T reg cells, and CD4+CD25(-). These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV-6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real-time PCR. The effects of virus infection on cytokine production from these T-cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV-6 replication. Virus infection enhanced the production of both Th1- and Th2-type cytokines from CD4+CD25(-) T cells; however, only Th2-type cytokine release was augmented from viral-infected CD4+CD25 high T cells. Further, while virusinfected CD4+CD25 high T cells shift their antiviral immunity toward Th2 dominance by producing IL-10, the role of virus-infected CD4+CD25(-) T cells remains obscure.     

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.     

High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.