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.     

TSC1/2 signaling complex is essential for peripheral naïve CD8+ T cell survival and homeostasis in mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral na?ve CD8(+) T cells but not CD4(+) T cells were severely reduced. Tsc1 KO na?ve CD8(+) T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8(+) T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO na?ve CD8(+)T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO na?ve CD8(+)T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral na?ve CD8(+) T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory

Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.     

Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B(-/-)) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B(-/-) C57BL mice infected with the more persisting virus, virus-specific CD8(+) T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B(-/-) BALB/c mice, virus-specific CD8(+) T cells are completely deleted, indicating that host genotype influences the severity of the T cell defect. In B(-/-) C57BL mice infected with the less persisting virus, CD8(+) T cell dysfunction was not as pronounced, although it was clearly present. Most importantly, the appearance of dysfunctional CD8(+) T cells clearly precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B(-/-) mice to produce neutralizing Abs. In contrast with CD8(+) T cells, which initially respond almost as in wild-type mice, the priming of virus-specific CD4(+) T cells was markedly impaired in B(-/-) mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects are likely to contribute to the chronic course of viral infection in B(-/-) mice.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.     

Divergent roles for CD4+ T cells in the priming and effector/memory phases of adoptive immunotherapy

The requirement for CD4(+) Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4(+) T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II beta-chain (MHC II KO), revealed that priming of therapeutic CD8(+) effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4(+) T cell help. The adoptive transfer of CD8(+) effector T cells, but not CD4(+) effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8(+) effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8(+) T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8(+) T cells were resistant to tumor challenge. Resistance was mediated by CD8(+) T cells in mice at 50 days, while both CD4(+) and CD8(+) T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4(+) Th cells are not required for the priming phase of CD8(+) effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.     

Type I IFN negatively regulates CD8+ T cell responses through IL-10-producing CD4+ T regulatory 1 cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.     

A regulatory T cell-dependent novel function of CD25 (IL-2Ralpha) controlling memory CD8(+) T cell homeostasis

A massive systemic expansion of CD8(+) memory T (T(M)) cells and a remarkable increase in circulating IL-2 were observed only in IL-2Ralpha (CD25) knockout (KO) mice but not in IL-2 KO and scurfy mice, although all three mutants lack regulatory T (Treg) cells. However, both phenotypes were suppressed by the transfer of Treg cells. The data presented indicate that Treg cell deficiency drives naive T cells to T(M) cells. The lack of high-affinity IL-2R in IL-2Ralpha KO mice increases circulating IL-2 that is then preferentially used by CD8(+) T(M) cells through its abundant low-affinity IL-2R, resulting in systemic CD8(+) T(M) cell dominance. Our study demonstrates the critical control of CD8(+) T(M) cell homeostasis by a Treg cell-dependent novel function of CD25 and resolves its mechanism of action.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

The antiviral efficacy of the murine alpha-1 interferon transgene against ocular herpes simplex virus type 1 requires the presence of CD4(+), alpha/beta T-cell receptor-positive T lymphocytes with the capacity to produce gamma interferon

Alpha/beta interferons (IFN-alpha/betas) are known to antagonize herpes simplex virus type 1 (HSV-1) infection by directly blocking viral replication and promoting additional innate and adaptive, antiviral immune responses. To further define the relationship between the adaptive immune response and IFN-alpha/beta, the protective effect induced following the topical application of plasmid DNA containing the murine IFN-alpha 1 transgene onto the corneas of wild-type and T-cell-deficient mice was evaluated. Mice homozygous for both the T-cell receptor (TCR) beta- and delta-targeted mutations expressing no alpha beta or gamma delta TCR (alpha beta/gamma delta TCR double knockout [dKO]) treated with the IFN-alpha 1 transgene succumbed to ocular HSV-1 infection at a rate similar to that of alpha beta/gamma delta TCR dKO mice treated with the plasmid vector DNA. Conversely, mice with targeted disruption of the TCR delta chain and expressing no gamma delta TCR(+) cells treated with the IFN-alpha 1 transgene survived the infection to a greater extent than the plasmid vector-treated counterpart and at a level similar to that of wild-type controls treated with the IFN-alpha 1 transgene. By comparison, mice with targeted disruption of the TCR beta chain and expressing no alpha beta TCR(+) cells (alpha beta TCR knockout [KO]) showed no difference upon treatment with the IFN-alpha1 transgene or the plasmid vector control, with 0% survival following HSV-1 infection. Adoptively transferring CD4(+) but not CD8(+) T cells from wild-type but not IFN-gamma-deficient mice reestablished the antiviral efficacy of the IFN-alpha 1 transgene in alpha beta TCR KO mice. Collectively, the results indicate that the protective effect mediated by topical application of a plasmid construct containing the murine IFN-alpha 1 transgene requires the presence of CD4(+) T cells capable of IFN-gamma synthesis.     

Cholinergically stimulated gastric acid secretion is mediated by M(3) and M(5) but not M(1) muscarinic acetylcholine receptors in mice

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M(1)-M(5)) remains unclear. This study examined the involvement of M(1), M(3), and M(5) receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M(3) KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M(1) KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulated by carbachol, histamine, and gastrin. Pirenzepine, known as an M(1)-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M(1) KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M(1)-receptor antagonism. Notably, M(5) KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M(5)-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M(3) (on parietal cells) and M(5) receptors (conceivably in the submucosal plexus), but not M(1) receptors.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.     

Role of TRAIL and IFN-gamma in CD4+ T cell-dependent tumor rejection in the anterior chamber of the eye

Although the anterior chamber of the eye expresses immune privilege, some ocular tumors succumb to immune rejection. Previous studies demonstrated that adenovirus-induced tumors, adenovirus type 5 early region 1 (Ad5E1), underwent immune rejection following transplantation into the anterior chamber of syngeneic mice. Intraocular tumor rejection required CD4(+) T cells, but did not require the following: 1) CD8(+) T cells, 2) B cells, 3) TNF, 4) perforin, 5) Fas ligand, or 6) NK cells. This study demonstrates that CD4(+) T cell-dependent tumor rejection does not occur in IFN-gamma-deficient mice. Ad5E1 tumor cells expressed DR5 receptor for TRAIL and were susceptible to TRAIL-induced apoptosis. Although IFN-gamma did not directly induce apoptosis of the tumor cells, it rendered them 3-fold more susceptible to TRAIL-induced apoptosis. Both CD4(+) T cells and corneal endothelial cells expressed TRAIL and induced apoptosis of Ad5E1 tumor cells. The results suggest that Ad5E1 tumor rejection occurs via TRAIL-induced apoptosis as follows: 1) tumor cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis, 2) IFN-gamma enhances TRAIL expression on CD4(+) T cells and ocular cells, 3) IFN-gamma enhances tumor cell susceptibility to TRAIL-induced apoptosis, 4) apoptotic tumor cells are found in the eyes of rejector mice, but not in the eyes of IFN-gamma knockout mice that fail to reject intraocular tumors, 5) CD4(+) T cells and corneal endothelial cells express TRAIL and induce apoptosis of tumor cells, and 6) apoptosis induced by either CD4(+) T cells or corneal cells can be blocked with anti-TRAIL Ab.