Acquired pMHC I Complexes Greatly Enhance CD4＋ Th Cell＇s Stimulatory Effect on CD8＋T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4＋ helper T （Th） cells play pivotal roles in induction of CD8＋ CTL immunity. However, the mechanism of CD4＋ T cell help delivery to CD8＋ T cells in vivo is still elusive. In this study, we used ovalbumin （OVA）-pulsed dendritic cells （DCovA） to activate OT-II mouse CD4＋ T cells, and then studied the help effect of these CD4＋ T cells on CD8＋ cytotoxic T lymphocyte （CTL） responses＋ We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter （RIP）-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex （pMHC） I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination ＋ ＋ ＋ with TCR transgenlc OT-I mouse CD8 T ceils, the tolerogenic dosage of CD4＋ Th cells with acquired pMHC I, but not CD4＋ （Kb-/-） Th cells without acquired pMHC I were able to cause diabetes in 8/10 （80%） RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4＋ T cell-mediated help in CD8＋ CTL induction. Cellular ＆ Molecular Immunology. 2008;5（6）：407-415.     

I L- 15 trans-presentation regulates homeostasis of CD4^＋ T lymphocytes

Interleukin-15 （IL-15） is essential for the survival of memory CD8^＋ and CD4^＋ T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4^＋ T cells. Adoptive transfer of splenocytes from non-obese diabetic （NOD） mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.II15^-/- mice when compared to NOD.scid recipients. The increased accumulation of CD4^＋ T cells is also observed in NOD.II15^-/- mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the I L- 15Ra chain, but not those lacking the common gamma chain, also show increased accumulation of CD4^＋ T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4^＋ T cells and requires trans-presentation of IL-15. CD4^＋ T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4^＋ T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-7 production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4^＋ T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.     

EBV-Induced Human CD8^＋ NKT Cells Synergise CD4^＋ NKT Cells Suppressing EBV-Associated Tumours upon Induction of Thl-Bias

CD8^＋ natural killer T （NKT） cells from EBV-associated tumour patients are quantitatively and functionally impaired. EBV-induced CD8^＋ NKT cells drive syngeneic T cells into a Thl-bias response to suppress EBV-associated malignancies. IL-4-biased CD4^＋ NKT cells do not affect either syngeneic T cell cytotoxicity or Th cytokine secretion. Circulating mDC1 cells from patients with EBV-associated malignancies impair the production of IFN-T by CD8^＋ NKT cells. In this study, we have established a human-thymus-SCID chimaera model to further investigate the underlying mechanism of EBV-induced CD8^＋ NKT cells in suppressing EBV-associated malignancies. In the human-thymus-SCID chimera, EBV-induced CD8^＋ NKT cells suppress EBV-associated malignancies in a manner dependent on the Thl-bias response and syngeneic CD3^＋ T cells. However, adoptive transfer with CD4^＋ NKT cells alone inhibits T cell immunity. Interestingly, CD4^＋ NKT cells themselves secrete high levels of IL-2, enhancing the persistence of adoptively transferred CD8^＋ NKT cells and T cells, thereby leading to a more pronounced T cell anti-tumour response in chimaeras co-transferred with CD4^＋ and CD8^＋ NKT cells. Thus, immune reconstitution with EBV-induced CD4^＋ and CD8^＋ NKT cells synergistically enhances T cell tumour immunity, providing a potential prophylactic and therapeutic treatment for EBV-associated malignancies.     

Th17 cells and IL-17 are involved in the disruption of vulnerable plaques triggered by short-term combination stimulation in apolipoprotein E-knockout mice

Considerable evidence indicates that type 1 T helper (Th1)- and Th17-mediated immune responses promote the formation of atherosclerotic plaques while that CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) have a protective effect. However, the functions of diverse CD4⁺ lymphocyte subsets in plaque rupture remain poorly understood because of a shortage of satisfactory plaque rupture models. Here, we established a murine model of atherosclerotic plaque rupture using a high-fat diet and collar placement on the carotid artery, and triggered plaque rupture by short-term stimulation with a combination of lipopolysaccharide, phenylephrine injection and cold in apolipoprotein E-knockout (ApoE^−/−) mice. We investigated the associations between Th1 cells, Th17 cells and Tregs and plaque rupture by PCR, flow cytometry, ELISA and immunohistochemistry. In total, 75% (18/24) of vulnerable plaques, but no stable plaques, showed rupture characteristics. The proportion of Th17 cells was increased among splenocytes after treatment, but the changes in the levels of Th1 cells and Tregs were not related to rupture. Furthermore, the treatment resulted in high levels of interleukin-17 (IL-17) in the serum and in the region of plaque rupture. In vitro, IL-17 increased the level of apoptosis, a major factor associated with plaque rupture, in cultured murine vascular smooth muscle cells. Th17 cells and IL-17 may be involved in the disruption of vulnerable plaques triggered by short-term stimulation with lipopolysaccharide, phenylephrine injection and cold in ApoE^−/−mice.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Deficiency of Mouse CD4^＋CD25^＋Foxp3^＋ Regulatory T Cells in Xenogeneic Pig Thymus-Grafted Nude Mice Suffering from Autoimmune Diseases

Xenogeneic thymus transplantation can efficiently induce specific immune tolerance to donor antigens in athymic recipients. However, many nude mice suffer from autoimmune diseases （AID） for over 10 weeks after xenogeneic thymus transplantation. CD4^＋CD25^＋Foxp3^＋ regulatory T （Treg） cells were recently determined to play a pivotal role in keeping immune tolerance in humans and mice. Thus, we investigated this subpopulation of Treg cells in the periphery of pig thymus-grafted nude mice suffering from AID. Our results showed that the expression of Foxp3, CTLA-4 and GITR on mouse CD4^＋CD25^＋ T cells and the ratio of CD4^＋CD25^＋Foxp3^＋ Treg cells to CD4^＋ T cells were significantly decreased in the periphery of pig thymus-grafted nude mice suffering from AID, compared with healthy pig or mouse thymus-grafted nude mice. Furthermore, mouse CD4^＋CD25^＋ T cells in pig thymus-grafted nude mice suffering from AID showed more severe deficiency in immunosuppressive function compared with the counterpart in xenogeneic pig or syngeneic thymus-grafted nude mice without AID. Thus, the decreased frequency, altered phenotype and functional deficiency of mouse CD4^＋CD25^＋ Treg cells in pig thymus-grafted nude mice may contribute to the development of AID in this model.     

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor‐deficient mice

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR^−/−) mice. The pIgR^−/− mice exhibited the accumulation of CD8αβ⁺ T-cell receptor (TCR)-αβ⁺ IELs (CD8αβ⁺αβ-IELs) after weaning, but no increase of CD8αβ⁺γδ-IELs was detected in pIgR^−/− TCR-β^−/− mice compared with pIgR^+/+ TCR-β^−/− mice. When 5-bromo-2′-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR^+/+ mice and pIgR^−/− mice. However, the proportion of BrdU-labelled CD8αβ⁺-IELs became higher in pIgR^−/− mice than pIgR^+/+ mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR^+/+ TCR-β^−/− mice and pIgR^−/− TCR-β^−/− mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αβ⁺αβ-IELs increased much more in the SI of pIgR^−/− TCR-β^−/− mice than pIgR^+/+ TCR-β^−/− mice 8 weeks after the transfer. αβ-IELs from pIgR^−/− mice could produce more interferon-γ and interleukin-17 than those of pIgR^+/+ mice, and intestinal permeability tended to increase in the SI of pIgR^−/− mice with aging. Taken together, these results indicate that activated CD8αβ⁺αβ-IELs preferentially accumulate in pIgR^−/− mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.     

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4⁺ T-cell help (CD4 help) plays a pivotal role in CD8⁺ T-cell responses against viral infections. However, the role in primary CD8⁺ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8⁺ T-cell responses to vaccinia virus (VACV) in MHC class II^−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8⁺ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8⁺ T cells in i.p. infected MHC II^−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II^−/− mice when a small viral dose was used. These data suggested that primary CD8⁺ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8⁺ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8⁺ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8⁺ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8⁺ T cells than i.p. infection.     

Relative importance of T-cell subsets in monocytotropic ehrlichiosis: a novel effector mechanism involved in Ehrlichia-induced immunopathology in murine ehrlichiosis

Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4⁺ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8⁺ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4⁺ and CD8⁺ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10³ and 10⁵ bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4⁺ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8⁺ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8⁺ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4⁺ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4⁺ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4⁺ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4⁺ and CD8⁺ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8⁺ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4⁺ T-cell apoptosis.     

TLR4 inactivation protects from graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation. To clarify the role of Toll-like receptor 4 (TLR4), which is a major receptor for bacterial lipopolysaccharides (LPS), in the development of acute GVHD, we used a TLR4-knockout (TLR4^−/−) mouse GVHD model and analyzed the underlying immunological mechanisms. When TLR4^−/− mice were used as bone marrow and splenocyte cell graft donors or recipients, GVHD symptom occurrence and mortality were delayed compared to wild-type (TLR4^+/+) mice. In addition, histopathological analyses revealed that in TLR4^−/−→BALB/c chimeras, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. In contrast to TLR4^+/+, TLR4^−/− mice dendritic cells did not express CD80, CD86, CD40, MHC-II or IL-12 during LPS induction and remained in an immature state. Furthermore, the ability of TLR4^−/− mice spleen dendritic cells to promote allogeneic T-cell proliferation and, in particular, T-helper cell 1 (Th1) development was obviously attenuated compared with TLR4^+/+ mice dendritic cells, and the levels of interferon-γ (IFN-γ) and IL-10, Th2-cell specific cytokines, were significantly higher in the serum of TLR4^−/−→BALB/c than in TLR4^+/+→BALB/c chimeric mice. Overall, our data revealed that TLR4 may play a role in the pathogenesis of GVHD and that targeted TLR4 gene therapy might provide a new treatment approach to reduce the risk of GVHD.     

Calcineurin deficiency decreases inflammatory lesions in transforming growth factor β1‐deficient mice

Transforming growth factor (TGF) β1) is an immunoregulatory cytokine involved in self-tolerance and lymphocyte homeostasis. Tgfb1 knock-out (KO) mice develop severe multi-focal autoimmune inflammatory lesions due to [Ca²⁺]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1^−/− thymocytes, and because calcineurin Aβ (CNAβ)-deficient mice do not reject allogenic tumours, we have generated Tgfb1^−/−Cnab^−/− mice to address whether CNAβ deficiency prevents T cell activation and inflammation in Tgfb1^−/− mice. Here we show that in Tgfb1^−/−Cnab^−/− mice inflammation is reduced significantly relative to that in Tgfb1^−/− mice. However, both CD4⁺ and CD8⁺ T cells in double knock-out (DKO) mice are activated, as revealed by up-regulation of CD11a lymphocyte function-associated antigen-1 (LFA-1), CD44 and CD69 and down-regulation of CD62L. These data suggest that deficiency of CNAβ decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1^−/− T cells can undergo activation in the absence of CNAβ, probably by using the other isoform of calcineurin (CNAα) in a compensatory manner. CNAβ-deficient T cells undergo spontaneous activation in vivo and are activated upon anti-T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.     

CD8α-Deficient Mice Are Highly Susceptible to 5-Fluorouracil-Induced Lethality

Intestinal intraepithelial lymphocytes (i-IEL) expressing CD8α are located in the intestine and may confer protection against invasion of intestinal microflora. We found that mice rendered deficient in CD8α molecules by homologous recombination were susceptible to 5-fluorouracil (5-FU)-induced lethality accompanied by translocation of members of the enterobacteria. The number of i-IEL was greatly reduced on day 6 after 5-FU administration in both CD8α^+/− mice and CD8α^−/− mice, whereas the recovery of the level of i-IEL thereafter was significantly impaired in CD8α^−/− mice compared with that in CD8α^+/− mice. The ability of i-IEL to produce gamma interferon in response to immobilized T-cell receptor (TCR) αβ or TCR γδ monoclonal antibodies was significantly lower in CD8α^−/− mice than in CD8α^+/− mice. Transfer of CD8⁺ i-IEL conferred significant protection against 5-FU-induced lethality in CD8α^−/− mice. The results suggest that CD8⁺ i-IEL play an important role in protection against 5-FU-induced lethality with translocation of Enterobacteriaceae.     

Ly49 receptors activate angiogenic mouse DBA＋ uterine natural killer cells

In humans, specific patterns of killer immunoglobulin-like receptors （KIRs） expressed by uterine natural killer （uNK） cells are linked through HLA-C with pregnancy complications （infertility, recurrent spontaneous abortion, intrauterine growth restriction and preeclampsia）. To identify mechanisms underpinning the associations between NK cell activation and pregnancy success, pregnancies were studied in mice with genetic knockdown （KD） of the MHC-activated Ly49 receptor gene family. B6.Ly49KD pregnancies were compared to normal control B6.Ly49z29 and C57BL/6 （B6） pregnancies. At mid-pregnancy （gestation day （gd9.5））, overall uNK cell （TCRI~-CD122＋DBA＋DX5- （DBA＋DX5-）） and TCRIβ-CD122＋DBA-DX5＋ （DBA-DX5＋）） frequencies in pregnant uterus were similar between genotypes. Ly49KD lowered the normal frequencies of Ly49＋ uNK cells from 90.3% to 47.8% in DBA-DX5＋ and 78.8% to 6.3% in DBA＋DX5- uNK cell subtypes. B6.Ly49KD matings frequently resulted in expanded blastocysts that did not implant （subfertility）. B6.Ly49KD mice that established pregnancy had gestational lengths and litter sizes similar to controls. B6.Ly49KD neonates, however, were heavier than controls. B6.Ly49KD implantation sites lagged in early （gd6.5） decidual angiogenesis and were deficient in mid-pregnancy （gd 10.5） spiral arterial remodelling. Ultrastructural analyses revealed that B6.Ly49KD uNK cells had impaired granulogenesis, while immunocytochemistry revealed deficient vascular endothelial cell growth factor （VEGFA） production. Perforin and IFNG expression were normal in B6.Ly49KD uNK cells. Thus, in normal mouse pregnancies, Ly49 receptor signaling must promote implantation, early decidual angiogenesis and mid-pregnancy vascular remodelling. Disturbances in these functions may underlie the reported genetic associations between human pregnancy complications and the inability of specific conceptus MHCs to engage activating KIR on uNK cells.     

Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3⁺ T cells of HIV-seropositive (HIV⁺) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV⁺ HCV-seropositive (HCV⁺), HIV- and HCV-seropositive (HIV⁺ HCV⁺), and HIV- and HCV-seronegative (HIV⁻ HCV⁻) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV⁺ individuals is upregulated and that its expression on T cells from HCV⁺ individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3⁺ T cells was seen in both HIV⁺ and HIV⁺ HCV⁺ individuals compared to HIV⁻ HCV⁻ individuals. HCV⁺ individuals had lower levels of RTF expression than HIV⁻ HCV⁻ individuals (P < 0.005 for CD4⁺; P < 0.0005 for CD8⁺). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV⁺ > HIV⁺ HCV⁺ > HIV⁻ HCV⁻ > HCV⁺. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.