TNF receptor signaling contributes to chemokine secretion, inflammation, and respiratory deficits during Pneumocystis pneumonia

CD8(+) T cells contribute to the pathophysiology of Pneumocystis pneumonia (PcP) in a murine model of AIDS-related disease. The present studies were undertaken to more precisely define the mechanisms by which these immune cells mediate the inflammatory response that leads to lung injury. Experimental mice were depleted of either CD4(+) T cells or both CD4(+) and CD8(+) T cells and then infected with Pneumocystis: The CD4(+)-depleted mice had significantly greater pulmonary TNF-alpha levels than mice depleted of both CD4(+) and CD8(+) T cells. Elevated TNF-alpha levels were associated with increased lung concentrations of the chemokines RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant. To determine whether TNFR signaling was involved in the CD8(+) T cell-dependent chemokine response, TNFRI- and II-deficient mice were CD4(+) depleted and infected with Pneumocystis: TNFR-deficient mice had significantly reduced pulmonary RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant responses, reduced inflammatory cell recruitment to the alveoli, and reduced histological evidence of PcP-related alveolitis as compared with infected wild-type mice. Diminished pulmonary inflammation correlated with improved surfactant activity and improved pulmonary function in the TNFR-deficient mice. These data indicate that TNFR signaling is required for maximal CD8(+) T cell-dependent pulmonary inflammation and lung injury during PcP and also demonstrate that CD8(+) T cells can use TNFR signaling pathways to respond to an extracellular fungal pathogen.     

Contribution of T cell subsets to the pathophysiology of Pneumocystis-related immunorestitution disease

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4(+) and CD8(+) T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4(+) cells were more abundant than CD8(+) cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8(+) cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4(+) T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8(+) T cell response was observed. In contrast, mice depleted of CD8(+) T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-gamma-producing CD4(+) cells, and a prolonged CD4(+) T cell response than mice with both CD4(+) and CD8(+) cells. These data suggest that CD4(+) T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8(+) T cells contributed to neither lung injury nor organism clearance when CD4(+) cells were present, but instead served to modulate CD4 function. In the absence of CD4(+) cells, CD8(+) T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4(+) and CD8(+) T cells affects the ultimate outcome of PcP-related IRD.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells

TNFR2 is predominantly expressed by a subset of human and mouse CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2(+) Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30-40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2(-) Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4(+)CD25(-)TNFR2(+) cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2(+) Tregs was considerably more potent than that of reportedly highly suppressive CD103(+) Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2(+) Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.     

Critical Role for Perforin-, Fas/FasL-, and TNFR1-Mediated Cytotoxic Pathways in Down-Regulation of Antigen-Specific T Cells during Persistent Viral Infection

Viral persistence following infection with invasive strains of lymphocytic choriomeningitis virus (LCMV) can be achieved by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset of infection drives responding cells into functional unresponsiveness (anergy) that can be followed by their physical elimination. In this report, we studied down-regulation of the virus-specific CD8(+)-T-cell response during persistent infection of adult mice with LCMV, with emphasis on the role of perforin-, Fas/FasL-, or tumor necrosis factor receptor 1 (TNFR1)-mediated cytolysis in regulating T-cell homeostasis. The results reveal that the absence of perforin, Fas-ligand, or TNFR1 has no significant effect on the kinetics of proliferation and functional inactivation of virus-specific CD8(+) T cells in the onset of chronic LCMV infection. However, these molecules play a critical role in the homeostatic regulation of T cells, influencing the longevity of the virus-specific CD8(+)-T-cell population once it has become anergic. Thus, CD8(+) T cells specific to the dominant LCMV NP(396-404) epitope persist in an anergic state for at least 70 days in perforin-, FasL-, or TNFR1-deficient mice, but they were eliminated by day 30 in C57BL/6 controls. These effects were additive as shown by a deficit of apoptotic death of NP(396-404) peptide-specific CD8(+) T cells in mice lacking both perforin and TNFR1. This suggests a role for perforin-, FasL-, and TNFR1-mediated pathways in down-regulation of the antiviral T cell response during persistent viral infection by determining the fate of antigen-specific T cells. Moreover, virus-specific anergic CD8(+) T cells in persistently infected C57BL/6 mice contain higher levels of Bcl-2 and Bcl-XL than functionally intact T cells generated during acute LCMV infection. In the case of proapoptotic factors, Bax expression did not differ between T-cell populations and Bad was below the limit of detection in all samples. As expression of the Bcl-2 family members controls susceptibility to apoptosis, this finding may provide a molecular basis for the survival of anergic cells under conditions of prolonged antigen stimulation.     

Pulmonary T cell activation in response to chronic particulate air pollution

The purpose of this study was to investigate the effects of chronically inhaled particulate matter <2.5 μm (PM(2.5)) on inflammatory cell populations in the lung and systemic circulation. A prominent component of air pollution exposure is a systemic inflammatory response that may exaggerate chronic diseases such as atherosclerosis and insulin resistance. T cell response was measured in wild-type C57B/L6, Foxp3-green fluorescent protein (GFP) "knockin," and chemokine receptor 3 knockout (CXCR3(-/-)) mice following 24-28 wk of PM(2.5) or filtered air. Chronic PM(2.5) exposure resulted in increased CXCR3-expressing CD4(+) and CD8(+) T cells in the lungs, spleen, and blood with elevation in CD11c(+) macrophages in the lung and oxidized derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine in wild-type mice. CXCR3 deficiency decreased T cells in the lung. GFP(+) regulatory T cells increased with PM(2.5) exposure in the spleen and blood of Foxp3-GFP mice but were present at very low levels in the lung irrespective of PM(2.5) exposure. Mixed lymphocyte cultures using primary, PM(2.5)-treated macrophages demonstrated enhanced T cell proliferation. Our experiments indicate that PM(2.5) potentiates a proinflammatory Th1 response involving increased homing of CXCR3(+) T effector cells to the lung and modulation of systemic T cell populations.     

TNF receptor 1 mediates dendritic cell maturation and CD8 T cell response through two distinct mechanisms

TNF-α and its two receptors (TNFR1 and 2) are known to stimulate dendritic cell (DC) maturation and T cell response. However, the specific receptor and mechanisms involved in vivo are still controversial. In this study, we show that in response to an attenuated mouse hepatitis virus infection, DCs fail to mobilize and up-regulate CD40, CD80, CD86, and MHC class I in TNFR1(-/-) mice as compared with the wild-type and TNFR2(-/-) mice. Correspondingly, virus-specific CD8 T cell response was dramatically diminished in TNFR1(-/-) mice. Adoptive transfer of TNFR1-expressing DCs into TNFR1(-/-) mice rescues CD8 T cell response. Interestingly, adoptive transfer of TNFR1-expressing naive T cells also restores DC mobilization and maturation and endogenous CD8 T cell response. These results show that TNFR1, not TNFR2, mediates TNF-α stimulation of DC maturation and T cell response to mouse hepatitis virus in vivo. They also suggest two mechanisms by which TNFR1 mediates TNF-α-driven DC maturation, as follows: a direct effect through TNFR1 expressed on immature DCs and an indirect effect through TNFR1 expressed on naive T cells.     

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.     

CD4+CD25+ T cell-dependent inhibition of autoimmunity in transgenic mice overexpressing human Bcl-2 in T lymphocytes

Regulation of lymphocyte survival is essential for the maintenance of lymphoid homeostasis preventing the development of autoimmune diseases. Recently, we described a systemic lupus erythematosus associated with an IgA nephropathy in autoimmune-prone (NZW x C57BL/6)F(1) overexpressing human Bcl-2 (hBcl-2) in B cells (transgenic (Tg) 1). In the present study, we analyze in detail a second line of hBcl-2 Tg mice overexpressing the transgene in all B cells and in a fraction of CD4(+) and CD8(+) T cells (Tg2). We demonstrate here that the overexpression of hBcl-2 in T cells observed in Tg2 mice is associated with a resistance to the development of lupus disease and collagen type II-induced arthritis in both (NZW x C57BL/6)F(1) and (DBA/1 x C57BL/6)F(1) Tg2 mice, respectively. The disease-protective effect observed in autoimmune-prone Tg2 mice is accompanied by an increase of peripheral CD4(+)CD25(+) hBcl-2(+) regulatory T cells (T(regs)), expressing glucocorticoid-induced TNFR, CTLA-4, and FoxP3. Furthermore, the in vivo depletion of CD4(+)CD25(+) T(regs) in (DBA/1 x C57BL/6)F(1) Tg2 mice promotes the development of a severe collagen type II-induced arthritis. Taken together, our results indicate that the overexpression of hBcl-2 in CD4(+) T cells alters the homeostatic mechanisms controlling the number of CD4(+)CD25(+) T(regs) resulting in the inhibition of autoimmune diseases.     

Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.     

Parenchymal,but not leukocyte,TNF receptor 2 mediates T cell-dependent hepatitis in mice

TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.     

TNF receptor-associated factor 1 expressed in resident lung cells is required for the development of allergic lung inflammation

TNF is a major therapeutic target in a range of chronic inflammatory disorders, including asthma. TNFR-associated factor (TRAF)1 is an intracellular adaptor molecule important for signaling by TNFR. In this study, we investigated the role of TRAF1 in an adoptive transfer model of allergic lung inflammation. Mice deficient in TRAF1 (TRAF1(-/-)) and wild-type (WT) control animals were adoptively transferred with WT OVA-immune CD4(+) T cells, exposed to an aerosol of LPS-free OVA, and analyzed for the development of allergic lung inflammation. In contrast to WT mice, TRAF1(-/-) recipients failed to display goblet cell hyperplasia, eosinophilic inflammation, and airway hyperresponsiveness in this model of asthma. Neither T cell recruitment nor expression of the proinflammatory cytokines IL-4, IL-5, IL-13, or TNF occurred in the lungs of TRAF1(-/-) mice. Although purified myeloid TRAF1(-/-) dendritic cells (DCs) exhibited normal Ag-presenting function and transmigratory capacity in vitro and were able to induce OVA-specific immune responses in the lung draining lymph nodes (LNs) following adoptive transfer in vivo, CD11c(+)CD11b(+) DCs from airways of TRAF1(-/-) recipients were not activated, and purified draining LN cells did not proliferate in vitro. Moreover, transfer of WT or TRAF1(-/-) DCs failed to restore T cell recruitment and DC activation in the airways of TRAF1(-/-) mice, suggesting that the expression of TRAF1 in resident lung cells is required for the development of asthma. Finally, we demonstrate that T cell-transfused TRAF1(-/-) recipient mice demonstrated impaired up-regulation of ICAM-1 expression on lung cells in response to OVA exposure.     

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.     

Lung T Lymphocyte Trafficking and Activation during Ischemic Acute Kidney Injury

Despite advances in renal replacement therapy, the mortality rate for acute kidney injury (AKI) remains unacceptably high, likely owing to extrarenal organ dysfunction. Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators that facilitate organ crosstalk and induce caspase-dependent lung apoptosis and injury through a TNFR1-dependent pathway. Given that T lymphocytes mediate local IRI in the kidney and are known to drive TNFR1-mediated apoptosis, we hypothesized that T lymphocytes activated during kidney IRI would traffic to the lung and mediate pulmonary apoptosis during AKI. In an established murine model of kidney IRI, we identified trafficking of CD3(+) T lymphocytes to the lung during kidney IRI by flow cytometry and immunohistochemistry. T lymphocytes were primarily of the CD3(+)CD8(+) phenotype; however, both CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocytes expressed CD69 and CD25 activation markers during ischemic AKI. The activated lung T lymphocytes did not demonstrate an increased expression of intracellular TNF-α or surface TNFR1. Kidney IRI induced pulmonary apoptosis measured by caspase-3 activation in wild-type controls, but not in T cell-deficient (T(nu/nu)) mice. Adoptive transfer of murine wild-type T lymphocytes into T(nu/nu) mice restored the injury phenotype with increased cellular apoptosis and lung microvascular barrier dysfunction, suggesting that ischemic AKI-induced pulmonary apoptosis is T cell dependent. Kidney-lung crosstalk during AKI represents a complex biological process, and although T lymphocytes appear to serve a prominent role in the interorgan effects of AKI, further experiments are necessary to elucidate the specific role of activated T cells in modulating pulmonary apoptosis.     

Enhanced neointima formation following arterial injury in immune deficient Rag-1-/- mice is attenuated by adoptive transfer of CD8 T cells

T cells modulate neointima formation after arterial injury but the specific T cell population that is activated in response to arterial injury remains unknown. The objective of the study was to identify the T cell populations that are activated and modulate neointimal thickening after arterial injury in mice. Arterial injury in wild type C57Bl6 mice resulted in T cell activation characterized by increased CD4(+)CD44(hi) and CD8(+)CD44(hi) T cells in the lymph nodes and spleens. Splenic CD8(+)CD25(+) T cells and CD8(+)CD28(+) T cells, but not CD4(+)CD25(+) and CD4(+)CD28(+) T cells, were also significantly increased. Adoptive cell transfer of CD4(+) or CD8(+) T cells from donor CD8-/- or CD4-/- mice, respectively, to immune-deficient Rag-1-/- mice was performed to determine the T cell subtype that inhibits neointima formation after arterial injury. Rag-1-/- mice that received CD8(+) T cells had significantly reduced neointima formation compared with Rag-1-/- mice without cell transfer. CD4(+) T cell transfer did not reduce neointima formation. CD8(+) T cells from CD4-/- mice had cytotoxic activity against syngeneic smooth muscle cells in vitro. The study shows that although both CD8(+) T cells and CD4(+) T cells are activated in response to arterial injury, adoptive cell transfer identifies CD8(+) T cells as the specific and selective cell type involved in inhibiting neointima formation.     

MHC-independent genetic factors control the magnitude of CD4+ T cell responses to amyloid-β peptide in mice through regulatory T cell-mediated inhibition

Accumulation of amyloid-β peptide (Aβ) is considered the triggering factor of pathogenic lesions in Alzheimer's disease (AD), and vaccines targeting Aβ are promising therapeutic options. However, the occurrence of meningoencephalitides attributed to T cell responses in 6% of Aβ-immunized patients underscores the need for a better understanding of T cell responses to Aβ. We characterized the parameters controlling the magnitude of Aβ-specific CD4(+) T cell responses in mice. T cell responsiveness to Aβ1-42 was highly heterogeneous between mouse strains of different H-2 haplotypes, with SJL/J (H-2(s)) mice displaying a strong response, mainly specific for Aβ10-24, and C57BL/6 (H-2(b)) mice displaying a weak response to Aβ16-30. Surprisingly, C57BL/6 mice congenic for the H-2(s) haplotype (B6.H-2(S)), which display a "permissive" MHC class II allele for presentation of the immunodominant Aβ10-24 epitope, showed a very weak CD4(+) T cell response to Aβ, suggesting that MHC-independent genes downmodulate Aβ-specific CD4(+) T cell responses in C57BL/6 background. Vaccine-induced CD4(+) T cell responses to Aβ were significantly enhanced in both C57BL/6 and B6.H-2(S) mice upon depletion of regulatory T cells (Tregs), whereas Treg-depleted SJL/J mice displayed unaltered Aβ-specific T cell responses. Finally, Treg depletion in C57BL/6 transgenic APPPS1 mice, a mouse model of AD, results in enhanced vaccine-induced CD4(+) T cell responses in AD compared with wild-type animals. We concluded that the magnitude of Aβ-specific CD4(+) T cell responses is critically controlled in both physiological and pathological settings by MHC-independent genetic factors that determine the overall potency of Aβ-specific Treg responses.     

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.