Splenic CD8α⁺ dendritic cells undergo rapid programming by cytosolic bacteria and inflammation to induce protective CD8⁺ T-cell memory

Memory CD8⁺ T lymphocytes are critical effector cells of the adaptive immune system mediating long‐lived pathogen‐specific protective immunity. Three signals – antigen, costimulation and inflammation – orchestrate optimal CD8⁺ T‐cell priming and differentiation into effector and memory cells and shape T‐cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α⁺ but not the CD8α^? cDCs most efficiently mediate CD8⁺ T‐cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I‐associated antigens, is most efficient at inducing naïve CD8⁺ T‐cell differentiation into pathogen‐specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α⁺ cDCs become endowed with all functional features to optimally prime protective memory CD8⁺ T cells in vivo within only a few hours post‐immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell‐based vaccine therapy.     

CD8α− conventional dendritic cells control Vβ T-cell immunity in response to Staphylococcus aureus infection in mice

Dendritic cells (DCs) are potent immune cells that control innate and adaptive immune responses. Previous studies have shown that the DCs are required for protection against Staphylococcus aureus infection. However, the role of conventional DC (cDC) subsets during S. aureus infection in vivo has not been well investigated. In this study, we examined the function of spleen DC subsets in the activation of immunity against S. aureus infection. C57BL/6 mice were infected intravenously with S. aureus and DC and T-cell activation were analyzed in vivo. We found that the spleen CD8α⁻ cDCs phagocytosed S. aureus more efficiently than type-1 conventional DCs (cDC1s) did. Moreover, the CD8α⁻ cDCs contributed to the production of pro-inflammatory cytokines in response to S. aureus infection, whereas the cDC1s did not. In addition, infection with S. aureus promoted an increase in the number of Vβ T cells. The CD4⁺ and CD8⁺ Vβ T cells up-regulated the production of interferon-γ (IFN-γ) and interleukin-17 (IL-17) in response to S. aureus infection. Importantly, the induction of IFN-γ and IL-17 production in CD4⁺ and CD8⁺ Vβ T cells was mediated by S. aureus-stimulated CD8α⁻ cDCs, whereas cDC1s failed to promote IFN-γ and IL-17 production in the cells. Therefore, these data suggested that the spleen CD8α⁻ cDCs are the main DC subsets for induction of S. aureus superantigen-specific immunity.     

CD8α⁺β⁻ and CD8α⁺β⁺ plasmacytoid dendritic cells induce Foxp3⁺ regulatory T cells and prevent the induction of airway hyper-reactivity

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8β as CD8α⁻β⁻, CD8α⁺β⁻, or CD8α⁺β⁺. In a mouse model of lung inflammation, adoptive transfer of CD8α⁺β⁻ or CD8α⁺β⁺ pDCs prevents the development of airway hyper-reactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3⁺ regulatory T cells compared with CD8α⁻β⁻ pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.     

IL4I1: an inhibitor of the CD8⁺ antitumor T-cell response in vivo

The L-phenylalanine oxidase IL4I1 inhibits T-cell proliferation in vitro through H(2) O(2) production, and is highly expressed in tumor-associated macrophages. IL4I1 is also detected by immunohistochemistry in neoplastic cells from several B-cell lymphomas and some non-lymphoid tumors. To evaluate IL4I1's effect on tumor growth, we developed a mouse melanoma model constitutively coexpressing IL4I1 and the GP33 epitope. After GP33 vaccination, tumors developed more frequently in mice injected with IL4I1-expressing cells in comparison with mice receiving control cells. Tumor escape was preceded by a rapid diminution of IFN-γ-producing cytotoxic antitumor CD8(+) T cells. Moreover, tumor incidence was already increased when only 20% of the injected cells expressed IL4I1. The minimal IL4I1 activities leading to tumor escape were close to those detected in human melanoma and mesothelioma. Thus, we demonstrate the immunosuppressive functions of IL4I1 in vivo and suggest that IL4I1 facilitates human tumor growth by inhibiting the CD8(+) antitumor T-cell response.     

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.     

CD103−CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4−CD8α− DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103−CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.     

Blockade of Tim-3 signaling restores the virus-specific CD8⁺ T-cell response in patients with chronic hepatitis B

Chronic hepatitis B (CHB) is characterized by functionally impaired virus-specific CD8(+) T-cell responses. However, the mechanism underlying this dysfunction has not been fully clarified. We examined the role of a newly identified protein, T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3), in regulating the antiviral CD8(+) T-cell response in CHB patients. Tim-3 expression on peripheral virus-specific CD8(+) T cells from 20 CHB patients and 20 healthy controls was determined by flow cytometry. The phenotypes and cytokine-producing capacity were compared between Tim-3(+) CD8(+) and Tim-3(-) CD8(+) T cells. The impact of Tim-3 signaling on cellular proliferation and cytokine-producing capacity was also studied. Tim-3 expression on hepatitis B virus (HBV)-specific CD8(+) T cells was higher than expression on cytomegalovirus (CMV)-specific CD8(+) T cells. Tim-3(+) CD8(+) T cells exhibited proliferative senescence phenotypes and decreased cytokine production upon antigen challenge. Finally, blocking the Tim-3 pathway significantly improved proliferation and antiviral cytokine secretion of CD8(+) T cells in response to HBV-specific antigen peptides. Tim-3 negatively regulates antiviral responses of CD8(+) T cells isolated from CHB patients, and this response is reversed by blocking the Tim-3 pathway.     

Plasmacytoid dendritic cells mature independently of MyD88 and IFN-αβR in response to Listeria and stimulate CD8 T cells

B cells not only play a pivotal role in humoral immunity, but also are involved in a broad spectrum of immune responses, including antigen presentation and T-cell function regulation. The identification of cell-surface CD molecules derived from a series of Human Leukocyte Differentiation Antigens (HLDA) Workshops has been instrumental to the discovery and functional characterization of human B-cell populations. Moreover, many events regulating B-cell development, activation, and effector functions are orchestrated by these cell-surface molecules. During the Ninth HLDA Workshop (HLDA9) eighteen new CDs were allocated to cell-surface molecules expressed on B cells: CD210a (IL10RA), CD215 (IL15RA), CD270 (TNFRSF14), CD307a (FCRL1), CD307b (FCRL2), CD307c (FCRL3), CD307d (FCRL4), CD351 (FCAMR), CD352 (SLAMF6), CD353 (SLAMF8), CD354 (TREM1), CD355 (CRTAM), CD357 (TNFRSF18), CD358 (TNFRSF21), CD360 (IL21RA), CD361 (EVI2B), CD362 (SDC2), and CD363 (S1PR1). Here we present their expression patterns on leukocytes, including T lymphocytes, NK cells, granulocytes, monocytes, plasmacytoid and monocyte-derived dendritic cells, and several B-cell subsets. These new CD molecules are expressed on B cells at various stages of differentiation; from bone marrow precursor pro-B cells to plasma cells. Three of them, CD307a, CD307b and CD307d, exhibit a B-cell restricted expression pattern, whereas the rest are also present on other leukocytes. In this paper we also review the structural characteristics, expression, and function of these new CD molecules. The availability of monoclonal antibodies directed against novel B cell-surface molecules will have broad implications not only for B-cell biology, but also for the development of new diagnostic and therapeutic tools.     

CD8α(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites

CD8α(+) dendritic cells (DCs) are important in?vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8α(+) DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T.?gondii infection. Toward this goal, we examined T.?gondii infection of Batf3(-/-) mice, which selectively lack only lymphoid-resident CD8α(+) DCs and related peripheral CD103(+) DCs. Batf3(-/-) mice were extremely susceptible to T.?gondii infection, with decreased production of IL-12 and interferon-γ. IL-12 administration restored resistance in Batf3(-/-) mice, and mice in which IL-12 production was ablated only from CD8α(+) DCs failed to control infection. These results reveal that the function of CD8α(+) DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T.?gondii infection.     

The earliest intrathymic precursors of CD8α(+) thymic dendritic cells correspond to myeloid-type double-negative 1c cells

The dendritic cells (DCs) present in lymphoid and non-lymphoid organs are generated from progenitors with myeloid-restricted potential. However, in the thymus a major subset of DCs expressing CD8α and langerin (CD207) appears to stand apart from all other DCs in that it is thought to derive from progenitors with lymphoid potential. Using mice expressing a fluorescent reporter and a diphtheria toxin receptor under the control of the cd207 gene, we demonstrated that CD207(+) CD8α(+) thymic DCs do not share a common origin with T cells but originate from intrathymic precursors that express markers that are normally present on all (CD11c(+) and MHCII molecules) or on some (CD207, CD135, CD8α, CX3CR1) DC subsets. Those intrathymic myeloid-type precursors correspond to CD44(+) CD25(-) double-negative 1c (DN1c) cells and are continuously renewed from bone marrow-derived canonical DC precursors. In conclusion, our results demonstrate that the earliest intrathymic precursors of CD8α(+) thymic DCs correspond to myeloid-type DN1c cells and support the view that under physiological conditions myeloid-restricted progenitors generate the whole constellation of DCs present in the body including the thymus.     

The response to TLR ligation of human CD16⁺CD14⁻ monocytes is weakly modulated as a consequence of persistent infection with the hepatitis C virus

Little is known about the frequency and function of CD16(+)CD14(-) monocytes from chronic HCV patients. We observed that the absolute numbers and ratio of CD16(+)CD14(-) to CD14(+)CD16(-) monocytes were similar between chronic HCV patients and healthy individuals. Functionally, we found that CD16(+)CD14(-) monocytes are more responsive to TLR8-ligation and only weakly responsive to LPS stimulation in producing TNF as compared to CD14(+)CD16(-) monocytes. We found no overt impairment of the function of CD16(+)CD14(-) monocytes from patients, except for an augmented induction of MIP-1β-producing CD16(+)CD14(-) monocytes upon TLR4-ligation. However, the increased frequency of MIP-1β-producing CD16(+)CD14(-) monocytes was not associated with viral load, ALT or fibrosis level. Our findings indicate that, different from other infectious diseases, the frequency and function of CD16(+)CD14(-) monocytes are only minimally altered as a consequence of the persistent state of HCV infections, and our findings therefore do not suggest a role for CD16(+)CD14(-) monocytes in HCV pathogenesis.     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.     

Analysis of the inflammatory response in HY-TCR transgenic mice highlights the pathogenic potential of CD4− CD8− T cells

Transgenic mice expressing a rearranged T cell receptor (TCR)-αβ prematurely at the double-negative stage develop an abnormal population of peripheral T cells that lack CD4 and CD8 expression and are hyper-reactive to anti-TCR antibody stimulation. One such example is the HY-TCR transgenic mice. These mice express a TCR transgenic specific for the HY antigen that is expressed in male but not in female mice. As a result, male mice have an abnormal population of HY⁺/CD4^? 8^? or HY⁺/CD4^? CD8^low T cells that are much lower in female mice. In this study, we investigated the potential patho/physiological function of these cells in vivo using a model of delayed-type hypersensitivity (DTH) reaction: the λ-carrageenan-induced paw edema. Interestingly, while both male and female HY-TCR mice develop a classical biphasic inflammatory response to λ-carrageenan, the degree of inflammation in the former was much higher than that in the latter. This was accompanied by a selective expansion of HY⁺/CD4^? 8^? and HY⁺/CD4^? CD8^low T cells in male mice and by a markedly increased production of typical DTH cytokines compared with cells from female mice. These results were specific since analysis of the inflammatory response of HY-TCR transgenic mice subjected to zymosan-induced peritonitis showed no differences between male and female mice. Together, these findings provide novel evidence for the pathological role of self-reactive CD4^? CD8^? T cells, previously described in several autoimmune strains and recently identified in patients suffering from autoimmune diseases such as systemic lupus erythematosus.     

Vaccinia virus infection of mature dendritic cells results in activation of virus-specific naïve CD8+ T cells: a potential mechanism for direct presentation

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of na?ve CD8(+) T cells. In order to optimally activate na?ve CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.     

Thymic but not splenic CD8⁺ DCs can efficiently cross-prime T cells in the absence of licensing factors

Cross‐presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross‐presentation, little is known about differences in cross‐presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady‐state thymic CD8⁺ DCs can efficiently cross‐prime naïve CD8⁺ T cells in the absence of costimulation. Surprisingly, cross‐priming by splenic CD8⁺ DCs was dependent on licensing factors such as GM‐CSF. In the absence of GM‐CSF, antigen–MHC‐class‐I complexes were detected on thymic but not on splenic CD8⁺ DCs, indicating that the cross‐presentation capacity of the thymic subpopulation was higher. The observed cross‐priming differences between thymic and splenic CD8⁺ DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide‐loaded splenic CD8⁺ DCs were able to induce CD8⁺ T‐cell proliferation. The observation that thymic CD8⁺ DCs are more efficient than splenic CD8⁺ DCs in T‐cell cross‐priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.     

Reduced tumor-antigen density leads to PD-1/PD-L1-mediated impairment of partially exhausted CD8⁺ T cells

Clinical progression of cancer patients is often observed despite the presence of tumor-reactive T cells. Co-inhibitory ligands of the B7 superfamily have been postulated to play a part in this tumor-immune escape. One of these molecules, PD-L1 (B7-H1, CD274), is widely expressed on tumor cells and has been shown to mediate T-cell inhibition. However, attempts to correlate PD-L1 tumor expression with negative prognosis have been conflicting. To better understand when PD-1/PD-L1-mediated inhibition contributes to the functional impairment of tumor-specific CD8(+) T cells, we varied the levels of antigen density and/or PD-L1 expression at the surface of tumor cells and exposed them to CD8(+) T cells at different levels of functional exhaustion. We found that the gradual reduction of cognate antigen expression by PD-L1-expressing tumor cells increased the susceptibility of partially exhausted T cells to PD-1/PD-L1-mediated inhibition in vitro as well as in vivo. In conclusion, chronically stimulated CD8(+) T cells become sensitive to PD-1/PD-L1-mediated functional inhibition upon low antigen detection; a setting which is likely involved during tumor-immune escape.     

The effects of advanced maternal age on T-cell subsets at the maternal–fetal interface prior to term labor and in the offspring: a mouse study

Women who conceive at 35 years of age or older, commonly known as advanced maternal age, have a higher risk of facing parturition complications and their children have an increased risk of developing diseases later in life. However, the immunological mechanisms underlying these pathological processes have yet to be established. To fill this gap in knowledge, using a murine model and immunophenotyping, we determined the effect of advanced maternal age on the main cellular branch of adaptive immunity, T cells, at the maternal–fetal interface and in the offspring. We report that advanced maternal age impaired the process of labor at term, inducing dystocia and delaying the timing of delivery. Advanced maternal age diminished the number of specific proinflammatory T-cell subsets [T helper type 1 (Th1): CD4⁺IFN-γ⁺, CD8⁺IFN-γ⁺ and Th9: CD4⁺IL-9⁺], as well as CD4⁺ regulatory T cells (CD4⁺CD25⁺FoxP3⁺ T cells), at the maternal–fetal interface prior to term labor. Advanced maternal age also altered fetal growth and survival of the offspring in early life. In addition, infants born to advanced-age mothers had alterations in the T-cell repertoire but not in CD71⁺ erythroid cells (CD3⁻CD71⁺TER119⁺ cells). This study provides insight into the immune alterations observed at the maternal–fetal interface of advanced-age mothers and their offspring.