Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells.

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.

Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells.

CD40L (CD154) on CD4(+) T cells has been shown to license dendritic cells (DCs) via CD40 to prime cytotoxic T lymphocyte (CTL) responses. We found that the converse (CD40L on DCs) was also important. Anti-CD40L treatment decreased endogenous CTL responses to both ovalbumin and influenza infection even in the absence of CD4(+) T cells. DCs expressed CD40L upon stimulation with agonists to Toll-like receptor 3 (TLR3) and TLR9. Moreover, influenza infection, which stimulates CTLs without help, upregulated CD40L on DCs, but herpes simplex infection, which elicits CTLs through help, did not. CD40L-deficient (Cd40lg(-/-)) DCs are suboptimal both in vivo in bone marrow chimera experiments and in vitro in mixed lymphocyte reactions. In contrast, Cd40lg(-/-) CD8(+) T cells killed as effectively as wild-type cells. Thus, CD40L upregulation on DCs promoted optimal priming of CD8(+) T cells without CD4(+) T cells, providing a mechanism by which pathogens may elicit helper-independent CTL immunity.

Id2 expression delineates differential checkpoints in the genetic program of CD8α+ and CD103+ dendritic cell lineages.

Dendritic cells (DCs) have critical roles in the induction of the adaptive immune response. The transcription factors Id2, Batf3 and Irf-8 are required for many aspects of murine DC differentiation including development of CD8α(+) and CD103(+) DCs. How they regulate DC subset specification is not completely understood. Using an Id2-GFP reporter system, we show that Id2 is broadly expressed in all cDC subsets with the highest expression in CD103(+) and CD8α(+) lineages. Notably, CD103(+) DCs were the only DC able to constitutively cross-present cell-associated antigens in vitro. Irf-8 deficiency affected loss of development of virtually all conventional DCs (cDCs) while Batf3 deficiency resulted in the development of Sirp-α(-) DCs that had impaired survival. Exposure to GM-CSF during differentiation induced expression of CD103 in Id2-GFP(+) DCs. It did not restore cross-presenting capacity to Batf3(-/-) or CD103(-)Sirp-α(-)DCs in vitro. Thus, Irf-8 and Batf3 regulate distinct stages in DC differentiation during the development of cDCs. Genetic mapping DC subset differentiation using Id2-GFP may have broad implications in understanding the interplay of DC subsets during protective and pathological immune responses.

Id2-mediated inhibition of E2A represses memory CD8+ T cell differentiation.

The transcription factor inhibitor of DNA binding (Id)2 modulates T cell fate decisions, but the molecular mechanism underpinning this regulation is unclear. In this study we show that loss of Id2 cripples effector differentiation and instead programs CD8(+) T cells to adopt a memory fate with increased Eomesodermin and Tcf7 expression. We demonstrate that Id2 restrains CD8(+) T cell memory differentiation by inhibiting E2A-mediated direct activation of Tcf7 and that Id2 expression level mirrors T cell memory recall capacity. As a result of the defective effector differentiation, Id2-deficient CD8(+) T cells fail to induce sufficient Tbx21 expression to generate short-lived effector CD8(+) T cells. Our findings reveal that the Id2/E2A axis orchestrates T cell differentiation through the induction or repression of downstream transcription factors essential for effector and memory T cell differentiation.

Interference with dendritic cell populations limits early antigen presentation in chronic γ-herpesvirus-68 infection.

A critical factor influencing the ability of the host to mount a robust immune response against a virus depends on the rapid recruitment of dendritic cells (DCs) presenting Ags. From the outset, this step sets the tempo for subsequent activation of virus-specific T cells. Despite this, how induction of the immune response might be modified by pathogens with the capacity to establish persistence is unclear. In this study, we have characterized the in vivo influence of murine gamma-herpesvirus K3-mediated interference with MHC class I in DCs that drive the initial adaptive immune response. We observed that gamma-herpesvirus could interfere with the very earliest phase of Ag presentation through K3 by directly targeting migratory and lymph node-resident DCs. These results show that a pathogen with the capacity to interfere with early Ag presentation can establish suboptimal conditions for rapid induction of the adaptive immune response and thus favor establishment of viral persistence.

Impairment of humoral immunity to Plasmodium falciparum malaria in pregnancy by HIV infection.

BACKGROUND: HIV infection increases the risk of malaria infection in pregnant women. Antibodies to variant surface antigens (VSA) on infected erythrocytes might protect against malaria in pregnancy. We postulated that HIV-induced impairment of humoral immunity to VSA mediates the increased susceptibility to malaria. METHODS: We compared serum concentrations of antibodies to VSA by flow cytometry or agglutination, and to merozoite proteins AMA-1 and MSP119 by ELISA, in 298 pregnant Malawian women, and related the findings to malaria and HIV infection, CD4-positive T-cell count, and HIV-1 viral load. FINDINGS: Concentrations of IgG to placental type VSA were lower in HIV-infected women than in HIV-uninfected women (median 8 units [IQR 4-23] vs 20 [12-30]; p<0.0001), among women with malaria (p=0.009) and those without malaria (p=0.0062). The impairment was greatest in first pregnancy. Agglutinating antibodies to placental VSA were present in a lower proportion of HIV-infected than HIV-uninfected women (58 [35.1%] of 165 vs 50 [53.8%] of 93, p<0.001). The degree of antibody binding by flow cytometry was correlated with CD4-positive T-cell count (r=0.16, p=0.019) and inversely with HIV-1 viral load (r=-0.16, p=0.030). Concentrations of antibodies to AMA-1 were lower in HIV infection (p<0.0001) but were not correlated with CD4-positive T-cell count or viral load. Responses to MSP119 were little affected by HIV infection. In multivariate analyses, HIV was negatively associated with amount of antibody to both VSA and AMA-1 (p<0.001 for each) but not MSP119. INTERPRETATION: HIV infection impairs antimalarial immunity, especially responses to placental type VSA. The impairment is greatest in the most immunosuppressed women and could explain the increased susceptibility to malaria seen in pregnant women with HIV infection.

Mouse models of viral infection: influenza infection in the lung.

Respiratory viral infections are a major cause of morbidity and mortality. Protection of the respiratory tract from pathogen infections, such as influenza virus, requires the orchestrated activation and trafficking of pulmonary dendritic cells (DCs) from the lung to the lymph node (LN) in order to ensure optimized T-cell responses. Gaining a better understanding of the cellular and molecular processes that protect the lung during infection is essential for future advances in vaccine strategies and treatments. Influenza viral infection in mice offers a very well-defined immunological system in which the underlying parameters regulating the generation of protective immunity can be elucidated. In this chapter, we review methods for quantitative analysis of DC and T-cell responses in a murine model infection of influenza. Antigen-specific tracking and quantitation of viral immune responses have been greatly facilitated by the advent of MHC tetramers and intracellular cytokine analysis, together with gentle isolation procedures for dendritic cells allowing detection of viral and endogenous antigens.

Dendritic cells: driving the differentiation programme of T cells in viral infections.

Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8(+) T cells. Successful memory cell generation begins with early interactions between naïve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8(+) T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8(+) T-cell immunity.

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation.

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

Bone marrow-derived cells expand memory CD8+ T cells in response to viral infections of the lung and skin.

While naive CD8(+) T cells have been shown to require bone marrow-derived dendritic cells (DC) to initiate immunity, such a requirement for memory CD8(+) T cells has had limited assessment. By generating bone marrow chimeras that express the appropriate antigen-presenting molecules on either radiation-sensitive bone marrow-derived or radiation-resistant non-bone marrow-derived compartments, we showed that both primary and secondary immune responses to influenza virus infection of the lung were initiated in the draining LN. This required cells of bone marrow origin, most likely DC, for optimal expansion within the secondary lymphoid compartment. This was similarly the case with HSV-1 infection of the skin. As Langerhans cells are radioresistant, unlike other DC populations, these studies also demonstrate that the radiosensitive DC responsible for secondary expansion of HSV-specific memory are not Langerhans cells.

CD4+ T cells specific for a model latency-associated antigen fail to control a gammaherpesvirus in vivo.

CD4(+) T cells play a major role in containing herpesvirus infections. However, their cellular targets remain poorly defined. In vitro CD4(+) T cells have been reported to kill B cells that harbor a latent gammaherpesvirus. We used the B cell-tropic murine gammaherpesvirus-68 (MHV-68) to test whether this also occurred in vivo. MHV-68 that expressed cytoplasmic ovalbumin (OVA) in tandem with its episome maintenance protein, ORF73, stimulated CD8(+) T cells specific for the H2-K(b)-restricted OVA epitope SIINFEKL and was rapidly eliminated from C57BL/6 (H2(b)) mice. However, the same virus failed to stimulate CD4(+) T cells specific for the I-A(d)/I-A(b)-restricted OVA(323-339) epitope. We overcame any barrier to the MHC class II-restricted presentation of an endogenous epitope by substituting OVA(323-339) for the CLIP peptide of the invariant chain (ORF73-IRES-Ii-OVA), again expressed in tandem with ORF73. This virus presented OVA(323-339) but showed little or no latency deficit in either BALB/c (H2(d)) or C57BL/6 mice. Latent antigen-specific CD4(+) T cells therefore either failed to recognize key virus-infected cell populations in vivo or lacked the effector functions required to control them.