Gabrielle T. Belz

Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens

Summary:  Cross‐presentation involves the uptake and processing of exogenous antigens within the major histocompatibility complex (MHC) class I pathway. This process is primarily performed by dendritic cells (DCs), which are not a single cell type but may be divided into several distinct subsets. Those expressing CD8α together with CD205, found primarily in the T‐cell areas of the spleen and lymph nodes, are the major subset responsible for cross‐presenting cellular antigens. This ability is likely to be important for the generation of cytotoxic T‐cell immunity to a variety of antigens, particularly those associated with viral infection, tumorigenesis, and DNA vaccination. At present, it is unclear whether the CD8α‐expressing DC subset captures antigen directly from target cells or obtains it indirectly from intermediary DCs that traffic from peripheral sites. In this review, we examine the molecular basis for cross‐presentation, discuss the role of DC subsets, and examine the contribution of this process to immunity, with some emphasis on DNA vaccination.

Virus-Specific CD8+ T Cells in Primary and Secondary Influenza Pneumonia

Virus-specific CD8+ effector T cells (eCTL) are enriched in the lungs of mice with primary influenza pneumonia, though later detection of memory T cells (mCTL) in the mediastinal lymph nodes (MLN) or spleen by peptide-based staining protocols is at the limits of flow cytometric analysis. Respiratory challenge with an H3N2 virus months after H1N1 priming induces a massive recall response, which reduces virus titers 2-3 days earlier than in nave controls. Influenza-specific mCTL produce interferon-gamma within 6 hr, but still take 4-5 days to localize to the infected respiratory tract. The delay reflects that the recall response develops first in the MLN, which contains relatively few mCTL. The response to a subdominant epitope is less obvious after secondary challenge.

The CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens.

We previously described a mechanism for the maintenance of peripheral self-tolerance. This involves the cross-presentation of tissue-associated antigens by a bone marrow–derived cell type that stimulates the proliferation and ultimate deletion of self-reactive CD8 T cells. This process has been referred to as cross-tolerance. Here, we characterize the elusive cell type responsible for inducing cross-tolerance as a CD8α+ dendritic cell (DC). To achieve this aim, transgenic mice were generated expressing yellow fluorescent protein (YFP) linked to CTL epitopes for ovalbumin and glycoprotein B (gB) of herpes simplex virus under the rat insulin promoter (RIP). Although tracking of YFP was inconclusive, the use of a highly sensitive gB-specific hybridoma that produced β-galactosidase on encounter with antigen, enabled detection of antigen presentation by cells isolated from the pancreatic lymph node. This showed that a CD11c+CD8α+ cell was responsible for cross-tolerance, the same DC subset as previously implicated in cross-priming. These data indicate that CD8α+ DCs play a critical role in both tolerance and immunity to cell-associated antigens, providing a potential mechanism by which cytotoxic T lymphocyte can be immunized to viral antigens while maintaining tolerance to self.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Blimp-1 Transcription Factor Is Required for the Differentiation of Effector CD8+ T Cells and Memory Responses

In response to viral infection, naive CD8(+) T cells proliferate and differentiate into cytotoxic and cytokine-producing effector cells. Here we showed that the transcription factor Blimp-1, a crucial regulator of plasma cell differentiation, was required for CD8(+) T cells to differentiate into functional killer T cells in response to influenza virus. Blimp-1 was not essential for the generation of memory T cells but was crucial for their efficient recall response upon reinfection. Antigen-specific Blimp-1-deficient CD8(+) T cells failed to appropriately regulate the transcriptional program essential for killer T cell responses and showed impaired migration to the site of infection. This study identifies Blimp-1 as a master regulator of the terminal differentiation of CD8(+) effector T cells and uncovers a conservation of the pathways that regulate the terminal differentiation of T and B cells.

Cutting Edge: Conventional CD8α+ Dendritic Cells Are Generally Involved in Priming CTL Immunity to Viruses

Dendritic cells (DCs) play a central role in initiating immune responses. Despite this, there is little understanding how different DC subsets contribute to immunity to different pathogens. CD8α+ DC have been shown to prime immunity to HSV. Whether this very limited capacity of a single DC subset priming CTL immunity is restricted to HSV infection or is a more general property of anti-viral immunity was examined. Here, we show that the CD8α+ DCs are the principal DC subset that initiates CTL immunity to s.c. infection by influenza virus, HSV, and vaccinia virus. This same subset also dominated immunity after i.v. infection with all three viruses, suggesting a similar involvement in other routes of infection. These data highlight the general role played by CD8α+ DCs in CTL priming to viral infection and raises the possibility that this DC subset is specialized for viral immunity.

The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells

Regulatory T cells (Treg cells) are required for peripheral tolerance. Evidence indicates that Treg cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of Treg cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector Treg cells. Thus, our study defines a differentiation pathway that leads to the acquisition of Treg cell effector functions and requires both IRF4 and Blimp-1.

Measuring the diaspora for virus-specific CD8+ T cells.

The CD8+ T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of DbNP366- and DbPA224-specific CD8+ T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8+ tetramer+ populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the “whole mouse” virus-specific CD8+ T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8+DbNP366+ and CD8+DbPA224+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 “activation marker” were detected consistently on virus-specific CD8+ T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69hi T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of “resting” CD8+ memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

Circulating Precursor CCR7loPD-1hi CXCR5+ CD4+ T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

Compromised Influenza Virus-Specific CD8+-T-Cell Memory in CD4+-T-Cell-Deficient Mice

ABSTRACT The primary influenza A virus-specific CD8+-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-Ab+/+ and CD4+-T-cell-deficient I-Ab−/− mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4+ subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of naïve and previously immunized I-Ab−/− mice. Thus, though the capacity to mediate the CD8+-T-cell effector function is broadly preserved in the absence of concurrent CD4+-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.