Alice O. Kamphorst

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Germinal Center Dynamics Revealed by Multiphoton Microscopy with a Photoactivatable Fluorescent Reporter

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage

The zinc finger transcription factor zDC is uniquely expressed by the cDC lineage among immune cells, and the insertion of diphtheria toxin receptor cDNA into the zDC locus allows specific ablation of the cDC lineage in mice.

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.

A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center

Entry into the germinal center requires antigen-bearing B cells to compete for cognate T cell help at the T–B border.

Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes

Dendritic cells (DCs), which maintain tolerance and orchestrate T cell immune responses, comprise a heterogeneous group of cells. For example, in the steady state, murine spleen contains pre-DC–derived CD8+ and CD8− conventional DCs. During inflammation, monocytes become activated and acquire some DC-like features, such as expression of CD11c and MHC class II. Although each of these cell types can present Ag, the relative efficiency of processing and presentation after Ag capture by different routes has not yet been systematically compared. To this end, we administered OVA to various conventional DCs and activated monocytes by receptor-mediated endocytosis, pinocytosis, or phagocytosis and measured internalization and presentation to MHC class I- and MHC class II-restricted T cells. We find that CD8− DCs are more efficient than any other type of APC tested in terms of presenting Ag to MHC class II-restricted T cells, irrespective of the route of Ag capture. In contrast, both subsets of splenic DCs are highly effective in cross-presenting Ags to CD8+ T cells. DCs and activated monocytes cross-presented Ags delivered by DEC205-mediated endocytosis and pinocytosis. However, DCs differ from activated monocytes in that the latter are several orders of magnitude less efficient in presenting Ags captured by phagocytosis to CD8+ or CD4+ T cells. We conclude that DCs derived from pre-DCs differ from monocyte-derived cells in that DCs process and present Ags efficiently irrespective of the route of Ag capture. Our observations have significant implications for understanding initiation of immune responses and vaccination strategies targeting DCs and activated monocytes.

Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent

Immunotherapeutic PD-1–targeted therapies require CD28 to promote cancer cell killing. CD28 is a critical target for PD-1 blockade PD-1–targeted therapies have been a breakthrough for treating certain tumors and can rejuvenate T cells to unleash the anticancer immune response (see the Perspective by Clouthier and Ohashi). It is widely believed that PD-1 suppresses signaling through the T cell receptor (TCR). However, Hui et al. find instead that the TCR costimulatory receptor, CD28, is the primary target of PD-1 signaling. Independently, Kamphorst et al. show that CD28 is required for PD-1 therapies to kill cancer cells efficiently and eliminate chronic viral infections in mice. Lung cancer patients that responded to PD-1 therapy had more CD28+ T cells, which suggests that CD28 may predict treatment response. Science, this issue p. 1428, p. 1423; see also p. 1373 Programmed cell death–1 (PD-1)–targeted therapies enhance T cell responses and show efficacy in multiple cancers, but the role of costimulatory molecules in this T cell rescue remains elusive. Here, we demonstrate that the CD28/B7 costimulatory pathway is essential for effective PD-1 therapy during chronic viral infection. Conditional gene deletion showed a cell-intrinsic requirement of CD28 for CD8 T cell proliferation after PD-1 blockade. B7-costimulation was also necessary for effective PD-1 therapy in tumor-bearing mice. In addition, we found that CD8 T cells proliferating in blood after PD-1 therapy of lung cancer patients were predominantly CD28-positive. Taken together, these data demonstrate CD28-costimulation requirement for CD8 T cell rescue and suggest an important role for the CD28/B7 pathway in PD-1 therapy of cancer patients.

Antigen-specific CD4 T-cell help rescues exhausted CD8 T cells during chronic viral infection

CD4 T cells play a critical role in regulating CD8 T-cell responses during chronic viral infection. Several studies in animal models and humans have shown that the absence of CD4 T-cell help results in severe dysfunction of virus-specific CD8 T cells. However, whether function can be restored in already exhausted CD8 T cells by providing CD4 T-cell help at a later time remains unexplored. In this study, we used a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection to address this question. Adoptive transfer of LCMV-specific CD4 T cells into chronically infected mice restored proliferation and cytokine production by exhausted virus-specific CD8 T cells and reduced viral burden. Although the transferred CD4 T cells were able to enhance function in exhausted CD8 T cells, these CD4 T cells expressed high levels of the programmed cell death (PD)-1 inhibitory receptor. Blockade of the PD-1 pathway increased the ability of transferred LCMV-specific CD4 T cells to produce effector cytokines, improved rescue of exhausted CD8 T cells, and resulted in a striking reduction in viral load. These results suggest that CD4 T-cell immunotherapy alone or in conjunction with blockade of inhibitory receptors may be a promising approach for treating CD8 T-cell dysfunction in chronic infections and cancer.

Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection.

T reg cells effectively maintain CD8 T cell exhaustion during chronic LCMV infection, but blockade of PD-1 is critical for elimination of infected cells.

A role for lipid bodies in the cross-presentation of phagocytosed antigens by MHC class I in dendritic cells

Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.