Tara Capece

Neutrophil trails guide influenza-specific CD8+ T cells in the airways

Neutrophils lay down the tracks T cells constantly circulate throughout the body until an invading pathogen calls them into action. Microbes often cause localized infections, so how do T cells know where to go? Lim et al. explore this question in a mouse model of influenza infection and find that immune cells called neutrophils help guide the way (see the Perspective by Kiermaier and Sixt). Upon infection, neutrophils quickly traffic to the trachea. There, they lay down “tracks” enriched in proteins called chemokines, especially the chemokine CXCL12, which guide CD8+ T cells to the infected organ. Mice whose neutrophils could not lay down such tracks exhibited defects in CD8+ T cell recruitment and viral clearance. Science, this issue 10.1126/science.aaa4352; see also p. 1055 Trails of chemokines left behind by neutrophils guide T cells to sites of viral infection. [Also see Perspective by Kiermaier and Sixt] INTRODUCTION Influenza virus infects the epithelial cells that line the respiratory tract. Therefore, cytotoxic CD8+ T cells must traffic to this site to eliminate infected cells. The functions of antiviral CD8+ T cell effector at tissue sites require a successful and early innate immune response. Neutrophils are an immune cell subset that helps organs initiate and maintain immune reactions and shapes the overall immune response by signaling to multiple immune cell types, including T cells. Under most inflammatory conditions, neutrophils are the first cell type that crosses the blood vessel endothelium into the tissue, often preceding a subsequent wave of effector T cells. Although neutrophils are known to recruit T cells into infected sites during both bacterial and viral infections and in chronic inflammatory diseases, the molecular mechanisms that link neutrophil and T cell migration remain unknown. RATIONALE The chemokine receptor family is the most potent tissue-specific family of homing receptors for T cells and is subset-selective. Therefore, it is widely assumed that the distinct migratory properties and distribution patterns of different subsets of specialized T cells result from the differential expression of the chemokines and their receptors. Although this idea has been verified experimentally in some settings, multiple chemokine receptors expressed on the effector T cells and the redundancy in their signaling pathways suggest the presence of a more complex mechanism that can confer specificity and selectivity to T cell recruitment. Furthermore, less is known about how chemokines released from newly recruited leukocytes act together with the local chemokines produced within the inflamed tissue. To address this, we performed intravital multiphoton microscopy imaging of the influenza-infected mouse trachea and explored how neutrophil-derived chemokines cooperate with the tissue-specific inflammatory cues to finely control the recruitment of CD8+ T cells to the influenza-infected trachea. RESULTS Here, we show that optimal CD8+ T cell–mediated immune protection requires the early recruitment of neutrophils into influenza-infected trachea. In particular, the relative motility of virus-specific CD8+ T cells in the trachea was determined by their localization to the epithelium, which was governed by the presence of neutrophils during early infection. Both in vitro and in vivo imaging showed that migrating neutrophils leave behind long-lasting trails from their elongated uropods (a protrusion at the rear of a cell) that are prominently enriched in the chemokine CXCL12. We observed that CXCL12 derived from the epithelial cells remained close to the epithelium, whereas CXCL12 derived from neutrophils was the main source of CXCL12 in the tissue interstitium during infection. Experiments with granulocyte-specific CXCL12 conditionally depleted (knockout) mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8+ T cell recruitment and antiviral effector functions. CONCLUSION The data presented here demonstrate that migrating neutrophils leave behind chemoattractant-containing trails, which result in the local accumulation of neutrophil-derived chemoattractant signals in inflamed tissues. As chemokines are small, diffusible molecules, perhaps these trails function to package the chemoattractant so that it can be preserved and survive severe mechanical perturbation during inflammation. Otherwise, the chemoattractant would be present only transiently, or it would immediately diffuse away from the site. Neutrophils trails guide virus-specific CD8+ T cell migration. In the influenza-infected trachea, tissue-infiltrating neutrophils (pink) deposit chemokine (CXCL12)–containing trails, which may serve like breadcrumbs or long-lasting chemokine depots to provide both chemotactic and haptotactic cues for efficient virus-specific CD8+ T cell migration and localization in the infected tissues. During viral infections, chemokines guide activated effector T cells to infection sites. However, the cells responsible for producing these chemokines and how such chemokines recruit T cells are unknown. Here, we show that the early recruitment of neutrophils into influenza-infected trachea is essential for CD8+ T cell–mediated immune protection in mice. We observed that migrating neutrophils leave behind long-lasting trails that are enriched in the chemokine CXCL12. Experiments with granulocyte-specific CXCL12 conditionally depleted mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8+ T cell recruitment and effector functions. Collectively, these results suggest that neutrophils deposit long-lasting, chemokine-containing trails, which may provide both chemotactic and haptotactic cues for efficient CD8+ T cell migration and localization in influenza-infected tissues.

Essential Role of Elmo1 in Dock2-Dependent Lymphocyte Migration

Elmo1 and Elmo2 are highly homologous cytoplasmic adaptor proteins that interact with Dock family guanine nucleotide exchange factors to promote activation of the small GTPase Rac. In T lymphocytes, Dock2 is essential for CCR7- and CXCR4-dependent Rac activation and chemotaxis, but the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. In this article, we show that endogenous Elmo1, but not Elmo2, interacts constitutively with Dock2 in mouse and human primary T cells. CD4+ T cells from Elmo1−/− mice were profoundly impaired in polarization, Rac activation, and chemotaxis in response to CCR7 and CXCR4 stimulation. Transfection of full-length Elmo1, but not Elmo2 or a Dock2-binding mutant of Elmo1, rescued defective migration of Elmo1−/− T cells. Interestingly, Dock2 protein levels were reduced by 4-fold in Elmo1−/− lymphocytes despite normal levels of Dock2 mRNA. Dock2 polyubiquitination was increased in Elmo1−/− T cells, and treatment with proteasome inhibitors partially restored Dock2 levels in Elmo1−/− T cells. Finally, we show that Dock2 is directly ubiquitinated in CD4+ T cells and that Elmo1 expression in heterologous cells inhibits ubiquitination of Dock2. Taken together, these findings reveal a previously unknown, nonredundant role for Elmo1 in controlling Dock2 levels and Dock2-dependent T cell migration in primary lymphocytes. Inhibition of Dock2 has therapeutic potential as a means to control recruitment of pathogenic lymphocytes in diseased tissues. This work provides valuable insights into the molecular regulation of Dock2 by Elmo1 that can be used to design improved inhibitors that target the Elmo-Dock-Rac signaling complex.

Regulation of T cell motility in vitro and in vivo by LPA and LPA2.

Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2−/− CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.

A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation

The integrin lymphocyte function–associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8+ T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8+ T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8+ T cells plays a key role in T cell activation and differentiation.

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites.