Jeremy A. O'Sullivan

NKG2D signaling on CD8 + T cells represses T-bet and rescues CD4-unhelped CD8 + T cell memory recall but not effector responses

CD4-unhelped CD8+ T cells are functionally defective T cells primed in the absence of CD4+ T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8+ T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8+ T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8+ T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4+ T cells in a CD4-dependent influenza model and rescues HIV-specific CD8+ T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8+ T cells from their pathophysiological fate and may provide therapeutic benefits.

Stimulation of the Glucocorticoid-Induced TNF Receptor Family-Related Receptor on CD8 T Cells Induces Protective and High-Avidity T Cell Responses to Tumor-Specific Antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4+CD25+ regulatory T (Treg) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to Treg cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by Treg cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.

Development of Tumor-Infiltrating CD8+ T Cell Memory Precursor Effector Cells and Antimelanoma Memory Responses Are the Result of Vaccination and TGF-β Blockade during the Perioperative Period of Tumor Resection

A main goal of cancer immunology research is the formation of Ag-specific memory T cell immunity capable of activation upon tumor re-encounter. The requirements necessary to overcome the inhibitory signals present in the tumor microenvironment and form such memory T cell responses are unknown. In contrast to previous studies targeting tumors expressing highly immunogenic model Ags, we demonstrate that alleviating tumor-induced suppression along with vaccination against authentic Ags during the perioperative period provides long-lasting protection against a highly suppressive and poorly immunogenic melanoma. In this study, we employed DNA vaccination with an immunologically optimized mouse melanoma-shared Ag, Trp1ee/ng, combined with systemic TGF-β blockade during the perioperative period of primary tumor resection, to confer protection against B16 melanoma, and against JBRH, an independently derived melanoma unrelated to B16. Importantly, we demonstrate that correlative to memory responses, perioperative immunotherapy increases the formation of tumor-infiltrating and tumor-reactive CD8+ T cells expressing low levels of the transcription factor T-bet, defined as memory precursor effector cells. We show that conditions for an immunologically fertile environment are met when TGF-β blockade and vaccination are applied during the perioperative period of primary tumor resection. These findings address limitations of current CD8+ T cell immunotherapies against cancer by generating effective CD8+ T cell memory recall responses.

Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells

Background Sialic acid–binding immunoglobulin‐like lectin (Siglec)‐8 is a cell‐surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec‐8 surface expression. Objectives We sought to determine whether Siglec‐8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec‐8–bearing cells to kill these cells. Methods Siglec‐8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec‐8–transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec‐8 intracellular trafficking was followed by confocal microscopy. The ribosome‐inhibiting protein saporin was conjugated to a Siglec‐8–specific antibody to examine the targeting of an agent to these cells through Siglec‐8 endocytosis. Results Siglec‐8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec‐8 localized to the lysosomal compartment. Maximal endocytosis in Siglec‐8–transduced HEK293T cells required an intact immunoreceptor tyrosine‐based inhibitory motif. Siglec‐8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec‐8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec‐8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC‐1.2. Conclusions Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec‐8 endocytic pathway.

Eosinophils and eosinophil-associated diseases: An update

The goal of this series is to offer a survey of the latest literature for clinicians and scientists alike, providing a list of important recent advances relevant to the broad field of allergy and immunology. This particular assignment was to cover the topic of eosinophils. In an attempt to highlight major ideas, themes, trends, and advances relevant to basic and clinical aspects of eosinophil biology, a search of articles published since 2015 in the Journal of Allergy and Clinical Immunology and other high-impact journals was performed. Articles were then reviewed and organized, and then key findings were summarized. Given space limitations, many outstanding articles could not be included, but the hope is that what follows provides a succinct overview of recently published work that has significantly added to our knowledge of eosinophils and eosinophil-associated diseases.

CD8+ T Cells Sabotage Their Own Memory Potential through IFN-γ–Dependent Modification of the IL-12/IL-15 Receptor α Axis on Dendritic Cells

CD8+ T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4+ T cells, leading to the tenet that CD8+ T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8+ T cell priming, we demonstrate that CD8+ T cells, themselves, actively limit their own memory potential through CD8+ T cell-derived IFN-γ–dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8+ T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8+ T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.

Priming with very low-affinity peptide ligands gives rise to CD8+ T-cell effectors with enhanced function but with greater susceptibility to transforming growth factor (TGF)β-mediated suppression

While the effects of TCR affinity and TGFβ on CD8+ T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8+ T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8+ T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFβ in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8+ T-cell-mediated cancer immunotherapeutic strategies.

Engagement of NK receptor NKG2D, but not 2B4, results in self-reactive CD8+T cells and autoimmune vitiligo

In this study, we demonstrate that engagement of two different natural killer receptors (NKRs) can lead to contrasting effects in the development of self-reactive CD8+T cells and autoimmune vitiligo. Specifically, using a mouse model, we show that CD8+T-cell targeting of a melanocyte antigen, tyrosinase-related protein-1 (TRP-1) in combination with delivery of the NKG2D ligands (Rae-1ϵ or H60), results in strong CD8+T-cell responses against TRP-1 and in the development of autoimmune vitiligo. In contrast, targeting of TRP-1 in combination with delivery of CD48, the natural ligand for the NKR 2B4, leads to reduced formation of TRP-1-reactive CD8+T-cell responses and decreased development of vitiligo. These data indicate that autoimmune vitiligo is limited by insufficient signals, despite plentiful self-reactive T cells in the peripheral immune system. To our knowledge, this is the first experimental evidence supporting the role of NKRs in modulating CD8+T-cell autoimmune vitiligo. This study supports the utilization of NKR signaling as a therapeutic avenue toward prevention of vitiligo and other autoimmune diseases.

Sialic acid–binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating β2-integrin–dependent function in human eosinophils

Background: Siglec‐8 is a CD33 subfamily cell‐surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec‐8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33‐related Siglecs function as inhibitory receptors, but the ability of Siglec‐8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec‐8 might instead function as an activating receptor. Objective: We sought to determine the role of IL‐5 priming and identify the signaling molecules involved in Siglec‐8 function for human eosinophils. Methods: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec‐8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec‐8 function for human eosinophils. Results: Cytokine priming was required to elicit the unanticipated finding that Siglec‐8 engagement promotes rapid &bgr;2‐integrin–dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec‐8–mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec‐8–mediated eosinophil apoptosis. Finally, engagement of Siglec‐8 on IL‐5–primed eosinophils resulted in increased phosphorylation of Akt, p38, and c‐Jun N‐terminal kinase 1 that was also &bgr;2‐integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec‐8–mediated eosinophil apoptosis. Conclusions: These data demonstrate that Siglec‐8 functions uniquely as an activating receptor on IL‐5–primed eosinophils through a novel pathway involving regulation of &bgr;2‐integrin–dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Frontline Science: Characterization of a novel mouse strain expressing human Siglec‐8 only on eosinophils

Sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐8 is a human cell surface protein expressed exclusively on eosinophils, mast cells, and basophils that, when engaged, induces eosinophil apoptosis and inhibits mast cell mediator release. This makes Siglec‐8 a promising therapeutic target to treat diseases involving these cell types. However, preclinical studies of Siglec‐8 targeting in vivo are lacking because this protein is only found in humans, apes, and some monkeys. Therefore, we have developed a mouse strain in which SIGLEC8 transcription is activated by Cre recombinase and have crossed this mouse with the eoCre mouse to achieve eosinophil‐specific expression. We confirmed that Siglec‐8 is expressed exclusively on the surface of mature eosinophils in multiple tissues at levels comparable to those on human blood eosinophils. Following ovalbumin sensitization and airway challenge, Siglec‐8 knock‐in mice generated a pattern of allergic lung inflammation indistinguishable from that of littermate controls, suggesting that Siglec‐8 expression within the eosinophil compartment does not alter allergic eosinophilic inflammation. Using bone marrow from these mice, we demonstrated that, during maturation, Siglec‐8 expression occurs well before the late eosinophil developmental marker C‐C motif chemokine receptor 3, consistent with eoCre expression. Antibody ligation of the receptor induces Siglec‐8 endocytosis and alters the phosphotyrosine profile of these cells, indicative of productive signaling. Finally, we demonstrated that mouse eosinophils expressing Siglec‐8 undergo cell death when the receptor is engaged, further evidence that Siglec‐8 is functional on these cells. These mice should prove useful to investigate Siglec‐8 biology and targeting in vivo in a variety of eosinophilic disease models.