Lorraine T. Tygrett

Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection

Infection of erythrocytes with Plasmodium species induces clinical malaria. Parasite-specific CD4+ T cells correlate with lower parasite burdens and severity of human malaria and are needed to control blood-stage infection in mice. However, the characteristics of CD4+ T cells that determine protection or parasite persistence remain unknown. Here we show that infection of humans with Plasmodium falciparum resulted in higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction. In vivo blockade of the PD-1 ligand PD-L1 and the inhibitory receptor LAG-3 restored CD4+ T cell function, amplified the number of follicular helper T cells and germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, and proper function can be restored by inhibitory therapies to enhance parasite control.

Elevated mitochondrial superoxide disrupts normal T cell development, impairing adaptive immune responses to an influenza challenge

Reactive oxygen species (ROS) are critical in a broad spectrum of cellular processes including signaling, tumor progression, and innate immunity. The essential nature of ROS signaling in the immune systems of Drosophila and zebrafish has been demonstrated; however, the role of ROS, if any, in mammalian adaptive immune system development and function remains unknown. This work provides the first clear demonstration that thymus-specific elevation of mitochondrial superoxide (O(2)(•-)) disrupts normal T cell development and impairs the function of the mammalian adaptive immune system. To assess the effect of elevated mitochondrial superoxide in the developing thymus, we used a T-cell-specific knockout of manganese superoxide dismutase (i.e., SOD2) and have thus established a murine model to examine the role of mitochondrial superoxide in T cell development. Conditional loss of SOD2 led to increased superoxide, apoptosis, and developmental defects in the T cell population, resulting in immunodeficiency and susceptibility to the influenza A virus H1N1. This phenotype was rescued with mitochondrially targeted superoxide-scavenging drugs. These findings demonstrate that loss of regulated levels of mitochondrial superoxide lead to aberrant T cell development and function, and further suggest that manipulations of mitochondrial superoxide levels may significantly alter clinical outcomes resulting from viral infection.

T regulatory cells participate in the control of germinal centre reactions.

Germinal centre (GC) reactions are central features of T‐cell‐driven B‐cell responses, and the site where antibody‐producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti‐glucocorticoid‐induced tumour necrosis factor receptor‐related protein (GITR) monoclonal antibody (mAb) to disrupt Treg‐cell activity. In anti‐GITR‐treated mice, the GC B‐cell pool was significantly larger compared with control‐treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti‐CD25 mAb treatment. Within the spleens of immunized mice, CXCR5+ and CCR7− Treg cells were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti‐transforming growth factor‐β or anti‐interleukin‐10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

B‐Cell Subsets Defined by the FcεRa

The data summarized herein demonstrate the utility of the low-affinity Fc epsilon R in delineating murine B-cell subsets. In the peritoneal cavity, the Fc epsilon R appears to be a reliable marker in distinguishing between conventional (Fc epsilon R+) and Ly-1/sister (Fc epsilon R-) B cells. In the spleens of normal animals, flow cytometric and histologic studies established that a distinct population of Fc epsilon R- B cells is also present and comprises the marginal zones. Thus in the spleen, the Fc epsilon R may be the first murine marker to allow for selective purification and analysis of marginal zone B cells. Although it is unlikely that splenic Fc epsilon R- B cells are directly related to peritoneal Fc epsilon R- Ly-1/sister B cells, further studies will be required to address this question. Analysis of autoimmune mice revealed that the splenic Fc epsilon R- subset is greatly expanded in these animals and indicates that the Fc epsilon R may be a sensitive indicator of abnormalities within the B-cell compartment. Additional studies compared the functional capacity of Fc epsilon R+ and Fc epsilon R- B cells and tested the ability of these populations to isotype-switch and respond to polyclonal stimuli. The results showed that Fc epsilon R+ and Fc epsilon R- B cells from both the peritoneum and spleen can switch to produce IgG, and all but the peritoneal Fc epsilon R- B cells can switch to the IgE class. This latter result is certainly interesting and demonstrates an important functional difference between peritoneal and splenic Fc epsilon R- B cells. Finally, experiments with B-cell mitogens showed further differences between the Fc epsilon R+ and Fc epsilon R- subsets. Whereas Fc epsilon R- B cells appeared to be more sensitive to LPS stimulation, Fc epsilon R+ B cells were clearly more responsive to an anti-IgM signal. Taken together, the results show that the Fc epsilon R is likely to be useful in separating B cells with different phenotypic, histologic, and functional characteristics.

Effect of pregnancy on thymic T cell development

PROBLEM: The thymus gland decreases in size during pregnancy. The significance of this alteration is not known.

18 F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib

18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression and treatment responses in genetically engineered mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development and new interventions in transgenic mouse models of human blood cancers such as multiple myeloma (MM) has not been demonstrated. Here we use BALB/c mice that contain the newly developed iMycΔEμ gene insertion and the widely expressed H2-Ld-IL6 transgene to demonstrate that FDG-PET/CT affords an excellent research tool for assessing interleukin-6- and MYC-driven plasma cell tumor (PCT) development in a serial, reproducible and stage- and lesion-specific manner. We also show that FDG-PET/CT permits determination of objective drug responses in PCT-bearing mice treated with the investigational proteasome inhibitor ixazomib (MLN2238), the biologically active form of ixazomib citrate (MLN9708), that is currently in phase 3 clinical trials in MM. Overall survival of 5 of 6 ixazomib-treated mice doubled compared with mice left untreated. One outlier mouse presented with primary refractory disease. Our findings demonstrate the utility of FDG-PET/CT for preclinical MM research and suggest that this method will play an important role in the design and testing of new approaches to treat myeloma.

B-Cell Studies in Chronic Ethanol Mice

Chronic alcohol abuse leads to multiple defects in the immune system, leading to an increased risk of infectious disease and malignancy. Immune lesions encompass both the innate and adaptive arms and include deficiencies in the B-cell compartment. Long-term alcoholics exhibit loss of B cells in the periphery and diminished ability to generate protective antibodies. To better mimic the chronic alcoholic patient, our group has used an ethanol-in-drinking-water mouse model. Mice consuming alcohol in this manner progressively develop a range of immune abnormalities, including defects in humoral immunity. To document and explore B-cell lesions in ethanol-consuming mice, our laboratory has used a broad panel of technologies. These include protocols to define the physical state of B cells in the bone marrow and periphery, in vitro approaches to test B-cell activation potential and in vivo experiments to document the humoral competence of the host. These key techniques are detailed in the present chapter.

Characterization of a novel mouse model with genetic deletion of CD177

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.

Delineation among eight major hematopoietic subsets in murine bone marrow using a two‐color flow cytometric technique

BACKGROUNDnMany methods have been developed specifically for identifying hematopoietic progenitor cells in murine bone marrow, but few methods allow rapid identification of multiple bone marrow populations. We describe a new, simple method for identifying simultaneously eight populations in murine bone marrow with two-color flow cytometry and phenotypically define these populations.nnnMETHODSnBone marrow was stained with anti-Ly-6C and anti-B220 (CD45R) in one fluorochrome and wheat germ agglutinin (WGA) in another fluorochrome. The eight populations identified in this way were defined further primarily by four-color flow cytometry.nnnRESULTSnSix of the eight populations were characterized phenotypically as containing erythroid, granulocytic, mast, early B, mature B, and stem cell populations. Two additional populations with phenotypic characteristics of partially differentiated precursor cells also were identified. One population was Ly-6C/B220+ and WGA-. It also expressed markers associated with early B, T, and/or dendritic cell differentiation. The second population was Ly-6C(hi)WGA(hi)Mac-1+ and was negative for numerous other lineage-specific and precursor markers. Its morphology suggested monocytic differentiative potential.nnnCONCLUSIONSnA two-color flow cytometric assay profiles six bone marrow populations with identifiable phenotypes and two additional unique, putative hematopoietic precursor populations.

The low affinity IgE Fc receptor (CD23) participates in B cell activation.

The low affinity IgE Fc receptor (FcɛRII or CD23) is expressed on all mature B lymphocytes (reviewed in 1 and 2). The FcʵRII is a 45 to 49 KD glycoprotein, and is categorized as a class II transmembrane molecule on account of its inverted membrane orientation. In addition to the transmembrane form of the receptor, a soluble form is readily released from B cells, a result of cleavage by a cell surface protease. cDNA cloning has revealed that the FceRII is a member of the hepatic lectin family, a somewhat unusual finding since all other characterized Fc receptors are known to be members of the immunoglobulin (Ig) gene superfamily.