David M. Gravano

Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Stability and function of regulatory T cells is maintained by a neuropilin-1–semaphorin-4a axis

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a–Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

Promotion and prevention of autoimmune disease by CD8+ T cells

Until recently, little was known about the importance of CD8+ T effectors in promoting and preventing autoimmune disease development. CD8+ T cells can oppose or promote autoimmune disease through activities as suppressor cells and as cytotoxic effectors. Studies in several distinct autoimmune models and data from patient samples are beginning to establish the importance of CD8+ T cells in these diseases and to define the mechanisms by which these cells influence autoimmunity. CD8+ effectors can promote disease via dysregulated secretion of inflammatory cytokines, skewed differentiation profiles and inappropriate apoptosis induction of target cells, and work to block disease by eliminating self-reactive cells and self-antigen sources, or as regulatory T cells. Defining the often major contribution of CD8+ T cells to autoimmune disease and identifying the mechanisms by which they alter the pathogenesis of disease is a rapidly expanding area of study and will add valuable information to our understanding of the kinetics, pathology and biology of autoimmune disease.

Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.

The epithelial cells (TECs) are microenvironmental niche cells which support T lymphocyte development in the thymus. Most studies of TEC biology have focused on TEC at the fetal stage of development, whereas the biology of adult-stage TECs is not as well-understood. Delineating the molecular mechanisms that control adult TEC differentiation has implications for the success of T-lymphocyte based therapies for autoimmune diseases and induction of immunological tolerance to stem cell-derived tissues. Detailed analysis of adult TECs is technically challenging due to their rarity, their diminishing numbers with age, and the limited number of markers to distinguish between unique TEC subpopulations. Here, we have devised an improved isolation protocol for adult mouse TECs and combined it with six-color multiparameter flow cytometry. Using these techniques, we have identified four distinct subsets of CD45- EpCAM+ TECs in adult mice: a) UEA1(low) CDR1(low) (UC(low)); b) UEA1(high) CDR1(high)(UC(high)); c) UEA1(low) CDR1(high) MHC(high) (cTEC); and d) UEA1(high)CDR1(low) MHC(int/high) (mTEC). PCR analysis verified that these TEC subsets differentially expressed known TEC genes. TEC subsets were further analyzed using high-throughput quantitative PCR arrays to reveal novel genes that could be important for TEC subset maintenance. Intracellular staining for keratin-5 and keratin-8 can also be added, but our results suggest that keratin expression alone cannot be used to distinguish adult TEC subsets. Our enhanced isolation allows for detailed analysis of rare TEC subpopulations in the adult mouse at the cellular and molecular levels.

ADAM17 Deletion in Thymic Epithelial Cells Alters Aire Expression without Affecting T Cell Developmental Progression

Background Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs) are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. Methodology/Principal Findings We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. Conclusions/Significance In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development remain to be identified.

Inhibition of proteolysis of Delta-like-1 does not promote or reduce T-cell developmental potential.

Notch signaling is critical for T‐cell generation in the thymus. Notch signaling is linear in nature and is highly regulated through differential gene expression and post‐translational modification. Upon ligand binding, the Notch receptor is sequentially cleaved, first via extracellular ADAM protease‐mediated cleavage, followed by an intracellular presenilin‐dependent cleavage to release the Notch intracellular domain and activate transcription. Delta‐like‐1 (Dll1) is a Notch ligand that positively regulates T‐cell development. Dll1 is proteolytically processed in a similar manner to the Notch receptor, and it has been speculated to participate in bidirectional signaling. We hypothesized that inhibition of Dll1 processing in Notch signal sending cells would lead to changes in their ability to support thymopoiesis. We used the OP9 in vitro co‐culture system, and transduced OP9s with full length, cleavable Dll1 or a non‐cleavable mutant (NC‐Dll1) lacking the ADAM protease cleavage site. OP9‐NC‐Dll1 cells were able to support T‐cell development with similar efficacy to OP9‐Dll1 cells. Interestingly, expression of the Notch target gene Hes5 was more highly induced in T‐cell progenitors by NC‐Dll1, whereas expression of Hes1, Deltex1, and pre‐Tα were similar to controls. Furthermore, a reduced ability of hematopoietic progenitors to assume the granulocyte cell fate in OP9‐NC‐Dll1 cultures was noted. Taken together, these findings show that proteolytic cleavage of Dll1 in Notch signal sending cells is dispensable for murine T‐cell development, differentially affects expression of Notch target genes, and might be a mechanism that regulates myelopoiesis.

CD8(+) T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure.

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response.

Globule Leukocytes and Other Mast Cells in the Mouse Intestine

Only 2 major mast cell (MC) subtypes are commonly recognized in the mouse: the large connective tissue mast cells (CTMCs) and the mucosal mast cells (MMCs). Interepithelial mucosal inflammatory cells, most commonly identified as globule leukocytes (GLs), represent a third MC subtype in mice, which we term interepithelial mucosal mast cells (ieMMCs). This term clearly distinguishes ieMMCs from lamina proprial MMCs (lpMMCs) while clearly communicating their common MC lineage. Both lpMMCs and ieMMCs are rare in normal mouse intestinal mucosa, but increased numbers of ieMMCs are seen as part of type 2 immune responses to intestinal helminth infections and in food allergies. Interestingly, we found that increased ieMMCs were consistently associated with decreased mucosal inflammation and damage, suggesting that they might have a role in controlling helminth-induced immunopathology. We also found that ieMMC hyperplasia can develop in the absence of helminth infections, for example, in Treg-deficient mice, Arf null mice, some nude mice, and certain graft-vs-host responses. Since tuft cell hyperplasia plays a critical role in type 2 immune responses to intestinal helminths, we looked for (but did not find) any direct relationship between ieMMC and tuft cell numbers in the intestinal mucosa. Much remains to be learned about the differing functions of ieMMCs and lpMMCs in the intestinal mucosa, but an essential step in deciphering their roles in mucosal immune responses will be to apply immunohistochemistry methods to consistently and accurately identify them in tissue sections.

Development and validation of a microbe detecting UAV payload

Airborne transport of microorganisms through the atmosphere has widespread implications for many atmospheric processes, ecological processes and human health. The proliferation of infectious disease-causing bacteria and fungi is of particular relevance, as many emerging diseases enter human populations via an atmospheric link to the surrounding environment. Coccidiodomycosis (Valley Fever), for instance, is a debilitating fungal disease contracted through the inhalation of Coccidiodes immitis and Coccidiodes posadasii of central California and elsewhere in the southwestern United States and northwestern Mexico. Recent studies suggest an increase in the incidence of Valley Fever throughout this region, but how the fungus is transported through the atmosphere is not well known. This is due in part to the fact that there is no effective and reliable standardized method for acquiring fungal spores at an elevated altitude, nor to do so rapidly-which would aid in limiting human exposure. This work fills the voids of sensing capability and rapid detection by means of small unmanned aerial systems (sUAS). The use of an sUAS enables low-altitude sampling, in addition to the low-cost development and operation of the payload. The payload consists of two coupled subsystems, which log environment data and extract a bioaerosol sample. The data and sample is analyzed and validated via a variety of molecular biological and microbiological techniques.

Th17/regulatory T cells balance is predictive of Coccidioides infection outcome in pediatric patients

Background Protective immunity against the fungal pathogen Coccidioides requires specific T helper responses. Mouse vaccine and infection studies have defined CD4+ T helper (Th)1 and Th17 cells in the resolution of infection and in effective protection. Patients with persistent Coccidioides infection demonstrate reduced cellular responses. Methods Peripheral blood and serum were collected from 30 pediatric Coccidioides-infected patients and 20 healthy controls in the California San Joaquin Valley. Samples were evaluated by flow cytometry for innate and adaptive immune populations and cytokines to define the early immune response and identify clinically useful biomarkers for predicting disease outcome. Clinical and flow data were evaluated according to disease outcome (resolved or persistent) using principal component analysis, high-dimensional flow cytometry analysis tools, chi-square automatic interaction detection, and individual cell population comparisons. Results Patients with persistent infection had lower Th17 and higher Treg frequencies, but similar Th1 responses, relative to patients that resolved disease. Treg frequency, eosinophil numbers and neutrophil numbers together distinguish patients that resolve infection from those that develop persistent infection. Conclusions The inability to resolve Coccidioides infection may be a result of elevated Treg frequency and functional capacity, and Treg frequency may predict patient disease outcome at diagnosis. In our study, Th1 responses were similar in persistent and resolved infection, in contrast to prior human studies. Instead, our data suggest that Th17 cells provide an effective protection during Coccidioides infection, and that elevated Treg frequency inhibits protective immunity.