Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification.

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-ß1).

Schnurri 3 promotes Th2 cytokine production during the late phase of T-cell antigen stimulation.

Th1 and Th2 polarization is determined by the coordination of numerous factors including the affinity and strength of the antigen-receptor interaction, predominant cytokine environment, and costimulatory molecules present. Here, we show that Schnurri (SHN) proteins have distinct roles in Th1 and Th2 polarization. SHN2 was previously found to block the induction of GATA3 and Th2 differentiation. We found that, in contrast to SHN2, SHN3 is critical for IL-4 production and Th2 polarization. Strength of stimulation controls SHN2 and SHN3 expression patterns, where higher doses of antigen receptor stimulation promoted SHN3 expression and IL-4 production, along with repression of SHN2 expression. SHN3-deficient T cells showed a substantial defect in IL-4 production and expression of AP-1 components, particularly c-Jun and Jun B. This loss of early IL-4 production led to reduced GATA3 expression and impaired Th2 differentiation. Together, these findings uncover SHN3 as a novel, critical regulator of Th2 development.

Siglec-5 is an inhibitory immune checkpoint molecule for human T cells.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5 suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of NFAT and AP-1. Furthermore, we show that GBS ß-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5 ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

IFN-ß reduces NRP-1 expression on human cord blood monocytes and inhibits VEGF-induced chemotaxis.

Type I interferons (IFNs) inhibit angiogenesis, the sprouting of new blood vessels, during tissue development, remodeling, and tumor growth. One of the major targets type I IFNs inhibit are circulating monocytes, which promote vascular development by secreting growth factors, chemokines, and proteases. This study tested the hypothesis that IFN-ß directly inhibits monocyte chemotaxis towards VEGF. We were interested in looking at chemotaxis towards VEGF because VEGF is known to create a pro-angiogenesis environment by acting as a stimulator and chemotactic factor for endothelial cells and monocytes. Here, we demonstrate that IFN-ß, a type I IFN, downregulates neuropilin-1 (NRP-1) expression by human monocytes and inhibits chemotaxis induced by vascular endothelial growth factor (VEGF), a NRP-1 ligand. Together, the data suggest that IFN-ß directly downregulates NRP-1 expression in monocytes, thus inhibiting monocyte chemotaxis toward a VEGF enriched environment.

Human CD36hi monocytes induce Foxp3+ CD25+ T cells with regulatory functions from CD4 and CD8 subsets.

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3+ regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3+ T-cell differentiation, the specific antigen-presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14+ monocytes expressing the scavenger molecule, CD36, can generate CD4+ and CD8+ T cells that coexpress Foxp3 and T-bet from both umbilical cord blood. These Foxp3+ T-bet+ T cells potently suppress T-cell proliferation and ameliorate xenogeneic graft-versus-host disease. CD14+  CD36+ monocytes provide known Treg-inducing signals: membrane-bound transforming growth factor-beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3+ T cells from both cord blood and adult peripheral naïve T cells. The induction of Foxp3+ T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36hi monocytes may contribute to the peripheral development of Foxp3+ T-bet+ T cells with regulatory functions in both neonates and adults.

TGF-ß suppresses RasGRP1 expression and supports regulatory T cell resistance against p53-induced CD28-dependent T-cell apoptosis.

Thymus-derived regulatory T cells (tTregs) play pivotal roles in immunological self-tolerance and homeostasis. A majority of tTregs are reactive to self-antigens and are constantly exposed to antigenic stimulation. Despite this continuous stimulation, tTreg and conventional T-cell populations remain balanced during homeostasis, but the mechanisms controlling this balance are unknown. We previously reported a form of activation-induced cell death, which is dependent on p53 (p53-induced CD28-dependent T-cell apoptosis, PICA). Under PICA-inducing conditions, tTregs survive while a majority of conventional T cells undergo apoptosis, suggesting there is a survival mechanism that protects tTregs. Here, we report that the expression of RasGRP1 (Ras guanyl-releasing protein 1) is required for PICA, as conventional T cells isolated from RasGRP1-deficient mice become resistant to PICA. After continuous stimulation, tTregs express a substantially lower amount of RasGRP1 compared to conventional T cells. This reduced expression of RasGRP1 is dependent on TGF-ß, as addition of TGF-ß to conventional T cells reduces RasGRP1 expression. Conversely, RasGRP1 expression in tTregs increases when TGF-ß signaling is inhibited. Together, these data show that RasGRP1 expression is repressed in tTregs by TGF-ß signaling and suggests that reduced RasGRP1 expression is critical for tTregs to resist apoptosis caused by continuous antigen exposure.

Activated human Foxp3+ regulatory T cells produce membrane-bound TNF.

TNF is a multifunctional cytokine that is critical to host defense against pathogens but can also drive the pathophysiology of inflammatory diseases. Inhibition of TNF occasionally causes exacerbation of some autoimmune diseases, suggesting a role for TNF in the regulation of immune homeostasis. Here, we demonstrate that human peripheral blood CD4+CD25+Foxp3+ regulatory T cells (Tregs) express membrane-bound TNF, a potent activator of the type 2 TNF receptor. While the type 1 TNF receptor can cause cell death and is expressed ubiquitously, the type 2 receptor promotes cell growth and its expression is limited mainly to immune and endothelial cells. When autocrine TNF is blocked in an in vitro culture without IL-2, activated Tregs stop proliferating. These data indicate a novel role for TNF as a Treg-derived autocrine growth factor.

Cell intrinsic characteristics of human cord blood naïve CD4T cells.

It has been generally considered that the perinatal immune system is less inflammatory compared to the adult system and type 2 responses predominate perinatal immune responses against antigens. Indeed, previous studies in mice showed that there are cell-intrinsic differences between neonatal and adult CD4T cells. However, studies on human cord blood and infant blood demonstrated that human perinatal T cells do not produce elevated levels of Th2 cytokines with the exception of IL-13. These data raise the question if human T cells in the perinatal blood fundamentally differ from adult T cells. To decipher differences between human perinatal and adult T cells, we performed a focused comparative analysis on purified naïve CD4T cells from umbilical cord blood (UCB) and adult peripheral blood. Our data demonstrate naïve CD4T cells from UCB differ from adult naïve CD4T cells in surface expression of CD26, dipeptidyl peptidase-4. While only a fraction of effector/memory T cells from adult blood express CD26, practically all T cells from UCB express high levels of CD26. We also determined that Th1/Th2 polarizing conditions induce UCB CD4T cells to produce higher levels of IFN-γ and IL-5 compared to adult CD4T cells, respectively. These data demonstrate intrinsic differences between UCB and adult naive CD4T cells and suggest that human perinatal immune responses involve more complex mechanisms than the previously thought Th2-dominant responses.

Hepatitis C virus-cross-reactive TCR gene-modified T cells: a model for immunotherapy against diseases with genomic instability.

A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability.

Cutting Edge: AhR Is a Molecular Target of Calcitriol in Human T Cells.

The immunoregulatory functions of vitamin D have been well documented in various immunological disorders, including multiple sclerosis, arthritis, and asthma. IL-10 is considered a chief effector molecule that promotes the vitamin D-induced immunosuppressive states of T cells and accessory cells. In this article, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol), has a profound inhibitory effect on the development of human Th9, a CD4 T cell subset that is highly associated with asthma, in an IL-10-independent manner. Our data show that calcitriol represses the expression of BATF, a transcription factor essential for Th9, via suppressing the expression of aryl hydrocarbon receptor, without an increase in IL-10. The data show a novel link between vitamin D and two key transcription factors involved in T cell differentiation.

IL-2 is a critical regulator of group 2 innate lymphoid cell function during pulmonary inflammation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear. OBJECTIVE: We sought to understand the role of IL-2 in the regulation of ILC2 function in the lung. METHODS: We used histology, flow cytometry, immunohistochemistry, ELISA, and quantitative PCR with knockout and reporter mice to dissect pulmonary ILC2 function in vivo. We examined the role of ILC2s in eosinophilic crystalline pneumonia, an idiopathic type 2 inflammatory lung condition of mice, and the effect of IL-2 deficiency on this disease. We determined the effect of IL-2 administration on pulmonary ILC2 numbers and function in mice in the steady state and after challenge with IL-33. RESULTS: We discovered an unexpected role for innate cell-derived IL-2 as a major cofactor of ILC2 function during pulmonary inflammation. Specifically, we found that IL-2 was essential for the development of eosinophilic crystalline pneumonia, a type 2 disease characterized by increased numbers of activated ILC2s. We show that IL-2 signaling serves 2 distinct functions in lung ILC2s, namely promoting cell survival/proliferation and serving as a cofactor for the production of type 2 cytokines. We further demonstrate that group 3 innate lymphoid cells are an innate immune source of IL-2 in the lung. CONCLUSION: Innate cell-derived IL-2 is a critical cofactor in regulating ILC2 function in pulmonary type 2 pathology.

Potential of targeting TGF-ß for organ transplant patients.

TGF-ß was originally considered as an immunoregulatory cytokine, but accumulating data demonstrate that it also plays a critical role in development of effector immunity. Since TGF- ß has a potent ability to alter immune responses, modulation of the TGF-ß pathway for treatment of transplantation patients could be effective if carried out in a target selective manner. This review will focus on the role of TGF-ß in T cell differentiation and discuss the prospect of TGF-ß as the therapeutic target of lung transplantation acceptance.

Ontogeny and localization of the cells produce IL-2 in healthy animals.

IL-2 is a growth factor for activated T cells and is required for maintenance of naturally arising regulatory T cells (nTregs). Mice defective in IL-2/IL-2 receptor signaling pathways have impaired nTregs and suffer from lymphoproliferative disorders, suggesting that IL-2 is present and functional in healthy animals. However, the cellular source of IL-2 is currently unknown. To determine which cells produce IL-2 in healthy animals, we established mice carrying cre gene knock in at the il-2 locus (termed IL-2(cre)). When IL-2(cre) mice were crossed with EGFP reporter mice, EGFP was exclusively expressed by a fraction of CD4 T cells present in both lymphoid and non-lymphoid tissues. Live imaging of IL-2(cre) mice that carry the luciferase reporter showed concentrated localization of luciferase(+) cells in Peyer's patches. These cells were not observed in new born mice but appeared within 3days after birth. Reduction of antigen receptor repertoire by transgene expression reduced their number, indicating that recognition of environmental antigens is necessary for generation of these IL-2 producers in healthy animals. A substantial fraction of EGFP(+) cells also produce IL-10 and IFN-γ, a characteristic profile of type 1 regulatory T cells (Tr1). The data suggest that a group of Tr1 cells have addition roles in immune homeostasis by producing IL-2 along with other cytokines and help maintaining Tregs.

Cellular basis of tissue regeneration by omentum.

The omentum is a sheet-like tissue attached to the greater curvature of the stomach and contains secondary lymphoid organs called milky spots. The omentum has been used for its healing potential for over 100 years by transposing the omental pedicle to injured organs (omental transposition), but the mechanism by which omentum helps the healing process of damaged tissues is not well understood. Omental transposition promotes expansion of pancreatic islets, hepatocytes, embryonic kidney, and neurons. Omental cells (OCs) can be activated by foreign bodies in vivo. Once activated, they become a rich source for growth factors and express pluripotent stem cell markers. Moreover, OCs become engrafted in injured tissues suggesting that they might function as stem cells.Omentum consists of a variety of phenotypically and functionally distinctive cells. To understand the mechanism of tissue repair support by the omentum in more detail, we analyzed the cell subsets derived from the omentum on immune and inflammatory responses. Our data demonstrate that the omentum contains at least two groups of cells that support tissue repair, immunomodulatory myeloid derived suppressor cells and omnipotent stem cells that are indistinguishable from mesenchymal stem cells. Based on these data, we propose that the omentum is a designated organ for tissue repair and healing in response to foreign invasion and tissue damage.

TGF-ß converts apoptotic stimuli into the signal for Th9 differentiation.

Naturally arising CD4(+)CD25(+)FoxP3(+) regulatory T cells (nTregs) have an essential role in maintenance of immune homeostasis and peripheral tolerance. Previously, we reported that conventional CD4(+) and CD8(+) T cells undergo p53-induced CD28-dependent apoptosis (PICA) when stimulated with a combination of immobilized anti-CD3 and anti-CD28 Abs, whereas nTregs expand robustly under the same conditions, suggesting that there is a differential survival mechanism against PICA between conventional T cells and nTregs. In this study, we demonstrate that TGF-ß signaling is required for nTregs to survive PICA. Conversely, when an active form of exogenous TGF-ß is present, conventional T cells become resistant to PICA and undergo robust expansion instead of apoptosis, with reduction of the proapoptotic protein Bim and FoxO3a. A substantial fraction of PICA-resistant T cells expressed IL-9 (T(H)9 cells). Moreover, the presence of IL-6 along with TGF-ß led to the generation of T(H)17 cells from conventional T cells. Together, the data demonstrate a novel role for TGF-ß in the homeostasis of regulatory T cells and effector T cell differentiation and expansion.

Generation of T cell hybridomas from naturally occurring FoxP3+ regulatory T cells.

Generation of regulatory T cells (or Treg) derived hybridomas offers a tool to study their antigen specificity. T cells hybridomas are produced by fusing TCR α-ß-thymoma BW5147 with highly dividing T cell population. In vitro anergy of Tregs is an obstacle in generation of highly dividing Treg population for their fusion. In this chapter, we describe a simple and efficient method to generate large number of blasting Treg and their successful fusion with thymoma BW5147. The resultant hybridomas lose Treg-specific transcription factor FoxP3, respond to antigenic stimulation by producing IL-2, and thus allow the evaluation of antigen specific, Tregs-derived TCRs.

Inhibition of bleomycin-induced pulmonary fibrosis through pre-treatment with collagen type V.

BACKGROUND: Tolerance to collagen structures has been shown to inhibit the progression of autoimmune scleroderma and rheumatoid arthritis. More recently, tolerance induction to collagen type V (colV) in experimental models of lung transplantation was shown to ameliorate the complex pathology known as "chronic rejection." The link between colV autoimmunity and progressive graft dysfunction and subsequent development of bronchiolitis obliterans syndrome (BOS) has been established in human lung transplant recipients. We hypothesized that intravenous injection of colV inhibits development of lung fibrosis in a bleomycin-induced lung injury mouse model. METHODS: Experimental animals were injected intravenously with saline or colV 10 days before intratracheal instillation of bleomycin. Pulmonary inflammation was monitored and quantified for the presence of cells in the bronchoalveolar lavage (BAL) fluid by flow cytometry and histology of lung tissue. RESULTS: ColV-pre-treated animals showed a significant reduction in lung inflammation compared with non-treated animals, according to histology and morphometry. The number of inflammatory cells in the BAL fluid was significantly reduced and associated with a lower proportion of gammadelta T cells and CD4(+) T cells in the colV-pre-treated group. Matrix metalloproteinase-2 and -9 (MMP-2 and -9; also known as gelatinase A and gelatinase B, respectively) levels in the BAL fluid were significantly reduced in colV-pre-treated mice compared with the non-treated mice. In addition, intravenous injection of colV was associated with a significant reduction in the relative expression of interleukin (IL)-6, IL-17 and IL-22 in cells present in BAL fluid at 7 and 14 days after bleomycin instillation. CONCLUSIONS: Pre-treatment by intravenous injection of colV inhibits bleomycin-induced pulmonary fibrosis by inhibiting IL-6 and IL-17 production. Fibrosis treatment in this context therefore should target induction of colV tolerance and Th17 development.

CD4(+)CD25(+) regulatory T cells resist a novel form of CD28- and Fas-dependent p53-induced T cell apoptosis.

Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2- and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4(+)CD25(-) and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4(+)CD25(+) T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4(+)CD25(+)FoxP3(+) regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.

hnRNP-K is a nuclear target of TCR-activated ERK and required for T-cell late activation.

Sustained extracellular signal-regulated kinase (ERK)-signaling plays a critical role in T-cell-mediated IL-2 production. Although many downstream targets are known for ERK, details remain unknown about which molecules play functional roles in IL-2 production. Here, we addressed this question using proteomic analysis of nuclear proteins from TCR-activated T cells and identified hnRNP-K as one of the ERK targets essential for IL-2 production. hnRNP-K was previously shown by others to be a direct substrate of ERK and form complexes with multiple signaling proteins as well as DNA and RNA. Our data showed a clear ERK-dependent increase in one form of hnRNP-K after TCR stimulation. Small interfering RNA-mediated gene knockdown of hnRNP-K expression abrogated IL-2 production by T cells. Moreover, reduction of hnRNP-K expression caused a notable increase in proteolysis of Vav1, a binding target of hnRNP-K. Since Vav1 is an essential molecule for T-cell activation, the data suggest that ERK signaling is required for T-cell activation partly by inhibiting activation-induced proteolysis of Vav1.