bluuebunny/crossref_metadata_2025 huggingface dataset:
Polychromatic Flow: MDR1 Expression on Th17/Th17.1 effector cells in Patients with Autoimmune Disease - Potential Role in Drug Resistance
Authors: Bisconte Angelina , Kisselbach Lynn , Neidinger Erik , Phippard Deborah
Published: January 2023

IL-17A secreting T cells represent a distinct lineage of CD4+ T effector cells (Th17) that have been suggested to play a role in the pathogenesis of inflammatory/autoimmune diseases such as Crohn’s disease. Precision for Medicine cryopreserves primary Th17 cells (AccuCell®) to provide an easy alternative to isolating fresh cells, for use in screening assays and functional immunological studies.

The human MDR1 gene encodes a membrane protein, P glycoprotein (Pgp), whose function is to export toxic substances or metabolites from the cell. MDR1 expresssion has been demonstrated in tissues of Crohn’s disease patients and on their circulating Th17 cells. Using AccuCell® Th17 cells from healthy and diseased patients we investigate how MDR1 expression differs in the disease state and highlight the challenges of screening drugs using healthy cells as opposed to the targeted disease state.

First we evaluated our cryopreserved Th17 cells to fresh isolations for surface marker, chemokines, intracellular cytokines (IL-17A, IFNγ, IL-22), and RORγt. Full retention of the tested markers were observed in the recovered cryopreserved cells. On our disease state PBMCs phenotypic and functional evaluation of MDR1+ Th17/17.1 cells included activation markers CD5, CD69, CD2, human leukocyte antigen-DR (HLA-DR) and co-stimulatory receptor CD28, compared to normal healthy donors. T-cell receptor dependent proliferation in MDR1+ Th17 cells was compared to healthy donor demographic matched Th17 cells. Utilizing AccuCell® cryopreserved Th17 cells which are fully functional empowered these studies to elucidate mechanisms as discussed above; thus eliminating fresh isolations so researchers can focus on studying the pathogenesis of these cells.
Plasma cell mitochondrial pyruvate import controls the duration of humoral immunity.
Authors: Lam Wing Yu , Becker Amy M , Kennerly Krista , Wong Rachel , Payne Elizabeth , Curtis Jonathan , McCommis Kyle , Fahrmann Johannes , Nunley Ryan , Wolfgang Michael , Patti Gary , Fink Brian , Pearce Erika , Bhattacharya Deepta
Published: January 2023

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells. Pathways that specifically allow long-lived plasma cells to persist remain unknown. Through bioenergetic profiling, we demonstrate that human and mouse long-lived plasma cells possess much greater mitochondrial spare respiratory capacity than do short-lived plasma cells. Inhibition of mitochondrial pyruvate import in long-lived plasma cells attenuates spare, but not basal respiration. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, leads to a progressive loss of long-lived plasma cells and vaccine-specific antibodies in vivo. Long-lived plasma cells uptake more glucose than do their short-lived counterparts, and subsequent catabolism to pyruvate is essential for spare respiratory capacity. IL-4 or IFNg treatment of activated B lymphocyte precursors to plasma cells promotes glucose uptake. Thus, signals provided to precursors, glucose uptake, and mitochondrial pyruvate transport allow long-lived plasma cells to provide enduring antibody-mediated immunity.
Checkpoint Regulator VISTA plays a role in Suppression of B-Cell Responsiveness by Monocytic Myeloid Derived Suppressor Cells from LP-BM5 retrovirus-infected Mice
Authors: Green Kathy A , Noelle Randolph J , Green William R , Wang Li
Published: January 2023

MDSC inhibition of tumor directed T cell responses is well described. We have reported an increase of monocytic MDSC (M-MDSC) during infection of B6 mice by LP-BM5 immunodeficiency causing retrovirus. These M-MDSCs suppressed T, and B cell responsiveness ex vivo. M-MDSC inhibition of stimulated T-cell proliferation and IFN-gamma production was ~100% iNOS/NO dependent; whereas suppression of B-cells was only ~50% dependent on iNOS/NO. An additional mechanism(s) for M-MDSC inhibition of B cell responsiveness involved V-domain Ig Suppressor of T cell Activation (VISTA), negative checkpoint regulator. Using anti-VISTA blocking mAb, ~50% of total MDSC suppression of B-cell responses was dependent on MDSC-expressed VISTA. The combination of iNOS/NO inhibitors and VISTA lead to additive, if not synergistic, blockade of M-MDSC suppression of B cell responsiveness. Regarding the LP-BM5-infection dependency of M-MDSCs, spleens from uninfected mice yielded ~3-fold fewer enriched M-MDSCs; and on a per-cell basis, the M-MDSCs from uninfected mice were also substantially less suppressive (4.3 fold); than those from infected mice. Consistent with a direct role of VISTA, in the absence of M-MDSCs, a VISTA-Ig fusion protein also partially inhibited B-cell responsiveness. Initial experiments show differential VISTA expression for the LP-BM5 M-MDSC CD11b+hiLy6C+hi subpopulations we have recently defined (O’Connor et al, 2015, Virology). Thus, LP-BM5 infected, when compared to analogous subsets from uninfected mice, contain substantially more VISTA+ cells. These results suggest an unique role for M-MDSCs in LP-BM5 induced immunodeficiency, and highlight multiple suppressive pathways in the area of MDSC suppression of B cell responses.
Protein tyrosine kinase Fyn regulates TLR4-elicited responses on mast cells controlling the function of a PP2A-PKC a/b signaling node leading to TNF secretion
Authors: Gonzalez-Espinosa Claudia , Ibarra-Sánchez Alfredo , Ávila Alejandro Martín , Medina-Tamayo Jaciel , Vázquez-Victorio Genaro , Castillo-Arellano Jorge Iván , Hernández-Mondragón Alma Cristal , Rivera Juan , Madera-Salcedo Iris Karina , Blank Ulrich , Macías-Silva Marina
Published: January 2023

Signaling pathways leading to cytokine production after TLR-4 triggering in mast cells (MC) are not well described. In this work, the participation of the Src family kinase Fyn on the production of TNF after stimulation with LPS was evaluated MCs from WT and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo synthesized TNF and the processes underlying that phenotype were investigated. In both cell types, TNF co-localized with VAMP3-positive compartments. Although LPS addition provoked coalescence of VAMP3 and the interaction of VAMP3 with SNAP23 in WT and Fyn−/− cells, the apparent size of VAMP3-containing aggregates was larger in Fyn−/− cells and increased formation of VAMP3-SNAP23 complexes was observed compared to WT. LPS-addition to Fyn−/− cells lead to higher activation of TAK-1, ERK1/2 and IKK, which was reflected on increased nuclear translocation of transcription factors such as NFkB and CEBPd. Increased LPS responsiveness in Fyn−/− cells was associated to a lower activity of Protein Phosphatase 2A(PP2A) and augmented phosphorylation of key signaling kinases, such as PKCa/b, which controlled TLR4-induced VAMP3 coalescence. Reconstitution of MC-deficient Wsh mice with Fyn−/− MC produced an increased LPS-dependent production of TNF in peritoneal cavity. Our data show that Fyn kinase is activated after TLR-4 triggering and exerts an important negative control on LPS-dependent TNF production in MC controlling the inactivation of PP2Ac and PKC a/b phosphorylation necessary for the secretion of TNF by VAMP3-positive carriers.

Supported by Grant ANR-Conacyt 188565 to UB, CGE and MMS.
Programming of RIG-I signaling through co-factor interactions
Authors: Dewey Elyse C , Tan Min Jie Alvin , Hibberd Martin , Gale Michael J
Published: January 2023

Retinoic acid-inducible gene I (RIG-I) functions as a cytosolic pathogen recognition receptor by binding the pathogen associated molecular patterns (PAMPs) in viral RNA. The molecular signature for RIG-I recognition has been identified as 5′-triphosphate-containing short double stranded or poly-uridine RNA (5′pppRNA) structures found in RNA species that amass in the cytoplasm during virus infection. PAMP-mediated activation of RIG-I triggers many signaling cascades that mediates its effector functions in innate immunity and apoptosis. In the innate immune response to virus infection, RIG-I sets off both IRF3/7 and NF-κB signaling to induce type I interferon (IFN) and pro-inflammatory cytokine production respectively. PAMP-induced RIG-I activation can also induce apoptosis in cancer cells. Since PAMP RNA can act as the activator of RIG-I’s pro-apoptotic and innate immune functions, we hypothesized that the induction of these separate pathways is programmed through differential RIG-I-cofactor interactions. We have identified and characterized novel co-factors of RIG-I by using a systems proteomic approach. These co-factors serve to program apoptotic versus innate immune signaling actions through their interaction with RIG-I. These co-factors and their biologic functions will be presented.
Natural TFH arise in the thymus and periphery of young naive mice
Authors: Adkins Elisabeth B , Wang Xulong , Sproule Thomas J , Christianson Gregory G , Park Giljun , Lane-Reiticker Sarah-Kate , Jain Shweta , Carter Gregory W , Morse Herbert C , Roopenian Derry C
Published: January 2023

T follicular helper cells (TFH) localize to B cell follicles and secrete Interleukin 21 (IL21), a cytokine that is vital for driving the proliferation of antigen-stimulated B cells within germinal centers and their subsequent differentiation into memory B cells and antibody-secreting plasma cells. Much of the current understanding of TFH is limited to studies following immunization of adult mice. Therefore, the ontological processes by which TFH naturally develop and express IL21 are poorly understood. Here we take advantage of a novel IL21-venus fluorescent protein reporter mouse to show that a major fraction of the earliest activated CD4 T cells that develop spontaneously in the thymi and the peripheries of young naive mice express IL21 within 2 weeks of birth. This population is rivaled in frequency only by FoxP3 natural T regulatory cells (nTREG) cells. Results from RNAseq profiling, their partial developmental dependence on IL6 and IL21 signaling and durability after adoptive transfers are all consistent with the generation of a novel, natural TFH (nTFH) population. This nTFH population is functionally defined by IL21 and is able to persist in that differentiation state in naive mice. We further show that thymic development of nTFH requires AIRE, while FoxP3 nTREG act in the periphery to restrain nTFH. We conclude that the precocious and robust presentation of nTFH in naive mice is caused by inherent self-reactivity that is developmentally driven by encounter with promiscuous self-antigens through AIRE. TREG are therefore the primary means by which nTFH are prevented from exercising their pathogenic potential in the periphery.
Sensitivity to restimulation-induced cell death is linked to glycolytic metabolism in human T cells
Authors: Larsen Sasha E , Bilenkin Abegail , Snow Andrew L
Published: January 2023

Restimulation-induced cell death (RICD) helps to regulate immune responses by setting an upper threshold for effector T cell expansion and limiting nonspecific damage to the host. RICD is triggered by re-engagement of the T cell receptor (TCR) on a cycling, activated T cell, resulting in apoptosis. However, it remains unclear how RICD sensitivity is calibrated in T cells derived from different individuals and subsets. As effector T cells expand and become responsive to RICD, they have also switched from a quiescent metabolic state into active aerobic glycolysis (Warburg effect). We hypothesized that in addition to facilitating proliferation and effector function, metabolic programming directly influences RICD sensitivity. We found that secretion of L-lactate, the end product of glycolysis, positively correlated with greater RICD sensitivity in CD8+ effector T cells from different human donors. Strikingly, replacing glucose in culture media for galactose, which results in almost exclusive use of oxidative phosphorylation (OXPHOS), rendered T cells significantly less sensitive to RICD. Consistent with this, blocking glycolysis with 2-deoxy-glucose (2-DG) significantly reduced RICD sensitivity, while inhibiting OXPHOS increased RICD sensitivity. Glycolysis promoted RICD sensitivity in part through mTOR signaling. Moreover, we found that active glycolysis facilitated induction of pro-apoptotic effector molecules like Fas ligand upon TCR restimulation, which was blocked with 2-DG treatment. Collectively, these data suggest that RICD susceptibility is dependent on glycolytic metabolism in effector T cells, and that switching back to OXPHOS may help shield cells transitioning into the memory pool from RICD.
ERK2 regulation of mitophagy is important for T cell activation
Authors: Tudyk Julie , Forsthuber Thomas
Published: January 2023

Critical signals necessary for T cell activation are transduced into the cell, in part, by the MAPK pathway, for which extracellular regulated kinase 2 (ERK2) is a key member. Upon activation, changes to mitochondria location and metabolism mediate T cell priming; however the signaling pathways that modulate mitochondria in T cells are largely unknown. A primary mechanism regulating mitochondria is a form of selective autophagy termed mitophagy. Importantly, ERK2 is required for mitophagy in other cell types and has been shown to co-localize at the autophagosome during formation. These data suggest that ERK2 may have a unique role regulating mitophagy in T cells. Notably, dysregulation of mitochondria in T cells can result in impaired immunity and has also been associated with autoimmune disease.

Here, we investigated the role of ERK2 for mitophagy in CD4+ T cells using a mouse model with T cell-specific deletion of ERK2. Our results show that Erk2−/− CD4+ T cells have impaired autophagosome formation and increased cell death upon activation, a phenotype consistent with mitophagy blockade. Further, we are working to identify key mitophagy proteins that are regulated by ERK2 and to characterize mitochondria signaling metabolites during T cell activation. By elucidating the signaling pathways that connect extracellular immune stimuli with mitochondria regulation, we will obtain important information that may lead to new therapeutic targets for T cell mediated diseases and dysfunction.
The microRNA-212/132 cluster regulates B cell development by setting a threshold for Sox4 expression
Authors: Mehta Arnav , Mann Mati , Baltimore David
Published: January 2023

MicroRNAs play unique roles as logic elements in B cell differentiation by allowing for fine-tuning of differentiation checkpoints. We have found that the miR-212/132 cluster is induced in B cells in response to anti-IgM stimulation and that it can regulate B cell differentiation by establishing a threshold of Sox4 expression necessary for progression of progenitor B cells. Over-expression of miR-132 blocks the transition of pre-pro-B cells to pro-B cells, in part by inducing apoptosis in primary bone marrow B cells. The deletion of miR-212/132 results in increased B cell output under conditions of inflammatory stress. We validate Sox4 as a novel target of miR-132, and show that co-expression of Sox4 with miR-132 can rescue the phenotype observed with over-expression of miR-132 alone. We further demonstrate that over-expression of miR-132 can inhibit the development of B cell leukemia in mice that have B cells expressing the c-myc oncogene. We have therefore described miR-212/132 as a novel regulator of B cell development through its regulation of Sox4 expression.
An intact C-terminus is required for the antimicrobial effects of CCL28 in host defense from oropharyngeal candidiasis
Authors: Huppler Anna R , He Jie , Volkman Brian F , Grayson Mitchell H
Published: January 2023

Oropharyngeal candidiasis (OPC) is an opportunistic infection caused by the commensal fungus Candida albicans. There is an urgent need for new therapeutic candidates to manage mucosal infection and prevent progression to high mortality disseminated candidiasis. The T helper 17 (Th17)-associated cytokines IL-23 and IL-17 are crucial for immunity to OPC. However, our recent work indicates the current model of IL-17-mediated defense is insufficient to completely explain susceptibility to OPC. CCL28 is a CC chemokine with antimicrobial peptide (AMP) activity that has a potential role in oral host defense. CCL28 is most abundant in the salivary gland and has in vitro antimicrobial activity against multiple pathogens including C. albicans. Little is known about the in vivo function or regulation of CCL28 in defense from infection. In the current study, we explore the motifs of CCL28 contributing to AMP activity and the relationship with OPC. We demonstrate that C. albicans killing by CCL28 at physiologic concentrations can be eliminated by truncation of the C-terminus. The N-terminus of CCL28, which is required for receptor binding, is dispensable for anti-fungal activity. In an in vivo model of OPC in mice, application of recombinant CCL28 reduces oral fungal burdens in immunodeficient mice. Ccl28 gene expression in salivary gland is suppressed at baseline in mice susceptible to OPC. Our results indicate that the CCL28 flexible C-terminal domain is an important functional motif for anti-Candida AMP activity, CCL28 treatment reduces mucosal fungal infection in vivo, and CCL28 may be a component of host defense from infection. CCL28 is a promising candidate for mucosal immunity and future development for treatment of Candida infections.
Nanoscopic characterization of immunogenic b-glucan exposure on Candida species pathogens
Authors: Neumann Aaron Kurt , Lin Jia , Graus Matthew , Wester Michael J , Lidke Keith
Published: January 2023

The cell walls of Candida spp. are composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell wall polysaccharide whose surface exposure is masked from the innate immunoreceptor Dectin-1 to evade immunity. Previous research suggested that the physical presentation geometry of β-glucan may determine whether or not it can be recognized by Dectin-1. We used super resolution imaging to measure the fine structure of glucan exposure on C. albicans cell walls before and after glucan unmasking with the antimycotic drug caspofungin. Most exposed glucan on C. albicans yeast and hyphae exists as single Dectin-1 binding sites. Caspofungin-mediated glucan unmasking results in nanoscale remodeling of glucan exposures, resulting in increased density and size of glucan exposure sites. The less studied pathogen C. glabrata exhibits significantly greater glucan exposure than C. albicans in type strains, but recent clinical isolates of C. glabrata have lower exposure, suggesting that C. glabrata also masks its glucan. We used C. glabrata cell wall mutants to define the role of N-mannan structure in maintaining glucan masking. The less complex N-mannan structures of mutant strains provided poorer glucan masking, increasing the density of nanoscale glucan exposure sites. a-(1,6)-mannan backbone length was a major structural determinant of glucan masking, while a-(1,2;1,3)-mannan side chain density was less critical to masking function. This work provides the first characterization of changing glucan exposure nanostructure at the pathogenic cell wall surface that participates in host-Candida interaction. Further studies will examine the functional impact of glucan nanostructure on Dectin-1 activation.
MyD88 suppresses IL-10 and IL-17 production in response to obligate intracellular Ehrlichia infection
Authors: Kader Muhamuda , Alaoui-El-Azher Mounia , Kode Bhushan , McArthur Morgan , Shinde Apurva , Wells Alan , Ismail Nahed
Published: January 2023

Ehrlichia is an obligate intracellular gram negative bacterium that target macrophages, and causes the potentially fatal human monocytic ehrlichiosis (HME). In murine models of fatal ehrlichiosis (FE), Ixodes ovatus Ehrlichia (IOE) inhibits autophagy, suppresses protective acquired immunity, and causes immunopathology and toxic shock syndrome. Innate Ehrlichia detection by TLR2 mediates host resistance, while NOD2 signaling confers host susceptibility to FE. Recently, we have shown that deficiency of type I interferon receptor (IFNaR) phenocopy NOD2 deficiency in IOE-infected mice, implying a contribution of both NOD2-IFNaR pathways to susceptibility to FE. However, how TLR2 mediates protective function against Ehrlichia is not clearly defined. Here, we investigated the role of MyD88, the main adaptor molecule for the TLRs in host response to virulent IOE using MyD88 knockout mice (MyD88−/−). Our data showed that, MyD88−/− mice have an impaired bacterial clearance, increased liver injury, and a heightened mortality when compared to infected wild type (WT) mice. Increased bacterial load in MYD88−/− mice correlates with decreased levels of TNF-α in both in vivo and in vitro {serum and IOE- infected bone marrow-derived macrophages (BMM)}, and increased frequency of splenic CD4+ T cells producing IL-10 and IL-17. Protective anti-Ehrlichia immunity and minimal pathology in WT mice infected with low virulent Ehrlichia species correlates with a high Th1 response, but a low frequency of IL-10 and IL-17 producing splenic T cells, suggesting that IL-10 and IL-17 may play pathogenic roles in host defense against Ehrlichia. Together these data suggest that MYD88 plays a protective role in ehrlichiosis via suppression of IL-10 and IL-17.
Endoplasmic Reticulum Aminopeptidase I-driven Production of IL-18 and IL-1β Protects from Severe Intestinal Inflammation and Mortality during Experimental Colitis
Authors: Aldhamen Yasser Ali , Alyaqoub Fadel S , Pepelyayeva Yuliya , Rastall David P.W. , Raedy Ashley , O’Connell Patrick , Pereira-Hicks Cristiane , Godbehere Sarah , Amalfitano Andrea
Published: January 2023

Altered endoplasmic reticulum aminopeptidase 1 (ERAP1) function has been linked to many pathological conditions that are characterized by chronic inflammation such as Ankylosing spondylitis (AS), however, a role for ERAP1 in colitis has not been described. Notably, AS patients are known to develop colitis. However, the exact molecular mechanism that predisposes AS patients to intestinal inflammation has not been defined. We previously demonstrated that ERAP1 deficiency alters innate immune responses during pathogen recognition. In addition, we also demonstrated that autoimmune disease-linked variants of ERAP1 activate the NLRP3/caspase-1 inflammasome pathway. Previous reports showed that mice deficient for NLRP3 inflammasome components are highly susceptible to dextran sodium sulfate (DSS)-induced colitis. Here, we hypothesized that ERAP1 may modulate the susceptibility of animals to DSS challenge, due to ERAP1’s impact on innate immune responses. We show that mice deficient for ERAP1 (ERAP1−/−) were highly susceptible to DSS-induced colitis as compared to WT mice. This process was associated with decreased IL-1β and IL-18 production in ERAP1−/− mice, resulting in increased mortality rates and severe intestinal inflammation. Assessment of the intestinal inflammation marker, Lcn-2 revealed potent increases in Lcn-2 in DSS-treated ERAP1−/− mice as compared to WT controls. Furthermore, utilization of ERAP1/β2m double-knockout mice revealed that these responses are mediated by a mechanism that was independent of antigen presentation by ERAP1. These results demonstrate that ERAP1 mediates a protective function during colitis, a function that we correlated with its ability to allow production of IL-18 and IL-1β.
Lineage-specific ablation of retinoic acid synthesis impacts on the development of gut-tropic regulatory T cells and oral tolerance
Authors: Lee Yu-Chi , Li Jiannan , Deng Jie , Noelle Randolph J
Published: January 2023

Oral tolerance is a multifaceted process that leads to systemic unresponsiveness to orally administrated antigens. Successful establishment of oral tolerance relies on induction of antigen-specific gut-tropic regulatory T cells (Tregs). Retinoic acid (RA), the active metabolite of vitamin A, is critical for the induction of gut-homing receptor on T cells and differentiation of Foxp3+ Tregs. Current paradigm, developed from in vitro experimentation, suggests that CD103+ RA-producing dendritic cells (DCs) are critical for mediating mucosal tolerance. However, in vivo evidence supporting this statement is lacking, due to the challenge of lineage-specific ablation of RA. We developed a novel mouse model to ablate RA synthesis in defined cell lineages and evaluate the requirement of RA for the migration, retention and stability of gut-tropic Tregs in different intestinal compartments. We found that RA regulates the development of gut-tropic Tregs cells in a step-wise process by lineage-specific up-regulation of gut-homing receptors. Furthermore, evaluation of the establishment of oral tolerance in an oral Listeria infection model revealed that suppression of immunity against Listeria was less efficient when RA synthesis is ablated. Collectively, this is the first study that elucidates the role of RA for oral tolerance in vivo, and offers novel insight into how RA fine-tunes the balance between gut immunity and tolerance.
The integrin αvβ8 promotes activation of TGFβ by human peripheral monocyte and intestinal macrophage populations
Authors: Travis Mark A , Kelly Aoife , Shuttleworth Elinor , Fenton Thomas , Smedley Catherine , Levison Scott
Published: January 2023

Transforming growth factor beta (TGFβ) plays a key role in maintaining immune homeostasis, both preventing inflammation and promoting tissue repair. The cytokine is secreted as an inactive complex, with the function of TGFβ tightly regulated at the level of its activation. Our previous work in mice has demonstrated a crucial role for an integrin, αvβ8, in activation of TGFβ in the immune system. This pathway appears especially important in the intestine, with important functions in regulating T-cell response via integrin αvβ8 expression in dendritic cells and regulatory T-cells. However, how TGFβ is activated to regulate the human immune system is poorly understood. Here, we show that, in humans, integrin αvβ8 is highly expressed on blood monocytes, which promotes activation of TGFβ by these cells. When monocytes are differentiated to macrophages, high integrin β8 expression and TGFβ-activating function is observed on the more tolerogenic (M2-like) macrophages versus inflammatory (M1-like) macrophages. In both monocytes and macrophages, expression of β8 is upregulated by specific bacterial TLR ligands, suggesting that stimulation of the cells may promote their ability to activate TGFβ. In addition to peripheral immune cells, intestinal lamina propria mononuclear cells express high levels of integrin αvβ8, with increased levels seen in patients with inflammatory bowel disease. Highest levels of integrin αvβ8 are seen on a population of CD14+CD33+CD64+ cells, corresponding to intestinal macrophages. Thus, our data suggest that expression of integrin αvβ8 on monocytes may play an important role in the induction and function of anti-inflammatory macrophages in the intestine via TGFβ activation.
Over-expression of CD200 protects mice from dextran sodium sulfate induced colitis
Authors: Gorczynski R. M , Chen Zhiqi , Yu Kai , Zhu Fang
Published: January 2023

CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of “knockout” of CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS) - induced colitis, a mouse model of inflammatory bowel disease (IBD).

Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA.

CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3+ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3+ regulatory T (Treg) cells was seen in the colon of CD200tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection.

We conclude that the CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice.
OASL1 deficiency promotes antiviral protection against genital herpes simplex virus type 2 infection by enhancing type I interferon production
Authors: Lee Heung Kyu , Oh Ji Eun
Published: January 2023

Type I interferon (IFN) interferes with virus replication, promotes antiviral responses, and controls innate and adaptive immune responses to certain viruses. Recently, we reported that 2′-5′ oligoadenylate synthetase-like 1 (OASL1) negatively regulates type I IFN production by inhibiting the translation of the type I IFN-regulating master transcription factor, IRF7. Notably, while OASL1-deficient mice induce robust production of type I IFN and are resistant to systemic viral infection, the effects of OASL1 during localized viral infection has not been studied. To this end, we investigated the role of OASL1 during mucosal HSV-2 infection of the genital tract. Oasl1−/− mice exhibited better survival rates than wild type (WT) mice following intravaginal HSV-2 infection, and suppressed virus replication more efficiently despite comparable recruitment of effector immune cells. Moreover, Ly6Chigh monocytes, and not pDCs or other cell types, displayed enhanced production of type I IFNs in Oasl1−/− mice in response to HSV-2 infection. Furthermore, cytotoxic T cell responses including IFN-γ production were accelerated in Oasl1−/− mice after mucosal HSV-2 infection. Collectively, these results demonstrate that OASL1 deficiency promotes antiviral immunity against local mucosal viral infection and suggest that OASL1 could be a therapeutic target for treatment of HSV-2 infection of the genital mucosa.
HIV infection impairs CD8+ resident memory T cell frequency in cervical mucosae
Authors: Pattacini Laura , Baeten Jared , Lingappa Jairam , Lund Jennifer
Published: January 2023

A critical challenge in establishing a cure for HIV is the elimination of reservoirs of infection. A proposed approach is to reactivate HIV replication in infected cells and eliminate them by eliciting functional CD8+ T-cells. Due to their location and rapid and efficient response, CD8+ resident memory T cells (TRM) represent an ideal candidate, but their frequency and localization in HIV-infected subjects had not been evaluated.

Therefore, we quantified frequencies of TRM cells by immunohistochemistry detection of CD8 and CD69 in cervical tissues from 5 HIV-infected and 5 non-infected women. In addition, migration assays of peripheral blood mononuclear cells (PBMC) from HIV-positive and negative donors were performed by plating one million PBMC on the top of a migration chamber placed in a well seeded with confluent HeLa cells. After 24 hours, cells that migrated to the bottom chamber were collected for flow cytometry evaluation.

In cervical tissues, TRM numbers and frequencies were significantly different in basal membranes (0.93% in HIV-negative versus 0% in HIV-positive subjects, p=0.006), whereas no differences were seen for total CD8+ T cells. Moreover, in samples from HIV-positive subjects, lower frequencies of CD8+ CD69+ CD103+ T cells were detected between the cells migrated to the bottom chamber in samples from HIV-infected subjects (0.86% versus 3.54%, p=0.002, in HIV-negative versus HIV-positive). In HIV-positive donors, the frequency of CD4+ CD69+ CD103+ expressing CCR5 was significantly lower than for HIV-negative, while expression of CXCR3 and CCR7 were comparable.

We propose that HIV impairs frequencies of TRM in the cervical mucosa at least in part by reducing CD8+ T cell migration through a CCR5-mediated mechanism.
Validation of Th17 Cell Differentiation from Peripheral Blood CD4+ T Cells Through Assessment of mRNA Expression and Cytokine Secretion using Microplate Reading and Cellular Imaging
Authors: Holderness Kathryn , Crossett Tracee , Larson Brad , Taubner Annegret
Published: January 2023

CD4+ T cells signal and regulate an immune response to pathogens after interacting with an antigen-MHC complex. This activation causes the cells to differentiate into distinct phenotypic and functional effectors, collectively referred to as T helper cells (Th). One such differentiated T cell subset includes the pro-inflammatory T helper 17 (Th17) cell subset. These cells can be beneficial to the host during infection, as they amplify ongoing inflammation by inducing expression of tumor necrosis factor-α. CD4+ T cells that differentiate into Th17 cells are uniquely characterized by production of the pro-inflammatory cytokine, IL-17.

T cells isolated from peripheral blood mononuclear cells and selected for CD4 are differentiated into Th17 cells using a cocktail of specific antibodies and cytokines. The procedure to create and confirm this differentiation can be labor and time intensive.

Here we demonstrate a validated, robust method to differentiate peripheral blood CD4+ T cells into functional Th17 cells using a bead-based activation technology. A novel, cell-imaging multi-mode reader can be used to image phenotypic differences between cells exposed to the antibody/cytokine cocktail and control cells as differentiation proceeds. Creation of fully functioning Th17 cells was then confirmed by assessing IL-17F mRNA levels using a fluorescence RNA in situ hybridization assay, in addition to IL-17 secretion using a homogeneous, bead-based immunoassay technology. The reader was able to perform brightfield and fluorescence imaging steps, as well as laser-based excitation for the secretion immunoassay. The combination provides a comprehensive solution for the creation and validation of this important class of helper CD4+ T cells.
Quantification of multiple human immune checkpoint molecules using antibody array technology
Authors: Huang Ruo-Pan , Peck Hannah , Yen Haw-Han , Huang Ruochun , Mao Yingqing
Published: January 2023

Immune checkpoint molecules play an important role in T cell functionality after TCR/MHC signaling. Blockade of two B7/CD28 family checkpoint molecules, CTLA-4 and PD-1 have already demonstrated superior efficacies in treatment for a variety of malignancies. Recently, the US FDA approved one anti-CTLA-4 monoclonal antibody (ipilimumab, 2011) and two anti-PD-1 monoclonal antibodies (pembrolizumab 2014, and nivolumab 2014) for the treatment of advanced melanoma. Other immune checkpoint molecules have been increasingly considered as new targets for cancer immunotherapy. To facilitate better identification of novel targets and elucidation of the checkpoint blockade, simultaneous detection of these pathway molecules in complex biological samples is essential. With antibody array technology, a novel checkpoint molecule array was developed for quantitative measurement 10 of the most important B7/CD28 family checkpoint molecules including B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2/ICOSL, B7-H3, CD28, CTLA-4, ICOS, PD-1, and PD-L2/B7-DC. Antigen-specific capture antibodies were fixed on glass slides with a non-contact arrayer, and multiplex ELISA detection of these proteins was achieved through laser fluorescence detection. All the markers could be detected at pg/ml level. The array was validated by cross reactivity and media recovery tests, and was further validated by conventional ELISA testing of normal human sera. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.
Biomarker Development for Cancer Immuno-oncology/Immunotherapy: Simultaneous Digital Counting of Nucleic Acids and Proteins at 800-plex
Authors: Warren Sarah , Dennis Lucas , Fling Steven , Danaher Patrick , Pekker Irena , D’Amico Leonard , Disis Nora , Geller Melissa , Jacquemont Celine , Kussick Steve , Shine Richard , Bailey Christina , Odunsi Kunle , Cesano Alessandra , Beechem Joseph
Published: January 2023

The ability to measure changes to the DNA, RNA, and protein is crucial to understanding both tumor and immune responses to therapy. NanoString® has pioneered 3D Biology™ – the ability to measure any combination of DNA, RNA, and protein simultaneously using a single detector from a small clinical sample – in order to characterize these complex interactions. Analytes of interest are labeled with fluorescent nucleic acid barcode (via direct hybridization for nucleic acids or antibody conjugated barcodes for protein) and detected via digital counting. This enables quantification of up to 800 analytes from a small sample (50,000 cells for DNA/RNA/protein, or 1–2 5μm FFPE slides for RNA alone). The cancer immunology profiling panel measures 770 mRNAs (unique signatures for 24 infiltrating immune cell types plus extensive immune-signaling pathways) plus 30 key IO proteins.

We present two examples of 3D Biology in action. First, a targeted BRAFV600E inhibitor induces expression changes of immune genes which can be detected at both the RNA and protein levels. Second, in work from ongoing collaborations with the Cancer Immunotherapy Trials Network, clinical samples from patients treated with either mono- or combination immunotherapy can be stratified by gene expression. We also show that gene signatures predict presence of immune cell populations with a high degree of concordance to flow cytometry and immunohistochemistry measurements.

Multiplexed RNA biomarkers have achieved success in predicting patient response to therapy, e.g. NanoString signatures predict response to pembrolizumab (anti-PD-1). Measuring DNA, RNA, and proteins in unison greatly expands the power of biomarker signatures to characterize responses to immunotherapy.
A fast live-cell assay to detect antigen-specific CD8+ T cells based on upregulation of integrin-mediated adhesion
Authors: Dimitrov Stoyan I , Besedovsky Luciana , Jensen Anja Tatiana Ramstedt , Born Jan , Gouttefangeas Cecile , Rammensee Hans-Georg , Lange Tanja
Published: January 2023

Antigen stimulation of T-cell receptors on T cells induces an immediate increase in ligand-binding affinity and clustering in the plasma membrane of beta2-integrins, resulting in a strong binding to intercellular adhesion molecules (ICAMs) on target cells. This firm adhesion, e.g. of cytotoxic T cells to virus-infected or tumor cells with formation of cytotoxic synapses is required for target cell lysis. Therefore, upregulation of integrin-mediated adhesion after stimulation identifies functional effector T cells. Here we present a novel flow cytometry assay to enumerate antigen-specific CD8+ T cells using soluble multimeric ICAM1-Fc-F(ab’)2 fluorescent complexes to identify integrin activation induced after short-term stimulation in vitro. By optimizing and validating this assay we were able to identify pathogen-, tumor- or vaccine-induced CD8+T cells, and show its feasibility for assessing and isolating live, functional antigen-specific CD8+T cells. Performing coexpression analyses, we found that antigen-stimulated cells that expressed cytokines and mobilized CD107a were predominantly highly adherent CD8+T cells. Finally, CD8+ T cells that upregulated integrins were shown to mediate cytolytic activity in an antigen-specific manner. The most striking advantage of this method is the short-time incubation (several minutes) that is necessary to identify and isolate viable antigen-specific cytotoxic T cells by assessing upregulation of T cell adhesiveness, a critical step for elaborating multiple effector functions, including cytotoxicity and cytokine production.
Immune Checkpoint and Immunostimulatory Reporter Bioassays to Advance the Development of Therapeutic Antibodies in Immunotherapy Research Programs
Authors: Somberg Richard , Cong Mei , Cheng Zhi-jie Jey , Grailer Jamison J , Stecha Pete , Wang Jun , Hartnett Jim , Fan Frank
Published: January 2023

Immunomodulatory biologics, which elicit their therapeutic effects by inducing, inhibiting, or modulating immune responses, have shown therapeutic utility in a variety of clinical conditions including cancer and autoimmune disease. As a result, biologics therapeutics, and monoclonal antibodies (mAb) in particular, are a dominant and increasing focus of pharmaceutical pipelines. However, due to their structural and biological complexity, mAb biologics and biosimilars require unique functional and analytical tools for development and characterization.

We have developed a suite of cell and reporter-based bioassays to advance the development and commercialization of mAb biologics. Specifically, we have developed cell-based bioassays in “thaw-and-use” format for the following Immune Checkpoint and Immunostimulatory receptor targets: PD-1/PD-L1, CTLA-4, LAG-3GITR, 4-1BB, CD40, OX40T Cell Activation

These reporter-based bioassays offer high specificity, sensitivity, and reproducibility, and demonstrate the performance required for use in antibody screening, potency determination, and stability studies.
TGF-β-induced regulatory T cells directly suppress B cell responses through a non-cytotoxic mechanism
Authors: Wang Julie , Olsen Nancy J , Zheng Song Guo
Published: January 2023

Foxp3+ regulatory T cells (Treg) playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases consist of thymus-derived naturally-occurring CD4+Foxp3+ Treg cells (nTreg) and those that can be induced ex vivo with TGF-β (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. The role of iTreg in regulating B cells remains unclear so far. The suppression assays of Treg subsets on activation, proliferation and antibodies production of B cells were measured using a Treg and B cell co-culture system in vitro. Transwell and antibody blockade experiments were performed to assess the roles of cell contact and soluble cytokines. Treg were adoptively transferred to lupus mice to assess in vivo effects on B cells. Like nTreg, iTreg subset also directly suppressed activation and proliferation of B cells isolated from C57BL/6. nTreg subset suppressed B cell responses through cytotoxic manner related to expression of Granzyme A, Granzyme B and Perforin; while the role of iTreg subset on B cells did not involve in cytotoxic action using Granzyme B and perforin KO mice, but depending on TGF-β signaling. Furthermore, iTreg subset can significantly suppress antibody produced by lupus B cells in vitro. Comparison experiments using microarray assay on autoantibodies demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo. Our data implicate a role and advantage of iTreg subset in treating B cell-mediated autoimmune diseases, boosting the translational potential of these findings.
IL-17 receptor A signaling impedes NF-κB p50/p50 repressor and subverts B-cell anergy in BXD2 mice
Authors: Hsu Hui-Chen , Hamilton Jennie A , Wu Qi , Yang PingAr , Luo Bao , Xie Shutao , Liu Shanrun , Li Jun , Mountz John D
Published: January 2023

B-cell fate decision checkpoint 2 at the transitional T2 stage represents a critical checkpoint defect in the development of autoimmune disease. We have found that there is a failure of anergy induction in the CD93+IgM+CD23+ transitional T2 B cells in the spleen of lupus prone BXD2 mice, which is associated with B-cell tolerance loss to anti-IgM or TLR7 in vitro. Interestingly, there was an enhanced BXD2-Il17ra−/− T2 B cell anergy phenotype in vivo, which is associated with a strong B cell anergic response to BCR or TLR7 stimulation in vitro. Abnormal survival of T2 B cells is thought to be regulated through a strong BAFF-R signaling. Surprisingly, despite normal expression of BAFF-R, BlyS cannot reverse the anergic response of BXD2-Il17ra−/− B cells to BCR stimulation. Analysis of the expression of IL-17RA in the B-cell subsets in BXD2 mice shows that only T2 and germinal center (PNA+Fas+) B cells, but not T1, T3 or mature (CD93−) B cells express surface IL-17RA. Single cell analysis of T2 B cells reveals the co-expression of Il17ra with B-cell activation gene, Bclxl. The strong anergic phenotype of BXD2-Il17ra−/− mouse B cells is associated with a dramatically enhanced nuclear expression of NF-κB1 (p50) and down-modulation of NF-κB phospho-p65. Our results suggest that, in BXD2-Il17ra−/− B cells, the anergy phenotype is established at the T2 stage. In these B cells, the stimulus-specific transcription repressor p50/p50 homodimer may act as a master transcriptional regulator to counteract pro-activation/survival NF-κB signaling provided by other major B-cell stimulators to enforce B cell anergy. Reagents that can promote the NF-κB p50/p50 repressome complex may be a novel strategy to enhance B-cell tolerance for autoimmunity.
Regulation of transcription factor IRF8 in myeloid progenitors is a critical checkpoint for formation of defective myeloid cells in cancer.
Authors: Messmer Michelle N , Netherby Colleen S , Abrams Scott I
Published: January 2023

Defectivemyeloid cells are commonly observed across a large range of solid cancers. These defects contribute to immune suppression and tumor progression tometastatic disease. Early studies focused on characterizing such myeloid-derivedsuppressor cells (MDSC) in peripheral tissues and the tumor microenvironment, but molecular processes underlying their development remained unclear. Bone marrowis the major site for normal myeloid production and is known to respond to signalsfrom the periphery. Thus, we hypothesizedthat tumors perturb bone marrow myelopoiesis, resulting in production of MDSC. During myelopoiesis, transcription factor IRF8 expression within granulocyte/monocyteprogenitors (GMP) determines differentiation such that high IRF8 favorsmonocytes while low IRF8 favors granulocytes. We previously revealed that IRF8 suppressionby tumor-derived factors leads to accumulation of MDSC, but exactly when andwhere IRF8 is compromised was unknown. Here we utilize a novel mouse model expressingan IRF8-EGFP fusion protein to investigate specific changes in IRF8 during myelopoiesisin both orthotopic and spontaneous mammary tumor progression. Our results show that:1) GMP can be divided into IRF8hi and IRF8lo expressingpopulations corresponding to monocytic and granulocytic progenitor phenotypes,2) IRF8lo GMP preferentially expand with increasing tumor size andincreasing G-CSF serum concentration, and 3) enforced IRF8 expression restrainsthis expansion and reduces the frequency of myeloid suppressors in theperiphery. Altogether, these data identify modulation of IRF8 in myeloidprogenitors as a consequence of tumor development and potential target fortherapeutic intervention.