bluuebunny/crossref_metadata_2025 huggingface dataset:
Multicolor flow cytometric analysis of CD4+ T-cells for CD25 and Foxp3 expression in human Prostate Cancer (50.21)
Authors: Perambakam Supriya , Peace David J
Published: January 2023

CD4+CD25+ T regulatory cells (Treg) are postulated to regulate immune responses against self-antigen, infectious agents, tumor antigens and transplantation antigens. The objective of the study is to examine whether PSA146–154 peptide vaccination impacts the frequency and/or phenotype of Treg in peripheral blood mononuclear cells (PBMC) of patients with prostate cancer (CaP). Frozen PBMC were rapidly thawed, enumerated, checked for viability and directly stained with fluorescent labeled antibodies to CD3, CD4, CD8, CD25 and Foxp3 and analyzed on a flow cytometer. Based on morphology, gates were set to include, (a) whole PBMC population but exclude debris and clumps or, (b) predominantly large lymphocyte population and then back gated on CD3+CD4+ T-cells. Quadrant analysis demonstrated the presence of 1–5% of CD4+CD25+Foxp3−, less than 1% of CD4+CD25−Foxp3+ and CD4+CD25+Foxp3+ cells in the whole PBMC population. While, 10–44% of CD4+CD25−Foxp3+, 1–2% of CD4+CD25+Foxp3−, 1–3% of CD4+CD25+Foxp3+ cells were observed in the large lymphocyte population. However, there was no significant difference between before and post vaccine samples. CD4+CD25−Foxp3+ cells may represent a distinct T-cell subset and warrants further investigation.

Funding: The Illinois Department of Public Health (IDPH# 4328301)
Macrophage functional plasticity: IL-12 rapidly alters the functional profile of tumor associated and tumor infiltrating macrophages in vivo. (50.4)
Authors: Watkins Stephanie Kaye , Egilmez Nejat K. , Suttles Jill , Stout Robert D.
Published: January 2023

Tumor-associated macrophages play a major role in promoting tumor growth and metastasis and in suppressing the anti-tumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific cytotoxic T cells. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated “suppressor” macrophages. Analysis of tumor infiltrating macrophages (TIMs) and distal tumor associated macrophages (TAMs) revealed that IL-12, both in vivo and in vitro, induced a rapid (< 90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, MIF, TGFâ production) and a concomitant increase in pro-inflammatory and pro-immunogenic activities (TNFá, IL-15, IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in TIMs isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen and peritoneal cavity. Therefore, although tumor associated macrophages display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to pro-inflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive anti-tumor immune response.

This research was supported by the Nat’l Cancer Institute, the KY Lung Cancer Research Fdn, the American Lung Assoc., and the KY Research Challenge Trust Fund.
Proteasome inhibition promotes tumor cell apoptosis by exogenous TRAIL without blocking anti-tumor immune effectors in vivo (50.6)
Authors: Shanker Anil , Brooks Alan D , Tristan Carlos A , Wine John , Sayers Thomas J
Published: January 2023

The resistance of tumors to cell death poses a major clinical problem. Bortezomib (Velcade/PS-341), a specific and reversible proteasome inhibitor, sensitized renal (Renca) and mammary (4T1) mouse carcinomas to apoptosis induced by the TNF family ligand TRAIL or its receptor (DR5) agonist antibody. The molecular basis of the tumoritoxicity of the combination of bortezomib and TRAIL seemingly involved amplification of the extrinsic cell death pathway as increased expression of DR5, activation of caspase-8 and -3, and decreased apoptosis inhibitory proteins cFLIP, cIAP-1 and XIAP were observed in bortezomib-treated tumors. In an established tumor setting, anti-DR5 administration to mice pre-treated with bortezomib not only reduced lung metastases of Renca and 4T1 tumors but also significantly increased long-term survival in mice bearing Renca as compared to either agent alone. Encouragingly, no evidence of any overt tissue damage was noted in the treated mice. This provides the first experimental evidence that proteasome inhibition can be combined with DR5 agonist antibody for successful cancer therapy. Moreover, bortezomib administration 24 hours prior to adoptive transfer of T cells did not block their anti-tumor effector function. This suggests that tumor sensitization with bortezomib may also be combined with tumor-specific T cell immunotherapy of cancer.

Funded in part by NCI contract N01-CO-12400.
CD18 Separates GVHD and GVL effect (50.9)
Authors: Liang Yaming , Yu Xue-Zhong
Published: January 2023

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. β2 integrins, a group of heterodimeric molecules including CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 and CD11d/CD18, are of critical importance for leukocyte extravasation through vascular endothelia and for T cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing graft-versus-leukemia (GVL) effect. In murine allogeneic BMT models, we found that recipients of CD18−/− donor T cells had significantly less GVHD morbidity and mortality compared with recipients of WT donor T cells. A cell-dose titration experiment indicated that the ability of CD18−/− T cells had more than 4-fold reduction compared to that of WT T cells in the induction of GVHD. Analysis of alloreactive showed that CD18−/− and WT T cells had comparable activation, expansion and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor-T cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that in vivo GVL effect of CD18−/− donor T cells was largely preserved, because mortality of the recipients transplanted with CD18−/− T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target β2 intergrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.
Evolution of antibody responses against carbohydrate and protein antigens of Pneumocystis carinii: Implications for antibody-based vaccine protection (51.12)
Authors: Rapaka Rekha R. , Zheng Mingquan , Kolls Jay K.
Published: January 2023

Pneumocystis (PC) pneumonia is a leading opportunistic infection among HIV+ individuals. Mice deficient in CD4+ T cells or B cells are susceptible to PC pneumonia, yet adoptive transfer of serum from PC challenged mice can prevent PC pneumonia. To evaluate factors critical for antibody-based resistance against PC, we assessed antibody responses to intratracheal PC challenge in Balb/c mice. The kinetics of anti-PC Ig induction are prolonged, peaking after the majority of PC is cleared from the lungs. Low anti-PC IgM titers(1:120) exist at baseline and are enhanced by vaccination(1:2600), and the induced anti-PC IgM is strongly cross-reactive with zymosan. Anti-PC IgG is absent at baseline but is rapidly induced through day 36 post-challenge (titer 1:38,000). We assessed the evolution of Igs directed against the PC cellular wall carbohydrates β-glucan and chitosan, which may potentially elicit B cell responses in a CD4+ T cell independent manner. Baseline IgM directed against the PC cellular wall antigen β-glucan is high, rapidly enhances 2 days post challenge (titer 1:5000), and diminishes, suggesting a potential role for B1 cells in this response, while PC induces anti-β-glucan IgG (titer 1:1000) with a significantly delayed timecourse, peaking at day 42 post-challenge; IgM and IgG responses directed against chitosan manifest similar kinetics. Finally, mice exposed to PC and subsequently depleted of CD4+ T cells after the primary response with the antibody GK1.5, mount similar boosting responses to CD4+ T cell intact mice. Our studies suggest that PC elicits antibodies against protein and carbohydrate antigens, implicating B1 and B2 cells in these responses, and suggest that components of anti-PC B cell memory may be maintained under CD4+ T cell deficiency.
The C2 receptor CRIT protects Trypanosoma cruzi against classical and MBL-directed, but not alternative pathway complement activation (51.14)
Authors: Inal Jameel Malhador , Cestari Igor dos Santos , Ingrid Evans-Osses , Juliana Freitas , Ramirez Marcel
Published: January 2023

We show that the sylvatic class I strains of Trypanosoma cruzi resist more effectively the classical (CP) and mannan-binding lectin (LP) mediated complement activation than the domestic class II strains. T. cruzi Colombiana (sylvatic class II) showed under 3% survival compared with 45% for the Y strain (domestic class I). The AP plays a minimal role in lysis mediated in the short-term. The complement C2 receptor CRIT gene is highly similar in T. cruzi class I and class II parasites. It is not expressed in log growth phase epimastigote insect forms of either class and is only expressed in late growth phase epimastigote (infective stage) forms of T. cruzi Y (class I ) but not Colombiana (class II) strains. When the Tc-CRIT gene of T. cruzi Y strain was overexpressed in Y and Colombiana strains, the transgenic parasites (pTEX-Tc-CRIT) showed 86% survival after 5 minutes in normal human serum (NHS) compared to 57.4 % (pTEX) and 53.4% (wild type). Once more in the presence of AP alone there was 80% survival after 5 and 10 minutes. As CRIT is a receptor which principally binds complement C2 it is presumed that overexpressed CRIT in epimastigote forms of T. cruzi Y strain resist CP and LP by depletion of C2 from NHS. Experiments in which an excess of C2 was added to NHS confirmed this mechanism by restoring hemolytic activity.
Diacylglycerol kinase zeta regulates microbial recognition and host resistance to Toxoplasma gondii (51.16)
Authors: Liu Cheng-hu , Machado Fabiana S. , Guo Rishu , Nichols Kim E. , Burks A. Wesley , Aliberti Julio C. , Zhong Xiao-Ping
Published: January 2023

Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting diacylglycerol (DAG) to phosphatidic acid (PA). Here, we demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mϕ) and dendritic cells (DC). DGKζ deficiency results in impaired IL-12 and TNFα production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the PI3K-Akt pathway and that inhibition of PI3K activity can restore LPS-induced IL-12 production by DGKζ deficient macrophages. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.
Mouse strain affects Stachybotrys chartarum induced alveolar macrophage cytotoxicity (51.9)
Authors: Lichtenstein Jamie H Rosenblum , Donaghey Thomas C , Molina Ramon M , Brain Joseph D.
Published: January 2023

The fungus Stachybotrys chartarum (S. chartarum) releases mycotoxins that can cause lung injury, inflammation, hemorrhage and cytotoxicity. We have shown that when BALB/c mice receive instilled spores, they have greater lung inflammation, hemorrhage, and cytokine production and less efficient spore clearance than C57BL/6J mice.

To investigate these mouse strain differences, we exposed alveolar macrophages (AMs) lavaged from these two strains of mice to spores in vitro. Then the cells were stained with calcein AM (live) and EthD1 (dead), and images were collected on a BD Pathway Bioimager. Both strains exhibited increasing AM death as doses increased from 200 to 5000 spores per 75,000 AMs (p=0.0001) with more C57BL/6J than BALB/c AMs dying (slopes significantly different at p=0.007). After one day, 84 ± 4% of C57BL/6J AMs versus 66 ± 5% of BALB/c AMs died at the 1000:75,000 spores:AMs dose. The spores killed C57BL/6J AMs sooner (p=0.04) than BALB/c AMs. Many of the dead AMs from both strains appear morphologically apoptotic.

In vivo, AM death may diminish cytokine levels and inflammation in C57BL/6J mice because dead cells do not synthesize cytokines. This increased cytotoxicity coupled with improved spore clearance, causing a lower effective spore dose, may reduce inflammation in C57BL/6J mice. Analogous differences may contribute to the range of sensitivity to S. chartarum seen among humans.
γδ T cells characteristic of the gut mucosa are stimulated by oligomeric tannins (52.11)
Authors: Holderness Jeff , Freedman Brett , Kimmel Emily , Jutila Mark A.
Published: January 2023

γδ T cells function, in part, to regulate the innate immune response and maintain tissue homeostasis at mucosal surfaces. Increasing the activity of the γδ T cells may be effective for treating a variety of pathogens that target the gut mucosa including rotavirus and Salmonella. Treatment with the phosphoantigen family of agonists stimulates only the Vδ2 subset of human γδ T cells and does not stimulate Vδ1 T cells, the primary γδ T cell population at the sites of pathogen entry. Here we define the biological activity of an agonist that stimulates both the Vδ2 and importantly, the Vδ1 subsets of human γδ T cell population. Initial examination of the effects in vivo suggests that the agonist may induce expansion of mouse intestinal γδ T cells after oral administration. The agonist is part of an unripe apple peel extract that contains a mixture of phenolics including monomeric and oligomeric tannins. Preliminary characterization of the extract suggests that the γδ T cell stimulation is due to the specific interaction of an oligomeric tannin.
Characterization of differential innate and adaptive immunity against Rickettsia conorii in resistant and susceptible mice (52.14)
Authors: Fang Rong , Ismail Nahed , Walker David H.
Published: January 2023

Spotted fever group (SFG) rickettsiae cause severe human diseases in many endemic areas of the world. In order to identify the critical factors in determining host susceptibilities or resistance to SFG rickettsiosis, we characterized cellular immunity against R. conorii in highly susceptible C3H/HeN (C3H) mice, relatively susceptible BALB/c mice and highly resistant C57BL/6 (B6) mice. A dose of rickettsiae lethal to C3H mice is cleared by all wild type (WT) B6 and BALB/c mice in 4 days with survival. Compared to C3H mice, significantly higher expansion of natural killer (NK) cells was detected in WT B6 mice. Similar to the respective WT mice, all RAG−/− and IL-12p40−/− knockout mice on B6 background and SCID on BALB/c background survived, whereas BALB/c SCID mice with deficiency of NK cells died. These findings suggest that NK cells play a major role in the innate immunity, which is IL-12p40- independent in the resistant host. Protection against sublethal rickettsial infection in C3H mice was associated with a CD4+ Th1 cell response, the presence of greater quantities of inducible CD4+CD25+ T regulatory cells and CTL than in B6 mice. These findings suggest that a cell-mediated Th1 immune response is critical for protection against rickettsial infection in the susceptible host and T regulatory cells may contribute to the outcome of rickettsial infection, most likely through balancing T cell mediated protective immunity and immunopathology.

This work was supported by a grant (AI021242) from the National Institute of Allergy and Infectious Diseases.
Neuroinflammatory pathways induced by TLR7 stimulation in the developing brain (52.3)
Authors: Butchi Niranjan Babu , Pourciau Susan , Peterson Karin E
Published: January 2023

Activation of astrocytes and microglia (gliosis) and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as autism and cerebral palsey. Neonates have poor adaptive immune responses to virus infections. Instead, innate immune response may play an important role in protection from virus mediated damage to the brain. Innate immune responses are often initiated by recognition of pathogen associated molecular patterns that stimulate toll like receptors. TLR7 and 8 were identified as eliciting anti viral effects when stimulated by viral single stranded RNA. To examine the potential of TLR7 to induce glial activation and neuroinflammation, we stimulated astrocyte cells (C8-DIA) with TLR7 ligands, Imiquimod and Loxoribine. Imiquimod induced the upregulation of Ifnb1 or Tnf á mRNA at 48 hrs post stimulation in astrocyte cultures. In contrast, Loxoribine did not induce Ifnb1 or Tnf á mRNA in astrocyte cultures but induce these responses in control macrophage cells. This showed the differential activation of astrocyte by different TLR7 ligands. To determine the effect of these TLR7 ligands in vivo, a single inoculum of either Imiquimod or Loxoribine was injected intracerebrally into new born mice. We are currently analyzing effect of TLR7 stimulation on induction of proinflammatory cytokines, chemokines, gliosis and adhesion molecule in the brain.
NTAL Negatively Regulates TREM-1/DAP12 Function (52.6)
Authors: Tessarz Anja S , Weiler Sandra , Zanzinger Kai , Angelisová Pavla , Horejší Václav , Cerwenka Adelheid
Published: January 2023

Engagement of Triggering Receptor Expressed on Myeloid cells (TREM-1) on macrophages and neutrophils leads to TNF-α and IL-8 production and enhances inflammatory responses to microbial products. For signal transduction, TREM-1 couples to the ITAM-containing adapter DNAX Activation Protein of 12 kDa (DAP12). In general, ITAM-mediated signals lead to cell activation, although DAP12 was recently implicated in inhibitory signaling in mouse macrophages and dendritic cells. So far, signals downstream of the TREM-1 and DAP12 complex in myeloid cells are poorly defined. By analyzing receptor-induced tyrosine phosphorylation patterns, we discovered that ligation of TREM-1 leads to tyrosine phosphorylation of the Non-T cell Activation Linker (NTAL/LAB) in a myelo-monocytic cell line and primary human granulocytes. Using RNA interference to decrease expression levels of NTAL, we demonstrate that in NTAL knock-down cell lines phosphorylation of ERK 1/2 is enhanced. In addition, low levels of NTAL are correlated with decreased and delayed mobilization of Ca2+ after TREM-1 triggering. Most importantly, we demonstrate that NTAL acts as a negative regulator of TNF-α and IL-8 production after stimulation via TREM-1. Our results show that activation signals delivered via DAP12 can be counterbalanced by the adaptor NTAL, identifying NTAL as gatekeeper of TREM-1/DAP12-induced signaling in myeloid cells.

Supported by a Marie Curie Excellence Grant (002739) to AC.
Polyanion-induced self-association of complement factor H: possible mechanism of host protection from an innate immune system (53.11)
Authors: Pangburn Michael K. , Rawal Nenoo , Ferreira Viviana P. , Atkinson Mark A.L.
Published: January 2023

Factor H is the primary soluble regulator of activation of the complement alternative pathway. It prevents activation of complement on host cells and tissues upon association with surface polyanions such as sialic acids, heparin and other glycosaminoglycans. We now show that interaction with polyanions causes self-association forming tetramers of the 155,000 dalton glycosylated protein. Monomeric human factor H is composed of twenty homologous CCP domains and in its extended form it exists as a flexible protein approximately 380 to 730 angstroms in length and 35 angstroms in width. Due to its extended shape, it exhibits an apparent size of 330,000 daltons, relative to globular standards, during gel filtration in the absence of polyanions. In the presence of dextran sulfate (5,000 daltons) its apparent Mr was found to be 1,400,000 daltons by gel filtration. Similar effects were seen with heparin. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a tetramer of 607,000 Mr in the presence of less that a two-fold molar excess of dextran sulfate. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H and the resulting inhibition of complement activation may involve formation of dimers and tetramers of factor H.

Support: NIH grant DK-35081, NIH grant HL-073804 and AHA grant 0265178Y.
Proteins involved in prophenoloxidase activating system and their roles in mud crab Scylla serrata (53.12)
Authors: Chen Jiann-Chu , Lin Yong-Chin , Vaseeharan Baskarakingam
Published: January 2023

Several proteins like proPO (prophenoloxidase), peroxinectin, serine proteinase and serine proteinase inhibitors such as pacifastin and a2-macroglobulin are involved in the proPO activating system. Isolation and purification of these proteins and cloning of these proteins have been widely studied in penaeid shrimp, crayfish and lobster. Recently we have cloned proPO, serine proteinase, and a2-macrglobulin from the haemocytes of mud crab Scylla serrata, an important commercial species in Asia and Oceania. The cDNA clone of proPO has a full-length of 2663 bp, with an open reading frame of 2019 bp, a 124-bp 5?untranslated region, and a 520-bp 3?untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5 kDa and with an estimated pI of 5.96. The cDNA clone of serine proteinase has an open reading frame of 1131 bp encoding a protein of 376 amino acids. The calculated molecular mass of the serine proteinase is 39.54 kDa with an estimated pI of 5.37. The cDNA clone of a2-macroglobulin has a full-length of of 5491 bp with an open reading frame (ORF) of 4986 bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2 kDa with an estimated pI of 8.41. Comparison of each protein of this species with that of other arthropod available in the GenBank, and phylogenetic analysis of each protein are discussed.
Upon ligation, CD35 (CR1) interacts with the Cdc42-binding protein Par6 in human neutrophils (53.13)
Authors: Ghiran Ionita Calin , Lawler Matthew Ryan , Nicholson-Weller Anne
Published: January 2023

Inflammation associated with infections is composed of well-choreographed sequences of pro- and anti-inflammatory responses. Among opsonins, the same paradigm is followed. While the signaling pathways mediated by the IgG receptors are well defined in neutrophils (PMN), little is known about the signaling pathway of CR1 (or CD35), the receptor for all nascent complement opsonins.

Our data provide evidence that in PMN, the cytoplasmic domain of CR1, which contains two PDZ motifs interacts with Par6 (partition defective protein) a PDZ domain-containing cytoplasmic protein, initially described in C. elegans and later in virtually all organisms from worms to humans. By virtue of its several protein-protein interacting domains, Par6 is involved in a wide range of cellular processes such as cell polarity, neuronal division and directed cell migration. Using its CRIB-like motif, Par6 binds Cdc42 and Rac1, GTPases involved in generating reactive oxygen species following FcR ligation. Our data show that the PDZ domain of Par6 interacts “in vitro” with the cytoplasmic tail of CR1 as assessed by protein array and BiaCore analyses and “in vivo” as assessed by co-immunoprecipitation of human PMN. We have demonstrated Par6 expression in PMN using flow cytometry, microscopy and western blot analyses. We hypothesize that in human PMN, CR1 attenuates pro-inflammatory response by binding Par6 and thereby preventing formation of the functional pro-inflammatory complex (Par6/Cdc42/Rac1) essential to Fc receptors signaling.
Complement C2 bypass mechanism involving the C3-convertase C4bBb (53.16)
Authors: Inal Jameel Malhador , Laich Andreas , Miot Sylvie
Published: January 2023

The majority of patients with a homozygous complement C2 (C2) deficiency show no or only minor disease, at most one third showing mild symptoms of systemic lupus erythematosus. This suggests that classical pathway activation is not solely dependent on C2. We show that factor B (FB) the alternative pathway homologue of C2 can substitute for C2. In ELISA, FB was found to complex with immobilised C4b. This was confirmed by surface plasmon resonance, giving an estimated binding constant of 2.0 x10−5 M. Furthermore Factor D was able to cleave factor B in the presence of C4b. The hybrid C3 convertase formed, C4bBb, also cleaved 125I-C3 at a low level, further supporting previous evidence of a C2-bypass mechanism.
Unique tissue distribution and molecular heterogeneity of human Fc receptor for IgA and IgM (Fcα/μR) (53.17)
Authors: Nishizaki Naoto , Grizzle William E. , Stockard Cecil R. , Kikuno Kaoru , Kubagawa Hiromi
Published: January 2023

Fcα/μR consists of an extracellular region with an N-terminal single Ig-like domain and a membrane proximal stalk region, a transmembrane (TM) segment and a cytoplasmic tail. Fcα/μR binds the polymeric IgM and IgA and is expressed by follicular dendritic cells in germinal centers, small intestinal Paneth cells, renal tubular epithelial cells and serous small epithelial cells of salivary glands in humans. Analyses of its transcripts and genomic DNA revealed the existence of an additional translation start site upstream of the previously isolated Fcα/μR cDNA and a splice variant in kidney tissues. The latter was generated from the direct splicing of exon 5 (Ig-like domain) to exon 7 (TM) and resulted in a reading frameshift in exon 7, generating a novel 48-aa hydrophilic C-terminal tail. To determine the roles of the upstream start site and the alternative splice variant, those cDNA were subcloned into an expression vector prior to establishment of the mouse cell lines stably expressing human Fcα/μR transgene. Transductants with the full-length Fcα/μR cDNA irrespective of the start sites expressed Fcα/μR on their cell surface, whereas those with the Fcα/μR splice variant expressed Fcα/μR associated with intracellular, not plasma, membranes. Protein blot analysis of these cell lysates revealed that the Fcα/μR proteins correspond to their predicted molecular masses.

(Supported by NIH/NIAID AI052243)
Cell surface complement regulatory functions of factor H are essential for the survival of normal and paroxysmal nocturnal hemoglobinuric (PNH) red blood cells (53.2)
Authors: Ferreira Viviana P. , Pangburn Michael K.
Published: January 2023

The complement system uses a complex set of cell-bound and plasma regulators to protect host cells from complement-mediated cytolysis. Although PNH red blood cells (RBCs) are partially (type II) or completely (type III) deficient in GPI-linked complement regulatory molecules CD59 and CD55, they are not rapidly lysed by complement. For instance, the in vivo lifespan of PNH III RBCs is 6–60 days, and is close to that of normal cells for PNH II RBCs. The plasma regulator factor H (fH) plays a key role in complement homeostasis. Here we show that a recombinant protein consisting of its two C-terminal domains (rH19-20) blocks its cell surface complement regulatory functions without affecting fluid phase control of soluble C3b. Also, addition of rH19-20 to normal human serum leads to moderate complement-mediated lysis of normal human RBCs, which possess membrane-bound complement regulators, and to aggressive lysis of PNH II and III cells. In agreement with this, the inhibition of CD59 or CD55 on normal RBCs results in their aggressive lysis only when combined with the loss of fH cell surface control. Taken together, the results highlight the importance of the cell surface protective functions of fH compared to other complement regulatory proteins and indicate that fH is essential for the 6–60 day survival of PNH RBCs, with possible implications in diagnosis and treatment.

Research support NIH DK35081.
Properdin: Cell Death’s Little Helper? (53.3)
Authors: Kemper Claudia , Mitchell Lynne M , Hourcade Dennis E
Published: January 2023

Apoptosis is a mechanism for the safe elimination of undesirable cells without evoking an immune response. C3b/C4b-opsonization of apoptotic cells through classical and lectin complement pathway activation is a major mechanism to mark these cells for clearance by scavenger cells. Recently we observed that the serum protein properdin bound to a biosensor surface leads to direct alternative pathway activation. Here we determine whether properdin can also direct complement activation to physiological targets i.e. human apoptotic T cells.

We found that:

Properdin (purified and in serum) binds apoptotic and necrotic CD4+ T cells, but neither non-activated nor viable activated T cells;Properdin bound to apoptotic cells initiates de novo complement activation and phagocytosis by dendritic cells/macrophages;Surprisingly, properdin also enhanced phagocytosis of apoptotic cells independently of complement activation.

We propose that properdin directly recognizes apoptotic T cells, promoting their targeted uptake by phagocytes. This mechanism represents a novel pathway for the identification and removal of undesired cells.

Funded by NIH R01 AI05143 (to DH).
The Neonate Fc Receptor (FcRn) For IgG Is Important For The Development of Rotavirus-Specific IgG Responses And Clearance of Rotavirus From The Intestines Of Mice (53.5)
Authors: Marcelin Glendie , Conner Margaret E
Published: January 2023

Rotavirus infection and virus-like particle vaccines induce intestinal rotavirus-specific IgG in mice that appears to play a role in protection. The mechanism by which rotavirus-specific IgG reaches the intestinal lumen is unknown. The neonatal Fc receptor for IgG (FcRn) is the only known transporter of IgG in the intestines in neonatal and adult mice. Additionally, FcRn is implicated in antigen presentation in the intestines of adult mice. We tested whether FcRn is important in protecting the intestine against rotavirus infection and in transport of rotavirus-specific IgG into the intestinal lumen. Naïve FcRn deficient (FcRn−/−) and wild type BALB/c controls were orally inoculated with 103ID50 ECWT rotavirus. Fecal samples were collected 0–12 days post inoculation (dpi) and tested for rotavirus antigen and rotavirus-specific IgG by ELISA. Virus clearance from the intestine was significantly delayed by 3 days in FcRn−/− compared to BALB/c mice. Rotavirus-specific intestinal IgG titers were also significantly lower in FcRn−/− versus BALB/c mice. These data indicate that FcRn plays a role in clearance of a primary rotavirus infection from the intestine and intestinal transport of rotavirus-specific IgG. Studies are underway to elucidate the mechanism(s) by which FcRn contributes to clearance of intestinal rotavirus infection.

VA Merit Review Grant, NIH RO1 AI24998
Presence of Id2− and RORγt-independent lymphoid tissue organogenesis (81.2)
Authors: Nagatake Takahiro , Fukuyama Satoshi , Kim Dong-Young , Takamura Kaoru , Kiyono Hiroshi
Published: January 2023

Id2 and RORγt have been shown to be the essential transcriptional regulators for the differentiation of CD3−CD4+CD45+ lymphoid tissue inducer cells (LTi). LTi migrate to the site of tissue genesis and provide the signal mediated by membrane-bound lymphotoxin (LT)α1β2, which results in the subsequent production of chemokines and adhesion molecules including CXCL13 and VCAM-1 by LTβR+ stromal cells. Organogenesis of lymphoid tissues such as Peyer’s patches are thus defective in Id2−/−, RORγt−/− and LTα−/− mice. Tear duct-associated lymphoid tissue (TALT) has been identified in human. However, the molecular and cellular requirements of TALT-genesis are totally unknown. Here we provide the first definitive evidence for the presence of Id2− and RORγt-independent tissue genesis program. We thus located the murine TALT in nasolacrimal duct and revealed that TALT-genesis did not require Id2, RORγt and LTα. TALT-genesis was initiated at postnatal days and developed in germ free and MyD88−/− mice. These results demonstrated that TALT has indeed developed using the unique pathway compared with other secondary lymphoid organs.
LIM Domain Binding Protein 1 (Ldb1) is Required for the Maintenance of Hematopoietic Stem Cells (81.4)
Authors: Li LiQi , Lee Jan , Cohen Tsadok , Gorivodsky Marat , Hayes Sandra M. , Zhao Yangu , El-Khoury Dalal , Grinberg Alexander , Westphal Heiner , Love Paul E.
Published: January 2023

Ldb1 is a ubiquitous nuclear protein that functions by interacting with and/or recruiting specific partners (including LMO2, SCL and GATA-1) to form multi-molecular DNA-binding transcription complexes. In the hematopoietic lineage, expression of Ldb1 is highest in lineage−Sca1+c-kit+ (LSK) multipotent progenitors. Ldb1 gradually decreases as cells mature within lymphoid or granulocyte/macrophage lineages, but remains high in erythroid lineage cells. We investigated the role of Ldb1 in hematopoiesis by conditional deletion of Ldb1 using a cre transgene driven by the Tie2 promotor; or by following the fate of Ldb1-null embryonic stem (ES) cells in mouse blastocyst chimeras. The number of Ter119+ (erythroid lineage) cells was decreased dramatically in Tie2Cre+Ldb1flox/flox mice, revealing a critical role for Ldb1 in erythropoiesis. Ldb1-null ES cells were capable of generating LSKs in the fetal liver and these cells could give rise to both myeloid and lymophoid lineage cells; however, the number of Ldb1−/− LSKs was markedly decreased at later stages of development. Following adoptive transfer, Ldb1-null fetal liver LSKs were rapidly lost indicating a failure of self-renewal or survival. Loss of Ldb1 was associated with an increase in multipotent progenitor (MPP) cells and a decrease in hematopoietic stem cells (HSCs), suggesting that Ldb1 participates in stem cell maintenance. These data provide the first evidence that LIM domain binding proteins function in hematopoiesis, and identify Ldb1 as a critical factor in regulating HSC maintenance/self-renewal.
Liver plasmacytoid dendritic cells stimulate NK1.1+ cells (82.12)
Authors: Nguyen Hoang Minh Lawrence , Kingham T. Peter , Plitas George , Burt Bryan M. , Chaudhry Umer I , DeMatteo Ronald P.
Published: January 2023

Introduction: Plasmacytoid dendritic cells (pDC) play a central role in innate immunity. While much is known about spleen pDC, liver pDC has only been recently identified and their function remains largely unknown. We sought to determine the ability of liver pDC to stimulate NK1.1+ cells in vitro in comparison to spleen pDC.

Methods: Liver and spleen pDC (CD11c+B220+NK1.1−CD19−CD11b−) from C57BL/6 mice were expanded in vivo with an adenovirus encoding murine Flt3L cDNA and isolated by fluorescence activated cell sorting. These cells were co-cultured with spleen NK1.1+ cells in the presence of CpG with or without a transwell insert. After 24 hours, supernatant IFN-γ level was determined by cytometric bead array. Additionally, α-GalCer –loaded liver and spleen pDC were stimulated with CpG and cultured with spleen NK1.1+ cells in the presence of CpG. IFN-γ production was measured after 24 hours.

Results: Flt3L-expanded liver pDC stimulated with CpG induced significantly higher CD69 expression on NK cells when compared to spleen pDC. CD69 expression by both groups was reduced by the presence of a transwell insert. Both liver and spleen pDC loaded with α-GalCer and stimulated with CpG induced large amounts of IFN-γ secretion by NK cells.

Conclusion: Upon expansion and stimulation, liver pDC are able to stimulate NK cells in vitro. This is partially mediated through cell-cell contact.

This work was supported by grant DK068346
NK cell phenotypes in Foxk1 deficient mice (82.13)
Authors: Moody Leslie A , Alexander Matthew S , Jiang Nan , Garry Daniel J , Bennett Michael
Published: January 2023

Foxk1 is a member of the forkhead/winged-helix transcription factor family. Foxk1−/− mice have a gross deficit of natural killer (NK) cells, roughly three-fold, in the spleen, peripheral blood and bone marrow. Both mature and immature NK cells are lacking in the affected tissues. NK cells are the only major lymphocyte compartment affected. Bone marrow chimera experiments between Foxk1−/− and B6 (control) mice have shown that the defect is NK cell intrinsic. The remaining NK cells in Foxk1−/− are functionally defective directly ex vivo as determined by YAC-1 killing. However, following in vitro culture with either IL-2 or IL-15, the Foxk1−/− NK cells regain lytic function. To further analyze the cytotoxic defect of Foxk1−/− NK cells, we have examined the expression of components of the lytic pathway. Foxk1−/− NK cells express lower amounts of Perforin, but increased amounts of FasL, compared to B6 mice. These levels of cytotoxic effector molecules still do not provide us with an answer as to why Foxk1−/− NK cells have a defect in lytic function. To examine the NK cell number deficit in Foxk1−/− mice we have used flow cytometry to assess the various developmental stages of NK cells in Foxk1−/− and B6 mice and have found that Foxk1−/− mice had altered cell surface marker expression at the earliest progenitor stage. Gene expression profiles of early precursors from B6 and Foxk1−/− mice are being examined by microarray analysis.
PLZF is essential for iNKT cell development (82.5)
Authors: Uche Olisambu U. , Sant’Angelo Derek B.
Published: January 2023

The promyelocytic leukaemia zinc finger (PLZF) protein is a member of the BTB/POZ zinc finger family of transcription factors. This family of transcription factors also include Th-Pok, BCL-6 and MAZR which are known to play important roles in the immune system. Here we demonstrate that PLZF deficient mice have a severe impairment in iNKT cell lineage development leading to a lack of iNKT cells in the thymus and periphery. Real-time PCR showed that PLZF mRNA is highly expressed in iNKT cells. This correlates with flow cytometry analysis which revealed exclusive expression of PLZF protein in iNKT cells within the immune system. Radiation chimeras showed that PLZF is required for iNKT cell development in a cell intrinsic manner. These results identify PLZF as a key factor in iNKT development. They also underline the BTB/POZ family of transcription factors as important factors in the immune system.
Natural killer dendritic cells are an intermediate of developing dendritic cells (82.6)
Authors: Gao Jian-Xin , Chen Li , Calomeni Edward , Wen Jing , Shen Rulong
Published: January 2023

Natural killer dendritic cells (NKDCs) appear to emerge as a distinct dendritic cell (DC) subset in human and rodents, which have the functions of both natural killer (NK) cells and DCs. However, the developmental relationship of NKDCs (CD11c+K1.1+) to CD11c+NK1.1− DCs has not been addressed. Herein, we show that NKDCs exclusively exist in the compartment of CD11c+MHC II− cells in the steady state, and express variable levels of DC subset markers, such as Interferon-producing killer dendritic cells (IKDC) marker B220, in a tissue-dependent manner. They can differentiate into NK1.1− DCs accompanied by the up-regulation of major histocompatibility complex (MHC) class II molecules and down-regulation of NK1.1 upon adoptive transfer. However, NK cells (NK1.1+CD11c−) did not differentiate into NK1.1+CD11c+ cells upon adoptive transfer. Bone marrow (BM)-derived Ly6C+ monocytes can be a potential progenitor of NKDCs, as some of them can differentiate into CD11c+NK1.1+ as well as CD11c+NK1.1− cells in vivo. The steady NKDCs have great capacity for lysis of tumor cells, but little capability to present antigens. Our studies suggest that NKDCs are an intermediate of developing DCs with the unique surface phenotype of CD11c+NK1.1+MHC-II−, strong cytotoxic function, and poor capability of antigen presentation in the steady state, and they probably play a critical role in linking innate and adaptive immunity.
Defects in early DN cell development and CD4 SP cell selection in Foxn1Lacz mice (82.7)
Authors: Xiao Shiyun , Chen Lizhen , Manley Nancy R
Published: January 2023

The Foxn1 gene is required for thymic epithelial cell (TEC) development and is expressed in the thymus from the fetal to adult stages. To track Foxn1 gene expression, we inserted an IRES-lacZ gene into the 3′UTR in exon 9 of the Foxn1 gene. We found that the postnatal thymus was progressively atrophic in Foxn1Lacz/LacZ mice and displayed a gradual disorganization. The total thymocyte cell number was reduced 10%, 56% and 80% at postnatal day 8, 14, and 28. The percent of CD4 SP cells in Foxn1Lacz/LacZ mice was significant reduced, but not CD8 SP cells compared to control Foxn1+/Lacz mice. The percent of DP cell was slightly increased, and the percent of DN cell was significant increased 2 to 3 fold in day 21 to 28 mice. The percent of DN1 cells was dramatically increased to more than 60% (around 20% in control mice), and more than 80% of these cells were CD19+ cells. The percent of DN2, DN3 cells were correspondingly reduced to 15% (around 60% in control mice), and the percent of DN4 cells was also reduced in Foxn1Lacz/LacZ mice during the period. Our preliminary data suggests that the expression of MHCII on surface of TSC and Foxn1 mRNA from the Foxn1Lacz allele was reduced; furthermore, the phenotype was more severe in Foxn1Lacz/nu mice, indicating that the defects depended on the dosage of the Foxn1 gene. Taken together, our results suggest that reduction of Foxn1expression in the postnatal thymus caused defects in TEC maintenance, resulting in defects in DN1 cells and in positive selection for CD4 SP cells, which ultimately greatly reduces production of T cells in the thymus. Furthermore, the resulting thymic microenvironment is favorable for B cells development in the postnatal thymus.

Supported by grant AI055001 NIH.
Categorical selection of the Ig CDR-H3 repertoire in splenic B cells (83.14)
Authors: Schroeder Harry W , Tanner Jason M , Zemlin Michael , Schelonka Robert L
Published: January 2023

H chain CDR-H3 serves as the focus of the diveristy of the antibody repertoire. Repertoire development in the bone marrow is associated with a reduction of specific categories of CDR-H3, particularly those with excessive hydrophobic or polar (charged) amino acids and those with a length of eight or fewer amino acids. To test whether CDR-H3 selection continues within the spleen, we sorted B cells into transitional (T1), marginal zone (MZ) and follicular (FO) populations; cloned 364 unique, in-frame, open V7183DJCμ transcripts; and compared them to 509 previously obtained sequences from immature (IM) and mature, recirculating (MA) bone marrow B cells. The T1 CDR-H3 repertoire shared extensive similarity of length and loop amino acid hydrophobicity to that of IM CDR-H3s, whereas FO CDR-H3s most resembled that of MA B cells. MZ CDR-H3s averaged one half-codon shorter than IM and T1 CDR-H3s, and a full codon shorter than FO and MA CDR-H3s (p<0.05 and p<0.005, respectively). 13% (20/151) of MZ CDR-H3s contained 8 or fewer amino acids, versus only 4% (9/254) MB CDR-H3s [p<0.001]. MZ CDR-H3s were also enriched for highly charged CDR-H3s, representing 5.3% (8/151) of the transcripts versu only 0.4% (1/254)among MB CDR-H3s. Unlike the splenic F0 and bone marrow MA CDR-H3 repertoires, the MZ appears to be a reservoir for short and highly charged CDR-H3s amino acid motifs, including those with two or more arginines. The latter in particular has been associated with self-reactive specificities. Our findings suggest that specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in central as well as peripheral development.

Supported by HD043327, AI42732, and AI48115.
Ag-engaged B cells express CD83, a sensitive marker for B cell activation (83.15)
Authors: Prazma Charlene M. , Tedder Thomas F.
Published: January 2023

CD83 is predominantly known as a surface marker that defines mature DCs. The generation of CD83−/− mice further demonstrates that thymic epithelial expression of CD83 plays a significant role in the efficient generation of CD4 single positive thymocytes. The altered phenotype and reduced number of peripheral CD4+ T cells in CD83−/− mice further suggests a role for CD83 in the peripheral lymphocyte function. With regards to the surface expression pattern of CD83, CD83 was detected in the thymic medullary compartment and was specifically detected on thymic DCs and B cells in the mouse. Peripheral CD83 expression is limited to low level expression by resting follicular B cells but is quickly upregulated and transiently expressed on nearly 90% of spleen B220+ cells within 4 h of in vivo stimulation. Additionally, the majority (> 70%) of adoptively transferred B cells, expressing a BCR specific to hen egg lysozyme (HEL), expressed CD83 within 4 h of administration of a low dose of soluble HEL. At this timepoint, and low dose of antigen, less than 20% of B220+ splenocytes expressed CD69. Thus, the restricted expression pattern of CD83 and its near immediate upregulation on B cells in response to various stimuli suggest that CD83 is a sensitive, early activation marker of B cells.
Complement C2 and CRIT are involved in the terminal differentiation of monocytes (83.17)
Authors: Inal Jameel Malhador , Eva Wintergerst , Lange Sigrun , Shamshiev Adijapar , Schifferli Juerg
Published: January 2023

The protein expression of the C2/FB binding complement regulatory receptor CRIT was monitored in myeloid cell differentiation using primary monocytes or pluropotentiated promonocytes. CRIT expression was markedly reduced in HL60 cells differentiated with retinoic acid or DMSO into neutrophils but was maintained on differentiation using vitamin C3 or phorbol esters into monocytes. Macrophages derived from primary monocytes have downregulated CRIT unlike monocyte-derived dendritic cells which retain expression levels. Coimunoprecipitation experiments show that CRIT associates with c-fes, a cytoplasmic tyrosine kinase involved in the terminal differentiation of monocytes and neutrophils, in THP-1 cells (but not Jurkat cells which lack c-fes). Blocking the binding of C2/FB to CRIT on THP-1 cells either by adding excess of CRIT-ed1 peptide or anti-C2 polyclonal antibody significantly limits growth and increases nitroblue tetrazolium reducing activity from 4 to 8 (A650/107 cells). With primary monocytes (but not with monocytes from a C2 deficient patient) ed1 blockage of C2/FB induced terminal differentiation to macrophages as observed by the increase in % CD11b+ cells from 25% to 40%, increase in % CD14+ cells from 32% to 40% and reduction in the relative mean fluorescence index (CD14+) from 108 to 80. Throughout, interferon-γ was used as a positive control for induction of monocyte to macrophage differentiation.