bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoimmunity developed in Hoxb8 knock-out mice (P4082)
Authors: Chen Shau Kwaun , He Xiao , Hu Dai , Jensen Peter , Capecchi Mario
Published: January 2023

Hoxb8 belongs to the homeobox transcription factor family. Hoxb8 knockout (KO) mice exhibit a compulsive grooming, similar to trichotillomania (an impulse control disease) in humans. Hoxb8 is expressed in multiple hematopoietic cells. Bone marrow (BM) cells from Hoxb8 KO are less competitive than wildtype (wt) BM cells in granulocyte/monocyte and T cell lineages, yet more competitive in B cell lineage. How these changes affect development and functions of lymphocytes remain unknown. Here we report that autoimmunity was developed in Hoxb8 KO. Hoxb8 KO sera contain autoantibody against several self-tissues, including brain, kidney and liver. Splenomegaly was found with a higher frequency in KO mice, associating with increased B and T cell numbers. The percentages of surface IgG+ B and activated T cells were significantly increased as well. Moreover, serum IgG levels showed an age-dependent increase: there were trends of increased IgG levels in 3 and 6 month old KO, compared to aged matched wt mice, but all IgG isotypes, except IgG3, were significantly increased in the 9-month-old KO mice. Finally, we showed that autoimmunity developed in Hoxb8 KO mice may not be the major cause of the excessive grooming behavior since compulsive grooming remains in Hoxb8/RAG2 double KO mice. This observation suggests that defects of Hoxb8 in the immune system may play a supportive but not a major role in the pathogenesis of compulsive behavior, which agrees with our microglia hypothesis.
Identifying modifier genes in Autoimmune Lymphoproliferative Syndrome (P4084)
Authors: Lo Bernice , Schäffer Alejandro , Paik Daniel , Davis Joie , Price Susan , Rao Koneti , Lenardo Michael
Published: January 2023

Autoimmune Lymphoproliferative Syndrome (ALPS) is an inherited disorder of defective lymphocyte apoptosis, characterized by chronic lymphadenopathy and/or splenomegaly, autoimmune cytopenias, and the expansion of a unique population of CD4-CD8- α/β T cells. ALPS is predominately caused by mutations in Fas with incomplete penetrance. In order to identify modifier genes and understand the mechanism of incomplete penetrance of disease in ALPS, we performed a linkage study using five large families with multiple ALPS patients and healthy Fas mutation-positive relatives (HMPRs) in each family. We identified a putative modifier locus shared by two of the families and a few potential linkage intervals shared by two of the other families. To screen large numbers of family members for an apoptosis defect, we developed a fast assay for testing lymphocyte Fas-mediated apoptosis susceptibility ex vivo, which we have named the FasT Kill assay. The conventional in vitro apoptosis assay takes over a week to obtain results whereas our assay using multi-parameter flow cytometry to specifically analyze apoptosis of the Fas-sensitive effector memory T cells in PBMCs can yield a diagnosis within a single workday. Using the FasT Kill assay, we could rapidly identify patients with apoptosis defects for the diagnosis of ALPS and compare the severity of apoptosis defects among ALPS patients or HMPRs. The results of these genetic analyses will be discussed.
Sex-specific effects of Sca-1/Ly-6A on development of B lymphocyte in the bone marrow (P4384)
Authors: Bamezai Anil , Vacharathit Vimvara , DeWolf Sean
Published: January 2023

Stem cell antigen-1 (Sca-1, also known as Ly-6A) is highly expressed on the hematopoietic stem cells (HSC) and on early lymphoid progenitors, but the role this cell surface protein plays in the development of B cells in the bone marrow is unknown. We report here that the absence of Sca-1/ Ly-6A protein in mice regulates B cell development in the bone marrow in a sex-specific manner. Significant impairment of B cell development was observed in female mice lacking the Sca-1/Ly-6A protein, while this impairment was not observed in Sca-1/Ly-6A deficient males. In contrast, higher percentages of NK1.1+ cells were observed in the thymus from Sca-1/Ly-6A deficient females, while the male mice lacking Sca-1/Ly-6A did not show any alteration when compared to the wild type controls. The deficiency of Sca-1/Ly-6A did not impair development of T cells in the thymus of mice of either sex. These findings indicate that Sca-1/Ly-6A plays an important role in B cell and NK1.1+ cell development and suggests a functional interaction between Sca-1/Ly-6A and female sex hormones in regulating development of B cells and NK1.1+ cells.
Central B-cell tolerance mediated by Aicda and Myd88 (P4404)
Authors: Kuraoka Masayuki , Snowden Pilar , Nojima Takuya , Liao Dongmei , Zhang Wenli , Kelsoe Garnett
Published: January 2023

Elimination of autoreactive B cells and establishment of self-tolerance is crucial for the prevention of autoimmunity, nonetheless, the mechanism(s) of B-cell tolerance are not fully understood. Recently, we and others have reported that activation-induced cytidine deaminase (AID) and myeloid differentiation primary response gene 88 (MyD88) play unexpected roles in establishing central B-cell tolerance in mice and humans. AID expression in bone marrow immature B cells is synergistically elevated by anti-IgM F(ab’)2 and oligo CpG DNA co-stimulation; reaches to near germinal center levels in 24 hours, but the synergistic effect was not observed by anti-IgM and lipopolysaccharide co-stimulation. These results imply that co-ordination between BCR- and intracellular Toll-like receptors/MyD88 signals regulate AID expression in immature B cells. This synergy was not seen in mature follicular B cells. To explore molecular pathway(s) downstream of AID in B-cell tolerance, we analyzed B-cell development in uracil-DNA glycosylase (UNG)-deficient mice. Unlike AID- or MyD88-deficient background, UNG-deficiency did not impair central B-cell tolerance; because UNG-deficiency severely impairs class-switch recombination (CSR) but not somatic hypermutation, our results imply CSR, e.g., “locus suicide recombination”, may not play a major role in central B-cell tolerance.
Bcr-Abl expression overcomes aging-associated functional declines in B-Lymphopoiesis leading to increased leukemogenesis (P4406)
Authors: Henry Curtis , Marusyk Andriy , Zaberezhnyy Vadym , Casas Matias , Serkova Natalie , Clambey Eric , DeGregori James
Published: January 2023

Current paradigms attribute the association between aging and cancers to the accumulation of oncogenic mutations. Alternatively, we hypothesize that aging-associated reductions in the fitness of stem and progenitor cell populations should be conducive for increased selection of adaptive oncogenic mutations which can partially restore fitness, thereby promoting the initiation of cancers. We have explored how aging affects B-cell progenitor fitness and subsequently leukemogenesis. Here we show that IL-7 receptor (IL-7R) signaling is defective in aged B-cell progenitors leading to reduced phospho-STAT5 and Akt levels. Aged B-cell progenitors also exhibit altered expression of IL-7R regulated genes, including reduced Pax5, Myc and HPRT, and increased TNF-α. Both decreasing IL-7R signaling and increasing inflammation phenocopied aging-associated B-lymphopoiesis defects in young mice. Decreasing IL-7R signaling also promoted selection for Bcr-Abl expressing B-cell progenitors coinciding with increased leukemogenesis in vivo. Importantly, Bcr-Abl restored defective IL-7R signaling and inflammation-induced defects in these cells. These studies elucidate how reduced IL-7R signaling and increased inflammation promote aging-associated reductions in B-lymphopoiesis. Furthermore, we show that the selection for cells harboring oncogenic mutations (such as Bcr-Abl) is increased in aged backgrounds due in part to their ability to correct aging-associated fitness defects.
mTORC2 modulates αβT-cell receptor processing and surface expression during thymocyte development (P4414)
Authors: Chen Po-Hao , Chou Po-Chien , Oh Won Jun , Wu Chang-Chih , Hall Michael , Jacinto Estela , Werlen Guy
Published: January 2023

An efficient immune response relies on the presence of T-cells expressing a functional T-cell receptor (TCR). While the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. Here we found that mTOR complex 2 (mTORC2) modulates T-cell ontogeny by controlling the quantity of TCR that reaches the cell surface. Deletion of the mTORC2 component rictor at early stages of T-cell development led to aberrant maturation and increased proteasomal degradation of nascent TCR polypeptides. Consequently, the levels of TCR as well as other receptors, including CD4, CD8 and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Since CD147 was also defectively glycosylated and expressed in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/post-translational processing of membrane receptors. Thus, this novel function for mTORC2 provides a rationale to target this complex in disorders caused by deregulated expression of surface receptors.
The role of the Bcl-2 BH3 domain in thymocyte apoptosis (P4416)
Authors: Burger Megan , Kang Chulho , Winoto Astar
Published: January 2023

Over-expression of Bcl-2 has been shown to protect thymocytes from apoptosis by many stimuli except strong T cell receptor signals during negative selection. This might be due to Bcl-2 taking on a pro-apoptotic function during negative selection mediated through its BH3 domain, which is exposed upon interaction with the nuclear orphan receptor Nur77. To test this, we generated a T cell-specific transgenic mouse over-expressing Bcl-2 with a mutated BH3 domain. Consistent with our hypothesis, we observed that thymocytes expressing the Bcl-2 BH3 transgene were better protected from apoptosis by TCR stimuli in vitro than those expressing a wild-type transgene. To evaluate TCR-induced apoptosis in vivo, the Bcl-2 BH3 mutant transgenic mice were crossed to F5 and HY TCR transgenic mice. Vibratome cut thymic slices were treated acutely with F5 and HY TCR-specific peptides, circumventing the peripheral cytotoxic effects of in vivo peptide injection. Bcl-2 BH3 mutant transgenic thymocytes were almost completely rescued from peptide-induced apoptosis, but surprisingly, thymocytes expressing the wild-type Bcl-2 transgene were equivalently protected. Thus our results indicate that Bcl-2 has some pro-apoptotic function, but in contrast to previously published data in TCR-transgenic models, we conclude that Bcl-2 over-expression affords substantial protection from apoptosis accompanying negative selection.
A subset of Vγ4+ γδ T cells are derived from the adult murine thymus (P4462)
Authors: Colpitts Sara , Sheridan Brian , Pham Quynh-Mai , Lefrançois Leo
Published: January 2023

γδ T cells are a phenotypically diverse subset of immune cells with equally diverse functions in innate and adaptive immunity. We have identified a population of Vγ4+ γδ T cells that play an important role in protective immunity against oral Listeria infection. To further examine these cells, we generated mixed chimeras using a combination of neonatal thymocytes (neoThy) and adult bone marrow (BM). Surprisingly, the Vγ4+ population (identified as lacking expression of all other known γδ TCRs) was derived from both neoThy and BM progenitors demonstrating that the development of Vγ4 cells is not strictly limited to early ontogeny. Indeed, Vγ4 cells were found in the adult murine thymus, and, similar to other subsets of BM-derived γδ T cells, were the progeny of c-kit+ early thymic progenitors and required an adult thymus for their development. However, unlike the neoThy-derived subset, the BM-derived Vγ4 cells had a CD44intCD27+CD25- phenotype and did not produce IL-17 in naïve mice. However, when challenged with oral Listeria, there was a significant increase in BM-derived Vγ4 cells producing both IL-17 and IFNγ, but the overall contribution of this population to the anti-Listeria response was not significant compared to that of the neoThy-derived cells. Thus, we have discovered a unique population of Vγ4-expressing γδ T cells that emerge in adulthood.
A multipotent mesenchymal stem cell-like population in the postnatal thymus (P4466)
Authors: Pezzano Mark , Osada Masako , Singh Varan , Trozky Yana , Sant’Angelo Derek
Published: January 2023

Thymic microenvironments are required for the proper development of T cells. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, we utilize a keratin 5 promoter-driven, Tet-regulated H2BGFP transgenic model to localize label retaining epithelial cells (LRCs) in the postnatal thymus. A subset of LRCs was then used to generate clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts supporting a mesenchymal stem cell-like phenotype. In culture, these cells maintained a diverse gene expression profile including expression of the stem cell markers Nanog, Oct4 and Sox2, genes associated with the maintenance of pluripotency, as well as genes associated with thymic epithelial cells including EpCAM, FoxN1, MHCII and Wnt4. Furthermore, the cells were found to express Sca1, PDGFRα, PDGFGRβ, CD29, CD44, CD49f, CD90 and CD106, similar to mesenchymal stem cells. Sorted GFP-expressing stroma that give rise to TMSCs contribute to thymic epithelial architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggest they may contribute to the maintenance of functional thymic microenvironments.
Ikaros regulates basophil and mast cell development and function (P4476)
Authors: Smuda Craig , Rao Kavitha , Brown Melissa
Published: January 2023

Mast cells and basophils are phenotypically similar but functionally distinct myeloid cells, originating from both common and distinct progenitors. Several transcription factors have been identified as important to development of these cells, but regulation of this process remains poorly understood. We have found Ikaros, a diversely spliced DNA-binding protein, to be critical to lineage selection in these cells. Ikaros-/- mice exhibit basophilia in peripheral blood and hematopoietic tissues and expansion of basophil progenitors. Mast cells and their precursors are absent in gut, though there are normal numbers in skin. This phenotype is reflected ex vivo in IL-3 driven bone marrow cultures: in Ikaros-/- cultures, basophils expand substantially and persist longer than in wild-type cultures, while mast cells exhibit an abnormal molecular and functional phenotype, losing expression of mRNA encoding adhesion molecules while inappropriately expressing basophil-associated transcripts and altering surface coactivation molecule expression. Ikaros acts in part by altering histone methylation at the Cebpa and Hes1 loci, genes associated with differential mast cell/basophil development. Restriction of Cebpa expression directly by Ikaros and by Hes1 inhibits basophil development. Ikaros thus restrains basophil development while promoting mast cell differentiation, and tuning of its activity may allow for selectivity between these lineages.
Type I IFN drives emergency myelopoiesis during chronic and acute TLR7 signaling and acute viral infection (P4478)
Authors: Hamerman Jessica , Buechler Matthew , Campbell Daniel , Teal Thomas , Elkon Keith
Published: January 2023

Mice overexpressing TLR7 (TLR7.1 mice) are a model of systemic lupus erythematosus pathogenesis and exhibit peripheral myeloid expansion. We show that TLR7.1 mice have a dramatic expansion of splenic cells that derive from granulocyte/macrophage progenitors (GMP) compared with wild-type mice. In the bone marrow, TLR7.1 mice exhibited hallmarks of emergency myelopoiesis and contained a discrete population of Sca-1+ GMP, termed emergency GMP (eGMP). The emergency myelopoiesis and peripheral myeloid expansion in TLR7.1 mice was dependent on type I IFN signaling. eGMP were found in a type I IFN-dependent manner during both acute and chronic TLR7 agonist administration and during acute LCMV infection. Using in vitro colony forming unit assays, we found that eGMP are superior myeloid precursors than GMP. In vivo, eGMP turnover more than GMP, suggesting that the superiority of eGMP to generate myeloid cells in vitro may stem from their increased ability to proliferate. This study shows that type I IFN can act upon myeloid progenitors to promote the development of emergency GMP, which leads to an expansion of their progeny in the periphery. Further studies are underway to understand the molecular mechanisms that differentiate eGMP from GMP and other myeloid progenitor cells.
Dendritic cell differentiation mediated by IRF4 and IRF8 requires PKCβII (P4480)
Authors: Spangler Haley , Farren Matt , Carlson Louise , Abrams Scott , Lee Kelvin
Published: January 2023

To better understand how Dendritic Cell (DC) subsets are differentiating, we investigated two essential arms of DC differentiation and how they interact to mediate this process. The transcription factors Interferon Regulatory Factor 4 and 8 (IRF4 and IRF8) are critical for DC differentiation, but have varying roles depending on the stimuli. While IRF8 is required for FLT3-L induced plasmacytoid DC differentiation, and IRF4 is required for GM-CSF induced conventional DC differentiation, it is not well characterized how these two molecules are differentially regulated. We found that Protein Kinase C βII (PKCβII) is activated by both FLT3-L and GM-CSF, and that it is required for DC differentiation when either of these stimuli is used to generate DCs from progenitor cells. Since PKCβII and these IRFs are contributing to DC differentiation we hypothesized they were interacting. We discovered that PKCβII upregulates IRF8 when activated by FLT3-L or phorbol ester (PMA, a known activator of PKCβII), and IRF8 upregulation is lost with the addition of a PKCβII inhibitor. Likewise, IRF4 upregulation is induced by PKCβII in cells treated with GM-CSF or PMA, and lost in the presence of a PKCβII inhibitor. We have also found that in IRF8 knockout DCs there are higer levels of PKCβII, suggesting a feedback mechanism by which these IRFs can be autoregulated. Thus, PKCβII regulating these IRFs may be the key to understanding and manipulating DC differentiation.
A type I IFN/Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors (P4484)
Authors: Lee Chien-Kuo , Chen Yi-Ling , Chen Ting-Ting
Published: January 2023

During infections and inflammation, plasmacytoid dendritic cells (pDCs) are the most potent type I interferon (IFN-I) producing cells. However, the developmental origin of pDCs and the signals dictating pDC generation remain incompletely understood. Here, we report a synergistic role for IFN-I and Flt3 ligand (FL) in pDC development from common lymphoid progenitors (CLPs). Both conventional DCs and pDCs were generated from CLPs in response to FL, while pDC generation required higher concentrations of FL and concurrent IFN-I signaling. An absence of IFN-I receptor, impairment of IFN-I signaling, or neutralization of IFN-I significantly impeded pDC development from CLPs. Furthermore, FL induced IFN-I in CLPs, resulting in Flt3 upregulation that promotes survival and proliferation of CLPs, as well as their differentiation into pDCs. Collectively, these results define a critical role for the FL/IFN-I/Flt3 axis in pDC differentiation from CLPs and identify a potential target for therapeutic intervention in inflammatory and autoimmune diseases associated with pDCs.
Unique role of Cbfβ2 variant in Langerhans cell development (P4492)
Authors: Tenno Mari , Naoe Yoshinori , Muroi Sawako , Taniuchi Ichiro
Published: January 2023

In mammals, Cbfβ protein, known as an essential subunit of Runx transcription factor complexes, are present as two RNA splicing variants, Cbfβ1 and Cbfβ2. To address in vivo roles of individual variant, we have generated mutant mouse strain lacking either Cbfβ1 or Cbfβ2. Here we show that a loss of Cbfβ2 variant results in a defect in Langerhans cell development. In the ear epidermis of adult Cbfβ2-deficient mice, no mature Langerhans cells expressing Langerin and EpCAM were detected. Instead, there were a few larger cells with characteristics of immature Langerhans precursors, such as the dendritic morphology and the expression of MHC class II and CX3CR1. Analysis of bone marrow chimera showed that these Langerin−EpCAM−classII+ cells were radio-resistant residential cells that could not be replaced by donor cells. Analyses of ontogeny of Langerhans cell development around neonatal stage revealed that seeding of CD45+ early progenitor cell to the skin occurred normally in Cbfβ2-deficient mice. However, the next transition into Langerin+EpCAM+ stage, which is know to require for TGFβ signals around day 3 after birth, was severely impaired in the absence of Cbfβ2. Interestingly, although immature Langerhans precursors disappear in adult epidermis by lack of TGFβR1 signals, our results indicate that those cells are stably maintained in the Cbfβ2-deficient mice, suggesting an unique role of Runx3/Cbfβ2 complexes in regulating Langerhans cell development.
TGFβ-signaling in T cells suppresses Tbet mediated CD4+ anti-tumor immune responses in a murine model of lung carcinoma (P2021)
Authors: Andreev Katerina , Soó-Becker Kinga , Engelhardt Marina , Koslowski Michael , Finotto Susetta
Published: January 2023

TGFβ is an important immunosuppression inducing factor in the lung tumor microenvironment and is suggested to facilitate the escape of tumor cells from immunosurveillance. In this project we investigate TGFβ-mediated effects on tumor infiltrating CD4+ T cells, also addressing the question of how TGFβ-induced T cell subtypes, such as Treg and Th17 cells, contribute to tumor cell tolerance. We found an upregulation of TGFβ in patients affected by lung carcinoma. In a murine model of lung carcinoma we also observed a significant reduction of tumor growth as well as prolonged survival of CD2TβRIIdN mice, which exhibit a defective TGFβ-signaling in T cells. This was accompanied by increased numbers of tumor infiltrating effector T cells, reduced induction of Tregs in the tumor tissue and enhanced Th1 cell responses. Similarly, reduced tumor load was observed after intranasal application of anti-TGFβ1 antibodies to tumor bearing wild-type mice. By contrast, Tbet -/- mice showed increased tumor growth. To further analyze the opposing effects of Tbet and TGFβ on CD4+ anti-tumor responses, we crossed CD2TβRIIdN and Tbet -/- mice and found that this newly generated mouse strain was not protected from tumor development. Moreover the results suggest a relation between tumor growth and Th17 cell responses in this context. Together, our data reinforce the importance of Tbet for anti-tumor immune responses and outline the manifold role of TGFβ for lung cancer.
CD28:a pro-survival signaling molecule in multiple myeloma as a novel therapeutic target (P2029)
Authors: Murray Megan , Nair Jayakumar , Lee Kelvin
Published: January 2023

Multiple myeloma (MM) survival is dependent on the bone marrow microenvironment and is mediated by the receptor CD28, expressed on MM cells. CD28 expression correlates clinically with poor outcomes, and we have shown that CD28 signaling protects MM from chemotherapy. However, the molecular pathway by which CD28 does this is not clear. Since CD28 signals via a PI3K-Akt-FoxO3a axis in T cells, we interrogated whether this pathway is critical in MM. We observed that blockade of PI3K or Akt abrogates CD28-mediated survival. Upon CD28 activation, transcription factor FoxO3a is phosphorylated and excluded from the nucleus. Being downstream of FoxO3a, we examined whether the pro-apoptotic molecule Bim is regulated by CD28. Indeed, CD28 activation downregulates Bim, blockade upregulates it, and Bim silencing abrogates sensitivity to CD28 blockade. To confirm that stromal cells protect MM, we cocultured MM and dendritic cells and observed increased survival. However, if CD28 or CD80/86 is blocked, protection is completely abrogated. To examine if this is relevant in vivo, we used the murine myeloma model Vk*MYC and a SCID/human model. In either case, treatment with CTLA4-Ig (to block CD80/86) in combination with a sub-therapeutic chemotherapy dose was able to significantly decrease tumor burden and extend survival. Taken together, these data suggest that CD28 is a direct mediator of chemotherapy resistance in vivo, and blockade of CD28 represents a novel target for clinical therapy.
Inhibition of the Heat Shock Protein receptor CD91 abrogates concomitant anti-tumor immunity (P2035)
Authors: Zhou Yu , Binder Robert
Published: January 2023

Immunosurveillance mechanisms of tumors depend on the host’s ability to efficiently cross-present tumor antigens. We provide evidence here that heat shock proteins (HSPs), when released into the tumor microenvironment, is the antigen carrier necessary for priming concomitant tumor specific immunity. These responses require CD91- the endocytic and signaling receptor for the immunogenic HSPs. In our study, we generated CD11c-specific CD91 knockout mice and showed that tumors grew faster in those mice, compared to CD91+/+ mice. In addition, we abrogated the interaction of tumor-derived HSP with CD91 in vivo by over-expression of Receptor Associated Protein (RAP), an endogenous CD91 antagonist. The data showed that: first, tumors expressing RAP grew with significantly faster kinetics than the non-RAP expressing counterparts in wild type mice, although this difference was non-existent in immunocompromised mice; second, RAP-expressing tumors, when used as an immunogen in tumor prophylaxis, were significantly less efficient in priming immune responses; third, in the presence of RAP, less amount of tumor-derived EGFP-labeled HSPs were taken up by APCs in draining lymph nodes. In summary, our study demonstrates that the HSP-CD91 pathway is critical for establishment of concomitant immunity. Considering that elevated RAP levels have been reported in cancer patients, we propose a novel mechanism of immune evasion for tumors expressing competing ligands for immune receptors such as CD91.
Tumor microenvironment induces selective migration of specific subsets of natural killer cells and inhibits their cytolytic function. (P2039)
Authors: Lal Girdhari , Paul Sourav , Singh Amit
Published: January 2023

Cytolytic function of NK cells plays an important role to regulate the cancer growth. We hypothesized that inflamed tumor microenvironment regulates the migration of specific subset of NK cells and inhibits their cytolytic function. To investigate the affect of tumor microenvironment on NK cell migration and function, B16F10 melanoma cells (1 X 106 cells/mouse) were injected (s.c.) into C57BL/6 mice. Depletion of NK cells showed 3-4 fold increased tumor growth compared to control IgG treated mice. Tumor transplanted (day 10) and naïve mice did not showed any significant difference in percentage and absolute number of CD11b+CD27+, CD11b+CD27- or CD11b-CD27+ NK cells in spleen and lymph nodes. However, CD11b+CD27+CD3-NK1.1+ cells were selectively recruited into the tumor. At day 5, increase CD31+ endothelial cell staining were observed. qRT-PCR analysis of tumor associated endothelial cells (CD31+CD45- cells) showed increased CCL2, CCL21, CXCL10, and CXCL12 mRNA expression compared to splenic blood endothelial cells. Tumor infiltrating NK cells (day 13) showed increased expression of CCR2, CXCR4, IL-6, FasL mRNA expression, and decreased perforin, IFNg and IL-21 expression compared to splenic NK subsets. These results demonstrate that inflamed tumor microenvironment selectively recruits a specific subset of NK cell and inhibit their cytolytic function. This suggests that modulating tumor microenvironment may be key to boost the NK cell function to control cancer progression.
Endotoxin tolerance in circulating monocytes of Chronic Lymphocytic Leukemia: a cross-tolerance mechanism (P2061)
Authors: Lopez-Collazo Eduardo , Jurado Teresa , Siliceo María , Cordoba Raul , Hernandez-Jimenez Enrique , Toledano Victor
Published: January 2023

Monocyte exposure to tumor cells induces a transient state in these cells rendering them refractory to further cancer exposure. This phenomenon, termed "tumor tolerance", is characterized by a reduction of pro-inflammatory cytokines production in response to tumors. In addition, exposure to consecutive endotoxin stimuli induces a similar refractory state called “endotoxin tolerance”. In the past, we found that these two phenomenons comprise IRAK-M up regulation and a marked down-regulation of MHCII molecules as well as of the MHCII master regulator, CIITA. These cells combine an impaired capability for antigen presentation with potent phagocytic activity and exhibit an M2-like phenotype. Herein we have demonstrated that after a first exposition to MEC1 (a CLL cell line), human monocytes failed to mount an inflammatory response against bacterial endotoxin, thus they showed a cross tolerance in vitro. We also tested the implication of miRNAs in this process. We confirmed our data in circulating cells isolated from Chronic Lymphocytic Leukemia suggest an explanation for the high frequency of infections in these kinds of patients.
Mast cell derived histamine promotes the activity of monocytic myeloid derived suppressor cells (P2065)
Authors: Saleem Sheinei , Martin Rebecca , Bear Harry , Conrad Daniel
Published: January 2023

Until recently, the interaction between mast cells (MCs) and CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) was limited to recruitment of MDSCs to the tumor site. However, we report that MCs are also needed for the activity of MDSCs. MDSCs failed to promote B16 melanoma metastasis in MC deficient mice. This was dependent on monocytic Ly6C+ MDSCs while granulocytic Ly6G+ had no effect. In vitro studies showed increased Th2 cytokine production by both cells. Given these observations and literature indicating histamine as a promoter of tumor expansion, myeloid cell proliferation, and Th2 skewed immunity; we investigated histamine in the MC/MDSC interaction. MDSCs were shown to express histamine receptor 1 (HR1). Furthermore, histamine enhanced MDSC survival and preferentially expanded monocytic LY6C+ MDSCs. This was abrogated with HR1 antagonization. Additionally, histamine upregulated Arg1 and iNOS enzyme expression, both of which are utilized by MDSCs to inhibit T cell activity. This was further supported by the finding that in the presence of MCs, MDSCs were more effective at inhibiting T cell proliferation. Taken together, our data indicates that beyond recruitment, MCs regulate MDSC activity at the tumor site via histamine release. These findings have important implications for the use of anti-histamines in cancer patients being simultaneously treated for allergies. Oncologists should consider this new MC/MDSC interaction when determining therapeutic interventions.
Opposing effects of myeloid vs. lymphoid deletion of adenosine A2A receptors on anti-tumor immune responses (P2007)
Authors: Cekic Caglar , Linden Joel
Published: January 2023

Extracellular adenosine is generated as part of immune escape programming orchestrated by tumor cells and regulatory cells. Adenosine A2A receptors (A2AR) on innate and adaptive immune cells are high affinity targets for adenosine-mediated immune suppression. In this study we demonstrate that myeloid-deletion of adenosine A2A receptor gene (adora2a) strongly inhibit tumor growth and metastasis. This was associated with increased APC function of macrophages and increased the numbers and activation of cytotoxic lymphocytes. Surprisingly, lymphoid deletion of adora2a markedly increased tumor growth and decreased the numbers of effector/memory T cells in the tumor. Mechanistic studies revealed that adora2a deletion in T cells severely impacts generation of the long/lived memory/effector T cell population within the tumor consistent with recent studies indicating that A2AR activation reduces activation-induced cell death. The results of this study have important implications for the development cell-based therapies targeting adenosine receptors.
Systemic lymphopenia results in loss of suppressive lymphocytes from precancerous skin (P2081)
Authors: Leggatt Graham , Mattarollo Stephen , Yong Michelle , Nissen Michael , Frazer Ian
Published: January 2023

We have previously demonstrated an immunosuppressive role for NKT cells that infiltrate hyperplastic skin epithelium in transgenic mice expressing HPV16E7 oncoprotein under a keratin 14 promoter (K14E7 mice)(Mattarollo et al.(2010) J. Immunol. 184:1242-1250). However, depletion of NKT cells is not easily achieved in either mouse or human. We hypothesized that systemic depletion of lymphocytes might result in turnover of NKT cells (and other lymphocytes) within the precancerous skin without replacement from the circulation. Without these locally suppressive populations, the precancer might then be more susceptible to a transferred immune system. When K14E7 skin was grafted onto RAG1-/- mice, which lack lymphocytes, the grafted skin became depleted of T cells (including NKT cells) within 7 days and remained depleted. Depletion of skin lymphocytes could also be achieved using cyclophosphamide and fludarabine, although with less efficiency. Preliminary data suggests that K14E7 skin grafts on RAG1-/- mice become susceptible to graft rejection after transfer of a new immune system. Together the data suggest that systemic lymphodepletion results in a loss of local, suppressive lymphocytes at the precancerous site and that this might enhance the immune mediated clearance of skin precancers.
The role of paracrine IL-6 in plasma cell neoplasia development (P2083)
Authors: Rosean Timothy , Tompkins Van , Waldschmidt Thomas , Janz Siegfried
Published: January 2023

Plasma Cell Neoplasms (PCN), such as multiple myeloma, are cancers of terminally differentiated B cells, and are the second most common blood cancers overall. PCN remains incurable, and therefore research into the development of disease is increasingly necessary. IL-6, a pro-inflammatory cytokine, has emerged as a key driver in PCN development, in part through its collaboration with the proto-oncogene c-Myc. Mice containing a gene insertion of c-Myc into the IgH locus (iMyc) and overexpressing human IL-6 (hIL-6) develop PCN at a significantly increased rate and incidence. While IL-6 is important for PCN development, it is unknown which source of IL-6, pre-malignant cells or the microenvironment, is more important. To examine this question, we developed an adoptive transfer system to investigate tumor development. Specifically, when premalignant B cells from iMyc mice are transferred into syngeneic recipients, PCN develops around 120 days post-transfer. To test whether the source of IL-6 affects PCN development, we performed two experiments monitoring tumor development. First, iMyc or iMyc/IL-6-/- premalignant B cells were transferred into syngeneic recipients. There was no significant difference in tumor onset. Secondly, iMyc/hIL-6 premalignant B cells were transferred into BALB/c or C.IL-6-/- recipients. Tumor onset was significantly delayed in C.IL-6-/- recipients, demonstrating for the first time the importance of paracrine IL-6 in PCN development.
Liver natural killer T cells enhance, rather than inhibit, liver metastases arising from intraocular melanomas in mice. (P2087)
Authors: Sadegh Leila , Chen Peter , Niederkorn Jerry
Published: January 2023

Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have shown that NKT cell-deficient mice develop significantly less liver metastasis from intraocular melanomas than wild-type (WT) mice. Here we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d-/- mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. Increased metastases in WT mice was associated with reduced liver NK cytotoxicity. However, liver NK cytotoxic activity was identical in non-tumor-bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with increased IL-10 production by bone marrow-derived liver cells and increased IL-10 receptor expression on liver NK cells. IL-10-/- mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. Impaired liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases.
Inflammatory chemokine CCL3 suppresses preferential tumor-associated accumulation of B cells and CD4 T cells in tumor-draining lymph nodes (P2017)
Authors: Allen Frederick , Bobanga Iuliana , Barkauskas Deborah , Myers Jay , Tsao Matt , Huang Alex
Published: January 2023

We have previously demonstrated that CCL3 (MIP-1α) and CCL4 (MIP-1β) were important in orchestrating cellular contacts in vaccinated lymph nodes (LNs) and can enhance memory T cell generation (Castellino et al). We hypothesize that incorporating CCL3 and CCL4 into tumor vaccines can enhance anti-tumor immunity. Using colon tumor model CT26, we generated tumors that secret CCL3, CCL4 or both, and inoculate them live into the footpad of naïve recipients. CCL3-secreting tumors failed to grow in immunocompetent mice, and enhance antitumor immunity when used as a vaccine. Flow cytometric analysis of draining popliteal LNs 3 days after live cell injection revealed that while the total LN cell number increased by 2.03 fold after wild type (WT) tumor challenge relative to contralateral LN (conLN), CCL3-secreting tumor injection failed to do so despite the presence of tumor cells in the LNs. CD4 cells in the WT draining LN were 1.58 fold higher compared to conLN, but remained unchanged in CCL3-secreting tumors. Striking differences were seen in B cells where WT tumor resulted in a 7.04-fold increased, but CCL3-secreting tumors only caused a modest 2.04-fold change. Dynamic intravital 2-photon microscopy and tissue histology studies further revealed that tumors occupy the B cell follicle during early metastasis, and exogenous CCL3 gradient may potentially alter lymphocyte homeostasis in tumor draining LN, resulting in immune activation rather than tolerance against CT26.
Pan-Shigella Surface Protein antigen 1 as potential cross-protective antigen for Shigella vaccine development. (P4286)
Authors: Kim Jae-Ouk , Kim Su Hee , Rho Semi , Kim Heejoo , Dey Ayan , Yang Jae-Seung , Nandy Ranjan , Czerkinsky Cecil , Kim Dong Wook
Published: January 2023

Shigellosis is a disease characterized by the destruction of the colonic epithelium in humans and one of the most severe diarrheal diseases, especially in children under 5 years of age in developing countries. We have identified several protein antigens that are shared by different shigella serotypes and species. Among these, one antigen, PSSP-1, induces cross-protection against experimental shigellosis induced by different species and serotypes. PSSP-1-induced protection requires (i) a mucosal route of administration; and (ii) the co-administration of a mucosal adjuvant (cholera toxin, mutant thermolabile enterotoxin (dmLT). When PSSP-1was formulated with dmLT, an adjuvant that has undergone human clinical testing, it induced cross-protection against S. flexneri 2a, 3a, 5a, 6, S. boydii and importantly also S. dysenteriae 1. While intradermally administered PSSP-1 adjuvanted with dmLT induced strong serum antibody responses, it failed to induce protection in the mouse lung pneumonia model (consisting of administering Shigella intranasally). In contrast, intranasal PSSP-1 + dmLT elicited efficient local (lung) and peripheral (spleen) antibody responses and regional and systemic IL-17 and IFNγ production. Interestingly, patients with recent onset shigellosis showed variable but significant antibody responses to most conserved Shigella protein antigens but not to PSSP-1. Although the above results are promising, these will have to be validated in humans.
Fc receptor-targeted vaccines: an adjuvant-independent approach for mucosal vaccination against intracellular and extracellular mucosal pathogens (P4290)
Authors: Gosselin Edmund , Bitsaktsis Constantine , Iglesias Bibiana , Pham Giang , Drake James , Snapper Clifford , Franz Brian
Published: January 2023

We previously demonstrated that immunization with inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with anti-F. tularensis mAb, enhances protection against F. tularensis challenge. We thus sought to determine the mechanism(s) involved, and if this strategy is compatible with recombinant vaccine technology. We found that inactivated F. tularensis combined with F. tularensis-specific mAb extends immunogen presentation to T cells via FcγR-mediated Ag persistence, enhances transport of immunogen to the NALT, increases recruitment of DCs to the lung, and increases the number of CD4 and IFN-γ producing effector memory T cells. Utilizing an extracellular infectious disease model (S. pneumoniae), we also demonstrate this strategy can be applied as a recombinant fusion protein composed of the immunogen PspA targeted to human FcγRI (constitutively expressed on DCs and macrophages). Furthermore, traditional adjuvant is not required to enhance protection in either vaccine model. In summary, for the first time, we utilize an established mucosal vaccine protection model to identify mechanisms involved in FcR-enhanced mucosal immunity. We also demonstrate this strategy is effective against both intracellular and extracellular mucosal pathogens and is compatible with the use of recombinant vaccine technology. These studies thus establish the broad applicability of FcR-targeted immunogens as an effective adjuvant-independent mucosal vaccine platform.
Efficacy of a prime/boost regimen including BCG and recombinant protein in an HN878 challenge of C57BL/6 mice (P4296)
Authors: Schaeffer Jacqueline , Laddy Dominick , Wachholder Robert , Anantha Ravi , Imam Zakari , Morris Sheldon , Derrick Steven , Hokey David
Published: January 2023

Tuberculosis remains a major concern despite the widespread use of BCG vaccine. Since BCG-induced protection against adult pulmonary TB has been highly variable, vaccines that confer better, more complete protection are needed and recombinant proteins in the presence of adjuvant are currently being evaluated in this context. C57BL/6 mice were used in an immunogenicity and aerosol challenge study to evaluate the efficacy of a heterologous prime-boost vaccination regimen. Adult females aged 6-8 weeks old were vaccinated with either BCG SSI alone, or in conjunction with a recombinant fusion protein expressing Ag85B, Ag85A and Rv3407 with a poly(I:C)-based TLR3 adjuvant. Prior to challenge, a subset of mice were used to evaluate immune responses by IFN-γ ELISA and flow cytometry. This combination elicited a strong Th1 response as evidenced by high levels of IFN-γ, IL-2 and TNF expression in CD4+ T-cells from splenocytes. Mice were challenged with 50-100 CFU of aerosolized M.tuberculosis strain HN878. Spleens and lungs were used to determine viable bacterial load (CFU) and lungs fixed for routine histopathology. We observed a statistically significant reduction in lung CFU at 12 weeks post infection in the prime-boost vaccine group compared to BCG alone. The use of a recombinant protein plus adjuvant as a boost to the widely used BCG SSI vaccine was shown to be immunogenic and to elicit an increased level of protection over BCG alone in the lungs of adult C57BL/6 mice.
In vivo targeting of human DC-SIGN drastically enhances CD8+ T cell-mediated protective immunity (P4298)
Authors: Berod Luciana , Hesse Christina , Ginter Wiebke , van Kooyk Yvette , Sparwasser Tim
Published: January 2023

Vaccination is one of the oldest and still most effective methods to prevent infectious diseases. Yet, eradication of intracellular pathogens and other diseases like cancer, requiring strong cytotoxic (CTL) T cell responses, remains a medical challenge. In mice, targeting antigens to dendritic cells (DCs) via specific antibodies against C-type lectin receptors represents a successful approach to induce strong cytotoxic immune responses if used in combination with adjuvants. However, a major drawback to use this strategy in the clinic is the lack of analogous targets in human DCs. DC-SIGN (DC specific ICAM3 grabbing non integrin/CD209) is a C-type lectin receptor with strong endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC-SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express the human DC-SIGN receptor under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti-DC- SIGN antibodies to target antigens to DCs and to induce protective immune responses against intracellular infectious agents like Listeria monocytogenes or Mycobacterium tuberculosis in vivo. We show that anti-DC-SIGN conjugated antibodies can induce strong and persistent antigen-specific CD4+ and CD8+ T cell responses and propose DC-SIGN as a promising candidate for novel vaccine strategies based on DC targeting against intracellular pathogens.
The role of Th17+ T cells and neutrophils in mucosal immunity against Staphylococcus aureus mastitis. (P4256)
Authors: Kanevsky-Mullarky Isis , Lehtimäki Mari , Wark Wendy , Mwangi Waithaka
Published: January 2023

Staphylococcus aureus is a common cause of mastitis, a disease estimated to cost the dairy industry ~$2 billion/yr. Public concern over the use of antibiotics in agricultural animals necessitates the identification of new mechanisms for controlling this pathogen. Our goal is to define the role of immune activating DC in the development of protective S. aureus-specific T cell memory and use this knowledge for identification of antigens for vaccine development. Our findings indicate IL-17 generated by PBMC in response to S. aureus loaded monocyte-derived DC that is greater in memory as compared to naïve animals. Lymphocytes stimulated with both live and gamma irradiated antigens presented by monocyte-derived DC released active Granzyme B. Cells from previously infected animals had higher Granzyme B response than animals with no previous infection. In animals with no previous clinical infections, irradiated S. aureus antigen induced lower response than live S. aureus. Furthermore, bovine neutrophils generate greater levels of cytokines when stimulated with supernatants from S. aureus induced T cells. This data indicates that previous infection can heighten the ability of cytotoxic T cells to produce Granzyme B and increase neutrophil function. The ability to increase presence of protective memory immune cells and innate immune cell function in the mammary gland will provide new vaccine targets for S. aureus.