bluuebunny/crossref_metadata_2025 huggingface dataset:
Hapten application to the skin induces an inflammatory program directing hapten-primed effector CD8 T cell interaction with hapten-presenting endothelial cells (60.6)
Authors: Kish Danielle , Volokh Nina , Baldwin William , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Effector CD8 T cell recruitment into the skin parenchyma to elicit CHS to hapten challenge requires prior CXCL1-directed neutrophil infiltration within 3-6 h after challenge and is dependent on IFN-γ and IL-17 produced by the hapten-primed CD8 T cells. Mechanisms directing CD8 T cell localization and activation in the challenge site to induce CXCL1 production in response to 2, 4-dinitrofluorobenzene were investigated. Both TNFα and IL-17, but not IFN-γ, mRNA was detectable within 1 h of hapten challenge of sensitized mice and increased thereafter. Expression of ICAM-1 was observed by 1 h after challenge of sensitized and non-sensitized mice and was dependent on TNFα. The induction of IL-17, IFN-γ and CXCL1 in the challenge site was not observed when ICAM-1 was absent or neutralized by specific antibody. During the elicitation of CHS, endothelial cells (EC) expressed ICAM-1 and produced CXCL1. EC isolated from challenged skin of naïve and sensitized mice had acquired the hapten and the ability to activate hapten-primed CD8 T cell cytokine production. These results indicate that hapten application to the skin of sensitized animals initiates an inflammatory response promoting hapten-primed CD8 T cell localization to the challenge site through TNFα induced ICAM-1 expression and CD8 T cell activation to produce IFN-γ and IL-17 through EC presentation of hapten.
Migrating out of the zone: the role of P-selectin glycoprotein ligand-1 on T follicular helper cell movement (60.8)
Authors: Hernandez Sairy , Weinstein Jason , Steiner Laurie , Bertino Sarah , Poholek Amanda , Gallagher Patrick , Craft Joseph
Published: January 2023

Follicular helper T cells (TFH) are essential for the formation of germinal centers (GC) during a T-dependent immune response, providing critical factors that promote memory B cell and plasma cell development. Differentiation and function of TFH cells are dependent upon their movement out of the T cell zone and into the B cell follicle, a process regulated by modulation of chemokine receptor expression. Here we explore the role of the secondary lymphoid organ homing molecule, PSGL1, as a T zone retention signal, demonstrating that its downregulation on TFH cells is necessary for their migration out of the T cell zone and into the follicle. Upon immunization of PSGL1-intact and -deficient mice, we found that the latter had an increase in TFH cells and GC B cells in lymph nodes and spleen, and enlarged GCs with enhanced numbers of T cell infiltrates, compared to wild type controls. Following adoptive transfer, a higher frequency of PSGL1-deficient T cells localized to B cell follicles and GCs compared to controls, whereas retroviral over-expression of PSGL1, preventing its downregulation, led to exclusion of T cells from follicles and GCs. Consistent with these observations was the finding in chromatin immunoprecipitation assays that Bcl6, the lineage-specific transcription factor of TFH cells, bound the psgl1 gene. These data reveal that PSGL1 downregulation on TFH cells is required for their migration within lymphoid organs and function during a TD immune response.
Type I IFN-mediated reversal of TLR tolerance in antigen presenting cells (61.13)
Authors: Poovassery Jayakumar , Bishop Gail
Published: January 2023

Many synthetic ligands for TLRs, especially TLR 7/8 and 9 agonists, induce potent innate and adaptive immune responses and are thus under consideration as vaccine adjuvants and as anti-cancer agents. In addition, TLR7 and 9 agonists are potent type I IFN inducers and play critical roles in defense against viral infections. Many TLR-mediated responses in antigen presenting cells are also modulated by Type I IFN receptor signals. However, the precise molecular mechanisms involved in cooperation between these two receptors are not clear. We have previously shown that repeated stimulation through TLR7 can induce a state of hyporesponsiveness (TLR tolerance) in B cells characterized by severe defects in major signaling pathways. In this study, we investigated whether simultaneous signals through type I IFN receptor can reverse tolerance in B cells and dendritic cells. We found that signals through type I IFN receptor can prevent and reverse TLR tolerance in mouse and human B cells and in mouse bone marrow derived dendritic cells. The reversal of hyporesponsiveness was associated with activation of p38 and ERK1, 2 and c-Jun phosphorylation. Moreover, Type I IFNR-mediated reversal of tolerance was dependent on the PI3K/Akt/mTOR pathway. A better understanding of how these important signaling pathways interact can significantly increase our ability to develop better vaccine adjuvants and immunotherapeutic agents against cancer.
CD1d expression by dendritic cells and CD40L or ICOS expression by NKT cells is dispensable for NKT-enhanced humoral immunity (61.16)
Authors: Lang Mark , Shah Hemangi , Joshi Sunil , Kovats Susan , Lang Gillian , Devera T.
Published: January 2023

CD1d-binding glycolipids have potent adjuvant effects on T-dependent humoral immune responses to co-administered protein antigen. We reported previously that cognate interactions between CD1d expressed on B cells and the T cell antigen receptor of natural Killer T (NKT)was critical for the adjuvant effect of the CD1d ligand α-galactosyl ceramide (α-GC). We have therefore tested the hypothesis that CD1d-expressing dendritic cells contribute to NKT-enhanced antibody production and that NKT cells provide co-receptor-mediated B cell help. Using bone marrow chimera approaches, we demonstrate that CD1d-expression by dendritic cells as well as CD40L and ICOS expression by NKT cells are dispensable for the adjuvant effects of α-GC. This reinforces the concept that the primary mode of NKT activation is through CD1d-expressing B cells and indicates that NKT cells utilize mechanisms distinct from that of helper T cells to provide specific B cell help.
Antigen altered idiotype induces immune regulation implications for cancer, autoimmunity and AIDS (61.17)
Authors: Williams R Michael
Published: January 2023

Antigen altered idiotype regulates specific immune responses. Mendelian segregation of a given +/- phenotype defines an immune response gene (IR-X) . Autoimmunity producing anti-self clones must be eliminated or specifically suppressed, so a large portion of the antigenic repertoire resembles allogeneic MHC molecules. Singer and Williams (Cell Immunol.1978: 4:1) proposed that exposure to many allogeneic HLA molecules could eventually overlap with important responses to pathogens, causing serious holes in the repertoire, some large enough to produce an acquired immunodeficiency syndrome (AIDS). The changes resulting from exposure to multiple incompatible allogeneic MHC molecules could also produce the opposite effect, an increase in a desirable immune response, or the allogeneic effect . IR-genes for histocompatible tumor resistance localized to the mouse MHC (Cancer Res. 1975;35:1586) The patient with HIV and AML, who was rendered free of both by transplantation of the right allogeneic stem cells (Blood,2010,Dec 8) supports our case. We propose that HIV is a unique and potent inducer of altered idiotype immune suppression. Explanation of the mechanism for synthetic antigens by Benacerraf (Nobel, 1980) and for viruses by Zinkernagel and Doherty (Nobel, 1986) followed from the idiotype network concept of Jerne (Nobel, 1984). Our attempt to relate these observations to the MHC and genetically controlled immune responses led us to the altered idiotype hypothesis.
Splenic FasL+CD5+ B lymphocytes are distinct from peritoneal B-1a cells and expand in response to interleukin-5 (61.2)
Authors: Lundy Steven , Alzua Brian , Reed Tamra , Klinker Matthew
Published: January 2023

A murine splenic B cell subset expresses the death-inducing molecule Fas ligand (FasL) and induces apoptosis of CD4+ T lymphocytes. The current study sought to further characterize FasL+ B cells and identify factors that control their growth. By flow cytometry, gating on FasL+ cells in the spleens of naïve mice demonstrated that >70% of constitutively FasL+ splenocytes were CD5+/CD1dhigh B cells. In naïve mice, splenocytes with a surface phenotype of CD19+/B220+/CD5+/CD11bneg/IgMhigh/CD1dhigh showed a higher expression of FasL than any other splenic cell population. Gene array analysis comparing sorted naïve CD5+ and CD5neg splenic CD1dhigh B cells indicated some significant differences in gene expression patterns including a 3.4 fold higher expression of IL-5Rα mRNA in CD5+/CD1dhigh B cells. Addition of recombinant IL-5 to CD40L-stimulated splenic B cell cultures resulted in the selective survival and proliferation of FasL+/IgM+/CD5+ killer B cells. In Xid mice, which have severely reduced numbers of peritoneal CD5+ B cells, we found no deficiency in splenic killer B cells, suggesting that killer B cells are distinct from peritoneal CD5+ B-1a cells. Splenic CD5+ B cells from Xid mice showed higher levels of surface FasL expression than all other splenic cell populations. Together, these data demonstrate that splenic killer B cells have a distinct surface phenotype compared to other B cells, proliferate in response to IL-5, and are present in B-1a cell deficient mice.
Class switch recombination appears to be regulated by BCR/idiotype-specific T cell action (62.10)
Authors: Lemke Hilmar , Hecht Oliver , Zemlin Michael , Trad Ahmad , Tanasa Radu , Schroeder Harry
Published: January 2023

The T cell-dependent processes of class switch recombination (CSR) and immune maturation are believed to be driven by affinity-dependent clonal selection. However, analysis of the anti-hapten 2-phenyloxazolone response reveals that (i) thymus-dependent IgM are as heterogene-ous as natural and thymus-independent antibodies, (ii) many IgM exhibit similar or greater affinities than the classic Ox1 clonotype that (iii) dominates only after CSR, (iv) CSR selects B cell clones with shorter and more uniform CDR-H3 lengths and (v) primary IgM encoded by VH1 family genes were almost completely eliminated from the repertoire by CSR. Thus, contrary to the current paradigm, CSR appears to be clonally regulated more on the basis of BCR-specific, e.g. CDR-H3 idiotope-related, T cell action than affinity-dependent clonal selection.
Estrogen receptors bind to and activate the promoter of the HoxC4 gene to potentiate HoxC4-mediated AID induction, immunoglobulin class-switch DNA recombination and somatic hypermutation (62.8)
Authors: Mai Thach , Zan Hong , Zhang Jinsong , Hawkins J. Seth , Xu Zhenming , Casali Paolo
Published: January 2023

Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen upregulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID promoter in B cells undergoing CSR. Rather, they bind to three evolutionary conserved and cooperative estrogen response elements (EREs) we identified in the HoxC4 gene promoter. By binding to these EREs, ERs synergized with CD154 or lipopolysaccharide (LPS) and IL-4-signaling to upregulate HoxC4 expression, thereby inducing AID and CSR, without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to NP-CGG. Ablation of HoxC4 (HoxC4-/-) abrogated the estrogen-mediated enhancement of AID gene expression, and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HoxC4 gene promoter and inducing the critical AID gene activator HoxC4.
Visualizing the requirement for homotypic T cell synapses in enhancing CD8+ T cell differentiation. (63.11)
Authors: Gerard Audrey , Omar Khan , Krummel Matthew
Published: January 2023

CD8+ T cells are functionally heterogeneous and mediate their effects through a variety of mechanisms. Effective adaptive immunity relies on the capacity of lymphocytes to differentiate and yet, as a cohort to respond as a single integrated entity. But the mechanism by which the “coordinated diversification” of CD8+ T cells is regulated is not fully understood. Here, we show that homotypic clustering of nascent effector cells facilitates and coordinates their differentiation. The dynamic nature of T cell clustering upon immunization was characterized by live 2-photon imaging of explanted lymph nodes. T-T interactions are highly dynamics and are driven by LFA-1 dependent interactions. These clusters are initiated by T cell activation and occur up to two days after immunization. Functionally, reducing T cell clustering inhibits the establishment of effector and memory functions. Surprisingly, after initial activation, CD8 differentiation does not require an ongoing contact with an APC, but does require T-T interactions. T cells exchange information through synaptic structures, where increased cytokine delivery and signaling is found and is required for T cells to influence each other’s fate. Altogether, we provide evidences that a ‘collective’ CD8 response is generated through direct cell-cell communication just after their elicitation. This data may explain how tolerance is maintained and how T cell responses may rapidly amplify as additional precursors are recruited.
STAT5 is a negative regulator of Bcl6-mediated TFH differentiation (63.2)
Authors: Johnston Robert , Diamond Jeffrey , Crotty Shane
Published: January 2023

Follicular helper T cells (TFH) constitute the CD4+ T cell effector subset that is specialized to provide germinal center B cell help. TFH differentiation is driven by the transcription factor Bcl6, and recent studies have identified cytokine and cell-cell signals that are required for optimal expression of Bcl6. However, the mechanisms underlying the negative regulation of TFH differentiation have been less clear. Here, we show that STAT5 was a key inhibitor of TFH differentiation and function. Constitutive STAT5 signaling in activated CD4+ T cells selectively blocked TFH differentiation, resulting in a collapse of the germinal center reaction. Conversely, STAT5-deficient CD4+ T cells (mature STAT5flox/flox CD4+ T cells transduced with a Cre-expressing retrovirus) rapidly upregulated Bcl6 expression and preferentially differentiated into TFH cells. STAT5 signaling failed to inhibit TFH differentiation in the absence of the transcription factor Blimp-1. We have previously shown that Blimp-1 is a direct repressor of Bcl6-mediated TFH differentiation (Science, 2009). These results demonstrate that STAT5 and Blimp-1 collaborate to negatively regulate TFH differentiation.
Two different pathways contribute to cytotoxicity of mouse cytotoxic T lymphocytes (63.25)
Authors: Dudenhöffer-Pfeifer Monika , Krause Elmar , Hoth Markus , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) are an important part of the immune system and kill cells which are infected by pathogens like viruses or bacteria (named antigen-presenting-cell or APC). Two different killing mechanisms are being used by CTLs - (1) exocytosis of lytic granules which contain cytotoxic substances and (2) the so called Fas/FasL pathway which relies on agonist/receptor interaction and induces apoptosis in the APC. So far the quantitative contribution of either pathway to the total killing capacity of mouse CTL was not known. To address this question we used a knock out mouse strain (Jinx) which lacks the SNARE associated protein Munc13-4. As a result CTLs from these mice display a severe defect in the secretion of cytotoxic substances, as we could demonstrate by a Granzyme B release assay (reduction of 85% ± 4%) and a FACS based degranulation assay (reduction of 70% ± 9%). Interestingly we found that despite the lytic granule release failure CTLs from Munc13-4 -/- have a remaining killing capacity of about 43%, arguing for a significant contribution of the Fas/FasL pathway. To strengthen this finding we established a single cell assay in which the increase of intracellular Ca2+ in the APC was used as a measure of apoptosis and found that about 50% of killing was induced by the Fas/FasL pathway alone. We conclude from these data that both killing mechanisms are equally important and function synergistically to maintain the cytoxic activity of a mouse CTL.
Embryonic stem cell-derived factors inhibit T effector activation and induce T regulatory cells by modulating PKC-θ activation. (63.29)
Authors: Mohib Kanishka , Al-Khamees Bodour , Allan David , Wang Lisheng
Published: January 2023

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. However, the mechanisms by which ESC inhibit immune activation remain mostly unknown. We have previously shown that ESC derived factors block dendritic cell maturation and thereby affect T cell activation indirectly. Our ongoing studies have elucidated that ESC derived factors also have a direct effect on T cell activation. In mixed lymphocyte reaction assays, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating IL-10 and TGF-β expression. Also, ESC derived factors suppressed the expression of Th1 transcription factor Tbet, while enhancing the expression of Treg transcription factor Foxp3. Furthermore, PBMC/purified T cells activated with CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Incubation of ESC derived factors with recombinant GST-PKC-θ resulted in the pull-down of four proteins. The purified proteins are currently being analyzed by mass spectrometry. In conclusion, ESC’s are able to directly impact T cell activation and polarization, likely by negatively regulating PKC-θ through potentially novel factors.
Activation of the protein kinase A pathway down regulates spleen tyrosine kinase in human T cells (63.30)
Authors: Ghosh Debjani , Kis-Toth Katalin , Juang Yuang-Taung , Tsokos George
Published: January 2023

Cyclic adenosine monophosphate (cAMP)-mediated activation of the protein kinase A (PKA) pathway has an overall negative impact on T cell proliferation and down regulates or neutralizes signaling pathways of many genes. Spleen tyrosine kinase (SYK) is an important gene expressed differentially during T cell development and functions by phosphorylating downstream effector molecules in both T and B lymphocytes. We report here that PKA inhibitors promoted, whereas cAMP analogues suppressed SYK expression. Overexpression of a catalytic subunit of PKA significantly abrogated SYK protein and mRNA expression as well as the levels of phosphorylated SYK. A complete cAMP response element (CRE) site on SYK promoter was found functional and capable of binding CREMα and CREB as detected by EMSA and chromatin immunoprecipitation. CREMα overexpression reduced, while disruption of the CRE site enhanced the basal promoter activity of SYK. Collectively, the cAMP-mediated PKA pathway functions as the negative regulator of SYK expression by promoting direct binding of a repressor element to the SYK promoter.
Tracking subcellular distribution of fluorescent fusion proteins during thymocyte migration and development (64.2)
Authors: Melichar Heather , Dzhagalov Ivan , Ross Jenny , Herzmark Paul , Robey Ellen
Published: January 2023

T cell receptors (TCRs) on developing thymocytes are screened for appropriate recognition of MHC molecules and self-reactivity before differentiating into CD4, CD8, or other non-conventional T cell subsets prior to exiting the thymus. However, how thymocyte interactions and T cell receptor signaling relate to developmental outcomes during these selection processes is unclear. Dynamic re-localization of proteins within cells can provide information about cell polarity and cell signaling. Here we use fluorescent fusion proteins to identify the subcellular localization of cell polarity proteins (PKCζ) and markers of TCR signaling (LAT) in conjunction with time-lapse two-photon microscopy of thymic lobes. In our current study, we have generated chimeras with bone marrow engineered to express fluorescent fusion proteins, and observe the re-localization of these proteins during thymocyte migration and intercellular interactions. We present a semi-automated approach to quantify fluorescent fusion protein distribution in cells of thymus tissue and show asymmetric distribution of the PKCζ-GFP fusion protein during migration as well as differential accumulation of the LAT-GFP fusion protein during developmental checkpoints. These observations may lead us to an understanding of how the quality of TCR interactions in the thymus affects developmental outcome.
mTOR complex 2 mediates Notch signaling in thymocyte and T-cell leukemia development (64.8)
Authors: Lee Keunwook , Gudapati Prathyusha , Volanakis Emmanuel , Cho Sunghoon , Boothby Mark
Published: January 2023

Mammalian target of rapamycin (mTOR) is an important component of the PI3-kinase (PI3K) pathway that regulates cell growth and proliferation. PI3K signaling cascades can be initiated by activation of Notch, a critical regulator for T lineage commitment and leukemia development. Here we show that mTOR complex 2 (mTORC2) is functionally vital as a downstream target of Notch in thymocytes and T-cell acute lymphoblastic leukemia (T-ALL). Conditional deletion of rictor, an essential component of mTORC2, results in impaired proliferation and differentiation of pre-T cells driven by Notch ligation. Moreover, mTORC2 is a primary kinase for Akt S473 and enhances Akt activity in thymocytes. Furthermore, mTORC2 regulates NF-κB activity, such that constitutively active Akt can restore nuclear NF-κB levels in the rictor-depleted pre-T cells, as well as normalizing Notch-initiated proliferation and differentiation. Strikingly, interruption of mTORC2 significantly delays T-ALL induced by Notch1 activating mutation and suppresses infiltration of leukemic cells into the non-lymphoid organs. Our study provides evidence of a key role of mTORC2 in relaying Notch signaling in thymocyte development, and reveals that mTORC2 promotes Notch-driven leukemia progression.
Monoclonal mouse antibodies against camel immunoglobulins (65.11)
Authors: Holzlöhner Pamela , Schliebs Erik , Maier Natalia , Pfister Friederike , Füner Jonas , Micheel Burkhard , Heilmann Katja
Published: January 2023

Camelid antibodies are of special interest because some of them belong to immunoglobulin subclasses (IgG2 and IgG3) which are build up only by heavy chains. To use such antibodies for practical application we need specific identification tools. Immunoglobulins llama (Lama glama) were isolated by Protein G (IgG3 and IgG1) and Protein A (IgG2) sepharose. Fab Fragments were generated from llama IgG1 by papain digestion. Balb/c mice were immunized against the three different IgG subtypes and the Fab fragments and monoclonal antibodies were produce by hybridoma technology. We were hitherto able to obtain the following purified monoclonal antibodies (moAb): - moAb reacting to all subtypes with identical binding strength, - moAb that preferentially bind the conventional IgG1, - moAb that bind only IgG1. All generated monoclonal antibodies react against immunoglobulins from new world (llama and alpaca) and old world camelids (bactrian camel and dromedary) but show no cross reactivity with immunoglobulins from other species (rat, mouse, sheep, rabbit, goat, chicken and human). The monoclonal antibodies are suitable to detect camelid antibodies by enzyme immunoassay (ELISA) but not in western blots, indicating that the binding epitops are discontinuous. The production of monoclonal mouse antibodies binding to camelid heavy chain only antibodies is subject for future experiments.
Immune repertoire high-throughput sequence analysis (IRAS) web service (65.20)
Authors: Wang Chunlin , Sanders Catherine , Yang Qunying , Han Jian
Published: January 2023

An immune repertoire is the sum of functionally diverse B and T cells, which is shaped by genetic polymorphism, antigen exposure history and regulation and modulation of the immune system. High-throughput sequencing techniques allow us to take a snapshot of the diversity of the immune repertoire, which is a fundamental determinant of the competence of the immune system. However, many researchers are overwhelmed by the task of managing and analyzing the flooding amount of sequence data generated by next-generation DNA sequencers. To make the immune repertoire high-throughput sequencing technology accessible to greater numbers of investigators, we have developed a web based immune repertoire sequence data analysis and management system (IRAS). IRAS is backed by a Linux cluster, where V, D, J germline sequences can be mapped onto reads using the Smith-Waterman algorithm in a high-throughput manner. Mapping results are loaded into a MySQL database and reads are annotated with CDR1, 2, 3 regions. We have developed a web portal to allow easy access to those data, where 2D-plot, 3D-plot, various summary tables, statistical results and mapping alignments can be generated on the fly. The IRAS is open to public and welcome to use. Users can submit their sequence data and relevant metadata through the web portal.
Multiplex immunoglobulin isotyping assays for human, mouse or rat serum samples (65.25)
Authors: Wiese Rick , Gessling Aliya , Hayes David
Published: January 2023

The determination of serum immunoglobulin isotype concentrations is an important indicator of immunological health. We have constructed several multiplex isotyping assays on the Luminex® assay platform. These assays allow for the analysis of human, mouse and rat serum, tissue culture or body fluid samples. The human isotyping assay enables the quantification of immunoglobulins A, M, G1, G2, G3 and G4 in a single assay well. While the mouse isotyping assay cover immunoglobulins A, M, G1, G2A, G2B, and G3, and the rat isotyping immunoglobulins A, E, M, G1, G2A, G2B, G2C. Since the concentrations of IgE in human and mouse samples are typically much lower than the other immunoglobulin isotypes we have also designed separate assays for mouse and human IgE analysis. This collection of assays allows for the rapid, multiplex analysis of major immunoglobulin isotypes in the human , as well as the two predominant research maodels, rat and mouse.
Skin-based soluble microneedle array mediated anti-malarial CSP vaccination (65.28)
Authors: Speaker Tycho , Chang Stella , Fairman Jeff , Kaspar Roger
Published: January 2023

A safe, effective, easily administered, and storage-stable anti-malarial vaccine delivery platform would improve vaccination efforts in populations at risk for malaria. Painless, self-blunting soluble microneedle arrays seem ideal for skin-based inoculation. To test the ability of adjuvanted microneedle arrays to induce an immune response, anti-malarial circumsporozoite surface protein (CSP) antigen was delivered directly to skin by water-soluble polyvinyl alcohol microneedles, inducing a robust and specific immune response in BALB/c mouse cohorts. Inclusion of JVRS-100 cationic lipid DNA complex adjuvant boosted immune response and dried arrays induced strong response even after exposure to accelerated thermal storage conditions. To assess storage stability of the needle array dosage form, loaded microneedle arrays and parallel antigen solutions were subjected to one-week storage at 20°, 37°, and 50°C, and subsequently used to inoculate mouse cohorts. Serum anti-CSP IgG titers indicated that while antigen solutions showed profound (10-fold) loss of activity with increasing storage temperature, the dry arrays exhibited no such degradation.
Magnetic bead-based 28-plex immunoassay for the quantification of rhesus and cynomolgus monkey cytokines (65.30)
Authors: Chitnis Vivek , Strange Christina , Sekella Phillip , Spencer James , Huuskonen Jarkko , Blankfard Martin
Published: January 2023

Non-human primates, such as rhesus and cynomolgus monkey share a similar biochemistry and metabolism to humans. These animals are therefore crucial model systems in a number of areas of biomedical research. To simplify cytokine profiling we have developed a 28-plex magnetic luminex bead-based cytokine immunoassay for the analysis of rhesus and cynomolgus monkey samples. Validated sample types include serum, plasma, and conditioned tissue culture medium. Analytes include: EGF, eotaxin, FGF basic, G-CSF, GM-CSF, HGF, IFN-γ, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-15, IL-17, I-TAC, MDC, MIF, MIG, MIP-1α, MIP-1β, MCP-1, RANTES, TNF-α, and VEGF. The utility of the assay is demonstrated by determining cytokine profiles obtained with LPS-stimulated or PMA plus A23187-stimulated peripheral blood mononuclear cells (PBMCs) from rhesus monkeys and cynomolgus monkeys. Linearity of dilution experiments with tissue culture medium and serum produce R2 values of 0.98 and greater, while sample recovery routinely falls between 80 and 120%. Intra-assay and inter-assay precision is ≤10% CV. This multiplex monkey cytokine assay provides a very powerful tool for addressing cytokine profiling in non-human primates. 28 cytokines can be simultaneously analyzed from a single sample greatly simplifying and cutting cost of monkey cytokine analysis.
Magnetic bead-based immunoassays for the quantification of human and mouse cytokines (65.31)
Authors: Chitnis Vivek , Strange Christina , Gu Jun , Curtis Daniel , Spencer James , Huuskonen Jarkko , Blankfard Martin
Published: January 2023

Multiplexing immunoassays have become a very powerful and economic way to measure multiple analytes from a single sample. This is especially important when sample volumes are limited, such as for rodent samples. Life Technologies has recently launched a set of luminex bead-based immunoassays that can conveniently quantify between 10 and 30 cytokines from human or mouse tissue culture, serum and plasma samples. These assays use magnetic beads which offer a convenient way to automate these assays using commercially available plate washers, such as BioTek ELx405 or Tecan HydroSpeed. The mouse analytes in these kits are: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p40/p70, TNF-α (10-plex), supplemented with FGF basic, IL-1α, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α and VEGF (mouse 20-plex). Human analytes are GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-α (10-plex), supplemented with IL-1RA, IL-2R, IL-7, IL-12 p40/p70, IL-13, IL-15, IL-17, IFN-α, MIP-1α, MIP-1β, IP-10, MIG, eotaxin, RANTES, MCP-1, VEGF, G-CSF, FGF basic and HGF (human 30-plex). All the kits have been extensively validated to include sample recoveries routinely between 80 and 120%, intra-assay and inter-assay precision of <10%, and linearity of dilution with tissue culture medium and serum with R2 values of 0.98 and greater. Benchmarking of the kits has been performed against our ELISA kits to ensure equal results between the two immunoassay platforms.
The role of PD-1 in human CD8 prostate infiltrating lymphocytes. (66.12)
Authors: Bruno Tullia , Durham Nicholas , Nirschl Christopher , Ceccato Christina , Getnet Derese , Pardoll Drew , Drake Charles
Published: January 2023

CD8 T cell tolerance to tumors is multi faceted and includes both extrinsic and intrinsic factors. Extrinsically, the tumor microenvironment seems to play a large role in the functional inertness of CD8 T cells; major contributors are persistent antigen exposure in the microenvironment as well as T regulatory cells. Intrinsically, lack of costimulation seems to play a role (anergy), and often, the presence of inhibitory checkpoints such as PD-1, CTLA-4, LAG-3, and 41BB can also facilitate the down regulation of CD8 T cell function. In CD8 prostate infiltrating lymphocytes (PILS) from cancer patients, we observe refractoriness to TCR stimulation accompanied by reduced cytokine production (IFN-γ and TNF-α) that cannot be reversed by addition of IL-2. Thus, in trying to decipher the exact intrinsic factor that could play a direct role on the CD8 PILS, we measured inhibitory checkpoints on the cell surface. PD-1 was a major checkpoint, and seemed to correlate with reduced Granzyme B production. Interestingly, the total CD8 PIL population was not deficient in Granzyme B production, just the PD-1+ CD8 PILS. Additionally, blockade with anti PD-1 restored proliferation and Granzyme B production in the CD8 PILS, which indicates a potential clinical implication of anti PD-1 immunotherapy.
Critical function of CCL1 in the maintenance of T regulatory cell’s suppressive role. (66.14)
Authors: Hoelzinger Dominique , Dominguez Ana , Lustgarten Joseph
Published: January 2023

T-regulatory cells (Tregs) create an immunosuppressive environment that reduces the efficacy of anti-tumor immunotherapy. Depletion of Tregs enhances anti-tumor immune responses, but also affects T-effector cells. Previously we showed that blockade of chemokine ligand 1 (CCL1) inhibits Treg conversion, their suppressive function, and in combination with intra-tumoral CpG-ODN results in complete rejection of tumors in BALB-NeuT tolerant mice. Most importantly, this therapeutic combination does not affect T-effector function and increases tumor-lytic CD8+NKG2D+ cells within the tumor. Here we expand on the role of blocking CCL1 in enhancing anti-tumor immunotherapy. Using CCL1 knockout (CCL1ko) mice, we found no impairment in TGFβ dependent de novo Treg conversion, however CCL1ko Tregs are less suppressive in vitro, than their wild type counterparts. CCL1ko mice treated with intratumoral CpG showed a marked reduction in tumor growth and a significant increase in tumor rejection as compared to similarly treated C57b control mice. Examination of lymphocyte populations within B16 and MucI tumors showed elevated levels of CD8+ cells. Finally the evaluation of levels of intra-tumoral cytokines and chemokines revealed elevated levels of IFNγ, CXCL9, CCL3 and CXCL10 in tumors grown in CCL1ko mice, suggesting a pro-inflammatory environment. Taken together these data support the role of CCL1as a critical factor for the maintenance of the suppressive function of Tregs.
STAT1 and IFNAR1 regulate Treg frequencies and IL-10 expression the tumor microenvironment (66.15)
Authors: Stewart Charles , Metheny Hannah , Dzutsev Amiran , Karp Christopher , Trinchieri Giorgio
Published: January 2023

The efficacy of cancer immunotherapy is limited by immunosuppression in the tumor microenvironment. Regulatory T cells (Treg) are found at high frequency in tumors, and have greater suppressive activity than Treg from spleen or lymph node tissues. In addition, IL-10 limits the activation of antigen presenting cells and production of proinflammatory cytokines at the tumor site. We examined the cellular sources of IL-10 in the tumor. Using IL-10GFP (VERTX) reporter mice and real-time PCR assays, IL-10 expression was detected in Ly6C+ myeloid cells, macrophages and approximately 50% of tumor Tregs. A similar portion of tumor Treg produced IL-10 following stimulation with PMA/ionomycin. Microarray analyses on IL-10 positive and IL-10 negative tumor Treg and total spleen Treg revealed an activation signature associated with IL-10 expression. This signature correlated strongly with expression of the transcription factor Maf and with induction a type I IFN/STAT1-signature. Using knockout mice we observed that both the type I IFN receptor, IFNAR1 and STAT1 were required for normal tumor Treg frequencies and IL-10 production. This indicates that the STAT1/IFNAR1 axis is required to drive immunosuppressive Treg and their production of IL-10 in the tumor microenvironment. Therefore the immunostimulatory effects of type I IFN are paralleled with an increase in Treg function and IFNAR1/STAT1 may constitute an important therapeutic target for tumor Treg.
Self-specific memory regulatory T-cells outrun tumor-specific effector T-cells at tumor emergence to impose tumor tolerance (66.18)
Authors: Podsypanina Katrina
Published: January 2023

Early responses of Tregs and effector T cells (Teffs) to their first encounter with tumor cells have been poorly characterized. We show, in both implanted and in situ-induced mouse tumor models, that the appearance of tumor cells is immediately sensed by CD44hi memory Tregs that are specific for self antigens. The rapid response of these Tregs preceded and prevented activation of naive antitumor Teffs. The relative speed of the Treg versus the Teff response within the first 2-4 days determined the outcome of the antitumor immune response: tolerance or rejection. If antitumor memory Teffs were present at the time of tumor emergence, both Tregs and Teffs were recruited and activated with memory kinetics; however, the Tregs were unable to control the Teffs, which eradicated the tumor cells. This balance between effector and regulatory responses did not depend on the number of Tregs and Teffs, but rather on their memory status. Thus, in the natural setting, dominant tolerogenic immunosurveillance by self-specific memory Tregs protects tumors, just as it protects normal tissues. More generally, our results reveal that the timing of Treg and Teff engagement, determined by their memory status, is an important mode of regulation of immune responses.
Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by CD70 signaling is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway (66.23)
Authors: Park Ga Bin , Kim Yeong-Seok , Lee Hyun-kyung , Song Hyunkeun , Cho Dae-Ho , Lee Wang Jae , Hur Dae Young
Published: January 2023

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70+ tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from mitochondria and cleavage of caspases. CD70-induced apoptosis was inhibited by ER stress inhibitor salubrinal, ROS quencher NAC, and Ca2+ chelator BAPTA. We supposed that ROS generation might be the first event because NAC blocked increases of ROS and Ca2+, but BAPTA did not block ROS. We also found that CD70 stimulation activated JNK and p38. JNK inhibitor and p38 inhibitor blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation blocked phosphorylation of JNK and p38 and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBVt- B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.
Effects of chronic alcohol consumption on B cells in B16BL6 melanoma-bearing mice (66.24)
Authors: Zhang Hui , Zhu Zhaohui , Meadows Gary
Published: January 2023

We previously found that chronic alcohol consumption inhibits CD8+ T cell immunity in B16BL6 melanoma-bearing mice and also decreases their survival. Recent reports indicate that lack of B cells, especially mature B cells, compromises CD8+ T cell anti-tumor immunity. It is unknown if alcohol consumption affects B cells in tumor-bearing hosts. Herein, we studied the effects of chronic alcohol consumption on B cell phenotypes in B16BL6 melanoma-bearing mice. Alcohol consumption did not alter the number of B cells in the bone marrow (BM), spleen or lymph nodes, but decreased B cells in the peritoneal cavity (PC) and blood. Notably, the B cell number decreased 3-4 fold in blood. The percentage of CD19+IgMhiIgDlo immature B cells slightly increased in the BM. Alcohol decreased marginal zone B cells and increased CD23-CD93+ immature B cells, and did not alter follicular B cells in the spleen. The percentage of CD19+CD5+ cells increased in the PC and CD19+CD5- cells decreased. The percentage of CD21+CD23-CD19+ B cells increased 2-fold in blood while CD19+CD23+ cells decreased. Alcohol consumption altered the gene expression of sphingosine-1-phosphate (S1P) receptor-1 (SIPR1) and S1P lyase. Collectively, these data indicate that alcohol decreases mature B cells in the blood and PC by impairing B cell circulation in melanoma-bearing mice. Altered S1P/S1PR1 signaling could contribute to impaired B cell circulation.
B cells infiltrating ovarian cancer exhibit a regulatory-like phenotype. (66.26)
Authors: Nunez-Cruz Selene , Quinn III William , Connolly Denise , Scholler Nathalie
Published: January 2023

IL-10, an immunosuppressive cytokine, is found in the tumor environment. While it is appreciated that Toll-Like Receptors (TLRs) in combination with signals through the B cell receptor and CD40 can modulate IL-10 production by B cells, the role that TLR stimulation plays on tumor-infiltrating lymphocytes B (TIL-B) remains unclear. To study this, we used a transgenic mouse model of serous ovarian cancer (Tg-MISIIR-TAg) and ascites of ovarian cancer patients; in vivo experiments in an IL-10 reporter mouse model are ongoing. We found that early tumors contained TIL-B phenotypically similar to B220hi CD21/35 med IgMmed follicular B cells, as demonstrated by flow cytometry. As the tumor progressed, phenotype evolved from fresh isolated TIL-B with up-regulation of CD1d and CD5, and overexpression of IL-10 compared to splenic B cells. In addition, freshly harvested TIL-B from Tg-MISIIR-TAg tumors and frozen B cells from ascites of ovarian cancer patients expressed high levels of TLR4, TLR2 and TLR9. Finally, TIL-B cultured in presence of TLR agonists increased further IL-10 production compared with fresh harvested TIL-B or splenic B cells cultured in the same conditions. Taken together, our findings indicate a direct link between the increased TIL-B production of IL-10 and stimulation through TLRs. We propose that chronic exposure of B cells to the tumor microenvironment may enhance their regulatory-like phenotype and increase their receptiveness to TLR-driven IL-10 production.
Defining the molecular mechanisms that control the development of tumor dendritic cells. (66.32)
Authors: Tang Michael , Diao Jun , Cattral Mark
Published: January 2023

Cancer-associated inflammation contributes to tumor progression and metastasis. Defective dendritic cell (DC) function causes impaired adaptive immunity to antigens expressed by tumors. We recently found that the intra-tumor inflammatory milieu recruits and skews development of DC precursors (pre-cDC) towards a novel Gr-1+ subpopulation, which has tolerogenic properties. This transformation reduced the proliferation and expansion of antigen-specific cytotoxic T cells in tumors. We sought to identify cytokines that regulate this differentiation. Isolated pre-cDC from mice spleen was incubated with tumor-conditioned medium (TCM) derived from Lewis lung carcinoma cell line. Supernatants from cultures were collected to analyze cytokine levels. Expression of Gr-1+ on DC was monitored by flow cytometry. We found that TCM induced pre-cDC to differentiate into Gr-1+ cDC. Neutralizing anti-cytokine Ab studies revealed that IL-6 play a role in promoting Gr-1+ cDC differentiation. Pre-cDC from IL-6-/- mice showed a reduced capacity to develop into Gr-1+ cDC. TCM contained no IL-6, but it induced IL-6 secretion by pre-cDC. These findings suggested that the release of autocrine IL-6 by pre-cDC created a positive feedback loop with tumor-derived factors in TCM to promote Gr-1cDC development. Elucidation of the mechanisms by which tumor-derived factors communicate with pre-cDC will advance our understanding of the mechanisms involved in the development of impaired immune response to cancer.
Tumor-derived G-CSF promotes granulocytic MDSC development in vivo (66.34)
Authors: Waight Jeremy , Abrams Scott
Published: January 2023

Myeloid-derived suppressor cells (MDSC) comprise a heterogeneous population of myeloid cells with the capacity to suppress tumor immunity. Consequently, these cells represent a significant impediment to effective cancer immunotherapy. While the mechanisms by which these cells mediate immunosuppression is well-established, details on how they develop remain poorly understood. Broadly speaking, it is known that tumor-derived factors play significant roles in diverse aspects of MDSC biology. Given that MDSC accumulation is a result of altered myelopoiesis and that a major subset is granulocytic, we tested the hypothesis that tumor-derived granulocyte-colony stimulating factor (G-CSF) promotes MDSC development. Using both implantable and autochthonous mouse mammary tumor models, we observed abundant amounts of G-CSF in vivo, which correlated with a massive accumulation of granulocytic MDSC. To determine a causal role of G-CSF in MDSC generation, we established both gain- and loss-of-function approaches. First, administration of recombinant G-CSF alone elicited immunosuppressive granulocytic MDSC. Secondly, over-expression of G-CSF in G-CSF-negative tumors led to granulocytic MDSC accumulation. Lastly, inhibiting G-CSF culminated in a significant reduction of granulocytic MDSC. Collectively, our studies demonstrate the importance of tumor-derived G-CSF in MDSC development, thus providing new insights into the identity of putative targets for therapeutic applications.
HMGB1: a regulator of myeloid-derived suppressor cell potency? (66.37)
Authors: Parker Katherine , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in individuals with cancer and inflammation and play a pivotal role in tumor immunity by suppressing T-cell activation and secreting proinflammatory molecules. The suppressive capacity of MDSC is mediated by immune suppressive factors such as arginase and reactive oxygen species (ROS). Nuclear protein, High Mobility Group Box1 (HMGB1), is present in nearly all cells and is released from myeloid cells as a danger response to sepsis, infection, or arthritis. Its release promotes inflammatory responses. HMGB1 signals through a multitude of receptors including TLR4, which is expressed by MDSC. In contrast to other inflammatory mediators which increase MDSC potency, HMGB1 reduced the suppressive capacity of TLR4(-/-) and wildtype MDSC, and reduced ROS levels in TLR4(-/-) MDSC. These findings suggest that HMGB1 may diminish MDSC function and may lead to new immunotherapeutic uses of HMGB1.