bluuebunny/crossref_metadata_2025 huggingface dataset:
NKG2D-NKG2D ligand interaction inhibits the outgrowth of low-grade B cell lymphoma (TUM9P.1019)
Authors: Markiewicz Mary , Raju Saravanan , Kretzmer Lena , Koues Olivia , Payton Jacqueline , Oltz Eugene , Cashen Amanda , Polic Bojan , Schreiber Robert , Shaw Andrey
Published: January 2023

Transgenic mice we generated with ubiquitous expression of the NKG2D ligand RAE1ε, and therefore low surface NKG2D expression, developed natural, spontaneous B cell lymphoma at a younger age than wild-type mice. Transplantation of lymphomas from RAE1ε transgenic mice into lymphodeficient mice resulted in expression of NKG2D on immune cells transferred along with the tumor and rejection of the lymphoma cells, suggesting NKG2D is important in controlling lymphoma outgrowth in wild-type mice. In the absence of overt tumor masses, older mice developed lymphoid aggregates in non-lymphoid organs and these aggregates occurred at a younger age in RAE1ε transgenic and NKG2D-deficient mice. The increased incidence of both these lymphoid aggregates and tumor masses in RAE1ε transgenic mice suggests these lymphoid aggregates are slow growing indolent lymphomas. Although NKG2D ligands were undetectable on wild-type tumors, ligands were present on splenic B cells in a similar proportion of 10 month-old wild-type and NKG2D-deficient mice as those with lymphoid aggregates present. These results suggest that 1) induction of NKG2D ligands is an early marker of B cell transformation, 2) NKG2D-NKG2D ligand interaction suppresses B cell lymphoma growth, and 3) loss of NKG2D receptor-ligand interactions leads to the transformation of indolent B cell lymphomas into aggressive tumors in this model. We are now exploring whether these results are relevant to human follicular lymphoma progression.
Inhibition of Treg suppression by GITR stimulation causes tumor regression selectively in the context of a pro-inflammatory tumor microenvironment (TUM9P.1005)
Authors: Zappasodi Roberta , Budhu Sadna , Decicco Emily , Schild Tanya , Draleau Kyle , Schaer David , Yang Xia , Merghoub Taha , Wolchok Jedd
Published: January 2023

GITR stimulation as immunotherapy has become increasingly important given the recent success of immune modulation in cancer therapy. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition with the agonist antibody DTA-1, and verified their functional changes in vitro. In vivo, Treg frequency was increased, Teff activation reduced and tumor antigen presentation impaired in more advanced tumors. DTA-1 reduced Treg frequency and expression of suppression markers in curative and refractory conditions. In contrast, Teff activation and tumor-associated MHCs were induced only in curative conditions. In vitro, DTA-1-pretreated Treg lost their suppressive activity without downregulating either Foxp3 or Helios, and increased Th1/Th17 cytokine release. Interestingly, co-culture with T cells was sufficient to up-regulate MHC-II on B16, but MHC-I was induced if both T cells and DTA-1 were present. GITR stimulation on human Tregs via GITRL consistently abrogated their functions, thus validating GITR as a target to revert immune tolerance also in this setting. Our data suggest that Treg inhibition by anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing in future trials.
Ptnp11 deletion in CD4+ cells causes cartilage tumors in a T cell independent manner (TUM9P.1006)
Authors: Miah S. M. Shahjahan , Salter Alexander , Reilly Emma , Fugere Celine , Jayasuriya Chathuraka , Yang Wentian , Chen Qian , Brossay Laurent
Published: January 2023

The ubiquitously expressed tyrosine phosphatase SHP-2 (encoded by Ptpn11) is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells and iNKT cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, are thought to dephosphorylate key players of TCR mediated activation. Using mice conditionally deficient for SHP-2in the T cell lineage, we show that classical T cells and iNKT cells develop normally in the thymus and exhibit proper maturation and functions in the periphery. These results suggest that SHP-2 serves a redundant role with other tyrosine phosphatases in the T cell lineage and is dispensable for T cell function. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Crerecombinase leads to cartilage tumors in a T cell independent manner, indicating that SHP-2 is a tumor suppressor.
Transcription factor slug-upregulated microRNA-221 suppresses E-cadherin expression and promotes breast cancer progression (TUM10P.1031)
Authors: Liu Yuan , Pan Yi , Gu Hongwei , li jing
Published: January 2023

E-cadherin protein expression is blocked during tumorigenesis due to slug/snail-mediated suppression of E-cadherin transcription. However, this mechanism of transcriptional blockade cannot explain why metastatic breast cancer cells maintain very low level of E-cadherin even with E-cadherin mRNA overexpression. In the present study, we reveal that E-cadherin is also post-transcriptionally regulated by slug-promoted microRNA-221 (miR-221), which serves as an additional lock preventing E-cadherin expression in metastatic cancer cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cell lines treated with/without TGFβ shows that the miR-221 level is significantly higher in metastatic breast cancer tissues and in MDA-MB-231 cells compared with the cancer-adjacent normal tissues or non-metastatic MCF-7 cells, and that miR-221 level is subjected to the TGFβ regulation. The level of miR-221 is inversely correlated with E-cadherin expression in breast cancer cells, and experimentally depleting or elevating miR-221 induces or decreases E-cadherin expression, consequently promoting or suppressing breast cancer progression. Moreover, we found that miR-221 suppresses E-cadherin translation via binding to the open reading frame of the E-cadherin mRNA and that miR-221 is specifically upregulated by Slug. In conclusion, our results demonstrate for the first time that Slug-upregulated miR-221 promotes breast cancer via targeting E-cadherin.
The NK killer receptor Ncr1 shapes tumor architecture to prevent metastasis (TUM10P.1034)
Authors: Glasner Ariella , Mandelboim Ofer
Published: January 2023

Natural killer (NK) cells were so-named because of their excellent ability to kill tumors and pathogens. NKp46 (Ncr1 in mice) is a prominent NK killer receptor, critically involved in controlling various pathogens as well as tumors and tumor metastases. We and others and have demonstrated the ability of the NK killer receptor Ncr1 to contain tumor growth, however, the mechanisms underlying the ability of Ncr1 to control tumor growth and metastases in vivo are yet undetermined Metastatic melanoma is an extremely aggressive disease and the leading cause of death from skin cancers. Here, we used two melanoma spontaneous metastases models: B16F10.9 (B16) and RET, the Ncr1 knockout mice (Ncr1gfp/gfp), Ifnγ-/- and Tnfα-/- knockout mice to visualize the tumors’ architecture in-vivo, using Reflectance Confocal Microscopy (RCM). We demonstrate that Ncr1, through Ifnγ prevents tumor metastasis by modulating the structural properties of the developing tumors, at the level of the tissue organization. Using RNAseq we have analyzed the transcriptome of Ifnγ treated B16 cells and reveled a 24 fold increase in the integrin binding ECM protein Fibronictin1 (FN1), the absence of which was reported to correlate with increased metastases. The finding that Ncr1, through Ifnγ, shapes the tumors’ structures and thus controls metastasis may lead to generation of new drugs aimed at targeting specifically the tumors’ architecture and new treatment options for melanoma patients.
Overexpression of LLT1 (CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A interaction (TUM10P.1052)
Authors: Mathew Stephen , Mathew Porunelloor
Published: January 2023

Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. In order to establish and grow, cancer cells must escape NK cell-mediated killing. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. We have identified LLT1 (Lectin-like transcript 1 or CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. Recently, we have found that LLT1 is overexpressed on DU145, LNCaP, and PC3 prostate cancer cells both at the mRNA and protein level. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb increased the killing of prostate cancer cells. NK-mediated killing of DU145 in the presence of anti-LLT1 mAb showed the maximum killing. Also, LLT1 was found to be overexpressed in primary prostate cancer tissues, whereas its level of expression was minimal in normal prostate tissue. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 gene to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.
Development of humanized mouse models to study human immune system-tumor interactions (TUM10P.1055)
Authors: Aryee Ken-Edwin , Shultz Leonard , Greiner Dale , Brehm Michael , Jurczyk Agata
Published: January 2023

Cancer progression involves interplay between tumor cells and non-malignant stoma, including immune cells. Our understanding of the interactions between immune systems and tumors is based mostly on mouse models. However many differences exist between murine and human biology, and translation of findings from mice to clinical application is challenging. The study of human immune systems and tumors is limited by logistical and ethical restrictions of working with primary specimens from patients. Here we describe a humanized mouse model to study the in vivo interactions between human immune systems and human tumors. In these studies NOD-scid IL2rgnull (NSG) mice expressing human SCF, GM-CSF and IL-3 (NSG-SGM3) were engrafted with human fetal thymus and fetal liver-derived hematopoietic stem cells (BLT). NSG-SGM3-BLT mice support high levels of human cell chimerism, including development of human myeloid cells, T cells and B cells. Engrafted NSG-SGM3-BLT mice were implanted with patient melanoma cells and monitored for tumor growth and immune system homeostasis. The human melanoma grew with similar kinetics in NSG-SGM3-BLT mice as compared to control NSG-SGM3 mice. Human immune cells, including human macrophage and T cells, infiltrated the melanoma. The T cell infiltrate was dominated by human FOXP3+ Treg and by PD1+ CD4 and CD8 T cells. These data show that NSG-SGM3-BLT mice, are a promising model to study human immune system-tumor interactions and to test novel therapies.
Exercise reduces tumorigenesis in a K-ras mouse model of non-small cell lung cancer (TUM10P.1057)
Authors: Lowder Thomas , Abadi Parisa , Bernardo Jenina , Pulicken Catherine
Published: January 2023

Exercise has been shown to reduce the prevalence of certain cancer subtypes, prolong survival rates, and improve quality of life of cancer patients. Exercise’s role in cancer includes not only prevention and post-treatment care, but also the reduction in cancer progression. Moderate-intensity exercise has been shown to have significant inhibitory effects on tumor initiating mechanisms in mice resulting in decreased tumor incidence for a variety of cancers including pancreatic, gastrointestinal, and mammary. This is the first report of the role that exercise plays in the suppression of spontaneous murine lung cancer. Sedentary mice (N = 14) lung tumor count was 40.57 ± 3.483 tumors (mean ± SEM). Lung tumor count in the exercised mice (N = 10) was significantly reduced to 21.80 ± 1.705 tumors (p < 0.001). Sedentary mice (N = 6) tumor percentage of lung volume was 12.34 ± 0.528%. Percent lung tumor volume in exercised mice (N = 8) was significantly reduced to 6.913 ± 0.262% resulting in a 44% reduction of tumors within lung tissue. IL-6 and TNF-alpha spleen data did not show any significant changes due to exercise in relation to lung cancer. These results demonstrate that moderate exercise can slow the progression of tumorigenesis in a mouse model of lung cancer.
IL-36 is required for the induction of therapeutic immunity by Tbet gene-modified dendritic cells. (TUM10P.1024)
Authors: Weinstein Aliyah , Chen Lu , Storkus Walter
Published: January 2023

Our lab has developed a dendritic cell (DC) vaccine as a means of overcoming immune dysfunction in the tumor microenvironment (TME) and generating a therapeutic anti-tumor immune response. As protective Type 1 immune responses are characterized by intrinsic expression of the Tbet transactivator in T cells and DC, we engineered DC with Tbet cDNA (ie, DC.Tbet) and injected these cells into the TME of syngeneic tumor-bearing mice as a therapy. Treatment of MCA205 sarcomas with DC.Tbet promoted the rapid (hours) and sustained (days-weeks) infiltration of Type 1-polarized CD4+ and CD8+ TIL into the TME and led to increased TME expression of factors characteristic of ectopic lymphoid structures (ELS), such as TNFSF14, CCL19, and CCL21. DC.Tbet cells revealed a 60-fold increase in the level of IL-36gamma mRNA over mock-transduced DC and inhibition of signaling through the IL-36 receptor (IL-36R) using natural antagonist IL-1F5 abrogated the ability of DC.Tbet therapy to inhibit tumor growth in vivo, concurrent with reduced recruitment of therapy-induced CD4+ and CD8+ TIL. The efficacy of DC.Tbet therapy was similarly negated in MCA205-bearing IL-36R-/- hosts. These data suggest a mechanism whereby treatment with DC.Tbet leads to recruitment of anti-tumor TIL via an IL-36R signaling-dependent mechanism that supports the development of ELS in the TME. These findings foreshadow the clinical use of IL-36gamma in cancer vaccine formulations as a means to promote therapeutic immunity.
Human antibody specificities that confer protective immunity to poxvirus infections (VAC11P.1109)
Authors: Gilchuk Iuliia , Gilchuk Pavlo , Hammarlund Erika , Slifka Mark , Lampley Rebecca , Sapparapu Gopal , Eisenberg Roselyn , Cohen Gary , Xiang Yan , Joyce Sebastian , Crowe James
Published: January 2023

Immunization with vaccinia virus (VACV) induces cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibodies (Abs). However, the specificity and breadth of protective human Abs to poxviruses are largely unknown. We used a highly-optimized human hybridoma technology to generate a panel of 90 anti-VACV monoclonal Abs from vaccinia-immunized subjects or from subjects who had a history of naturally-acquired monkeypox infection. Then, we assessed the neutralizing and protective capabilities of the Abs in vitro and in a lethal respiratory VACV challenge in a mouse model. A large fraction of Abs from the panel (>40%) possessed neutralizing activity against VACV, cowpox and/or monkeypox viruses. Antibodies to a variety of envelope proteins in VACV extracellular virions (A33, B5) or mature virions (L1, A27, D8, H3) contributed substantially to neutralizing activity in vitro. For protective immunity in vivo, L1, A27, A33 and B5 antibodies conferred levels of protection that were comparable to those induced by live viral vaccination, especially when used in combination. Such antibody cocktails have promise for use in as immunotherapeutics in humans.
B cell memory but not antibody response is maintained in the elderly after repeated influenza vaccine immunizations and aged murine plasma cell generation is defective (VAC11P.1066)
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Landin Ana Marie , Frasca Daniela
Published: January 2023

The success of a vaccine depends on both plasma cells and memory B cells. We previously showed that serum antibodies and the in vitro AID response to a new influenza vaccine are decreased in elderly individuals. We hypothesized that even though AID is decreased in the elderly, the ability to generate memory B cells might be improved by a repeated vaccine. To test this hypothesis, we recruited young and elderly individuals who had been immunized in at least two consecutive influenza vaccine seasons in which the H1N1 strain was the same. Results showed that frequencies of influenza vaccine-specific memory B cells were similar in young and elderly individuals but the levels of serum antibodies were lower in the elderly. These findings suggest that multiple re-exposures to the vaccine cause a divergence between memory B cell frequencies and antibody levels with age, which could be due to impaired memory B cell to plasma cell differentiation in the elderly. To test the hypothesis of defective plasma cell development in old mice, we measured the percentage of plasma cells and Blimp-1 in LPS-stimulated B cells and found them significantly lower in old than in young cultures. The average fold reduction in the secreted amount of IgM and IgG3 in aged cultures compared to young cultures is higher than the average reduction in CD138+ plasma cells (10-fold vs 2-fold) suggesting qualitative as well as quantitative plasma cell defects with age.
In MCA-induced sarcoma models, booster vaccination with GM-CSF-secreting whole-cell tumor vaccines maintain or improve efficacy but may alter trafficking of tumor-specific T cells (VAC13P.1128)
Authors: Messenheimer David , Jensen Shawn , Fox Bernard
Published: January 2023

Previously, we reported that while a single vaccination effectively generated therapeutic tumor-specific T cells for adoptive immunotherapy (AIT), repeated vaccination with the poorly immunogenic B16BL6 melanoma, modified to secrete GM-CSF, induced high levels of CD4+FoxP3+ T regulatory (Treg) cells and AIT with spleen cells was non-therapeutic. The effect could be overcome by transient depletion of CD4+ T cells, confirming the effect of Treg cells. Here experiments were performed to test whether this suppressive effect was observed in active-specific protection in 2 sarcoma models, MCA-304 and MCA-310. Mice were vaccinated once or thrice with either irradiated parental sarcoma or that sarcoma expressing GM-CSF. We did not observe significant changes in percentage or total count of splenic Treg cells under any conditions, however, tumor-specific IFN-γ release decreased in mice thrice vaccinated with GM-CSF-secreting tumors compared to parental (p < 0.001). Importantly, multiple vaccination offered tumor protection that was as good or better than single vaccination. In the most strongly immunogenic tumor, MCA-304, both single and triple vaccination with parental or GM-CSF-secreting tumors provided 100% protection. However, in the weakly immunogenic, MCA-310 sarcoma, multiple vaccinations improved effectiveness with the greatest effect seen with the GM-CSF-secreting MCA-310. Our results provide a rationale for the continued use of GM-CSF as a cancer vaccine adjuvant.
G-CSF induced neutrophil invasion of the CNS is inhibited by Interferon γ (VIR9P.1150)
Authors: Chandran Ramakrishna , Cantin Edourad
Published: January 2023

Inflammation of the brainstem (BS) of wildtype (wt) 129 mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils is causally implicated in fatal HSV induced encephalitis (HSE). Anti-viral treatment with acyclovir following virus entry into BS cannot protect mice, as inflammation continues unabated. We showed previously that IL-10 regulates IM infiltration into BS. We determined the role of IFNγ, a key pleiotropic cytokine that directs the host immune response by inducing potent inflammatory or regulatory activities, in HSE. IFNγ knockout (GKO) mice developed HSE much quicker than wt or Rag mice. Surprisingly, mortality did not correlate with the ability to control virus replication but rather with massive expansion and infiltration of neutrophils into the BS. Neutrophil expansion in the absence of IFNγ was not due to increased IL-17 but rather to augmented secretion of G-CSF especially in CNS. Depletion of G-CSF by administration of anti G-CSF Ab or 25 mg IVIG (high dose), which contains anti G-CSF Ab, but not 4 mg (low dose) inhibited progenitor proliferation and neutrophil output from bone marrow and protected GKO mice. Importantly, reduction of serum G-CSF levels resulted in renewed IL-10 secretion by ICOS+ CD4 T cells and Tregs, which limited IM expansion. This study reveals that the complex interplay of IFNγ with G-CSF and IL-10 determines the ability to skew neutrophil or monocyte output from the bone marrow in response to infection.
Hepatic retinoic acid regulates group 3 innate lymphoid cells and modulates viral hepatitis (VIR9P.1151)
Authors: Sun Jiaren , Liang Yuejin , Jie Zuliang , Zhang Kangling
Published: January 2023

IL-22 is important for hepatoprotection in hepatitis; however, the source and regulation of this cytokine in the liver microenvironment are not well defined. Here, we present evidence for a significant expansion of IL-22+ intrahepatic cells in mice during adenovirus and LCMV infection. In addition to gamma/delta and double-negative T cell, a subset of IL-22+ cells belong to group 3 innate lymphoid cells (ILC3s). These cells contributed significantly to the establishment of the early cytokine milieu in virus-infected liver. Interestingly, retinoic acid, a metabolite of vitamin A, increased in the liver following viral infection. Moreover, retinoic acid administration promoted IL-22 production by ILC3s and protected liver from inflammatory response-related injuries. Retinoic acid inhibited Th1/CTL responses through down-regulating DC activation and T cell priming in vitro and in vivo. Mass spectrometric studies suggested that retinoic acid induced arginase 1 expression of DC and dampened its function. Lastly, hepatic IL-7 was important for ILC3-derived IL-22 production. Blockade of IL-7Ra in vivo significantly decreased IL-22 levels and exacerbated hepatic inflammation. Collectively, this study has demonstrated the importance of a unique intrahepatic subpopulation ILC3 and its cytokine production during the initial stages of viral infection in the liver.
STING-dependent sensing of polyomavirus infection (VIR9P.1158)
Authors: Shwetank .. , Barber Glen , Lukacher Aron
Published: January 2023

Polyomaviruses (PyV) are nonenveloped DNA viruses that persist as low-level asymptomatic infections in a variety of vertebrates, including humans. In immunosuppressed individuals, the human BK and JC PyVs may cause transplant-associated nephropathy and progressive multifocal leukoencephalopathy, respectively. Mouse polyomavirus (MPyV) allows investigation of PyV immunity in a natural host. After caveolae-/clathrin-mediated endocytosis, PyV virions traffic in microtubule-guided microvesicles from endosomes to the ER, where uncoating is initiated. Unknown is whether complete uncoating occurs in the cytosol, exposing the viral DNA to cytosolic DNA sensors, before it is transported to the nucleus where viral replication and assembly occur. Here, we report that persistent MPyV infection in mice is associated with chronically elevated IFN-β transcripts, and that WT primary mouse embryo fibroblasts (MEFs) infected with MPyV upregulate IFN-β, TNF-α and ISG (PKR and MDA-5) mRNAs. However, MPyV infection failed to induce expression for these transcripts in MEFs from STING-/- mice. Interestingly, MPyV acute infection activated NK cells in WT, but not in IFNαβR-/- mice or in STING-deficient (Goldenticket) mice. Together, these data suggest that MPyV-induced activation of NK cells requires Type I IFN and a STING-dependent cytosolic DNA sensing pathway(s). Moreover, these data provide strong evidence that PyV DNA is exposed in the cytosol and activate innate immune sensors.
HIV elite controllers have lower frequencies of CD14dimCD16+ inflammatory monocytes with greater CX3CR1-dependent tissue homing potential than viremic controllers (VIR9P.1138)
Authors: Palmer Christine , Romero-Tejeda Marisol , Scully Eileen , Jost Stephanie , Seaman Michael , Goldenthal Ariel , Piechocka-Trocha Alicja , Pereyra Florencia , Walker Bruce
Published: January 2023

Without antiretroviral therapy, a small fraction of HIV-infected patients does not progress to AIDS. Such elite controllers (EC) or viremic controllers (VC) maintain undetectable or low levels of viremia, respectively. Generation of broadly neutralizing antibodies (bNAb) does not correlate with viral control, but might enhance antibody-dependent cellular phagocytosis by monocytes. Monocytes are classified into CD14+CD16-, CD14+CD16+ and CD14dimCD16+ monocytes. The latter show increased inflammatory responsiveness and are elevated during chronic HIV-1 infection. We measured markers of T cell activation and monocyte subset distribution and quantity, and investigated the relationship between these parameters and production of bNAb to identify markers for the development of bNAbs. We showed that VC display increased frequencies of CD38+ CD4+ and CD8+ T cells compared to EC. Our data also show that elevated frequencies of CD8+CD38+HLA-DR+ T cells and CD14dimCD16+ monocytes were associated with both viremic control status and production of bNAbs. bNAb production was also strongly associated with increased frequency of CD57+HLA-DR+ CD8+ T cells, as well as decreased expression of chemokine receptors CCR2 and CX3CR1 on CD14dimCD16+ monocytes in VC compared to EC. Further studies are required to establish the cause and effect of alterations in monocyte populations during HIV-1 infection and whether these could tip the scale towards viral control below detectable levels.
Vicilin and 11s globulin cashew allergens share promiscous CD4+ T cell epitopes with other tree nuts (APP2P.100)
Authors: Archila Diaz Luis Diego , McGinty John , Chow I-Ting , Jeong David , Farrington Mary , Robinson David , Kwok William
Published: January 2023

Allergic reactions to cashew can be life threatening. While IgE-specific cashew epitopes have been studied, CD4+ T-cell specific epitopes for cashew remain uncharacterized. Additionally, cross-reactivity amongst tree nuts has not been examined in humans at the T-cell level. We sought to provide a thorough phenotypic analysis of cashew reactive T-cells in allergic and non-allergic subjects as well as to identify promiscuous epitopes among tree nut allergens. CD4+ T-cell responses by CD154 up-regulation assays indicate that cashew allergens Ana o 1 and Ana o 2 play a major role in cashew allergy. Multiple Ana o 1 and Ana o 2 T-cell epitopes were identified with the Tetramer-Guided Epitope Mapping approach. Direct ex vivo staining showed that the majority of Ana o 1 and Ana o 2-specific T-cells have a CCR4+ TCM (central memory) phenotype while a subset of these T-cells express CCR4+CCR6+ in allergic subjects. Conversely, in non-allergic subjects a CXCR3+ population was observed. Cashew-specific T-cell clones for several epitope regions responded robustly to hazelnut and pistachio but not to walnut, while some epitopes responded to cashew exclusively and were found to be species specific. Allergic responses to cashew could be triggered by cross-reactive tree nut epitopes, therefore, restricted consumption of tree nuts should be advised for cashew allergic subjects.
Comparison of systemic lupus erythematosus and healthy anti-nuclear antibody positive African-Americans reveals distinct differences in CD4+ and CD8+ T cell populations (BA3P.117)
Authors: Lu Rufei , Slight-Webb Samantha , Maecker Holden , Utz Paul , Guthridge Joel , James Judith
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disorder arising from genetic and environmental factors that likely affect phenotypic and functional characteristics of multiple cell lineages. Almost all SLE individuals are antinuclear antibody positive (ANA+) although ANA positivity does not obligate autoimmune disease. The differences in cell physiology between ANA+ healthy individuals and individuals that go on to develop SLE remains a critical goal in understanding SLE development. We visualized the expression of 33 cellular markers through the hierarchical clustering of SPADE to provide a comprehensive view of the immune system between ANA- African Americans (n=8), ANA+ healthy controls (n=8) and ANA+ patients with SLE (n=8). In addition, the levels of 51 cytokines were measured in plasma using a multiplex immunoassay. Compared to healthy controls, SLE patients had lower frequencies of cytotoxic CD8+ T cells and regulatory CD4+ T cells (P<0.05). Concentrations of serum sCD40 ligand were reduced in patients with SLE compared with ANA+ and ANA- healthy controls (p<0.05). Interestingly, sCD40L production positively correlated with a decrease in regulatory CD4+ T cell populations (p<0.01). IL-7 levels were lower in both ANA+ controls and SLE patients compared with ANA- healthy individuals (p<0.05). Our results indicate that early differences in IL-7 may contribute to decreased levels of regulatory CD8+ and CD4+ T cells important for controlling systemic inflammation.
Type I interferons promote lupus through disruption of the lymphotoxin receptor-mediated mechanosensing MKL1 pathway in marginal zone macrophages (BA4P.124)
Authors: Mountz John , Li Hao , Hsu Hui-Chen , Zhou Yong , Wu Qi , Yang PingAr , Li Jun , Luo Bao , Morel Laurence , Ware Carl , Fu Yang , Yagita Hideo
Published: January 2023

Defective clearance of apoptotic cells (ACs) is a central pathogenic event in systemic lupus erythematosus (SLE). Here, we describe an underlying mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleen of lupus BXD2 mice disrupts marginal zone macrophages (MZMs), which normally clear apoptotic debris and prevent entry of the AC antigens (AC-Ags) into the follicles. The phagocytosis of ACs by the spleen MZMs is enabled by a megakaryoblastic leukemia 1 (MKL1) transcription factor-mediated mechanosensing pathway. The MZ B cells maintain this pathway through their expression of membrane lymphotoxin-α1β2 (mLT). Follicular shuttling of mLT+ MZ B cells disengages their interaction with the LTβR+ MZMs, thereby downregulating MKL1 in the MZMs. This leads to defective F-actin polymerization, loss of their ability to clear ACs and eventually MZM dissipation. Type I interferon (IFN) signaling, a well-established clinical characteristic of SLE and is commonly considered as a downstream event of AC-Ag stimulation, is identified to play a key role in MZM erosion via promoting follicular shuttling of mLT+ B cells. Follicular translocation of MZ B cells, erosion of MZMs and loss of MKL1 were similarly identified in lupus B6.Sle1.Sle2.Sle3 mouse and human SLE spleens. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signals in the MZM barrier to prevent follicular entry of AC-Ags.
Venetoclax (ABT-199), a potent and selective BCL-2 inhibitor, is efficacious in NZB/WF1 mouse model of lupus nephritis and reduces human lymphocyte lifespan in vitro (BA4P.127)
Authors: Grebe Kristie , Perper Stuart , O'Connor Liz , Schwartz Annette , Goess Christian , Hartman Dawna , Westmoreland Susan , Graff Candace , Souers Andrew , Leverson Joel , Elmore Steven , Olson Lisa , Wang Lichun
Published: January 2023

Proteins in the BCL-2 family are key regulators of apoptosis, or programmed cell death. We have conducted studies to evaluate a highly potent and orally available BCL-2 selective inhibitor, venetoclax (ABT-199), for efficacy and mechanism of action in the lupus prone NZB/W F1 mouse model and in human cells in vitro. Venetoclax treatment dose-dependently reduced the incidence of severe proteinuria and prolonged survival compared to vehicle controls and attenuated glomerulonephritis, tubular dilatation, immune cell infiltrates and IgG deposition in the kidney. Venetoclax mediated a significant reduction in the numbers of splenic T cells but conferred a preferential reduction in select B cell subsets. Interestingly, venetoclax did not impair the number of CD138+ long-lived plasma cells in the bone marrow, which was consistent with unaltered circulating anti-dsDNA titers in these animals. Venetoclax efficacy also correlated with a dose -dependent reduction of lymphocytes in peripheral blood of NZB/WF1 mice. Consistent with these findings, human lymphocytes from both healthy donors and systemic lupus erythematosus (SLE) patients treated with venetoclax in vitro have reduced lifespan. Taken together, these data underscore the essential role of BCL-2 in the pathogenesis of lupus and support further exploration of selective BCL-2 inhibition in autoimmune diseases such as SLE/Lupus Nephritis. The design, study conduct, and financial support for this research were provided by AbbVie.
Involvement of CD137L in peripheral nerve injury-induced neuropathic pain behaviors (CAM1P.161)
Authors: Malon Jennifer , Cao Ling
Published: January 2023

CD137L, also known as 4-1BBL, is a co-stimulatory molecule involved in many immune functions. CD137L-mediated signaling can promote monocyte/macrophage proliferation, migration, and production of proinflammatory factors; however, CD137L-mediated microglial responses have not been studied extensively. Here, we sought to investigate the role of microglial CD137L in the development of peripheral nerve injury-induced neuropathic pain using a murine model, spinal nerve L5 transection (L5Tx). First, compared to wild type (WT) controls, B6_CD137L knockout (KO) mice displayed significantly decreased L5Tx-induced mechanical and heat hypersensitivity starting at day 3 post-L5Tx until day 35 post-L5Tx when the experiment was terminated. To further determine the role of microglial CD137L, an anti-CD137L neutralizing antibody (Ab, clone TKS-1) was intrathecally injected into WT BALB/c mice daily from day 0 (before surgery) to day 7 post-L5Tx. The neutralizing Ab significantly reduced L5Tx-induced mechanical hypersensitivity but not the heat hypersensitivity. Our data indicate that spinal cord CD137L contributes to the maintenance of peripheral nerve injury-induced neuropathic pain-like behaviors. Future study will examine the involvement of microglial CD137L in L5Tx-induced lumbar spinal cord microglial and astrocyte activation.
Reciprocal regulation of c-Maf tyrosine phosphorylation by TEC and PTPN22 in TH2 cells (CCR3P.210)
Authors: Miaw Shi-Chuen , Liu Chih-Chun , Lai Chen-Yen , Lin Yu-Hsien , Chang Hui-Hsin , Ho I-Cheng
Published: January 2023

c-Maf plays an important role in regulating cytokine production in TH cells. It contains an N-terminal transactivation domain, which is connected to the C-terminal DNA binding domain through a hinge domain. c-Maf’s transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. Here, we demonstrate that Tec kinase family member TEC, but not RLK or ITK, is a tyrosine kinase of c-Maf and that TEC enhances c-Maf-dependent IL-4 promoter activity. This effect of TEC is counteracted by PTPN22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.
Dexamethasone suppresses IL-33-mediated mast cell activation (CCR3P.215)
Authors: Chernushevich Oksana , Paranjape Anuya , Ryan John
Published: January 2023

Dexamethasone, a glucocorticosteroid known for its anti-inflammatory and immunosuppressive activities, has been shown to inhibit IgE-mediated mast cell activation. The present research investigates the role of this drug in suppressing IL-33-mediated mast cell activation. IL-33 is a novel cytokine of the IL-1 family that elicits mast cells to produce inflammatory cytokines independently of IgE-FceRI signals. We have found that micromolar concentrations of dexamethasone are capable of suppressing IL-33-mediated cytokine production, regardless of genetic background. Dexamethasone also suppressed IL-33-induced cytokine production in mouse peritoneal mast cells. Inhibition was maximal when dexamethasone was added simultaneously with IL-33. Intracellular staining demonstrated that dexamethasone significantly reduces T1/ST2 expression in mast cells. However, cytokine suppression was shown to be independent of this downregulation of the IL-33 receptor. Thus, dexamethasone is capable of suppressing IL-33-mediated mast cell activation during the innate and adaptive immune responses. Further studies on the mechanism of dexamethasone are necessary for the understanding of those pathways and the development of more effective strategies for the treatment of inflammatory conditions.
RNA-binding protein HuR controls IL-2 homeostasis in activated T cells by regulating CD25 expression (CCR3P.202)
Authors: Glascock Jacqueline , Techasintana Patsharaporn , Magee Joseph , Atasoy Ulus
Published: January 2023

Posttranscriptional gene regulation by RNA binding proteins (RBPs) and their microRNA (miRNAs) partners is known to affect 50% of genes during T cell activation. The RBP, HuR regulates mRNAs by binding to AU-rich elements (AREs) in transcript 3’UTRs. Many Th2 cytokines and IL-2 possess AREs. We hypothesized that HuR may play a critical role in T cell activation and differentiation via regulation of IL-2 signaling pathways. To test this, we generated a conditional HuR KO mouse, distal-lck Cre ROSA HuRfl/fl, in order to ablate HuR prior to T cell activation. HuR KO T cells developed and egressed from thymus normally. However, they displayed an inability to shut off IL-2 and maintain stable cell surface expression of IL-2Ra (CD25). HuR KO T cells had striking increases in IL-2 mRNA and protein but decreases in Th2 cytokines. They also had proliferation defects, decreased p-stat5 and reductions in CD25 and prdm-1 transcription. We determined that HuR protein binds to IL-2 and CD25 mRNAs by two independent methods. CD25 mRNA stability was unchanged despite increased IL-2. We quantitated polysomal loading in HuR KO T cells compared to controls and discovered significant reductions in CD25 mRNA recruitment to heavy polysomes. Therefore, HuR regulates T cell activation and IL-2 homeostasis by controlling CD25 expression and enabling translational efficiency. We conclude that HuR plays an indispensible role in normal IL-2 homeostasis and T cell activation.
Mir-128-2 inhibits the development of B cell (HEM1P.218)
Authors: Yang Yi , Zhang Jinping
Published: January 2023

Accumulating evidence suggest that miRNAs play important roles in immune system. At present study, we first found that miRNA expression profiles were significantly different among vary immunocytes by microarray. Here, We focused on miR-128-2 which was highly expressed in DP T cells, but lower in other detected cells. In order to further study the function of miR-128-2 in lymphocytes development, we established the miR-128-2 chimeric and transgenic mice, confirmed by FACS analysis and PCR. We found B cell development appeared profoundly defects in both mice models, however, the development of T cells had no significant changes after over-expressing mir-128-2. Further analysis suggest that CLP was significantly higher in mir-128-2 TG mice compared to WT mice, but HSC and MMP cells were comparable in mir-128-2 TG mice and WT mice. Brdu and annexinV staining showed that over-expressing mir-128-2 did not affect the proliferation and apoptosis of CLP. These suggested that miR-128-2 inhibited the CLP cells to develop into B cell. Finally, we try to seek for the targets of miR-128-2 through software analysis and molecular biology methods. We found that miR-128-2 might down-regulate a group of genes including BCL11a, MALT1, A2b et. al. to inhibit B cell development confirmed by luciferase assay, real time PCR and western blot. Thus, our present study suggests that miR-128-2 mainly blocks CLP to differentiate into PreProB cells by down-regulating the BCL11a, MALT, A2b et al. genes.
Systemic adjuvant inflammation rapidly reorganizes bone marrow B cell populations, and their niche, to shape B cell function and repertoire development (HEM1P.224)
Authors: Moreau Joshua , Paige Christopher
Published: January 2023

Maturing B cells migrate through the organized bone marrow parenchyma toward sinusoid vessels before entering circulation. Niche components rigorously control this process. Infecting mice with pathogens, or injecting inflammatory agents, is known to produce perturbations in B lymphopoiesis, however; it is poorly understood how the B cell niche responds to these challenges. Moreover, the impact on subsequent B cell function remains unknown. Here, we report that injection of Freund’s incomplete adjuvant in mice induced a large and rapid (within hours) mobilization of B cells to the bone marrow, mediated by increased sensitivity to the chemokine CXCL12. Notably, localization within the marrow is altered with all B cell subsets showing a reduced affinity for the sinusoids. Reorganization of B cell positioning occurred together with relevant modification of their supportive niche including increased expression of BAFF and MIF mRNA, expansion of DX5+Thy1loAPRIL+ basophils, and elevation of several important cytokines. This altered marrow milieu may have a role in shaping B cell fate, as we observed a decrease in lambda light chain usage in immature cells. Further, disruption of homing dynamics with a CXCR4 inhibitor attenuated the formation of IgM secreting cells. Together these results demonstrate an exquisite sensitivity of the marrow B cell compartment to peripheral inflammation and suggest that initial inflammatory signaling regulates subsequent B cell effector responses.
Pre-B cells suppress RAG expression in response to DNA double-strand breaks (HEM1P.225)
Authors: Fisher Megan , Bassing Craig
Published: January 2023

The ability of the Rag1/Rag2 (RAG) endonuclease to assemble antigen receptor (AgR) genes is essential for adaptive immunity. However, aberrant induction or repair of RAG-induced DNA double strand breaks (DSBs) can lead to oncogenic AgR translocations. We have previously shown that RAG-induced DSBs in pre-B cells activate the ATM kinase to prevent RAG cleavage of the homologous allele, and to inhibit expression of Rag1 and Rag2. These breaks also suppress expression of Gadd45α, which promotes Rag1 and Rag2 transcription. Since DSBs induced by ionizing radiation (IR) signal through ATM to increase Gadd45α expression in all other cell types including mature B cells, these results indicate that RAG DSBs and/or pre-B cells use unique mechanisms to regulate Gadd45α expression. We now demonstrate that IR-induced DSBs in pre-B cells signal through the ATM kinase to down-regulate Gadd45α expression. We also demonstrate that these IR DSBs signal ATM-dependent suppression of Rag1 and Rag2 transcription, without affecting stability of Rag1 or Rag2 mRNA. Because the presence of multiple DSBs in a cell greatly increases the risk of translocation, we hypothesize that pre-B cells have developed a unique developmental stage-specific DSB response to suppress the induction of RAG DSBs in the presence of other DSBs. Our ongoing studies will determine the role of this pre-B cell specific DSB response in suppressing AgR translocations and enforcing mono-allelic assembly of immunoglobulin genes.
HEB plays a critical role in the installment of IL-17 program in fetal thymic γδ T cells (HEM2P.231)
Authors: In Tracy , Anderson Michele
Published: January 2023

IL-17 producing γδ T cells that arise during fetal development form an integral part of the immune system of various lymphoid and mucosal tissues in mice. Here we aim to better understand the transcriptional networks that drive the functional programming of IL-17 producing γδ T cells. Specifically, we investigated the role of the transcription factor HEB in this process using mice that carry the HEB null allele. To evaluate the role of HEB in fetal γδ T cell development, we utilized fetal thymic organ culture from HEB+/+ and HEB-/- embryos at E14. We found that the absence of HEB did not have a significant impact on γδ T cell numbers, and that all functional subsets of γδ T cells were present as indicated by expression of CCR6, CD27, CD44 and CD62L. However, the fetal thymic γδ T cells from HEB knockout mice exhibited a profound deficiency in their ability to produce IL-17, especially in the CD44+ γδ T cell population. In addition, HEB-deficient fetal γδ T cells expressed significantly lower levels of SOX13 and RORγt, factors associated with IL-17 producing γδ T cells, while expression of IFNγ, Tbet and Egr3 were unchanged. Furthermore, HEB conditional knockout mice on a Vav-Cre background had significantly lower frequencies of IL-17 producing γδ T cells in the spleen, lymph nodes and lungs. Collectively, our work shows for the first time that HEB plays an important role in installing the IL-17 program in γδ T cells.
Bim expression in early DP thymocytes limits development of TCRαβ+DN thymocytes and CD8αα intestinal intraepithelial lymphocytes (HEM2P.243)
Authors: Li Kun-Po , Roy Eron , Cuda Carla , Grimes H , Perlman Harris , Hildeman David
Published: January 2023

Thymocyte negative selection is thought to cull T cells with strong self-reactivity and maintain T cells with non-self-specificity. While initial work implicated Bim as a critical mediator of thymocyte negative selection, its role in the process has been controversial. Thus, we re-examined thymocyte development in mice with global and T cell specific loss of Bim. We found that global Bim-/- mice had a striking accumulation of DN4 thymocytes expressing high levels of surface TCR, which failed to generate DP cells in OP9-DL1 culture. Interestingly, CD4Cre-driven deletion of Bim promoted DN4 cells, but later, dLckCre-driven deletion of Bim did not. Combined, these data show that the aberrant DN4 cells in Bim-/- mice were once DP cells, but had lost co-receptor expression. Investigating the fate of these cells, we found that splenic DN T and gut CD8αα T cells were increased in Bim-/- mice. While IL-15 is critical for both DN/CD8αα T cells, the additional loss of Bim partially restored DN/CD8αα T cells in IL-15 deficient mice, suggesting that IL-15 signals antagonize Bim to promote DN T and CD8αα T cells. Finally, TCR or IL-15 signals were sufficient to promote expression of both CD8αα and the gut homing receptor CCR9 on splenic DN T cells. In conclusion, these data further delineate the role of Bim on thymocyte fate and DN/CD8αα T cell development; and show that dLckCre-Bimf/f mice can isolate the effects of Bim on peripheral T cell survival without affecting thymic selection.