bluuebunny/crossref_metadata_2025 huggingface dataset:
B7-H1 blockade plus regulatory T cell depletion improves clinical efficacy over either strategy alone in a melanoma model (P4268)
Authors: Curiel Tyler , Murthy Kruthi , Kious Mark , Sun Lishi , Zhang Bin , Pandaswara Srilakshmi , Hurez Vincent
Published: January 2023

Denileukin diftitox (DD) is an FDA-approved conjugate of human IL-2 + diphtheria toxin. It depletes Tregs and improves clinical and immune outcomes, but Treg generation can reduce efficacy. B7-H1 is an immune co-signaling molecule that promotes Treg generation and hinders anti-cancer immunity. αB7-H1 or DD each slowed tumor growth and improved anti-tumor immunity in B16 melanoma. DD reduced Treg numbers but αB7-H1 also reduced Treg suppression. In vitro, αB7-H1 reduced Treg generation from naïve CD4+CD25- T cells. We thus hypothesized that αB7-H1 would synergistically improve clinical efficacy of Treg depletion with DD by slowing regeneration or function of Tregs after depletion. We treated mice 7 days after B16 challenge when detectable tumors were present. DD+αB7-H1 significantly slower tumor growth at 25 days (avg tumor ~600 mm3 vs either single agent treatments (avg 1500-2000 mm3; p <0.05). Spleen CD4+ and CD8+ T cells with combined therapy showed significantly more activated CD44+CD62L- cells vs individual treatments, but we found no significant difference in T cell interferon-γ or IL-17 in treatment groups. Strikingly, we also did not detect reduced Treg function or numbers in DD + αB7-H1 combination vs individual treatments in spleen cells. Preliminary data suggest that effects are primarily in tumor draining lymph nodes, which are under current study. We use FDA approved agents like DD or agents in clinical trials such as αB7-H1 for rapid clinical translation.
Adiponectin deficiency suppress murine fibrosarcoma growth via modulation of tumor-associated macrophages polarization (P2127)
Authors: Peng Jiao , Ho Derek , Chan Vera , Tam Paul , Chen Yan
Published: January 2023

Adiponectin was reported to modulate macrophage polarization towards an anti-inflammatory phenotype (M2-type), but it remains unclear whether APN deficiency could modulate tumor development via tumor-associated macrophages. In this study, effects of APN on TAMs polarization and tumor development were investigated in MN/MCA1 inoculated mouse fibrosarcoma model. Our results indicated that APN deficiency exerted suppressive activities on the development of MN/MCA1 fibrosarcoma in vivo, whereas no pronounced APN-induced cyto-toxicity was observed in vitro. Phenotypic analysis demonstrated that M1-like TAMs were accumulated in tumor bearing APN-/- mice, which is evidenced by expression of M1-like marker, MHCII molecule. Moreover, M1-like markers, such as iNOS, TNF-α and IL-12 were up-regulated and the M2-like makers IL10, YM1, IL4Rα, MgI-1and MMP-9 mRNA were down-regulated in TAMs isolated from APN-deficient mice. Immunostimulatory effects were also depicted by increasing tumor infiltrated cytotoxic T lymphocytes and natural killer cells. In summary, adiponectin deficiency could promote TAMs polarization towards M1-like phenotype which might provide insight of modulation of TAMs in cancer therapy.
Caloric-restriction maintains tumor immunity and CD4 T cells priming during aging: role of an inflammatory DC population (P2175)
Authors: Ruby Carl , Farazi Michelle , Goldufsky Josef , Cohn Zachary , Linnane Stephanie , Nguyen Justine , Lukaesko Lisa , Weinberg Andrew
Published: January 2023

Immune responses progressively wane during aging, posing significant challenges to the protection and treatment of cancer. We previously demonstrated that in the context of OX40 costimulation older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. We hypothesized that anti-tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed by challenging with either sarcoma or breast tumor cells followed by agonist OX40 treatment. Our results show CR, but not RES, maintained OX40-mediated anti-tumor immunity, during aging. In addition, CR maintained tumor-specific CD4 T cell activation in aged mice. This observed effect of CR on T cell activation, lead to an assessment of DCs. We found that the accumulation of an inflammatory DC subset (CD11c+CD11b+Ly6C+), critical for the activation of CD4 T cells, was significantly decreased in the LNs of older control mice compared to young mice. In contrast, CR aged mice had more inflammatory DCs in the priming LNs compared to aged controls. Thus, CR appears to maintain immunological fitness of a DC subset during aging that is critical for antigen-specific CD4 T cell activation and tumor immunity.
Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis (P2181)
Authors: Gerstein Rachel , Ramkumar Charusheila , Cui Hang , Kong Yahui , Jones Stephen , Zhang Hong
Published: January 2023

Current treatment of diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin’s lymphoma, has improved patient survival significantly. However, 40-50% of DLBCL patients still die from this disease. There is a critical need to better understand the specific molecular pathways that are perturbed in DLBCL for the development of new therapeutic approaches. Using a mouse model deficient in the E3 ubiquitin ligase Smurf2, we show that Smurf2 deficiency increases the susceptibility of these mice to spontaneous B-cell lymphomagenesis. Lymphomas developed in Smurf2-deficient mice resemble human DLBCL with molecular features of germinal center B cells. We discover that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Importantly, loss of Smurf2 enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, we find that expression of Smurf2 is significantly decreased in primary human DLBCL samples compared to normal B cells. Importantly, low levels of Smurf2 expression correlate with poor survival prognosis in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, and identifies Smurf2 as a new therapeutic paradigm for DLBCL.
Autophagy in tumor cells and the host STING pathway are critical for innate immune sensing of tumors and bridging to an adaptive immune response (P2183)
Authors: Woo Seng-Ryong , Fuertes Mercedes , Furdyna Michael , Leung Michael , Duggan Ryan , Gajewski Thomas
Published: January 2023

Spontaneous T cell responses against tumors occur frequently. However, the mechanisms by which innate immune responses become induced in response to cancer, and how they can bridge to T cell priming against tumor antigens, have remained poorly defined. We recently reported that host type I IFN signals are required for a spontaneous T cell response against tumors. In the current work, we have mapped the sensing mechanism to one involving STING/TBK1/IRF3 which drives IFN-β transcription by host APCs. The tumor-derived ligand that triggers this pathway appears to be DNA. Using the DNA-specific dye DRAQ5 or EDU labeling, we demonstrated transfer of tumor DNA to host APCs via single cell ImageStream analysis; this was confirmed using PCR specific for tumor DNA. This was associated with IFN-β mRNA induction and nuclear translocation of IRF-3, which were dependent upon host STING. We hypothesized that autophagosome provide a mechanism for delivery of protected tumor DNA into APCs. By inhibition of tumor cell autophagy using the chemical 3-MA, or shRNA silencing of Becn or ATG5, we found marked reduction in the induction of host IFN-β production and spontaneous T cell responses in vivo. Our data suggest that tumor cell autophagy is required for optimal T cell priming against tumor antigens in vivo, that occurs through DNA-mediated activation of host IFN-β production via a STING-dependent pathway.
IL-17D, natural killer cells, and macrophages collaborate to promote tumor rejection (P2097)
Authors: Bui Jack , O'Sullivan Timothy , Saddawi-Konefka Robert , Gross Emilie
Published: January 2023

The process of cancer immunoediting generates a repertoire of cancer cells that can persist in immune competent hosts. In its most complex form, this process begins with the elimination of highly immunogenic unedited tumor cells followed by the eventual escape of less immunogenic, edited cells. However, it is not known whether editing can also change the global gene expression profile of developing tumor cells. Here we show using a gene microarray study that the novel cytokine interleukin 17D (IL-17D) is highly expressed in certain unedited but not edited mouse tumor cell lines or certain human tumors. Moreover, forced expression of IL-17D in edited tumor cells induced rejection by stimulating monocyte chemotactic protein 1 (MCP-1, aka CCL2) production from tumor endothelial cells leading to the recruitment of natural killer (NK) cells. We go on to show that NK cells can promote the accumulation of M1-type macrophages. Altogether, these results identify IL-17D as a novel cytokine and describe one mechanism by which IL-17D can induce tumor rejection.
Human multi-cancer gene expression analysis defines an inflammatory cancer-associated molecular pattern with prognostic significance (P2209)
Authors: Rachidi Saleh , Qin Tingting , Sun Shaoli , Zheng Jim , Li Zihai
Published: January 2023

Chronic inflammation is a hallmark of cancer. We propose that a “signature” of dysregulated inflammatory molecules exists across multiple cancer types. Genome-wide cDNA expression data was analyzed for 221 normal and 324 cancer specimens in 7 cancer types: breast, prostate, lung, colon, gastric, oral and pancreatic. 44 elevated and 52 repressed genes were identified and protein expression was confirmed by immunohistochemistry for some of these genes. This “Inflammatory Cancer-Associated Molecular Pattern” includes proteins whose roles in cancer are known and others (BST2, TNFAIP6, TNFSF4 and LEAP2) whose functions are yet to be appreciated. Clinical analysis in colon and ovarian cancer patients showed strong associations with tumor grade, stage and/or survival. In ovarian cancer, 18 genes predicted overall survival and 15 genes predicted recurrence. In colon cancer, 13 and 17 genes predicted 5-year overall and relapse-free survival, respectively. In both diseases, better prognosis correlated with high CXCL13 and lower level of GREM1, LOX, TNFAIP6, CD36, and EDNRA. An “Inflammatory Gene Integrated Score” was further developed from these genes, which also predicted survival in an independent cohort of ovarian cancer patients. This work defines an inflammatory signature shared by seven epithelial cancer types, which not only has significant biological implications, but also may serve as a novel biomarker for prognostication and individualized treatment of cancer.
The roles of tumor associated macrophages and interferon gamma in immune surveillance in CNS lymphoma (P2211)
Authors: Gao Hua-Xin , Wong Valerie , Chen Lingjing , Locksley Richard , Lowell Clifford , Rubenstein James
Published: January 2023

The distinctive roles of macrophages in cancer are believed to reflect selective M1 or M2 states of differentiation. Central nervous system lymphomas (CNSL) are aggressive tumors with poor prognosis. We generated murine CNSL models to elucidate the role of the immune microenvironment, especially macrophages, in this disorder. We determined that the majority of tumor-associated macrophage (TAM) expressed the mannose receptor CD206, an established M2 marker, and showed phagocytic activity of lymphoma. Surprisingly, this phenotype of TAM was also observed in mice deficient in IL-4, IL-4Ra, IL-10, or STAT6, suggesting that the M2-like feature of TAM was not driven by Th2 pathways. Few TAM were noted to express arginase I, another M2 marker. We also found upregulation of M1 markers MHCII and nitric oxide on TAMs, indicating a M1/M2 intermediate phenotype. CNSL progressed more rapidly In IFNγ-/- Rag-/- mice than in Rag-/- mice. Furthermore, macrophage depletion significantly exacerbated CNSL progression, but failed to do so in IFNγ deficient mice, suggesting that IFNγ-primed macrophage inhibits tumor growth in CNSL. Reduction of macrophage infiltration to tumor site promoted CNSL progression. Distinct from the CD45loCD11blo microglias, TAMs were CD45hiCD11bhi and maintained Ly6C, suggesting a monocytic origin of these cells. Based upon this data we report that CNSL TAMs present both M1 and M2 features and may mediate immune surveillance against lymphoma progression in brain.
Anti-nuclear IgM antibodies protect against lymphoma/lymphocytic leukemia (P2219)
Authors: Jiang Chuancang , Zhao Ming-Lang , Hobbie Kristen , Herbert Ronald , Diaz Marilyn
Published: January 2023

Our previous work showed that AID-deficient MRL/lpr (AID-/-) mice which had high levels of autoreactive IgM experienced a longer lifespan than muS-/-AID-/-MRL/lpr (muS-/-) mice whose B cells did not secrete any antibodies. Histology results indicated that these mice died of Lymphoma/lymphocytic leukemia (LLL). Further studies showed that at 6 months of age, all muS-/- mice developed LLL, while only 24% AID-/- mice had LLL. At 12 months of age, eventually all the mice from both strains developed LLL, but the LLL grade of muS-/- mice was higher than that of AID-/- mice (P = 0.004). When bone marrow cells from AID-/- mice were transferred to muS-/- mice, the mice with successful reconstitution of IgM-secreting B cells (showing high levels of IgM) displayed a lower grade of LLL than both the mice with poor reconstitution of IgM-secreting B cells (showing low levels of IgM) and PBS control mice. In vitro experiments demonstrated that autoreactive IgM-rich serum from AID-/- mice induced apoptosis of lymphoma cell line TK-1 cells, while antibody-absent serum from muS-/- mice did not had the effect. This led to the finding that some anti-nuclear monoclonal IgM antibodies also induced the apoptosis of TK-1 cells. Treatment of p53-/-mice with the mAb reduced the lymphoma grade compared to control mice (P = 0.021). These data demonstrate that anti-nuclear IgM antibodies can protect against LLL, probably through induction of tumor cell apoptosis. (CJ and MZ are equal contributors)
Immune regulation by CD11b+ myeloid cells in the intestine (P3195)
Authors: Murai Masako , Krause Petra , Shui Jr-Wen , Madan Rajat , Karp Christopher , Kronenberg Mitchell
Published: January 2023

We found that a subset of CD11b+ myeloid cells in the intestinal lamina propria constitutively produce IL-10 mRNA, and adoptive transfer of these IL-10 producing cells to recipients helped to maintain Foxp3 expression and the function of Treg. Although much emphasis has been placed on the regulatory role of T cell derived IL-10, our data show that IL-10 synthesis by intestinal myeloid cells has a nonredundant and dramatic effect on maintaining Treg function and preventing T cell-mediated colitis in mice. Here, we further investigate the role of intestinal myeloid cell derived IL-10 in the regulation of gut inflammation. IL-10 producing intestinal myeloid cells expressed higher CD11b, CD11c, F4/80, Gr-1 and CX3CR1 messenger RNA and protein, compared to their non-IL-10 producing intestinal myeloid counterparts. Myeloid cell specific (LysM-Cre driven) IL-10 deficient mice had a similar Treg population in the large intestine compared to controls. C. rodentium infection caused a decrease in the number of Treg in the large intestine, but not in the spleen, and moreover, this defect was more pronounced in mice with a myeloid cell specific IL-10 deficiency. Inflammation in colitis models or following acute infections leads to decreased intestinal Treg. IL-10 producing myeloid cells has a role in maintaining the Treg population in the intestine under inflammatory conditions, as exemplified by C. rodentium infection.
The protective capacity of CD8+ TRM cells in distant skin after cutaneous virus infection (P3201)
Authors: Jiang Xiaodong , Kupper Thomas
Published: January 2023

We have previously shown that repetitive infections of skin at different sites leads to an accumulation of protective T cells that encompass all sites of skin, including those distant from the infected sites. Additional central memory cells also accumulate in lymph nodes. These protective skin homing T cells presumably move out of lymph nodes draining the infected skin, enter the blood stream, and are distributed throughout the body, where they preferentially home to sites that express low constitutive levels of their ligands—E selectin, CCL19, and ICAM-1, all of which are expressed on normal skin. In order to test this theory, we repetitively infected a mouse, and thirty days later we parabiotically joined it to a normal mouse. After four weeks, parabionts were separated, rested for two weeks, and challenged with vaccinia virus (VACV) on distant skin. The previously infected parabiont rapidly cleared VACV infection from distant skin, while the uninfected parabiont, although it contained abundant central memory T cells and antibody, could not efficiently clear infection. This indicates that prior skin infection at different sites in the same organ by the same pathogen multiple times through a subject life leads to subsequent resistance to infection throughout that organ, in a process mediated by skin-resident memory T (TRM) cells generated by multiple infections.
Assessment of interferon responses in intact human placental explants using a novel whole mount immunofluorescence technique (P3205)
Authors: Bushway Meghan , Gerber Scott , Miller Richard , Lord Edith , Murphy Shawn
Published: January 2023

Pro-inflammatory cytokines required for immunity against non-self, such as interferons (IFNs), are expressed at the maternal-fetal interface during normal pregnancy. However, neither the placental cell targets nor the functions of these cytokines have been defined. We hypothesized that semi-allogeneic trophoblast cells (TBCs), which are the only cells derived from the fertilized egg that are in direct contact with maternal tissue, are hypo-responsive to IFNs in order to evade maternal immune recognition. Therefore, we developed a novel whole mount immunofluorescence technique to identify cellular targets of IFN-γ and IFN-α/β by examining expression of phosphorylated STAT-1 (pSTAT-1) in intact human placental explants. Using this technique we were able to identify the major cell populations present in the placental explants, including fetal macrophages and distinct subpopulations of TBCs. Neither the syncytiotrophoblast layer in direct contact with maternal blood, nor the underlying cytotrophoblast cells expressed pSTAT-1 in response to either IFN. In contrast, extravillous trophoblast cells, which are in direct contact with maternal decidual tissue, and fetal macrophages induced strong expression of pSTAT-1 in response to IFNs. The identification of differential IFN responsiveness in distinct human trophoblast cell subpopulations provides important new insights into the roles of IFNs in normal pregnancy and placental pathology and infection.
CD103-CD11b+ mucosal DCs control CD4 T cell responses to bacterial flagellin (P3179)
Authors: Atif Shaikh , McSorley Stephen
Published: January 2023

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received less attention. Here, we demonstrate that TLR5 expression by DCs allows a 1000-fold enhancement of T cell sensitivity to flagellin, and this enhancement did not require expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of OVA-specific T cells. In the spleen, the exquisite T cell sensitivity to flagellin was regulated by CD4-CD8α- dendritic cells and was blocked by a monoclonal antibody to TLR5. In mesenteric lymph nodes, flagellin-specific T cell responses are controlled by a population of CD103-CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DCs regulates the adaptive immune sensitivity to flagellated pathogens.
Multifunctional and protective memory γδ T cells in intestinal tissues (P3227)
Authors: Sheridan Brian , Romagnoli Pablo , Pham Quynh-Mai , Fu Han-Hsuan , Alonzo III Francis , Schubert Wolf-Deiter , Freitag Nancy , Lefrancois Leo
Published: January 2023

Recently, studies in humans have suggested that γδ T cells may play a role in adaptive immunity. Surprisingly, oral Listeria monocytogenes (Lm) infection of mice induced a unique population of CD27neg CD44high Vγ4+ γδ T cells in the mesenteric lymph nodes (MLN) and lamina propria that was maintained into memory and capable of rapid expansion following secondary infection. Lm-elicited γδ T cells were mobilized into the blood and upregulated the gut homing receptor α4β7 but failed to migrate into the epithelium. The responding γδ T cells produced IL-17A and IFNγ and expressed T-bet and RORγt. Cytokine expression from memory γδ T cells could be induced by MLN from 2 dpi mice and was reduced by depletion of MHC II-expressing cells. Moreover, batf3-/- mice failed to induce γδ T cell expansion suggesting a role for migrant APCs in the activation of Lm-elicited γδ T cells. Interestingly, Lm-elicited memory γδ T cells failed to expand to i.v. Lm infection or oral Salmonella infection suggesting contextual specificity to the priming pathogen. In the absence of αβ T cell control, memory γδ T cells are the predominate source of protection and limit bacterial burden through enhanced expansion and cytokine production. While depletion of αβ T cells or γδ TCR internalization had little effect on protection, coupling treatments completely abrogated protection in intestinal tissues. Thus, γδ T cells are capable of generating a protective mucosal memory population following oral Lm infection.
Life history of cognate antigen exposure indicates skewing of mucosal lymphocyte populations during aging (P3229)
Authors: Vogel Laura , Moitra Samrat , Heidenreich Byron
Published: January 2023

The impact of aging on mucosal immune responses, particularly IgA production, is controversial. Many previous studies haven’t separated effects of the aged microenvironment (i.e. aged T cells, homing molecule expression, antigen exposure) from intrinsic B cell defects. We hypothesized that life-history of cognate antigen exposure influences function and distribution of mucosal B cell populations during aging. These studies used immunoglobulin transgenic mice, whose cognate antigen nitrophenyl is not naturally encountered. Mice were periodically immunized orally from birth with T cell-dependent or -independent antigens. At various ages, intestines were analyzed for relative abundance, size, and cellular composition of Peyer’s Patches and isolated lymphoid follicles. Results revealed significantly more isolated lymphoid follicles in the distal region of aged mice when compared to young. Comparing immunized and control mice, flow cytometry data reveals a significant decrease in percentages of transgenic B cells in both tissues, and in animals receiving T cell independent antigen there were changes in CD4 and CD8 T cell distribution in aged mice. There was also a significant decrease of transgenic B cells in aged mice immunized with a T cell-dependent antigen for both Peyer’s Patches and isolated lymphoid follicles. Hence, preliminary data suggests significant role of cognate antigen exposure and further elucidates the mechanisms of age-related immune dysfunction.
Epithelial T cell gene expression is altered by obesity and Type 2 diabetes causing T cell exhaustion and barrier dysfunction (P3185)
Authors: Jameson Julie , Costanzo Anne , Clementsmith Zerlina , Cheung Kitty
Published: January 2023

Intraepithelial lymphocytes in tissues such as the skin and intestine play key roles in maintaining barrier integrity via the production of growth factors and cytokines. Obesity and type 2 diabetes induce defects in tissue repair and barrier function causing chronic nonhealing wounds and more severe cases of colitis. Our studies show that as obesity and type 2 diabetes progress, animals exhibit a dramatic decline in the number of epithelial T cells due to impaired T cell homeostatic proliferation. To understand how obesity impacts gene expression in the epithelium, we performed gene chip analysis and qPCR on cells isolated from the epithelium of obese mice. Our studies show that the normal gene expression program in both epithelial cells and intraepithelial T cells becomes disrupted. While T cells exhibit a gene profile indicative of exhaustion with a reduced ability to make growth factors and cytokines, the neighboring epithelial cells downregulate cell adhesion molecules and members of the IL-1 family such as IL-1F6. Returning the animals to a low fat diet does not rapidly restore the ability of T cells to recover their numbers and improve their function. However, blocking the low level of chronic inflammation that occurs with obesity improves T cell function in repair. These data indicate that controlling the myriad of epithelial complications associated with obesity will require turning down chronic inflammation and restoring the normal cellular program in intraepithelial T cells so immune responses at the critical barrier tissues are preserved.
Fli-1 transcription factor affects inflammatory cell infiltration into the kidneys independent of autoantibodies (P4150)
Authors: Sato Shuzo , Williams Sarah , Zhang Xian
Published: January 2023

Background and Purpose: Expression levels of Fli-1, a member of the Ets family of transcription factors, are a mitigating factor in the development of nephritis in lupus model mouse. In this study, we examined the effects of reduced expression of Fli-1 on inflammatory cell infiltration into the kidneys in vivo. Methods: We generated MRL/lpr transgenic mice expressing green fluorescent protein (GFP), and isolated spleen cells from these mice. One million cells were injected into 9 Fli-1 heterozygous (+/-) and 13 wild-type (WT) MRL/lpr mice at the age of 4 to 8 months. Mice were sacrificed 18 hours after injection, kidneys were removed and frozen sections were made. Inflammatory cells with GFP were counted by immunofluorescence microscopy. The expression of chemokines in the kidney tissue was analyzed by real-time PCR. Results: The GFP positive cells were significantly higher in kidneys from WT MRL/lpr mice compared to Fli-1+/- MRL/lpr mice despite similar autoantibody titers found. We identified the majority of these cells as T cells, B cells and macrophages. Fli-1+/- MRL/lpr mice had less proteinuria and the expression level of chemokines in the kidneys had lower trends after GFP cells injection. Conclusion: Our data indicate that Fli-1 expression impacts lupus nephritis development by modulating expression of inflammatory chemokines in the kidney and infiltration of inflammatory cells independent of autoantibodies in MRL/lpr mice.
Transition from acute to persistent infection: Antigen persistence and cytotoxic T cells response in infectious bursal disease virus infected SPF chickens (P6129)
Authors: Rauf Abdul , Vlasova Anastasia , Murgia Maria , Jung Kwonil , Saif Yehia
Published: January 2023

Described by Cosgrove about a half century ago, infectious bursal disease (IBD) is as an acute, highly contagious viral infection of young chickens, which last for 7 to 10 days and leads to immunosuppression. Here, we are reporting for the first time that in IBD virus (IBDV) -infected, specific pathogen free (SPF) chickens bursa, the virus antigen, macrophages, CD4+, CD8+ cells, Fas, Fas ligand (FasL), and caspase-3 were consistently detected by immunohistochemistry up to 56 days post inoculation (DPI). Two-week-old SPF chickens were inoculated intraocularly with standard challenge strain (STC) using a 104embryo infectious dose. Histopathology of bursal tissues revealed severe lymphoid follicular depletion at DPI 5-28. A distinctive pattern of bursal follicles in the form of small and large follicles was detected from DPI 35 to 56. At 42 and 56 DPI a characteristic clustered pattern of IBDV antigen, macrophages, CD4+, CD8+ cells, Fas, FasL, and caspase-3 were detected in or around the small bursal follicles as compared to large bursal follicles. This is the first report on the persistence of IBDV antigen causing a latent infection, in bursal tissues of SPF chicken. Detection of persistent IBDV infection is highly significant in the elucidation of virus pathogenesis, immunology and epidemiology and it raises many questions that need to be answered in future studies.
N-acetylcysteine modulation of JAK2 and JAK3 results in decreased STAT3 activation in human B lymphocytes (P1473)
Authors: Bonnaure Guillaume , Néron Sonia
Published: January 2023

Activation of STAT3 is crucial for human B lymphocyte differentiation and inhibition of its phosphorylation in CD40-activated B lymphocytes results in impairment of in vitro generation of immunoglobulin-secreting cells. Conversely, redox homeostasis plays a role in B lymphocyte maturation. Thus, the presence of antioxidants can potentially modulate their outcome. We showed that N-acetylcysteine (NAC), an antioxidant molecule, can inhibit STAT3 phosphorylation, which is paralleled with strong inhibition of immunoglobulins secretion. Western analysis reveals that NAC-treatment of CD40-activated human B lymphocytes, in the presence of IL-2, IL-4 and IL-10, causes a reduced phosphorylation of JAK2 and JAK3, which are important activators of STAT3. Furthermore, this inhibition appears to be dose-dependant. Our results indicate that other antioxidant molecule such as α-tocopherol and trolox do not affect these two kinases phosphorylation. Other kinases or their inhibitors associated to STAT3 phosphorylation do not seem affected by the presence of antioxidant in the media. In conclusion, these observations reveal that STAT3 phosphorylation down regulation in NAC conditions is due to its association with JAK2 and JAK3.
Metabolic strategies used by long-lived bone marrow plasma cells (P1474)
Authors: Minges Wols Heather , Witte Pamela
Published: January 2023

Maintaining effective humoral immunity is an important aspect of healthy aging and the goal of most vaccines. Evidence from multiple laboratories agrees that long-lived humoral immunity derives from long-lived plasma cells (LLPC) that reside in the bone marrow. Because serum antibodies have a relatively short half-life and must be constantly produced, an urgent need is a clearer understanding of how LLPC maintain secretion of protective antibodies for years or a lifetime. PC secrete an estimated 1000 molecules of antibodies/sec, and some investigators make a compelling argument that the only recourse for a PC is a short lifespan, limited by oxidative and proteosomal stress. Our work begins to address how these important cells survive the extreme metabolic demands of constant antibodies secretion that is essential to lasting, protective immunity. We identified PC using the fluorescent probe ER Tracker - as highly-secretory cells, PC have large amounts of rough ER. In parallel, we stained cells with a reduced MitoTracker probe that fluoresces upon oxidation in respiring mitochondria. Our data show that ER Tracker-hi cells were found in both MitoTracker-hi and -lo populations, suggesting PC may use different metabolic strategies: aerobic respiration and glycolysis. Further studies will assess what governs each metabolic strategy, providing a deeper understanding of the cellular metabolic phenotype of long-lived plasma cells.
Analysis of the differentiation of single antigen-specific B cells early in the primary response (P1477)
Authors: Taylor Justin , Pape Kathryn , Jenkins Marc
Published: January 2023

Following activation with antigen, naïve B cells undergo a robust proliferative expansion and differentiation that results in the production of numerous distinct cell types with different roles in protective immunity. While some of the transcriptional regulators and extrinsic factors directing differentiation are known, these mechanisms cannot account for the heterogeneity of the response. In this study we tracked the response of single naïve B cells and found that individual cells do not differentiate into all of the cell types found within a polyclonal response. Thus, the heterogeneity found within the polyclonal response is in part the result of different naïve B cells differentiating differently. Ongoing work is focused on determining the contributions that B cell receptor affinity and stochastic competition play in differentiation.
The translesion DNA synthesis polymerase Rev1 recruits uracil DNA glycosylase to switch regions and enhances dU glycosylation for immunoglobulin class switch DNA recombination (P1478)
Authors: White Clayton , Zan Hong , Thomas Lisa , Mai Thach , Li Guideng , Xu Zhenming , Zhang Jinsong , Casali Paolo
Published: January 2023

Immunoglobulin (Ig) class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response by diversifying the biological effector functions of antibodies. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine (dC) deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in Ig switch (S) region DNA. Translesion DNA synthesis (TLS) polymerases, such as Rev1, play important roles in somatic hypermutation (SHM), but their role in CSR has not been defined. We found that Rev1 directly interacts with Ung to recruit it to S region DNA and enhance dU glycosylation for CSR. Rev1 targets S regions undergoing CSR in an AID-dependent and Ung-independent fashion. Ung recruitment to S regions, DNA-dU glycosylation and CSR are reduced in Rev1-/- B cells. Together with a mutation spectrum in S region DNA that is similar to that of Ung-/- B cells, our findings suggest that Rev1 operates in the same pathway as Ung during CSR, as emphasized by further decreased CSR in Rev1-/- Msh2-/- B cells. Rescue of CSR by a catalytically inactive Rev1 mutant in Rev1-/- B cells shows that the role of Rev1 in CSR is mediated by Rev1’s scaffolding function, not its enzymatic function. Thus, we describe a novel and important scaffolding function for Rev1 in CSR, which together with its known enzymatic function in SHM highlights a central role of this TLS polymerase in the maturation of the antibody response.
Increased autoimmunity in men with Klinefelter’s syndrome: Sex chromosome dosage effect (P3309)
Authors: Okorogu Rita , Surampudi Prasanth , Valera Isela , Halder Ramesh , Okereke Chetachi , Wang Christina , Swerdloff Ronald , Singh Ram
Published: January 2023

Autoimmune diseases are more common in women than in men. Studies in animal models suggest a role of sex chromosome genotype in modulating autoimmune susceptibility. Here, we begin to translate these murine findings onto humans. Our hypothesis is that X chromosome dosage, rather than the female sex itself, imparts susceptibility to autoimmune diseases. Our results show that levels of IgG anti-chromatin and anti-nucleosome autoantibodies were significantly higher in men with sex chromosome aneuploidy (47,XXY) compared to 46,XY men. XXY men, however, did not have non-specific B cell hyper-reactivity, as the levels of anti-thyroid peroxidase antibody that is associated with autoimmune thyroid disease were similar between the two groups. A preliminary analysis of completed questionnaire revealed an increase in autoimmune and inflammatory conditions when compared with known population prevalence of these diseases. Analysis of immune cell phenotype and responses conducted thus far showed a significant reduction in the frequency of natural killer T cells in 47,XXY men as compared to 46,XY men. These data suggest a role of X chromosome dosage in inducing autoimmunity in humans. Since animals and humans with autoimmune diseases have reduced numbers and/or functions of natural killer T cells, our observation of reduced natural killer T cells in 47,XXY men may be related to increased autoimmunity in these men.
Dual signals of TCR and Notch promote antigen-specific T cell development from pluripotent stem cells (P4370)
Authors: Lei Fengyang , Song Jianxun
Published: January 2023

The pivot in conducting a successful adoptive T-cell immunotherapy for cancer is the obtaining of a large number of naïve phenotyped, stem-cell like tumor antigen specific T cells. Previously we have reported that both conventional and antigen-specific T cells could be generated from induced pluripotent stem (iPS) cells by different methods. Here, we observed that dual signals of TCR and Notch played a significant role in antigen-specific T lineage differentiation from pluripotent stem cells. We used a murine MHC-I restricted, tyrosinase-related protein 2 (TRP2) specific TCR and Notch ligands Delta-like (DLL) 1 and 4 as a model system for inducing iPS cells to differentiate into melanoma-specific CD8+ T cells. Murine iPS cells were retrovirally transduced with TRP2-specific TCR (iPS-TRP2), and then co-cultured on OP9 bone marrow stromal cells overexpressing both DLL1 and DLL4 ligands (OP9-DL1/DL4) in the presence of Flt3L and IL-7. This approach has a better effect in terms of directing an in vitro iPS-TRP2 differentiation towards TRP2-specific CTLs. In addition, adoptive transfer of in vitro developed iPS-TRP2-derived precursor T cells into C57BL/6J mice bearing xenografted B16 melanoma significantly increased anti-tumor immunosurveillance and protected animal from tumor challenge. These data suggest that signals from TCR and Notch can cooperatively promote antigen-specific T cell development from pluripotent stem cells.
Quantitative single-cell functional characterization of CD19-specific chimeric antigen receptor T cells (P4344)
Authors: Varadarajan Navin , Liadi Ivan , Romain Gabrielle , Singh Harjeet , Cooper Laurence
Published: January 2023

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL). In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells, by employing a novel methodology single-cell nanowell screening (SNS).We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells within the 6 hour window of observation. Hierarchical clustering indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity of T cells. CAR+ T cells that secreted IFNγ upon target ligation did not undergo activation-induced cell death (AICD) whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells.
Poly(dG) conjugated protein immunization targets dendritic cells and induces T cell immunity in vivo. (P4528)
Authors: Toussaint Helene , Erdos Geza , Larregina Adriana , Carey Cara , Falo Louis
Published: January 2023

The efficacy of current vaccines results primarily from the induction of protective antibodies. However, we still lack efficacious vaccines against many infectious agents that, like cancer, require durable and protective T cell immunity. Efficient and selective delivery of antigen and adjuvant to antigen presenting cells in vivo remains a critical challenge to vaccine design. Therefore, we have designed an antigen and adjuvant delivery platform based on simple and direct coupling of Poly(dG) oligo deoxynucleotides to protein antigens. In initial studies we found that immunization of naïve animals by direct injection of conjugates of Poly(dG) and Ovalbumin, Poly(dG)-OVA induces efficient priming of potent antigen specific CTL responses, differentiation of naïve CD4 T cells into Th1 cells, induction of specific memory T cell responses and generation of antigen-specific antibody responses. Applying this strategy to HIV, we immunized mice with conjugated Poly(dG)-SIV-Gag and demonstrated potent induction of Gag-specific CTL responses in vivo. Further, in human models we show that intradermal injection of Poly(dG)-antigen conjugates result in efficient uptake by human skin migratory DCs, preferentially dermal DCs, that also acquire potent T cell stimulatory functions. Our data suggests that the Poly(dG)-protein conjugate immunization platform can both selectively target antigens to skin APC and function as an APC-directed adjuvant to induce broad based humoral and T cell immunity.
Disrupted T cell central memory following vaccination in patients with sickle cell disease (P4536)
Authors: Adami Alexander , Szczepanek Steven , Lewis Deirdre , Farrington Nayre , Bracken Sonali , Natarajan Prabitha , Secor, Jr. Eric , Jellison Evan , Thrall Roger , Andemariam Biree
Published: January 2023

Increasing evidence suggests that a systemic state of disrupted immunity and inflammation accompanies sickle cell disease (SCD) despite the erythrocytic nature of the SCD mutation. Frequent infection plagues patients, who require aggressive vaccination from an early age. However, recent experimental and clinical evidence suggest diminished vaccine efficacy in SCD. We hypothesize that patients with SCD exhibit disrupted T cell memory in response to vaccination. We collected blood from nine healthy adult controls and eleven SCD patients before and 4-6 weeks after routine vaccination against influenza virus (IRB #11-026-1). Peripheral blood mononuclear cells were isolated and analyzed by flow cytometry. As previously reported, relative percentages of CD8+ central memory (CM) T cells significantly contracted following vaccination in control subjects (from 48.2 to 39.3, p < 0.01). Conversely, relative percentages of CD8+ CM T cells did not change in SCD patients (44.3 to 44.4, p > 0.05). Further, CD4+ CM T cells were significantly higher in patients with SCD versus controls before vaccination (58.7 vs. 44.7, p < 0.02) and trended higher following vaccination (56.8 vs. 40.7, p = 0.08). These data indicate that patients with SCD exhibit both disrupted CD8+ CM kinetics and altered CD4+ CM levels in the context of vaccination. Our findings suggest that at least a portion of the poor clinical response to vaccination in SCD may arise from disrupted T cell memory.
Metabolic engineering of Salmonella vaccine bacteria to boost human γδ T cell immunity (P4538)
Authors: Workalemahu Grefachew , Wang Hong , Puan Kia-Joo , Kuzuyama Tomohisa , Jones Bradley , Morita Craig
Published: January 2023

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C -methyl-D-erythritol-4-phosphate (MEP) pathway used by most Eubacteria and Apicomplexan parasites, and self isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with synthetic prenyl pyrophosphates or aminobisphosphonates elicit short-term expansion with rapid anergy and deletion. We hypothesized that a live attenuated bacterial vaccine metabolically engineered to overproduce HMBPP would elicit Vγ2Vδ2 T cell immunity by mimicking natural infections. Here we report on an aroA- Salmonella enterica serovar Typhimurium SL7207 strain metabolically engineered by deleting the gene for lytB (the downstream enzyme from HMBPP) and complementing with genes encoding mevalonate pathway enzymes. lytB- SL7207 had extremely high HMBPP levels, infected human cells like the wild type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells in all 6 human donors tested. Moreover, vaccination of rhesus monkeys with live lytB- SL7207 expanded Vγ2Vδ2 T cells without significant side effects. Thus, these studies provide proof-of-principle for the use of metabolic engineering to derive Salmonella vaccines that boost human γδ T cell immunity.
IL-27Rα is required for shaping the magnitude and repertoire of T cells responses to subunit immunization (P4500)
Authors: Pennock Nathan , Kedl Ross
Published: January 2023

A goal of cellular immune vaccines is the generation of large numbers of antigen specific T cells in response to subunit immunization. A broad spectrum of cytokine and cell surface costimulatory molecules are known to shape the magnitude and repertoire of T cells responding to vaccination. We show here the majority of TLR agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4 and CD8 T cells responses. This is in sharp contrast to infectious challenge which generates T cell responses that are IL-27 independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell intrinsic, requiring T cell expression of IL-27Rα. Further, we show that IL-27 dependency is not only necessary for the maximal expression of vaccine elicited T cell responses, but also critical for representation of high affinity T cells in the responding T cell pool. This observation complements the biology from other reports demonstrating dependence of low affinity T cells on the TNFR family member CD27. Collectively our data highlight the unexpected central importance of IL-27 in the generation of robust, high affinity cellular immune response to subunit immunization.
Anti-viral vaccines license T cell responses by suppressing granzyme B levels in human plasmacytoid dendritic cells. (P4510)
Authors: Fabricius Dorit , Nussbaum Bendikt , Busch Danie , Panitz Verena , Mandel Birgit , Vollmer Angelika , Kaltenmeier Christof , Lunov Oleg , Tron Kyrylo , Nienhaus Ulrich , Debatin Klaus-Michael , Jahrsdörfer Bernd
Published: January 2023

Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. Here, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis (TBEV). We show that pDC GrB levels inversely correlate with the proliferative response of co-incubated T cells and that GrB suppression by a specific antibody or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrBhigh, but not GrBlow pDC transfer GrB to T cells, and may degrade the zeta-chain of the T cell receptor in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both anti-viral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.