bluuebunny/crossref_metadata_2025 huggingface dataset:
B cells are required for CD8 T cell recovery after antibody-mediated depletion (TRAN3P.895)
Authors: Ayasoufi Kathy , Wang Xi , Valujskikh Anna
Published: January 2023

Antibody-mediated lymphoablation is used as an induction therapy in transplant patients. We investigated the requirements for T cell recovery following lymphoablation in a mouse model of heart transplantation. Memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts and in non-transplanted B6 mice. Treatment with blocking anti-CD154 mAb plus mATG inhibited CD8 T cell recovery, while agonistic anti-CD40 mAb restored memory CD8 T cell expansion after mATG treatment of CD4 T cell depleted recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFα suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.
Comparative immunological study of of lamellar and penetrating keratoplasty in murine model (TRAN3P.906)
Authors: Lee Hyun Soo , Choi Min-Young , Joo Choun-Ki
Published: January 2023

Lamellar keratoplasty (LK) has been considered an acceptable alternative surgery for penetrating keratoplasty (PKP), because LK has the advantage of lower risk of graft rejection and intraocular complications, according to recent clinical outcomes. But the relative immunological mechanism of graft rejections for the two procedures is uncertain. We have developed a murine model of lamellar keratoplasty to try to understand the immunological mechanisms of those two procedures. Our results demonstrate that LK mice showed less graft rejection than PK mice and LK led to less DTH response and IFN-γ secretion in vitro recall assay of T cells from drainage cervical lymph nodes (LNs) at day 21, as compared to PK (acceptance; acc). There was no difference of mRNA expressions of IL-1β, IL-6, and IFN-γ in the corneas between two groups at day 5, but mRNA expressions of IL-1β and IFN-γ in PKacc mice were significantly increased at day 28. In addition, LK showed less angiogenesis and lymphangiogenesis in grafted cornea than PK, and LK led to decreased number of mature MHC-IIhighCD11c+ antigen presenting cells (APCs) in the draining LNs of transplanted mice, compared with PKacc. In conclusion, these results show that LK presents less graft rejection than PK through lower angiogenesis and lymphangiogenesis, which could make pathologic T cells and APC less migration into drainage LNs and grafted corneas, respectively.
Inhibition of indoleamine 2,3-dioxygenase enhances graft-versus-leukemia effect after donor leukocyte infusion in mice: differential efficacy of 1-methyl tryptophan isomers (TRAN3P.907)
Authors: Lim Ji young , Park Gyenogsin , Choi Eun Young , Min Chang-Ki
Published: January 2023

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through kynurenine (KYN) pathway, has an important role in immunomodulation. Moreover, it was shown that treating mice with 1-methyl tryptophan (1-MT), a small-molecule inhibitor of IDO, could break the tolerogenic state, providing antitumor responses. Despite the role of IDO in the induction of tumor immune tolerance, the direct role of IDO inhibition on GVL and GVHD still remains obscure. We established a murine GVL model. Recipient mice were sublethally irradiated and were transplanted with donor bone marrow cells on day 0. Recipient mice were receiving with or without DLI on day 14. Two isomers of IDO inhibition (D-1MT and L-1MT, 100mg/kg) were administered for 5 days per week from the day of P815 cell inoculation until 4 weeks. Both 1-MT isomers administration reduced leukemia growth in mice with CC. On the other hand, L-1MT but not D-1MT reduced leukemia growth in mice with MC. IDO expressions was increased in TDLN and tumor tissues of the CC mice but not in the MC mice. Accordingly plasma concentrations of KYN were increased in the CC mice on day 14 and treatment of both 1-MT isomers abrogated its production. These results demonstrate both isomers of 1-MT enhanced DLI-induced GVL after transplantation, possibly mediated by inhibition of IDO activity in the tumor microenvironment. L-1MT but not D-1MT may directly abrogate leukemia growth regardless of the expressions of IDO.
Islet allograft destruction in NOD mice is independent of donor MHC expression (TRAN3P.870)
Authors: Burrack Adam , Beilke Joshua , Kupfer Tinalyn , Beard K. Scott , Coulombe Marilyne , Gill Ronald
Published: January 2023

It is unclear whether autoimmune disease recurrence overlaps with allograft immunity in the autoimmune diabetic recipient of a pancreatic islet transplant. We grafted autoimmune NOD mice with either syngeneic NOD or allogeneic C57Bl/6 (B6) islets that were genetically deficient in MHC class I, MHC class II, or both MHC class I and MHC class II. NOD.RAG-/- or MHC II-deficient NOD islets were acutely destroyed, but depletion of CD8+ T cells significantly prolonged graft survival. Most NOD MHC I-deficient or MHC I/II-deficient islets survived long-term (>100 d). In contrast, B6 allografts that were MHC sufficient, or deficient in MHC class I, class II, or both were acutely rejected. Allograft rejection was CD4+ T cell dependent and CD8+ T cell independent. Of note, islet allograft rejection in non-autoimmune recipients requires donor islet MHC I or MHC II expression. Lastly, autoimmune disease recurrence was accelerated by depletion of CD25+ cells. We hypothesize that autoimmune disease recurrence is primarily a CD8+ T cell mediated response restricted by donor MHC class I molecules and is delayed by CD4+CD25+ regulatory T cells. In contrast, results suggest that CD4+ T cells play a predominant role in allograft rejection in the autoimmune recipient. We propose the lack of down-regulation of the anti-allograft response and the presence of primed autoreactive T cells synergize to produce the rapid allograft rejection observed in the autoimmune islet transplant recipient.
Oral rapamycin (eRapa) safely prevents carcinogen-induced dermal carcinogenesis through an interferon-γ-dependent mechanism (TUM7P.931)
Authors: Dao Vinh , Hurez Vincent , Pandeswara Sri Lakshmi , Cardenas Kim , Sun Lishi , Liu Aijie , Hasty Paul , Sharp Z. Dave , Curiel Tyler
Published: January 2023

eRapa extends life in mice and cancer prevention could be a mechanism. Rapamycin inhibits mTOR, which has significant immune effects that are surprisingly unstudied in cancer therapy or prevention. We hypothesize that cancer prevention by eRapa is mediated by improved cancer immune surveillance, which we tested in the well-established DMBA/TPA carcinogen-induced skin cancer model. Mice got eRapa or control chow, and skin tumors were induced with DMBA/TPA over 24 weeks. eRapa reduced benign (p=.001) and malignant (p=.05) tumors in WT mice. T cells and IFN-γ mediate cancer immune surveillance. eRapa reduced skin tumors in βδ KO mice lacking all T cells (p=.04), but not in IFN-γ KO mice (p=.13), consistent with loss of beneficial eRapa-induced, non-T cell IFN-γ. In support, WT or IFN-γ KO T cell transfer into IFN-γ KO mice did not alter eRapa cancer prevention in DMBA/TPA. In WT mice on DMBA/TPA, eRapa increased IFN-γ-producing natural killer cells (p=.01) that could mediate skin cancer immune surveillance, and decreased CD34+CD49fint skin cancer stem cells (p=.01) and CXCR3+ T cells (p<.001) that contribute to cancer in this model. eRapa reduced skin pAKT with divergent mTORC1/2 effects needing more study. eRapa appeared safe (no increased Tregs or reduced protection in infection and autoimmunity models). A widely applicable, safe and tolerable cancer prevention agent would be highly useful. Understanding its immune mechanisms could improve efficacy and widen applications.
Characterization of a novel anti-CD20 StradobodyTM with potent in vitro anti-tumor activity (TUM7P.935)
Authors: Zhang Xiaoyu , Burch Erin , Chen Shaodong , Sallin Michelle , Olsen Henrik , Block David , Strome Scott
Published: January 2023

Despite the efficacy of anti-CD20 mAb in the treatment of B-cell malignancies, a proportion of patients are non-responsive or relapse after mAb therapy. Therefore, developing more effective mAbs represents a major area of interest. In this study, we assessed a novel anti-CD20 antibody (stradobodyTM from Gliknik Inc.) consisting of an unmodified Fab sequence and a multimerized Fc domain. In vitro, the anti-CD20 stradobodyTM mediates significantly enhanced inhibition of cell proliferation on select B lymphoma cell lines relative to the unmodified anti-CD20 mAb. In addition, B lymphoma cell lines exhibit increased susceptibility to Complement Dependent cytotoxicity in the presence of a low concentration of the anti-CD20 stradobodyTM. However, anti-CD20 stradobodyTM did not elicit enhanced anti-tumor efficacy in vivo in a Raji-scid xenograft model, in comparison with unmodified anti-CD20 mAb. This discrepancy of in vitro and in vivo anti-tumor efficacy of anti-CD20 stradobodyTM might be attributable to less avidity of the modified human Fc domain to the murine receptors compared with human receptors and highlights the challenges with validating novel targeted mAb therapeutics in vivo.
Role of IL-13 and its receptors in the pathogenesis of cutaneous T-cell lymphomas (TUM7P.961)
Authors: Fuschiotti Patrizia , Devlin Jason , Stolz Donna , Viragova Sara , Falo Lou , Geskin Larisa
Published: January 2023

Cutaneous T-cell lymphomas (CTCL) affect primarily skin and are characterized by proliferation of mature CD4+ T-helper cells. Diagnose is difficult in the absence of specific markers for malignant lymphocytes. The pattern of cytokine production in the skin and blood is considered to be of major importance for the pathogenesis of CTCL. Abnormal cytokine expression in CTCL may be responsible for enhanced proliferation of the malignant cells and/or the depression of the anti-tumor immune response. Cytokine IL-13 promotes growth or survival of several tumors as well as suppresses immunosurveillance. Here we show that IL-13 and its receptors IL-13Rα1 and IL-13Rα2 are highly expressed in the clinically involved skin of CTCL patients. We also show that the tumor cells, identified by the expression of CD4 and TOX (thymus high-mobility group box), in the skin and blood of CTCL patients produce IL-13 and express both receptors. This finding is intriguing since CTCL is a T-cell origin tumor yet T cells do not express IL-13 receptors. Thus IL-13 is implicated as a possible autocrine factor for CTCL. Furthermore expression of the decoy IL-13Rα2 by CTCL cells may represent a novel marker of CTCL malignancy. Soluble human IL-13Rα2 and an anti-IL-13 monoclonal antibody are being tested for inhibition of tumor cell proliferation in the peripheral blood of patients. Altogether these results identify potential tumor markers for the early diagnosis of CTCL as well as novel therapeutic targets.
Constitutive signaling through Myd88 contributes to tumor-derived CCL2 expression and nuclear translocation of NFkB p65, p52, Fos and ATF2 (TUM7P.923)
Authors: Kurt Robert , Serrano Antonio , Boateng Kofi , Parsons Victoria , Phuong Tiffany , Ricca Jacob , Tucker Kyle , Eidelman Alec , Carey Maureen
Published: January 2023

Previous data in our lab suggested that there may be constitutive signaling through the Myd88 signaling cascade in murine mammary carcinoma. Here we extend these findings by showing that the myddosome complex is preformed in the tumor cells. Using co-immunoprecipitation we found Myd88 associated with IRAK2 and IRAK4. After screening many different products of the TLR signaling cascade we found that the most clear consequence of this Myd88 signaling in the tumor cells was expression of CCL2. Targeting Myd88 expression using RNA interference, or function using an inhibitory peptide, resulted in reduced CCL2 expression in a dose dependent manner. Coinciding with Myd88 dependent expression of CCL2 we also detected presence of the CCL2 transcriptional factors NFkB p65, p52, Fos and ATF2 in the nucleus of the tumor cells, and the levels of these proteins in the nucleus decreased upon treatment with the Myd88 inhibitory peptide. Targeting Myd88 using the inhibitory peptide, constitutive shRNA expression, or tetracycline inducible shRNA expression also resulted in a reduced tumor growth rate in vitro. Despite this we found limited therapeutic potential of targeting Myd88 in vivo using the inhibitory peptide alone. Collectively, these data imply that constitutive Myd88 signaling contributes to tumor-derived CCL2 expression, and suggests that alternative mechanisms to target Myd88 in vivo warrants investigation.
ATM-deficiency in the absence of T cells promotes the development of B cell lymphomas with dependence on NF-kB (TUM7P.927)
Authors: Hathcock Karen , Padilla-Nash Hesed , Camps Jordi , Triner Daniel , Shin Dong-Mi , Maul Robert , Gearhart Patricia , Morse Herbert , Ried Thomas , Hodes Richard
Published: January 2023

The protein product of the ataxia-telangiectasia mutated gene, ATM, is a kinase that plays a central role in maintaining genomic integrity and protecting cells from oncogenic transformation. Mutations of the human ATM gene cause ataxia-telangiectasia, an autosomal recessive disorder that is associated with increased incidence of malignancies, especially lymphoid tumors. ATM-deficient mice have been useful tools to study thymic lymphomas, which occur at high frequency in these mice, but the early onset of these tumors has prevented analysis of other types of malignancies. In this study we generated ATM- and CD3ε-deficient mice that lacked T cells. These mice do not develop thymic lymphomas, but routinely develop early onset B cell lymphomas that express surface markers characteristic of mature activated B cells. These B cell lymphomas resemble human diffuse large B cell (DLBC) lymphomas and possess complex karyotypes with genetic amplifications, deletions and unbalanced translocations. Interestingly all lymphomas (10/10) contain an amplification of a 5Mb region on chromosome 18 containing MALT1. These lymphomas also express NF-kB and are dependent on MALT1 and NF-kB signaling for in vitro survival. As such they represent a mouse model for NF-kB- dependent human DLBC lymphomas of the ABC- subtype.
Activation of natural killer T cells by liposomal glycosphingolipids increases the efficacy of liposomal doxorubicin against DMBA-induced tumors in mice (VAC10P.968)
Authors: Khan Masood , Aljarbou Ahmad , Aldebasi Yosef
Published: January 2023

Natural Killer T (NKT) cells show potent antitumor activity upon activation with their specific ligands. Glycosphingolipids (GSLs) isolated from Sphingomonas paucimobilis were incorporated into DPPC-liposomes to prepare GSL-incorporated liposomes (GSL-liposomes). Treatment of tumor-bearing mice with a combination of GSL-liposomes and doxorubicin liposomes resulted into slowing down of the progression of tumors and increase in the survival of mice. Splenocytes obtained from mice treated with GSL-liposomes produced higher levels of IFN-γ and IL-12 compared to mice that did not receive GSL-liposomes. Mice pretreated with GSL-liposomes showed low frequency of tumors after DMBA exposure. These findings suggest that co-administration of NKT cells activating GSL-liposomes and anti-cancer drug loaded liposomes may be proved a better option for the treatment of cancer.
PR1-specific cytotoxic T lymphocytes expanded from cord blood are functional in vitro (VAC11P.1002)
Authors: Wan Liping , St. John Lisa , He Hong , Clise-Dwyer Karen , Cooper Laurence , Sphall Elizabeth J , Bollard Catherine , Molldrem Jeffrey
Published: January 2023

Introduction Neutrophil elastase (NE) and proteinase 3 (P3) are serine proteases often found overexpressed in myeloid leukemic cells. PR1 is a nine amino acid, HLA-A2 restricted peptide derived from NE and P3. The frequency of PR1-specific T cells (PR1-CTL) is very low in healthy adult peripheral blood (0.0005 to 0.05% of CD8+ T cells), but much higher in umbilical cord blood (UCB) (0.007 to 0.345% of CD8+ T cells, N=57). We have previously shown sort-purified UCB PR1-CTL to be active against AML in a murine model. We now investigate the potential of UCB PR1-CTL to undergo expansion while maintaining the anti-leukemic properties of the cells. Methods HLA-A*0201 transfected K562 cells were loaded with PR1 peptide and irradiated with 3000 cGy. UCB mononuclear cells were stimulated with K562/A2/PR1 cells once a week for three weeks. CD8+ T cells were then negatively isolated from the expanded population by magnetic beads and underwent intracellular cytokine staining. Results Prior to expansion, the median percentage of PR1-CTL in the 9 UCB units tested was 0.211% (0.00%-0.348%) of CD8+ T cells. After stimulation this percentage increased to 0.958% (0.346%-2.1%). The expanded UCB PR1-CTL was of memory phenotype (CD45RA-/CCR7-). In addition, expanded UCB PR1-CTL produced both IFN-γ and TNF-α in response to PR1 peptide-loaded T2 cells. Conclusion PR1-CTL can be expanded from UCB and these expanded cells produce cytokines in a specific manner in response PR1-peptide loaded targets.
Lack of p53 augments anti-tumor functions of cytolytic T cells (VAC11P.1005)
Authors: Banerjee Anirban , Thyagarajan Krishnamurthy , Kesarwani Pravin , Mehrotra Shikhar
Published: January 2023

Various effects of altered p53 expression are known with reference to uncontrolled cell proliferation, senescence and cell death. Loss of p53 also induces spontaneous inflammatory lesion and Treg deficiency. Only minor differences in proliferation, apoptosis, and maintenance of ‘non-self’ viral antigen specific T cells have been previously reported. However, the role of p53 in controlling T cell response to ‘self’ tumor epitope has not been comprehensively investigated. The h3T TCR transgenic mice with human tyrosinase epitope reactive T cells were thus developed on p53 deficient background and in vitro/in vivo analysis performed. Our data shows that TCR transgenic T cells from p53 KO mouse in comparison to wild type T cells exhibited increased proliferation, higher IFN-g secretion, enhanced cytolytic capacity, higher expression of stemness gene signature and were less susceptible to cell death upon restimulation with the cognate antigen. The effect of p53 inhibition on increased glycolytic effector functions was mirrored when treating human TCR transduced T cells with p53 inhibitor pifithrin-µ. Finally, the increase in effector function after p53 inhibition also correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of T cells. Thus, our data highlights the key role of p53 in metabolically regulating the tumor epitope specific T cell response and has potential translational significance in adoptive T cell therapy.
Comparative combination cancer immunotherapy with vaccination, checkpoint inhibition and TNFRSF stimulation (VAC12P.1021)
Authors: Schreiber Taylor , Podack Eckhard
Published: January 2023

Therapeutic tumor immunity requires the presence and activation of tumor antigen specific CD8+ T cells and migration of activated tumor-antigen specific CD8+ T cells into a tumor microenvironment where immunosuppressive barriers have been eliminated. Antibody mediated blockade of CTLA-4 and PD-1 is a clinically effective strategy to dampen tumor mediated immunosuppression in a minority of patients with advanced cancer, and this approach may be potentiated through vaccination to prime additional CTL clones and through direct stimulation of T cell costimulatory molecules of the TNF superfamily. Here we provide a systematic comparison of anti-tumor vaccination (3-MCA sarcoma and B16 melanoma) with either heat shock protein gp96-Ig or traditional peptide/adjuvant vaccines given alone or in combination with CTLA-4, LAG-3 or PD-1 blockade and direct T cell costimulation via OX40, 4-1BB and TNFRSF25. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine induced CD8+ T cell proliferation following both primary and secondary antigen challenge. When combined with checkpoint inhibition in therapeutic murine orthotopic tumor models, synergistic anti-tumor activity was observed. These studies highlight the complementary activity of vaccination, checkpoint inhibition and direct T cell costimulation, which may guide the application of combination immunotherapy for effective anti-tumor immunity.
Targeting multiple ErbB family members by EGFR peptide immunization. (VAC12P.1016)
Authors: Doyle Hester , Koski Raymond , Bonafé Nathalie , Bruck Ross , Tagliatela Stephanie , Gee Renelle , Mamula Mark
Published: January 2023

A major breakthrough in cancer treatment has been the development of monoclonal antibodies targeting tumor antigens. A majority of solid tumors overexpress the epidermal growth factor receptor (EGFR). Current treatments for EGFR overexpressing tumors include anti-EGFR monoclonal antibodies alone or in conjunction with standard chemotherapy or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in some patients, drawbacks include the high cost of treatment, side effects associated with multiple drug infusions, and the development of antibody-resistant tumors. As an alternative to monoclonal antibody treatment, we have focused on peptide-based vaccination to trigger long lasting natural anti-tumor antibodies. Mice immunized with membrane-proximal epitopes in the extracellular domain of EGFR generate antibodies against EGFR as well homologous regions of the EGFR family members HER2 and HER3. HER2 and HER3 form heterodimers with EGFR and compensate for the lack of signaling through EGFR. EGFR peptide immune serum inhibits in vitro growth and promotes tumor killing of both human EGFR+ and HER2+ EGFR- tumor cells. A multi-specific tumor vaccine that targets multiple EGFR family members will provide greater tumor targeting than single agents alone while providing long-lasting immunity.
Bovine central memory T cells are highly proliferative in response to bovine tuberculosis infection. (VET2P.1033)
Authors: Fernanda Maggioli Mayara , Palmer Mitchell , Vordermeier H. Martin , Whelan Adam , Waters Wade Ray
Published: January 2023

Long-term (i.e., 14 days) cultured IFN-γ ELISPOT assays measure central memory T cell (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-γ ELISPOT responses correlate with protection. In other species, Tcm’s posses low activation threshold and are highly proliferative. It has been shown that Tcm’s accounts for 75% of long-term cultured cells. The objective of this study was to access the proliferative capability of Tcm cells compared to 6 day cultured cells (n = 6) in response to aerosol Mycobacterium bovis infection. Long-term cultured PBMCs from infected cattle were stimulated with M. bovis purified protein derivative (PPDb), rESAT-6:CFP10 (E:C), rTB10.4 and rAg85A for 13 days. Next, cells were stained with CellTrace dye and re-stimulated with E:C in the presence of fresh autologous adherent cells for an additional six days (20 days). In response to E:C, long-term cultured were mainly CD4+ cells and were more proliferative (~ 48%) than 6d cultured cells (27%; p=0.001). Long-term cultured cells were: ~30% Tcm (CCR7+, CD45RO+), ~48% Tem (CCR7-, CD45RO+), ~8% effector (CCR7-, CD45RO-) and <1% of naïve cells (CCR7+, CD45RO-). Six day cultured cells were: ~16% Tcm (p=0.01), ~47% Tem, ~42% effector (p=0.001) and < 1% naïve cells. These findings demonstrate that bovine Tcm cells present high proliferative capability as compared to short-term cultured cells.
Sequence and structural models of a bovine monoclonal antibody Fv region specific for bovine herpesvirus 1 glycoprotein D (VET2P.1037)
Authors: Levings Randall , Roth James
Published: January 2023

The cDNA of heterohybridoma mRNA encoding the variable domains of the light (VL) and heavy (VH) chains of an IgG1 bovine monoclonal antibody (MAb) neutralizing bovine herpesvirus 1 was commercially synthesized, cloned and sequenced. The variable (V) light chain sequence had highest identity with members of the V lambda family, 1a subfamily, and the joining (J) gene JL3, both commonly expressed in cattle. The V heavy chain sequence had highest identity with members of the VH1 gene family and JH1, also both commonly expressed in cattle. The VH nucleotide sequence had highest identity with the bovine diversity (D) gene DH3. The derived amino acid sequences were used to examine the complementarity-determining regions (CDRs) according to multiple systems. CDRL1-3 and CDRH1-2 were identified as belonging to or being similar to known canonical classes, and results from “H3 rules” were determined for CDRH3. Models of the Fv structure were derived using three on-line software packages. Overlapping sets of rodent and human MAb were used by the packages as templates for each framework and CDR structure individually. Predictions of the CDRL3 and CDRH3 configurations were more variable than that of other CDRs, and resulted in different paratope models. The results suggest sequences and crystal structures of MAb from a variety of species may be useful in describing additional canonical structures of immunoglobulins.
Impaired antibody effector function during persistent viral infection (VIR7P.1049)
Authors: Yamada Douglas , Elsaesser Heidi , Brooks David
Published: January 2023

Persistent viral infections are associated with dysfunctional B cell responses including polyclonal B cell activation, non-virus-specific hypergammaglobulinemia, and ultimately the inability to control infection. Using the lymphocytic choriomeningitis virus (LCMV) system of murine infection, we addressed whether similar to other antiviral mechanisms, the suppressive immune environment generated during persistent infection impacted antibody effector function. We demonstrate that high levels of circulating antibodies generated during persistent infection (termed hypergammaglobulinemia) impede antibody dependent phagocytosis and cell killing. As a result, otherwise highly efficacious cell killing antibodies are ineffective during persistent virus infection. Since antibodies are essential to the control of persistent infection, these observations have important implications immunologically for understanding the complex control of viral replication by antibodies as well as therapeutically for antibody immunotherapies aimed at viral clearance and secondary co-infections. Thus, we have identified a previously unrecognized mechanism of immune dysfunction during viral persistence with important implications for understanding the mechanisms of antibody function/dysfunction and affecting their ability to kill infected cells to control persistent virus infection.
Polyclonal CD4 TCR Affinities are not altered in chronic viral infection (VIR7P.1058)
Authors: Andargachew Rakieb , Martinez Ryan , Evavold Brian
Published: January 2023

The range of polyclonal CD4 T cell receptor (TCR) affinities that participate during the response to viral infection is currently unknown. Utilizing a highly sensitive two-dimensional micropipette adhesion frequency assay (2D), we determined the TCR affinity and frequency of CD4 T cells that expand and survive throughout multiple phases of the chronic and acute lymphocytic choriomeningitis virus (LCMV) T cell response. We discovered consistently more (4-5 fold) CD4 T cells specific for the IAb GP66-77 LCMV epitope than detected with pMHC II tetramer. The 2D identified lower affinity cells participate in the anti-viral response and are apparent at all time points of acute and chronic infection. Average TCR affinities measured at peak and later stages of the chronic response remained constant and equivalent to corresponding acute response measurements. However, programmed death 1 (PD-1) expressing CD4+ T cells late in chronic infection are functionally unresponsive despite static affinities. In contrast, memory cells in the acute response have a higher functional avidity compared to peak effectors (produce IFNγ in response to lower amount of peptide antigen) yet T cell memory is not restricted to select TCR affinities. Our findings suggest long term antigen exposure and functional enhancement/impairment exert no selective pressure in the survival or deletion of specific CD4 TCR affinities and TCR affinity alone does not always predict biological response.
Interaction of structurally diverse HSPs with ligand binding domains of CD91 (APP2P.110)
Authors: Mizote Yu , Pawaria Sudesh , Binder Robert
Published: January 2023

Select members of the Heat Shock Proteins (HSPs) are molecular chaperones, carrying diverse cargo including nucleotides, lipids, misfolded proteins and peptides. Immunization of mice or humans with HSPs leads to priming of T cell responses specific for the peptide antigens that they chaperone. HSPs, with the chaperoned peptides, in the extracellular environment have been shown to associate with the cell surface receptor CD91 expressed on antigen presenting cells (APCs). CD91 on APCs allow for endocytosis of HSP-peptide complexes and cross-presentation. Recently, we discovered that the same receptor CD91 acts as a signaling receptor for HSPs. HSPs were shown to cause the phosphorylation of the cytoplasmic tail of CD91, and thereby dictate the release of cytokines in a CD91-dependent manner. In this study, we aim to identify the domains on CD91 that are necessary for HSP binding. CD91 is expressed as a single polypeptide chain in the ER that is cleaved into the extracellular 515 kDa α-chain, included four ligand-binding domains, and the 85 kDa β-chain. By creating mini-gene constructs representing the ligand binding domains of CD91, the association of each HSP with each construct can be examined. The completion of this study will explain how the structurally diverse HSPs bind to a single receptor, necessary for priming immune responses.
T cell cross-reactivity to self peptide:MHCII shapes the foreign peptide: MHCII-specific CD4+ T cell repertoire (APP2P.112)
Authors: Nelson Ryan , Beisang Daniel , Jenkins Marc
Published: January 2023

The sizes of naïve CD4+ T cell populations specific for different foreign peptide:major histocompatibility complex II (p:MHCII) ligands vary by several orders of magnitude. This variability is biologically significant because large naïve populations generate more effector cells during an immune response than small ones. We hypothesized that the size of a given foreign p:MHCII-specific population is influenced by negative selection due to cross-reactivity between the foreign peptide and self peptides. We found that the frequency of naïve T cells specific for a given foreign p:MHCII ligand was inversely related to the number of self peptides predicted to bind MHCII and sharing 4 TCR contact amino acids with the foreign peptide. In addition, foreign p:MHCII-specific populations that were very small in normal mice were preferentially increased in mice with defective negative selection. Furthermore, gene-targeted mice lacking individual self peptides predicted to be involved in negative selection showed increased responsiveness to cross-reactive foreign peptides. These results suggest that immunodominant foreign peptides have this property because the corresponding naïve CD4+ T cell population has been subjected to minimal negative selection on cross-reactive self peptides.
A unique human-like CD8+ iNKT cells in a humanized mouse model (APP2P.113)
Authors: Wen Xiangshu , Kim Seil , Lawrenczyk Agnieszka , Xiong Ran , Rao Ping , Casorati Giulia , Dellabona Paolo , Yuan Weiming
Published: January 2023

The distinct differences in CD1d/NKT lipid presentation systems in humans and mice largely account for the minimal success of Natural Killer T (NKT) cell-based anti-tumor clinical trials in humans, contrasting their strong anti-tumor effects in conventional mouse models. We aim to humanize mice at the molecular level to better model human CD1d/NKT lipid presentation in vivo. Previously, we reported a novel human CD1d knock-in (hCD1d-KI) mouse, which contain a human-like abundance of iNKT cells yet possess potent anti-tumor immunity by the iNKT cells (Proc. Natl. Acad. Aci. USA 110: 2963-8, 2013). To further humanize the CD1d/NKT lipid presentation system, we have introduced the human Vα24 TCR transgene into the KI mice. These mice have comparable numbers of iNKT cells in thymus and spleen, but significant reduced numbers in liver comparing to Vα24Tg mice. These iNKT cells predominantly express mouse Vβ8 TCR, the homolog of human Vβ11, with reduced CD4 expression. Importantly, we detected in these mice a unique population of CD8+ iNKT cells, which exist in human, but not in wild type mice. Bone marrow transfer experiments suggested that CD8+ iNKT cells are hCD1d-dependent. These CD8+ iNKT cells produce cytokines upon activation and are cytotoxic to B16 melanoma cells in vitro. The hCD1d KI-Vα24Tg mice will allow more accurate in vivo modeling of the human CD1d lipid presentation to human iNKT cells and will facilitate the preclinical assessment of iNKT cell-based therapies.
Unusual antigen recognition provides clue to pathogenicity of diabetogenic CD8 T cell (APP2P.101)
Authors: Mukherjee Gayatri , Lamont Deanna , Prakash Rajesh , Samanta Dibyendu , Almo Steven , DiLorenzo Teresa
Published: January 2023

The defined length restriction as well as preference for amino acids at particular positions for each MHC allele is imposed by the physical as well as chemical nature of the peptide binding groove of the MHC molecule. Accordingly, the preferred peptide for the mouse MHC class I allele H-2Db is a 9mer with anchor residues N at P5 and M/I or L at position 9. However, in this study, we found that AI4, one of the most pathogenic CD8 T cell clones that participates in type 1 diabetes pathogenesis, recognizes an unusual 7mer peptide from the β cell specific protein insulin (InsA14-20) in the context of H-2Db. Further investigation showed that a majority of NOD mice had islet infiltrating T cells that recognized InsA14-20 or mimotopes derived from it, making the AI4-like T cells a very prevalent population. Crystallographic analysis revealed that InsA14-20 shares two solvent-exposed residues with all previously identified AI4 ligands which could explain the promiscuous nature of the AI4 T cell receptor. In addition, accessory anchoring residues were observed to compensate for the lack of the C-terminal anchor residue. In sum, the study demonstrates that less than ideal peptide-MHC interaction can still trigger T cell reactivity and contribute to disease pathogenesis, aided further by lack of thymic negative selection due to weak binding affinity.
Interaction of complement-coagulation factors in ischemia/reperfusion-induced injury in autoimmune prone (B6.MRL/lpr) mice (BA3P.205)
Authors: Moratz Chantal , Peng Suzette
Published: January 2023

Studies in the B6.MRL/lpr strain using the ischemia/reperfusion tissue injury (IR) model have demonstrated that complement activation is a key modulator of inflammatory tissue damage. The role of various components of the complement pathway (activation vs. deficiency) and the use of complement inhibition as therapeutic agents are areas of active research for systemic lupus erythematous (SLE). C1q inhibition is acutely effective in attenuating tissue injury in B6.MRL/lpr strain. Studies in our laboratory identified a C3-independent mechanism of complement C5a generation, terminal tissue injury, and systemic inflammation. The studies suggest a pathway which bypasses C3 by interacting with the coagulation cascade. Using the mesenteric IR model, we compared the extent of intestinal injury after IR when C1q inhibitors or coagulation inhibitors were administered. In our model C1q inhibition resulted in decreased tissue damage, thrombin generation, and C5a generation. Tissue Factor pathway inhibitor (TFPI) and anti-thrombin III attenuated tissue injury, thrombin generation, and C5a production but not C3b production. Interaction between thrombin and the complement pathway in the autoimmune mouse has significant clinical implications - especially in autoimmune diseases in which inflammation and hypercoagulability are known manifestations.
Involvement of necroptosis in the development of imiquimod-induced psoriasis-like dermatitis (BA3P.135)
Authors: Honda Tetsuya , Kabashima Kenji
Published: January 2023

Necroptosis is a form of programed cell death, in which receptor-interacting protein (RIP)1 and subsequent RIP3 kinase activation is a central pathway for its development. Necroptotic cells induce inflammation through the release of various cytokines or necrotic danger-associated molecular patterns. Recent reports indicated that mice with keratinocyte-specific necroptotic cell death exhibit severe chronic skin inflammation, which suggests the involvement of necroptosis in the pathogenesis of chronic skin inflammatory diseases, such as psoriasis. In this study, we investigated the involvement of necroptosis in the pathogenesis of psoriasis using an imiquimod (IMQ)-induced mouse psoriasis model. Daily application of IMQ to skin caused skin inflammation with psoriariform scaly erythema with epidermal and dermal thickening. In the skin, we detected positive signals of TUNEL both in the epidermis and dermis, suggesting the induction of programmed cell death in skin by IMQ application. In addition, RIP3 expression was significantly upregulated both in mRNA and protein level in the skin lesions. Administration of necrostatin-1, a specific inhibitor of RIP1 kinase, significantly reduced the severity of IMQ-induced psoriasis-like dermatitis. Immunohistochemical analysis revealed positive signals for RIP3 in the human psoriasis skin lesion. These results indicate the involvement of necroptosis in mouse psoriasis model, and suggest it's involvement in the pathogenesis of psoriasis.
Novel, critical roles of autophagy in dendritic cells and neutrophils to mediate experimental autoimmune encephalomyelitis (EAE) in mice (BA3P.201)
Authors: Bhattacharya Abhisek , Eissa N Tony
Published: January 2023

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, affects more than a million people worldwide. Insufficient understanding of pathogenesis has precluded curative therapy for MS. Autophagy, a conserved bulk degradation process, is important in several immune functions, such as antigen presentation and infection control, but the potential roles of autophagy in regulating autoimmune diseases including MS are not well understood. To test our hypothesis that autophagy in innate immune cells is required to mediate MS, we generated conditional autophagy deficient mice where Atg7 or Atg5, essential autophagy genes, were deleted from dendritic cells (DC) or myeloid cells, induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, and monitored disease severity. We found that autophagy deficiency in neutrophils ameliorated EAE by reducing neutrophil degranulation, resulting in failure of breakdown of blood-central nervous system barrier, a process required for EAE progression. Moreover, autophagy deficiency in neutrophils resulted in global reduction in neutrophil mediated-inflammation. Autophagy deficiency in DC, on the other hand, reduced both incidence and severity of EAE, secondary to decreased antigen presentation to T cells in the periphery. Our data show that autophagy is required for neutrophil-mediated inflammation. Our data further suggest that autophagy is a potential target for treating autoimmune diseases such as MS.
Dysregulated CD8+ T cells promote B cell activation and antibody-mediated autoimmune disease (BA2P.123)
Authors: Hoyer Katrina , Gravano David , Sadiqi Sharif , Sanders P.
Published: January 2023

IFNγ-producing CD4 and CD8 T cells accumulate during autoimmune disease in IL-2-KO mice. CD4 T cells are necessary for disease progression, and we have previously shown that IL-2-KO CD4 T cells can transfer autoimmunity. However, the importance of CD8 T cells to initiation and propagation of disease is unclear. Several groups have demonstrated a protective role for CD8 T cells in inflammation and autoimmunity, while others have suggested a pathogenic role. To define the function of CD8 T cells during spontaneous autoimmune disease, we depleted CD8 T cells in IL-2-KO mice and evaluated the pathogenesis of autoimmunity. Elimination of CD8 T cells for only two weeks resulted in a significant augmentation of survival, reduction in the number and activation state of the CD4 T cells and B cells, and a delay in the development of RBC-specific antibodies. In untreated IL-2-KO mice, both CD4+ and CD8 T cells produced IFNγ in response to RBC antigens, indicating that RBC-specific CD8 T cells are present during disease. Our results suggest that autoreactive CD8 T cells contribute to the initiation or propagation of lymphoproliferation and autoimmunity. We are addressing the relative importance of autoreactive CD8 T cells to that of the CD4 T cells, and are evaluating the mechanisms by which CD8 T cells influence the autoimmune disease process. Early data indicate that IL-2-KO CD8 T cells produce elevated levels of perforin, granzyme B, and IFNγ, and induce B cell proliferation.
T follicular helper cells contribute to autoimmunity through the BCMA-BAFF axis (BA2P.117)
Authors: Coquery Christine , Loo William , Wade Nekeithia , Bederman Annelise , Tung Kenneth , Lewis Janet , Hess Henry , Erickson Loren
Published: January 2023

T follicular helper (TFH) cells are critical for the development of protective antibodies via germinal center B-cell responses; however, uncontrolled TFH cell expansion activates autoreactive B-cells to produce antibodies that cause autoimmunity. Although increased circulating level of the cytokine BAFF has been linked to loss of B-cell tolerance in systemic autoimmunity, the potential role for BAFF in TFH cell homeostasis is not known. B-cell maturation antigen (BCMA) is a receptor for BAFF and is critical for the development and survival of bone marrow plasma cells (PCs). Paradoxically, BCMA deficiency exacerbates the formation of autoantibody-secreting PCs in spleens of autoimmune-prone mice. Here, we show that TFH cells uniquely express BCMA and that the absence of BCMA in TFH cells enhances their numbers and responsiveness to BAFF, and in turn, drive autoantibody production. Moreover, circulating CD4+ T cells and TFH-like cells that express another BAFF receptor BR3 (but not BCMA) are elevated in SLE patients and positively correlate with disease severity. These results identify a new BCMA-BAFF axis in controlling TFH cell homeostasis and suggest that the balance between BCMA and BR3 signaling in TFH cells serves as an additional checkpoint of immune tolerance.
Decreased inflammatory dendritic cells in lupus prone ERαKO mice correlate with increased survival (BA4P.214)
Authors: Cunningham Melissa , Wirth Jena , Eudaly Jackie , Gilkeson Gary
Published: January 2023

Lupus is a disease that disproportionately affects females. We previously showed that a functional knockout of estrogen receptor alpha (ERαKO) resulted in significantly reduced renal disease and increased survival in murine lupus. Dendritic cell development, which requires both estrogen and ERα is impacted, as is activation status and cytokine production. Due to altered hormonal feedback loops, ERαKO mice have hypergonadism and partial endocrine sex reversal. Elevated estrogen (E2) and testosterone levels may have immunomodulating effects. Thus, we studied the phenotype of the lupus-prone ERαKO mouse following ovariectomy (OVX) +/- E2 replacement to preserve a physiologic hormonal state. We found that NZM2410 ERαKO mice were protected from lupus disease expression (no early deaths; no proteinuria at 32 weeks) if they were either unmanipulated or if they were both ovariectomized and E2-repleted. These mice also had fewer inflammatory and activated cDCs (CD11c+/CD11b+/MHCII+ cells from Flt3L-cultured bone marrow, or ex vivo spleen or kidney cells), which correlated with increased survival. Interestingly, protection was lost after ovariectomy if no E2 pellet was administered, suggesting that the protective effect requires E2 in the system (despite the lack of a functional ERα). A protective effect was not observed in lupus-prone Ex3a mice (ERα−/−) suggesting that the functional mutant in ERαKO mice, in the presence of estrogen, potentially modulates disease.
Mouse CD300b (CLM-7) regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition. (BA4P.215)
Authors: Coligan John , Tian Linjie , Margulies David , Krzewski Konrad , Murakami Yousuke
Published: January 2023

The CD300 receptor family emerges as group of important molecules that modulate a variety of immune cell processes.We show that mouse CD300b (CLM7/LMIR5), expressed by myeloid cells, recognizes outer membrane-exposed phosphatidylserine (PS) and does not, as previously reported, directly recognize TIM1 or TIM4. CD300b conveys activation signals through its association with adaptor molecules, like DAP12, and accumulates in the phagocytic cups at apoptotic cell contact sites, thereby facilitating the engulfment of such cells. Engulfment requires a functional DAP12 ITAM motif. Importantly, silencing CD300b expression by macrophages diminishes Syk, PI3K and Akt phosphorylation in the presence of apoptotic cells and impairs phagocytosis of these cells. Collectively, our data shows that CD300b recognizes PS as a ligand, and regulates the phagocytosis of apoptotic cells via the DAP12 signaling pathway.
Ste20-like proline/alanine-rich kinase (SPAK) deficiency exacerbates experimental colitis in mouse (CAM1P.239)
Authors: Yan Yutao , Hong Yu , Cao Yifei , Xu Xianrong , Tan Xiaohua , Yang Lei
Published: January 2023

Inflammatory bowel diseases (IBDs) are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Here, SPAK knockout (KO) C57BL/6 mice exhibited significant increase in translocation of intestinal bacteria into mucosa, accompanying with marked elevation in paracellular permeability to FITC-dextran and altered apical side tight junction sodium ion selectivity, compared to wild type (WT) mice. In murine models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfuric acid (TNBS), KO mice were more sensitive than WT mice, as demonstrated by colonoscopy features, histological characteristics and MPO activities. Consistent with these findings, KO mice showed decreased IL-10 levels and increased proinflammatory cytokine secretion. Co-immunoprecipitation and immunohistochemical assays demonstrated that SPAK is associated with PKCtheta and CD46, regulators of regulatory T cell differentiation and activation. Flow cytometry analysis found that KO mice had significant lower amount of T regulatory 1 (Tr1) cells in comparison with WT mice. In conclusions, SPAK deficiency compromises intestinal innate immune homeostasis, implying its important role as a potential intervention target against pathological responses in IBD.