bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the cytotoxic T lymphocyte response of MHC-defined swine to foot-and-mouth disease virus (170.14)
Authors: Patch Jared , Pedersen Lasse , Kenney Mary , Pacheco Juan , Glabman Raisa , Pandya Mital , Nielsen Morten , Buus Soren , Golde William
Published: January 2023

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed animals that remains a threat to the livestock industry. FMD virus (FMDV) causes an acute infection characterized by fever and the formation of vesicles on the feet and tongue. This picornavirus is classified into seven serotypes and the current killed-virus vaccines provide antibody-mediated protection that is serotype specific. A next-generation vaccine that relies on non-replicating recombinant human adenovirus 5 (Ad5) has been shown to induce neutralizing antibodies. To date, the role of cytotoxic T lymphocytes (CTLs) has been marginally explored. We have shown that swine vaccinated with an Ad5 vectored vaccine that targets the CTL response exhibited greater CTL killing of MHC-matched target cells compared to swine given the aforementioned Ad5 FMDV vaccine. In two trials with the T cell vaccine, the CTL response showed limited epitope specificity by class I MHC tetramer staining. Using these assays, we now show that FMDV infection also elicits a CTL response with broader epitope specificity than that induced by vaccination. These data provide a basis for evaluating whether CTLs can protect against disease following virus challenge.
Entry of diabetogenic T cells into islets induces changes to amplify the cellular response and the entry of non-specific T cells (44.11)
Authors: Calderon Boris , Carrero Javier , Miller Mark , Unanue Emil
Published: January 2023

We previously showed that the early localization of activated diabetogenic T cells into the islets of Langerhans required the identification of its specific antigen presented by the resident antigen presenting cells. Instrumental in the specific localization was the presence of intra-islet dendritic cells bearing the β-cell-peptide-MHC complex. The entry of diabetogenic CD4 T cells rapidly triggered inflammatory gene expression changes in the islets and blood vessels by upregulating chemokines (CXCL9, 10 and 11) and adhesion molecules (ICAM-1 and VCAM-1). Blood vessels expressed VCAM-1 and ICAM-1. ICAM-1 was also found on β-cells. Interferon-inducible genes were upregulated as early as 8 hours post T cell transfer. These expression changes resulted in the entry of non-specific T cells. Non-specific T cells did not localize unless diabetogenic T cells were also injected. In contrast to the entry of diabetogenic CD4 T cells, the entrance of non-specific T cells required a chemokine response and VCAM-1 expression by the islets. Interferon-gamma was also important for the early gene expression changes in the islets. Mice lacking the IFN-γ receptor showed a reduced islet T cell entry and no gene or adhesion molecule changes were detected. In conclusion, these studies establish that entry of diabetogenic T cells induces a state of receptivity of islets to subsequent immunological insults.
Differential role of nucleic acid-sensing toll-like receptors in autoantibody production. (44.29)
Authors: Koh Yi , Gahan Jennifer , Fink Kathren , Silverman Gregg , Beutler Bruce , Nemazee David , Theofilopoulos Argyrios , Kono Dwight
Published: January 2023

SLE is associated with a diversity of autoAbs, of which some are relatively specific for SLE, whereas others are also found in other autoimmune diseases. Nucleic acid sensing-TLRs (TLR3, 7-9; NA-TLRs) through the engagement of NA-containing material alone or in immune-complexes have been shown to play a critical role in lupus pathogenesis. However it is unclear to what extent NA-TLR signaling also promotes autoAbs to self-antigens (Ags) unrelated to NA. Here, we studied the role of NA-TLRs in MRL-lpr mice using the 3d mutation that abolishes NA-TLR signaling. In 3d mice, IgG and IgM antibodies to nuclear and RNA-associated Ags were essentially completely abolished, whereas other autoAbs with potential to cross-react to NAs (cardiolipin, and β2-GP1) were reduced to a lesser extent, and Abs unrelated to NAs (desmoglein3) were unaffected. Using a novel model that addresses loss of tolerance to B cells that acquire self-reactivity after class-switch recombination, it was found that NA-TLRs did not play an essential role in this process. Absence of NA-TLR signaling reduced lymphoproliferation, splenomegaly, and glomerulonephritis, but did not affect the incidence or severity of skin disease consistent with no reduction in cryoglobulins. Thus, in the MRL-lpr model, NA-TLRs are necessary for nucleic acid-associated autoantibody specificities and glomerulonephritis, but to varying degrees for other autoAb specificities and cutaneous disease.
Titration of CD45 expression regulates sequential tolerance checkpoints during B cell development (45.2)
Authors: Zikherman Julie , Doan Kristin , Raschke William , Weiss Arthur
Published: January 2023

The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor (AgR) signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases (SFKs). Here we take advantage of an allelic series of mice in which CD45 expression is titrated across a broad range (0-180%). Although high expression of CD45 inhibits TCR signaling, here we show that CD45 plays a purely positive regulatory role during BCR signaling. In concert with enhanced BCR signaling, mice expressing very high levels of CD45 exhibit marked loss of follicular and marginal zone B cells in a cell intrinsic manner. Although we do not see B cell loss at earlier stages of bone marrow development, we do find increasing lambda light chain usage across the CD45 allelic series suggesting that receptor editing is triggered by high CD45 expression. In the context of the IgHEL/sHEL model of B cell tolerance, such high CD45 expression transforms anergy into deletion. However, elimination of autoreactive ligand in this system fails to fully rescue survival of mature B cells in these mice. Indeed, we find that B cell survival upon exposure to BAFF is severely impaired by high CD45 expression, while low CD45 expression promotes response to BAFF. Titration of BAFF responsiveness by CD45 is cell intrinsic and correlates with BAFFR surface expression. This constitutes a novel mechanism by which B cells exhibiting hyper-responsive AgR signaling are kept in check during splenic B cell development.
A B cell subset uniquely responsive to innate stimuli accumulates in aged mice (45.7)
Authors: Hao Yi , O'Neill Patrick , Naradikian Martin , Scholz Jean , Cancro Michael
Published: January 2023

We have discovered a functionally distinct mature B cell subset that accumulates with age, comprising up to 30% of all mature B cells by 22 months. Despite sharing some features with other mature B cell subsets, these cells are refractory to BCR and CD40 stimulation. Instead, they respond to TLR-9 stimulation and divide maximally upon combined BCR and TLR ligation. Although similar to follicular B cells in both BLyS receptor expression and BLyS binding capacity, these cells do not rely upon BLyS for survival. Further, they are not the result of intrinsically altered B lymphopoiesis in aged bone marrow, but instead appear to be gradually generated from a small subset of mature B cells that exhaustively expand throughout the individual’s lifetime. Thus, while the magnitude of the mature primary B cell niche is maintained with age, it is increasingly populated by cells refractory to BCR-driven activation yet responsive to innate receptor stimulation. This shift may contribute to the diminished capacity for adaptive humoral responses and increased propensity for autoimmunity associated with aging.
Selective gene regulation by glucocorticoid receptor translational isoforms in T cells. (46.10)
Authors: Shin Soon Cheon , Schleimer Robert , Lu Nick
Published: January 2023

Glucocorticoids are indispensable in treating inflammatory diseases including asthma and hay fever. The antiinflammatory actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). We previously found that alternative translation mechanisms produce multiple GR isoforms. To determine the functions of the translational isoforms of GR, we generated clones of Jurkat T cell lines expressing individual GR isoforms. Microarray analysis indicated that GR isoforms have unique gene targets. Realtime RT-PCR experiments confirmed that some genes are regulated by the GR-C but not by the GR-A isoform. These genes have a wide range of physiologic functions in T cell differentiation (eomesodermin, EOMES; and phosphoinositide-3-kinase interacting protein 1, PIK3IP1), proliferation (tumor necrosis factor superfamily member 8, TNFSF8, and B-cell translocation gene 1, BTG1), cell adhesion (junctional adhesion molecule 3, JAM3), virus-killing (interferon stimulated exonuclease gene 20kDa, ISG20), NF-B signaling (5-azacytidine induced 2, AZI2), and transcription regulation (runt-related transcription factor 2, RUNX2). Furthermore, we found that mitogens induced the GR-C but not other GR isoforms in purified CD3+ cells. Thus, GR-C selective genes may mediate glucocorticoid actions in activated T cells. These genes may also serve as biomarkers for selective GR isoform activation and provide new targets for the development of novel anti-inflammatory agents.
The expression of FcγRIIB by effector and memory CD8 T cells (46.14)
Authors: Starbeck-Miller Gabriel , Badovinac Vladimir , Harty John
Published: January 2023

In response to infection, antigen-specific naïve CD8 T cells proliferate and differentiate, eventually becoming long-lived memory cell populations. It has been well documented that throughout this process, CD8 T cells undergo both phenotypic and functional changes. However, the expression of FcγRIIB (CD32) by effector or memory CD8 T cells has not been reported at the molecular level. Using OT-I TCR-tg cells and OVA expressing attenuated L. monocytogenes (LM) infections, we found that CD32 mRNA was increased in memory relative to naïve populations. Surface expression of CD32 is undetectable on naive CD8 T cells, but can be observed at effector and memory time points in endogenous populations following either LM or LCMV Arm-infection. At memory, CD32+ antigen-specific CD8 T cells are CD127hiCD122hi and undergo more homeostatic proliferation relative to CD32- cells. CD32 KO antigen-specific CD8 T cells expand similarly to WT following LM challenge. However, CD32 KO cells undergo a higher degree of contraction and, thereafter, undergo less homeostatic proliferation than WT cells. These data suggest that the expression of CD32 positively correlates with the maintenance of CD8 T cells from effector to memory differentiation.
Cognate CD4+ T cells enhance CTL survival and memory programming via IL-2, CD40L and acquired pMHC-I signaling (46.16)
Authors: Sokke Umeshappa Channakeshava , Xu Shulin , Hebbandi Nanjundappa Roopa , Xiong Sidong , Jim Xiang
Published: January 2023

A hallmark of cellular immunity is the generation of memory CD8+ cytotoxic T lymphocytes (CTL) that escape activation-induced cell death (AICD) during the effector phase. Factors governing effector CTL survival in CD4+ T helper1 cells (Th1) that lead to functional memories are poorly understood. Here, we determined the role of Th1 cells with acquired antigen-presenting machineries on effector CTL fates. Contrary to previously known immune-regulatory mechanisms among Th1 or CTL interactions, our study establishes a novel co-operative role of cognate Th1-CTL interactions. Th1’s help mediated enhanced CTL survival, and functional memory CTL development, capable of protecting against lethal tumor challenge, via CD40L, IL-2, and acquired peptide-MHC-I signaling. Helped CTL showed survival advantages by displaying mRNA profiles similar to naïve CD8+ T cells, and increased NF-kB and Akt1 activation. In contrast, unhelped CTL underwent rapid AICD-mediated contraction through increased NFATc1 activation, and up-regulation of pro-apoptotic and down-regulation of anti-apoptotic genes known to mediate FasL- and Trail-associated apoptotic pathways. This form of Th1-CTL cooperation could explain why memory CTL generated under cognate Th1’s help show survival and recall advantages, a phenomenon frequently observed in organ transplantations, cancers, and many autoimmune and infectious diseases, and may impact effective adoptive CTL therapy of cancers.
FoxO1 represses mTOR dependent T-bet expression to facilitate CD8+ T cell memory (46.21)
Authors: Shrikant Protul , Rao Rajesh , Li Qingsheng
Published: January 2023

The ability of mTOR to regulate T-bet expression is central to its role in CD8+ T cell differentiation for type 1 effector functions. However, the mechanisms underpinning mTOR mediated T-bet expression are not completely understood. We demonstrate that instructional programming of naïve CD8+ (OT-1) T cells for mTOR and T-bet dependent type I effector functions, induces Akt dependent FoxO1 phosphorylation; leading to its nuclear exclusion and loss in expression of its target genes; KLF2 and CD62L. Inhibition of mTOR by rapamycin blocks FoxO1 phosphorylation, whereby increasing its expression, nuclear translocation for enhanced expression of KLF2, CD62L and for transitioning type 1 CD8+ effector to memory precursor cells. The rapamycin mediated FoxO1 expression is required to inhibit T-bet expression (mRNA, protein), switching the transcriptional profile of OT-1 cells from T-bet to Eomesodermin. Furthermore, transgenic Myr-Akt (active) or FoxO1 knockdown (siRNA) or T-bet re-expression restores type 1 effector functions in rapamycin treated FoxO1+ memory precursor OT-1 cells, indicating the essential role FoxO1 mediated T-bet repression for memory. These results reveal a novel role for FoxO1 in regulating T-bet dependent type 1effector maturation and identify duality of FoxO1 functions in regulating CD8+ T cell memory.
Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP expression (46.4)
Authors: von Rossum Anna , Krall Randall , Escalante Nichole , Choy Jonathan
Published: January 2023

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affect their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokines IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death. Human CD8 T cells activated in the presence of IL-1/6/23 underwent significantly less cell death after activation as compared to those activated in IL-12. This was due to reduced susceptibility to Fas-mediated activation-induced cell death (AICD). CD8 T cells activated in IL-1/6/23 expressed less FasL and more c-FLIPs as compared to those activated in IL-12, and were also less sensitive to cell death induced by a Fas agonistic antibody. Inhibition of c-FLIP expression by siRNA increased cell death of CD8 T cells activated in both conditions. When the effect of IL-1, IL-6, and IL-23 individually was examined, IL-1 or IL-6 alone was sufficient to inhibit CD8 T cell death that occurs after activation in IL-12 while the effect of IL-23 was variable. Our findings show that the presence of IL-1 and/or IL-6 during activation of human CD8 T cells attenuates Fas-mediated AICD while IL-12 increases the susceptibility of activated CD8 T cells to this form of cell death.
Phenotypic characterization of mice carrying a PTPN22 single nucleotide polymorphism associated with autoimmunity in humans (47.13)
Authors: Beauchemin Hugues , Piccirillo Ciriaco , Peterson Alan , Polychronakos Constantin
Published: January 2023

Type 1 diabetes (T1D) is a strongly heritable autoimmune disease due to the destruction of pancreatic β-cells by self-reactive T-cells. The third strongest genetic locus for T1D involves a single-nucleotide polymorphism (SNP) causing an arginine(R) to triptophan(W) substitution at codon 620 (620R>W) in PTPN22, the gene encoding the tyrosine phosphatase LYP, an important negative regulator of T-cell receptor (TCR) activation. However, the cellular effects of the 620R>W risk allele remain controversial, as different studies report either a gain or a loss of function in its ability to inhibit TCR signaling. To study this variant in an animal model, we created a knock-in mouse by introducing the 620R>W substitution in, the murine LYP orthologue, PEP encoded by Ptpn22. The knock-in analyzed on the autoimmune-resistant C57Bl/6 background showed a mild but significant increase in CD4+ helper T-cell activation and proliferation upon weak TCR activation. Conversely, upon strong TCR stimulation, the tendency seemed to be reversed as the 620W allele showed a slightly decreased responsiveness compared to the 620R allele. Phenotypic analysis of other immune cellular compartments, including memory T cells, showed no significant difference between genotypes. There was no evidence of autoimmunity in this genetic background. This dependence of allelic effect on the strength of the TCR signal may reconcile previous observations and indicate a complex role of R620>W in autoimmunity.
A spontaneous mutation leads to impaired regulatory T cell functions and inflammatory bowel disease (47.19)
Authors: Chabod Marianne , Pedros Christophe , Lamouroux Lucile , Vergnolle Nathalie , Andreoletti Olivier , Saoudi Abdelhadi , Fournie Gilbert , Dejean Anne S.
Published: January 2023

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders affecting genetically predisposed hosts. The current challenge lies on the identification of the causal gene variants and of their contribution to disease physiopathology. We identified a spontaneous mutation in Brown-Norway rats localized in a 1.2 cM locus containing only 6 genes. We showed that rats bearing the mutation present a CD4 T cell lymphopenia that originates in the thymus and is T cell autonomous. Interestingly, mutated rats spontaneously develop inflammatory lesions that share many pathological features with Crohn’s disease. Histological analysis revealed multifocal cellular infiltrations mainly composed of polymorphonuclear cells, macrophages and presence of granuloma. These lesions are characterized by increased myeloperoxidase activity and proinflammatory cytokine levels such as IL-1β, IL-2, IL-6, IL-17, TNF, CCL2 and CCL3. Further functional studies revealed an impaired suppressive activity of regulatory CD4 T cells (Treg) from mutated rats. IBD development is likely due to this defect, since complementation of mutated rats with wild-type Treg decreased disease incidence and severity. Thus, we report that a gene or a set of genes included within the 1.2 Mb interval should be considered henceforth as a candidate susceptibility locus for IBD and that it may act through its effect on Treg suppressive function and therefore on immune system homeostasis.
Reduced expression of cyclin-depedent kinase inhibitor Cdkn2c reduced IL-2 production by CD4+ T cells expressing the murine NZB-derived Sle2c1 susceptibility locus (47.22)
Authors: Xu Zhiwei , Potula Hari-Hara , Vallurupalli Anusha , Morel Laurence
Published: January 2023

Sle2c1 is a sublocus from the NZB-derived Sle2 locus found in the NZM2410 lupus-prone mouse. In addition to expanding the peritoneal cavity B1a cells, Sle2c1 significantly reduced IL-2 production by activated CD4+ T cells in vitro and is associated with a reduction of the Treg compartment, especially in Fas-deficient mice. The characterization of three Sle2c1 recombinants mapped the IL-2 deficiency to a short interval on chromosome 4 that contains the Cdkn2c gene. Cdkn2c encodes for p18INK4c (p18), a cyclin-dependent kinase inhibitor that regulates G1 cell cycle arrest. We have found a novel SNP in the Cdkn2c promoter that is associated with a significantly reduced expression of this gene in CD4+ T cells. Activated CD4+ T cells from B6.p18-/- mice produced significantly less IL-2 than B6 controls, demonstrating that a dysregulated cell cycle interferes with IL-2 production. Interestingly, we have also found that p18 reduced expression, either in B6.Sle2c1 or B6.p18-/- mice, is also associated with an expansion of the B1a cells compartment. Overall these results show that a dysregulated cell cycle affects CD4+ T cell and B cell homeostasis and is associated with lupus susceptibility, either through an expansion of the b1a cells, a reduced IL-2 production, or both.
The de novo revision of TCRα but not TCRβ is responsible for generating autoantibody-inducing CD4+ (aiCD4+) T cell that causes systemic autoimmunity, i.e., SLE (47.26)
Authors: Uto Kenichi , Tsumiyama Ken , Takimoto Mai , Shiozawa Shunichi
Published: January 2023

Objective: The induction of autoantibody-inducing CD4+ (aiCD4+) T cell after repeated immunization with antigen is the key for the induction of autoantibodies including anti-dsDNA antibody and immune tissue injury in SLE in our ‘self-organized criticality theory’. Here we investigated the mechanism for generating aiCD4+ T cell. Methods: BALB/c mice were immunized 8x with staphylococcul enterotoxin B (SEB). RAG1/2, TdT and pTα were detected using RT-PCR. Expression of GFP was studied in RAG-1/GFP knock-in mice. The rearranged intermediates of TCRα joining regions from 1 to 61 and all TCRβ variable and diversity regions were detected using LM-PCR. Histone modification of TCR loci was examined using ChIP assay. Results: RAG1/2, TdT pTα and were re-expressed in splenic T cell and expression of RAG1 gene was confirmed in vivo in RAG1/GFP knock-in mice after immunization 8x with SEB. The rearranged intermediates of TCRα joining region 12 were selectively induced in splenic Vβ8+CD4+ T cell after immunization 8x with SEB. Instead, rearranged intermediates of all TCRβ regions were never observed. The histone H3 of the TCR loci of splenic CD4+ T cell was heavily acetylated and trimethylated to the extent identical to thymocytes. Conclusion: As to the reason why TCRα gene was solely rearranged, since TCR gene loci were found to be always accessible and ready to TCR revision, we assume that TCR revision related to autoimmunity may be governed structurally by TCRβ molecule.
The TNFAIP3 haplotype associated with SLE leads to altered A20 expression and enhanced cellular activation of primary human B cells (47.6)
Authors: Humphrey Mary Beth , Templeton Amanda , Hu Yanqing , Wen Daniel , Gaffney Patrick
Published: January 2023

TNFAIP3 encodes an ubiquitin modifying enzyme, A20, required to limit NF-kB activity downstream of TLR and TNF receptors. We have recently identified a novel TT>A polymorphism ~25 kb downstream of TNFAIP3 as the best candidate polymorphism responsible for the association of TNFAIP3 with SLE (Adrianto, Wen, Templeton, et al. Nature Genetics, in press). Here we investigate the functional consequence of expression of the risk haplotype defined by the minor -/A allele at position chr6:138,271,732-733 in peripheral B cells isolated from subjects expressing one copy of the risk haplotype compared to controls. With IRB approval, blood samples from 11 paired risk and controls were collected through the Large Lupus Family Registry at OMRF. Resting B cells from risk samples had significantly less A20 expression compared to controls. CD40L stimulation significantly increased A20 expression in control samples but not risk samples. CD40L induced higher NF-kB p52 activation and induced significantly more CD86 expression in risk compared to control samples. Overall, these results confirm the risk haplotype defined by the TT>A polymorphism confers functional consequences of lower A20 expression, enhanced NF-kB signaling and increased cellular activation of primary B cells. Inappropriate B cell activation as a result of reduced TNFAIP3 expression in cells carrying the TT>A variant likely contributes to increased autoimmunity and SLE risk.
Promigratory secreted protein Collagen triple helix repeat containing 1 is involved in sex-biased regulation of inflammatory arthritis (47.8)
Authors: Shekhani Mohammed , Ramez Mohamed , Forde Toni , Lyons Elizabeth , Lindner Volkhard , Adarichev Vyacheslav
Published: January 2023

Molecular mechanisms of sex disparities in autoimmune inflammatory diseases, including rheumatoid arthritis (RA), are not well defined. To model human disease, collagen antibody-induced arthritis (CAIA) was induced in BALB/c.DBA/2-Pgia8 congenic mice. Chromosome interval Pgia8 showed anti-inflammatory effect in congenic males, while females exhibited CAIA that was similar to wild-type mice. Transcriptome analysis indicated that expression of three locus-specific genes was tightly correlated (r=0.87-0.91) with inflammation: Collagen triple helix repeat containing 1 (Cthrc1), R-spondyn 2 and Syndecan 2. The link between Cthrc1 and arthritis was reproduced in F1(BALB/c x FVB-Tg[Cthrc1]) mice, since mice overexpressing Cthrc1 transgene showed 1.2-1.4-fold stronger inflammation than tg-negative F1 hybrids. Importantly, naïve F1(BALB/c x FVB-Tg[Cthrc1]) mice exhibited pre-inflammatory phenotype of neutrophilia (30% upregulated, p<0.014) accompanied with monocytopenia (50% downregulated, p<0.025) in peripheral blood. Cellular mechanisms of arthritis regulation via Cthrc1 were addressed using immunodetection techniques. Present study indicates that promigratory protein Cthrc1 effects hematopoiesis and is also implicated in sex-biased regulation of inflammatory arthritis.
The importance of be(ginn)ing naïve: implications for cancer immunotherapy (48.18)
Authors: Cieri Nicoletta , Camisa Barbara , Provasi Elena , Magnani Zulma , Mondino Anna , Bondanza Attilio , Bonini Chiara
Published: January 2023

Manipulation required to expand or engineer tumor-specific lymphocytes often induces terminal differentiation, resulting in poor in vivo efficacy. We hypothesized that initial targeting of Naive T cells (TN) could overcome this limitation. We activated and efficiently transduced FACS-sorted TN, TCM and TEM cells by viral vectors. Activation of TN with anti CD3/CD28 beads resulted in a predominant population of highly proliferative CD45RA+CD62L+CCR7+ T cells, that did not produce γIFN nor cytotoxic molecules and required IL7 and IL15 for their generation and maintenance. Manipulated-TN were post-mitotic, and expressed CD45RO, CD122, CXCR3 and CD95. Compared to manipulated-TCM and -TEM, naïve-derived T cells expressed higher levels of CD127, c-Kit and CXCR4 and lower levels of CCR5, HLA-DR and PD-1. To verify their differentiation potential, TN TCM and TEM were transduced to express a WT1-specific TCR and stimulated with WT1 peptide. In this context, TN expanded at higher numbers, differentiated into both CD45RA-CD62L+ and CD45RA-CD62L- cells and generated a mixed population of CD127± cells. When infused in NOD/Scid mice, manipulated TN showed a degree of xenoreactivity comparable to that of unmanipulated lymphocytes. In contrast to manipulated TCM cells, manipulated TN cells engrafted in secondary and tertiary recipients indicating that persistence, self-renewal abilities and expansion potential are preserved in this novel T cell subset after genetic manipulation.
Tri-component MUC1 glycopeptide vaccine induced both humoral and cellular immune responses in MUC1.Tg mice with MMT tumor. (48.20)
Authors: Lakshminarayanan Vani , Thompson Pamela , Wolfert Margreet , Bradley Judy , Pathangey Latha , Madsen Cathy , Cohen Peter , Boons Geert-Jan , Gendler Sandra
Published: January 2023

Effective immunotherapy for cancer depends on both cellular and humoral immune responses to tumor antigens. MUC1, which is expressed at increased levels on breast cancer, also exhibits altered glycosylation that contributes to the formation of novel antigens. The identification of MHC class I and II binding peptides derived from tumor-associated MUC1 has facilitated the development of MUC1 based cancer vaccines. We have shown that the MHC class I binding epitopes from MUC1 tandem repeat, when given as emulsification with adjuvants, has always resulted in strong cellular response with no antibody response. It is possible that better immunogenicity would be obtained using glycopeptides more representative of the novel forms of MUC1 as seen in cancer to which individuals may be less tolerant and that direct linking of the vaccine components would result in a superior immune response than delivering them as a mix. Here we show that a three-component vaccine composed of a TLR2 agonist, a helper epitope and a T cell epitope, which is also a B cell epitope, derived from the MUC1 can break tolerance and elicit both humoral and cellular immune responses. Immunization with the MUC1 glycopeptide vaccine led to a significant reduction in tumor burden compared to mice treated with adjuvants only and empty liposomes. The tri-component vaccine activated MUC1 glycopeptide-specific cytotoxic CD8+ T cells and elicited robust titers of IgG antibodies that lyse relevant tumor cells by ADCC.
Interaction between tumor and myeloid cells induce/expand Th17 cells in malignant gliomas (48.28)
Authors: Parajuli Prahlad , MIttal Sandeep , Chinni Sreenivasa , Lum Lawrence , Thakur Archana , Guthikonda Murali
Published: January 2023

Th17 cells, a recently discovered inflammatory T cell subtype, have been reported in several tumors, where they are implicated with either pro- or anti-tumor activity, depending on the tumor type. Th17 cells have not been studied with respect to brain tumors. We have been studying the mechanism of induction and role of Th17 cells in malignant gliomas using primary tumor as well as glioma cell lines. We report here that: 1) There is considerable prevalence of Th17 cells and myeloid (CD11b) cells among glioma-infiltrating immune cells; 2) Glioma cells express the cytokines TGF-β1 and IL-6, while the infiltrating myeloid cells express IL-1β and IL-23; 3) Naïve T cells cultured with anti-CD2/CD3/CD28-loaded microbeads in the presence of glioma culture supernatants differentiate into Th17 cells, which is inhibited by antibodies to TGF-β1 and IL-6; 4) Supernatant from glioma-monocyte co-culture generates significantly higher frequency of Th17 cells compared to glioma supernatant, which is abrogated by antibodies to IL-1β and IL-23. Moreover, 5) Th17 cells generated in the presence of supernatant from glioma-monocyte co-culture have low IFN-γ and high IL-10 expression, suggesting that glioma-associated Th17 cells may be immune suppressive. Further studies on the mechanism of tumor-infiltration, developmental pathways and pro-/anti-tumor functions of Th17 cells will provide rationale for developing novel adjuvant immunotherapeutic strategies for malignant gliomas.
Thymic stromal lymphopoietin mediates breast cancer progression and metastasis (48.9)
Authors: Bodogai Monica , Olkhanud Purevdorj , Rochman Yrina , Malchinkhuu Enkhzol , Wejksza Katarzyna , Gress Ronald , Hesdorffer Charles , Leonard Warren , Biragyn Arya
Published: January 2023

Breast cancer escape and metastasis is an active process that requires inflammation and participation of immune cells. We have previously reported that this is a primary tumor-controlled process which activates lungs to produce CCL17 and CCL22 to facilitate lung metastasis together with the recruitment of CCR4+ regulatory T cells (Tregs). To understand the mechanism of this process, we performed expression array analysis in both metastatic and non-metastatic cancer cells and found that metastatic cells produced thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs. Moreover, TSLP was also abundantly expressed in variety of human solid tumors, including breast, lung, colon and ovarian cancers. To gain further insights into the role of TSLP in cancer progression, we have blocked TSLP expression in cancer cells and found that TSLP is required for efficient breast cancer escape and metastasis. Moreover, utilizing adoptive transfer studies in TSLPR deficient mice, we demonstrated that by targeting CD4+ T cells to induce Th2-type inflammatory responses, cancer-produced TSLP promoted cancer escape. Taken together, our data indicate that TSLP is an important factor that needs to be controlled to effectively combat with cancer escape.
Early cytokine dysregulation contributes to mortality during lethal influenza infection. (49.18)
Authors: Brown Deborah , Vogel Alexander , Condon Shirley
Published: January 2023

Recent studies have suggested that pandemic influenza viral strains induce high pro-inflammatory cytokine levels in the lung that may contribute to increased mortality. However, the mechanisms linking highly virulent virus infection and mortality have not been fully elucidated. Here we compare a sublethal dose of highly pathogenic H1N1 virus, A/Puerto Rico/34/8 (PR8) with a lethal dose to determine the extent by which viral load affects inflammatory responses in the lung. We observed a 5-10 fold increase in viral load that correlated with a 10 fold increase in TNF-α, IL-1β and MIP-1α secretion in lethally infected mice compared to sublethally infected mice at day 2 post infection (p. i.). Surprisingly, this difference was not apparent at later time points after infection indicating early cytokine dysregulation may contribute to mortality. IL-6, MCP-1 and MIP-1β were also upregulated at day 2 p. i. and peaked at day 6 suggesting sustained levels of certain cytokines may also contribute to pathology. Furthermore, we find that RIG-I and TLR-3 are upregulated in CD45- epithelial cells leading to high expression of IFN-β and IL-6 while TLR-7 and TLR-9 are highly upregulated in CD45+ immune cells correlating with expression of TNF-α. Thus, differential PRR usage and cytokine secretion may be a contributing factor in the balance between protective immunity and pathogenicity.
Memory CD4 T cells drive emergence of viral escape mutants during influenza challenge in the absence of CD8 T cells and B cells (49.6)
Authors: McKinstry Karl , Strutt Tara , Swain Susan
Published: January 2023

How T cells contribute to protection against influenza virus during heterosubtypic challenge is not fully understood. Here we investigate the potential for memory CD4 T cell-mediated protection against influenza. We find that transfer of influenza-primed, Th1-, or Th17-polarized T cell receptor transgenic memory CD4 T cells recognizing influenza can completely protect otherwise naïve hosts against an otherwise lethal influenza challenge in the absence of either B cells or CD8 T cells. However, in the absence of both B cells and CD8 T cells, monoclonal memory CD4 T cells fail to clear virus, leading to a chronic infection characterized by high viral titers, systemic inflammation, and ultimately death. During such chronic infection, memory CD4 T cells exert substantial selective pressure on the challenge virus as we demonstrate the emergence of T cell receptor epitope-specific viral escape mutants. In contrast to the failure of monoclonal memory CD4 T cells to clear influenza in the absence of CD8 T cells and B cells, transfer of polyclonal memory CD4 T cells isolated from influenza-primed mice completely clear virus. These studies demonstrate that memory CD4 T cells can not only provide protection against influenza, but also that they can exert substantial selection pressure. Furthermore, our results support the concept that heterosubtypic immunity to influenza can take several forms.
Complementary functions of FADD and RIP1 critical for embryogenesis and T cell proliferation (50.8)
Authors: Zhang Jianke
Published: January 2023

FADD is a common adaptor shared by several death-receptors (DRs) for signaling apoptosis through recruitment and activation of caspase 8. DRs are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, FADD-/- mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation. How FADD regulates embryogenesis and lymphocyte responses has been a long standing enigma. FADD could directly bind to RIP1, a serine/threonine kinase which mediates both necrosis and NF-kB activation. Our data indicate that FADD plays a role in regulating RIP1 expression and necrosis in embryos. To investigate a potential in vivo functional interaction between RIP1 and FADD, we generated mice lacking both FADD and RIP1and performed analyses of the resulting double knockout mutant mouse embryos and lymphocytes. The data from these studies have revealed an unexpected cell type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis, necrosis and NF-kB activation during embryogenesis and lymphocyte function.
Adenosine 2A Receptor agonists-induced tolerogenic dendritic cell protected kidney from ischemia/reperfusion injury (52.13)
Authors: Li Li , Huang Li-ping , Song Steven , Ye Hong , Porcelli Steven , Okusa Mark
Published: January 2023

NKT cell activation initiates the kidney ischemia/reperfusion injury (IRI) immune response. There are no effective treatments available on acute kidney injury (AKI) to date. DCs represent a new and promising immunotherapeutic approach for autoimmune diseases. Adenosine 2a receptor (A2aR) agonist, ATL313, protected kidney from IRI. We hypothesize that ATL313 programs DCs into tolerogenic DCs during a-galactosylceramide (aGC) priming in vitro, which protected the kidney from IRI by suppressing NKT cell activation. Both kidney pedicles were clamped in B6 mice for 24 mins and then released for 24 hrs. BMDCs were cultured and loaded with aGC(0.1nM) w or w/o ATL313(1nM). Cells were adoptively transferred to naive WT mice 2 days prior to the surgery. ATL313-treated aGC loaded WT DC (WT DC-aGC-ATL) significantly protected kidney IRI with lower plasma creatinine compared with DC-aGC control (P<0.001), which was not seen in A2aRKO DC-aGC-ATL treated mice (P>0.05). Mice receiving WT DC-aGC had significantly increased plasma IFN-g level (P<0.01), while mice receiving WT-DC-aGC-ATL had significantly lower level (P<0.01). ATL313 has no effect on CD1d/aGC complexes (L363) on DCs, but decreased IA, CD86 and CD40 expression on WT not A2aRKO DC-aGC. ATL313-induced tolerogenic DCs have increased IL-10 mRNA expression compared with DC-aGC (P<0.05). A2aR agonist-induced toerogenic DCs is a unique and potent cell-based strategy to attenuate IRI by blocking DC-NKT cell immunological synapses.
Anti-microbial immunity augmented by extracellular matrix adjuvant from porcine small intestinal submucosa (52.22)
Authors: Roy Chowdhury Roshni , Ghosh Swapan
Published: January 2023

Developing a long-lasting protective vaccine against Staphylococcus aureus has been a challenge, since S. aureus has several mechanisms to evade the immune system. The issue is whether appropriate adjuvant(s) could overcome this evasion and elicit a robust immune response. Porcine small Intestinal submucosa (SIS) exhibits adjuvanticity against pathogenic antigens and bacterial toxins (Suckow, USPA 20100136050). However, there is as yet no report on the adjuvanticity of SIS against the intracellular pathogen S. aureus. Hence, this study was designed to assess SIS as an adjuvant, relative to alum, in terms of specific antibody response, T-cell infiltration, inflammasome activation and TLR involvement, in BALB/c mice against heat-killed S. aureus. Our results showed that SIS helped evoke high-titer antibody, but alum was ineffective in augmenting antibody response. Both SIS and alum up-regulated the mRNA expression of GATA-3 and FOXP3. Our inflammasome microarray analysis showed that unlike alum, SIS did not involve any inflammasome pathway (Nlrp1a/Nlrp3/IPAF). In comparison with Staph alone, alum significantly upregulated the mRNA expression of chemokines Ccl12, Ccl7 and Cxcl3 but SIS did not. Alum although is TLR-independent, influenced the up-regulation of the mRNA expression of TLRs 2,3,5,7 and 8. SIS also showed a similar effect. In summary although SIS was more effective than alum in antibody production against S. aureus, its mode of action needs to be explored further.
Alternative mechanism for glycoconjugate vaccine activation of the adaptive immune system (52.25)
Authors: Avci Fikri , Li Xiangming , Tsuji Moriya , Kasper Dennis
Published: January 2023

Glycoconjugate vaccines, which are currently made by covalently coupling a bacterial capsular polysaccharide to a carrier protein, have been used to immunize many millions of people and have enormously reduced disease burden, particularly among children. However, the immunogenicity and efficacy of these vaccines have been relatively low and heterogeneous in some high-risk populations. The classic antigen presentation hypothesis for glycoconjugates proposes that helper CD4+ T cells recognize a peptide derived from the carrier protein. We investigated mechanisms of processing and presentation of a prototypic glycoconjugate vaccine in which group B streptococcal type III polysaccharide is coupled to a carrier. Upon uptake into the endosome of an antigen-presenting cell, the polysaccharide undergoes oxidative depolymerization, which, in conjunction with acidic protease-mediated digestion of the carrier protein, creates a carbohydrate T cell epitope conjugated to an MHCII binding peptide. These epitopes formed in the endosome bind to MHCII through the peptide and are subsequently presented on the APC surface, with the hydrophilic carbohydrate positioned for easy recognition by the T-cell receptor. Recognition of the carbohydrate in the presence of MHCII drives T-cell help for antibody-producing B cells. An enhanced understanding of immune mechanisms such as T-cell recognition of carbohydrates may lead to markedly improved vaccines against infectious diseases.
The inhibition of T cell proliferation in a mixed lymphocyte reaction by Itolizumab (T1h) is associated with reduction in pro inflammatory cytokines and CD6 internalization. (52.27)
Authors: Nair Pradip , Ghorai Abhijit , Adhikary Laxmi , Melarkode Ramakrishnan , Montero Enrique
Published: January 2023

We have shown previously that the CD6-ALCAM interaction in vitro induced a synergistic co-stimulation of normal human peripheral blood mononuclear cells (PBMCs), defined by Bliss analysis. The use of a CD6 membrane-distal domain (SRCR1)-specific non-depleting monoclonal antibody Itolizumab, inhibited the CD6-ALCAM induced proliferation of PBMCs. This is associated with reduction in pro inflammatory cytokines at the protein and mRNA level. In this study, we show that the antibody inhibits T cell proliferation in a typical mixed lymphocyte reaction wherein the matured dendritic cells over express ALCAM. The inhibition is associated with reduction in CD4 and CD25 surface expression in these T cells. A marked reduction in interferon-γ, tumour necrosis factor-α and IL-17 production is also observed. Using confocal microscopy we show that the receptor CD6 internalization occurs within minutes in T cells in the presence of Itolizumab and is not localized to the immunological synapse. Intriguingly, a deglycosylated antibody showed reduced inhibition of T cells in the mixed lymphocyte reaction. The deglycosylated antibody did not show any difference in binding to HuT 78 cell expressing CD6 as compared to the normal antibody. These results would suggest that the monoclonal antibody Itolizumab also prevents T cell proliferation induced in mixed lymphocyte reactions and there is a role for the Fc region glycosylation of the antibody to elucidate this function.
CD4 T-helper cell cytokine phenotypes following tetanus toxoid immunization (52.3)
Authors: Livingston Kimberly , Jiang Xiaowen , Stephensen Charles
Published: January 2023

Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify TT-specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vaccination. Blood was drawn before, 3, 7, 14, and 28d after vaccination. PBMC cultures were stimulated with TT or PBS. Activation markers (CD25 and CD69) were measured after 44h (n=8), cytokines in supernatant after 3 and 7d of culture, and intracellular cytokine staining (ICS) was performed in proliferated cells (PKH67 dye dilution) after 7d of culture (n=6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3+CD4+ lymphocytes, and TT-specific proliferation at 7, 14 and 28d post vaccination. Vaccination induced TT-specific Th1 (IFNγ, TNFα, and IL2) Th2 (IL13, IL5, and IL4), Th17 (IL17A) and IL10+ cells as measured by ICS. TT-specific Th1 cells were the most abundant (12-15% of all CD3+CD4+ T-cells) while IL10+ (1.8%) Th17 (1.1%) and Th2 cells (0.2-0.6%) were less abundant. TT-specific cytokine concentrations in PBMC supernatants followed the same pattern. A TT-specific IL-9 response was also seen in the supernatants. In conclusion, TT booster vaccination induced a broad T-helper cell response. This method of evaluating cytokine phenotypes may be useful in examining the impact of nutrition and environmental conditions on the plasticity of T-helper cell memory responses.
B cell depletion impairs both CD4+ and CD8+ T cell activation during acute viral infection in mice (52.33)
Authors: DiLillo David , Weimer Eric , Tedder Thomas
Published: January 2023

Therapeutic B cell depletion in mice using CD20 mAb significantly reduces CD4+ T cell activation in response to model and self-antigens, but not CD8+ T cell activation. However, the effects of B cell depletion on acute viral infections and the consequences of long-term B cell depletion on immune system function are unknown. To assess this, B cells were depleted in wild type mice using a potent CD20 mAb that depletes >98% of tissue and circulating mature B cells. Short-term (2 week) B cell depletion did not affect tissue T cell numbers. However, when B cells were depleted 1 week before LCMV-Armstrong infection, splenic CD44hiCD62Llo CD4+ and CD8+ effector-memory T cell numbers were reduced by 51-57%. The numbers of LCMV-specific CD4+ T cells producing IFNγ and TNFα were reduced by 64% and 80%, respectively. Virus-specific CD8+ T cells producing IFNγ and TNFα were also reduced by >56%. Even without pathogen challenge, similar changes were observed in mice treated chronically with CD20 mAb for 6 months, where CD4+ and CD8+ effector-memory T cell numbers were reduced by 76% and 39%, respectively. Chronic B cell depletion also reduced IFNγ- and TNFα-producing CD4+ T cell numbers by 76% and 35%. Nonetheless, naïve T cell numbers were not affected by either short-term or chronic B cell depletion. Thus, B cells are required for optimal CD4+ and CD8+ T cell activation, expansion, and function in vivo, particularly during cellular responses to pathogens.