bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid composition impacts the assembly of MHC class II molecules in nanodiscs
Authors: Becker Tynan A , Osan Jaspreet , Rivera Kristian , Polanco Jonilee , Ferrante Andrea , Kuhn Thomas
Published: January 2023

Class II major histocompatibility complex molecules (MHCII) are transmembrane glycoproteins that display peptides generated and selected by intracellular antigen processing and presentation mechanisms to CD4+T cells, initiating an adaptive immune response. MHCII is expressed on the cell surface and in endosomal compartments, each of which have their own signature membrane characteristics. The traditional strategy to investigate peptide binding to MHCII has relied on the use of soluble MHCII, in which the transmembrane portion has been removed. This work studied MHCII in its native, membrane-embedded form. To this end, we used nanodisc, a synthetic model membrane device, to evaluate the effect of membrane lipid composition on MHCII assembly. The full-length human MHCII allele HLA-DR1 (DR1) was isolated from B-lymphoblastoid cell lines via immunoaffinity chromatography. Three types of nanodisc were generated: simple, fluid disordered and rigid ordered, each of them characterized by a unique lipid composition. Nanodiscs were separated using fast protein liquid chromatography (FPLC), the readings from which showed clear differences between nanodisc types. We observed that the fluid disordered nanodisc showed DR1 assembly as a cluster, with one major FPLC-generated peak. In contrast, simple and rigid nanodiscs were distinguished by multiple peaks. As DR1 tends to form aggregates, we inferred that in simple and rigid nanodiscs DR1 tetramers and dimers form in separate nanodisc assemblies. Taken together, our results indicate that membrane lipid composition has a strong impact on native MHCII assembly. As such, we predict that MHCII conformation and activity are function of the cell compartment they reside at any point in time.
Immunomagnetic cell separation is a suitable method for T cell electrophysiology and ion channel pharmacology
Authors: Tajti Gabor , Szanto Tibor G. , Racz Greta , Csoti Agota , Hajdu Peter , Panyi Gyorgy
Published: January 2023

Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation.

PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration.

The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells.

Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.
Lupus mice treated with Lupresan decreased auto-antibodies titers and improve facial lesions
Authors: Baeza Carlos Wong , Mora Albany Reséndiz , Pensado Monserrat Figueroa , Patiño Sandra Vela , Estrada Griselda Nava , Ramírez Carlos Wong , Ramírez Isabel Baeza
Published: January 2023

The nonbilayer phospholipid arrangements (NPA) are lipid associations different from the bilayer that are transient, so they are not immunogenic. But if they are stabilized by some drugs such as chlorpromazine, they become immunogenic and induce anti-NPA antibodies, which are found in patients with Lupus and are the ones that trigger a disease similar to Lupus in mice. Chloroquine, a drug used in the treatment of malaria, has been widely used to treat patients with Lupus; however, the mechanism of action by which it works in the treatment of Lupus is not known with certainty. In our research group, we have shown that chloroquine prevents the formation of NPA or reverts the formed, so it could be its mechanism in the treatment of Lupus. On the other hand, we have also shown that the polyamine spermidine is more effective than chloroquine in preventing the formation of NPA or in reverting the formed ones. Probably because this polyamine better stabilizes the lipid bilayer, because it has 3 positive charges in its structure at physiological pH, unlike the chloroquine that only has two. However, although polyamines participate in many biological functions, they cannot be used as drugs because when they have been administered in humans, they produce hematuria, proteinuria, renal failure and nephrotoxicity. In addition, in laboratory animals, they produce seizures, coma and death. So, in this work we design and synthesize Lupresan, an analogue of chloroquine with three positive charges at physiological pH. Lupresan was more effective in reverting or preventing the formation of NPA than chloroquine or spermidine, and as a result, it decreased auto-antibodies titers and healed the malar rash in mice with lupus to a greater extent than chloroquine.
Improved antimalarial delivery to inflammatory cells driving lupus pathogenesis using shape-based targeting nanoparticles
Authors: Allen Marilyn , Karabin Nicholas , Golding Amit , Scott Evan , Szeto Gregory
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic disease that attacks healthy organs via self antigen-autoantibody immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) via (toll-like receptor) TLR7 and 9, driving inflammatory type I interferon (IFN). Antimalarials, hydroxychloroquine (HCQ) and chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE patients, but require ~3+ months before therapeutic efficacy. Recent work reports 7.5% patients treated with HCQ are at risk of irreversible retinopathy after 5 years, which increases to 1 in 5 patients with duration of therapy. We hypothesized that PEG-b-PPS filomicelles (FMs) will improve drug activity and reduce toxicity by directly delivering HCQ or CQ to pDCs via passive, shape-based targeting. Human peripheral blood mononuclear cells (hPBMCs) were treated with soluble CQ or CQ in FMs, stimulated with CpG-A ODN 2216 and quantified for type I IFN via myxovirus-resistance 1 (MX1) gene expression using real-time RT-PCR. Our results show that 50 μg CQ/mg FM decreased MX1 expression 20 fold to soluble CQ and 10 fold to CpG-A ODN 2216 control, while CQ in control polymer PLGA spheres had no effect. Additionally, cellular-level distribution of FMs in hPBMCs show that FMs accumulate in pDCs more than B and T cells over a 48 hour time course. These results show polymer shape impacts drug delivery, CQ-FMs inhibit SLE-relevant pathways and CQ-FMs may be effective and more targeted than CQ.
Response Gene to Complement -32 exerts proinflammatory and profibrotic effects in immune complex mediated glomerulonephritis
Authors: Rus Violeta , Tatomir Alexandru , Nguyen Vinh , Talpos-Caia Ana , Papadimitriou John , Atamas Sergei , Luzina Irina , Sung Sun-Sang J , Badea Tudor , Rus Horea
Published: January 2023

Response Gene to Complement (RGC)-32 is a cell cycle regulator induced by complement activation, growth factors and cytokines. RGC-32 mediates TGF-β dependent profibrotic pathways, while in immune cells promotes the differentiation of Th17 cells in vitro and in vivo. RGC-32 expression is increased in tubules and glomerular infiltrating cells in kidney biopsies of patients with lupus nephritis. We used the nephrotoxic nephritis (NTN) mouse model to assess whether RGC-32 plays a local role in immune complex mediated glomerulonephritis (GN).

NTN was induced in WT and RGC-32−/− mice. Proteinuria, blood urea nitrogen and kidney histopathology were determined to assess kidney function and damage, IgG and C3 deposition. Single cell suspension of kidneys and spleens were analyzed by flow cytometry. Expression of IL-17A, collagen I, III and IV was determined by RT-PCR.

RGC-32−/− mice exhibit attenuated renal damage as shown by decreased proteinuria and glomerular scores, as well as decreased frequency of infiltrating PMN and decreased mRNA expression for IL-17A, collagen I, III and IV compared to WT mice. RGC-32 deficiency did not interfere with the induction of NTN as mouse anti-sheep IgG titers, percentage of splenic germinal center B cells, plasma cells, effector CD4+ T cells, Tregs, IL-17A, IFNg secreting cells and kidney deposition of autologous antibodies did not differ between RGC-32−/− and WT mice. These results suggest that RGC-32 contributes to the pathogenesis of immune complex mediated GN by promoting proinflammatory and profibrotic pathways. These data support further efforts to examine the mechanisms by which RGC-32 modulates these pathways and suggest that RGC-32 is a potential novel therapeutic target in the treatment of LN.
Targeting T cell activation and lupus autoimmune phenotypes with a novel glucose transporter inhibitor
Authors: Li Wei , Qu Ganlin , Choi Seung-Chul , Cornaby Caleb , Titov Anton , Teng Xiangyu , Wang Haiting , Morel Laurence
Published: January 2023

CD4+ T cells have numerous features of over-activated cellular metabolism in lupus patients and mouse models of the disease, including a higher glycolysis. We have previously shown that treatment of lupus-prone mice with 2-deoxy-D-glucose, which inhibits the first step of glycolysis, was sufficient to prevent autoimmune activation. Because 2DG has potential off-target effects, we sought to directly inhibit glycolysis by blocking glucose transporters. Here, we show that CG-5, a novel glucose transporter inhibitor, ameliorated lupus-like symptoms in a spontaneous lupus-prone mouse model, B6.NZM2410.Sle1.Sle2.Sle3 (Triple-congenic, TC), and in a chronic graft-versus-host-disease (cGVHD) model of induced lupus. Moreover, CG-5 treatment reduced lupus phenotypes including the expansion of germinal center B cells, as well as the production of autoantibodies in both TC mice and cGVHD models. In vitro, CG-5 blocked glycolysis in CD4+ T cells, and limited the expansion of CD4+ T cells induced by alloreactive stimulation. CG-5 also modulated CD4+ T cell polarization by inhibiting Th1 and Th17 differentiation and promoting regulatory T (Treg) induction. Finally, CG-5 blocked glycolysis in human T cells. Overall, our data suggest that blocking glucose uptake with a small molecule inhibitor ameliorates the symptoms of lupus, at least partially due to its inhibition of glycolysis in CD4+ T cells.

Supported by grants from the NIH (RO1 AI128901) and the Lupus Research Alliance (LRA-TIL 416522) to LM, and by an Experimental Pathology Innovative Grant from the Department of Pathology, Immunology and Laboratory Medicine, University of Florida to WL.
Human gingival tissue-derived MSC suppress osteoclastogenesis and bone erosion via CD39-adnosine signal pathway in autoimmune arthritis
Authors: Zheng Song Guo , Luo Yang , Wang Julie
Published: January 2023

Bone destruction is one of many severe complications that occurs in patients with rheumatoid arthritis (RA) and current therapies are unable to cure this manifestation. This study aims to determine whether GMSC can directly inhibit osteoclast formation and eventually attenuate osteoclastogenesis and bone erosion in inflammatory milieu. GMSC were co-cultured with osteoclast precursors that had been pretreated with or without the inhibitors of CD39 inhibitor, CD73 inhibitor or adenosine receptors and osteoclast formation were evaluated in vitro. 2×106 GMSC were transferred to CIA mouse and pathology scores, frequency of osteoclasts, and bone erosion in joints were assessed in vivo. We demonstrated that GMSC but not fibroblasts, markedly suppressed human or mice osteoclastogenesis in vitro. GMSC treatment also resulted in a dramatically decreased level of NF-κB p65/p50 in osteoclasts in vitro. Infusion of GMSC to CIA significantly attenuated the severity of arthritis, pathology scores, frequency of osteoclasts, particularly bone erosion, as well as a decreased expression of RANKL in synovial tissues in vivo. Blockade of CD73-CD39-adenosine receptors has significantly abrogated the suppressive ability of GMSC in vitro and therapeutic effect of GMSC on bone erosion during CIA. Thus, GMSC inhibit osteoclast formation in vitro and in vivo partially via CD73-CD39-adenosine signals. Manipulation of GMSC may have a therapeutic implication on rheumatoid arthritis and other bone erosion related diseases.
Inhibition of dendritic cells and Th17 cells by a flavonoid compound attenuates clinical severities in a murine collagen-induced arthritis model
Authors: Yen Hung-Rong , Pai Fu-Tzu , Lu Cheng-You
Published: January 2023

Rheumatoid arthritis is a common autoimmune disease, which leads to severe disabilities and affecting approximately one percent of the worldwide population. It is manifested with significant infiltration of immune cells, synovial inflammation, bone erosion and progressive disability. Despite the use of conventional therapies and biological agents, there are still some unmet needs and concerns. We aimed to develop novel therapeutic agents for the treatment of rheumatoid arthritis. We first identified that flavonoid compound CD001 derived from plants possesses suppressive effects on the maturation of dendritic cells and reduced the production of cytokines by dendritic cells. CD001 inhibited the Th17 differentiation in a dose-dependent manner in vitro. CD001 also inhibited the IL-6 secretion by SW982 synovial cells stimulated by IL-17 in vitro. Furthermore, in a murine model of collagen-induced arthritis, administration of CD001 significantly ameliorated paw edema and clinical scores in a dose-dependent manner. IL-17, IL-22 and TNF-a production of CD4 T cells decreased after treatment with CD001, while the percentage of Tregs increased. Immunohistochemistry stains also showed that CD001 decreased IL-17+ cells in the joints. In summary, pharmacological approach to inhibit dendritic cells and Th17 cells may be a promising intervention to treat rheumatoid arthritis.
The potential role for berberine, an alkaloid dietary supplement, in mitigating the subclinical pathology and delaying onset of rheumatoid arthritis
Authors: Vita Alexandra Adorno , Pullen Nicholas A , Lyons David
Published: January 2023

Berberine (BBR), a dietary supplement, is primarily marketed for the alleviation of polycystic ovary syndrome, type II diabetes, and inflammatory bowel disorders, with evidence from successful clinical trials. However, evidence also suggests that BBR might alleviate symptoms of rheumatoid arthritis in vivo, and have a potential prophylactic role from in vitrostudies. We examined BBR’s prophylactic potential for delaying onset of collagen-induced arthritis (CIA) in DBA/1J mice. To induce arthritis, mice were injected with a collagen/CFA emulsion on day 0 and a booster injection of collagen/IFA on day 18. Mice were either treated with 1 mg/kg of BBR daily (CIA+BBR group) or a vehicle control (CIA+PBS) via IP injections from day 0 to day 28, were left untreated (CIA group), or were in a non-arthritic control group. Incidence of arthritis by day 28 in CIA+BBR mice was 40%, compared to 90% in the CIA and CIA+PBS groups. Populations of B cells and T cells from the spleens and draining lymph nodes were examined from random mice across treatment groups during the pre-clinical stage of arthritis (day 14), and from the remaining mice on day 28 when arthritic signs and symptoms were expected to be apparent. Expression of co-stimulatory molecules CD80, CD86, MHC Class II, and CD40 was quantified on CD19+ B cells, and CD40L and CD28 on CD4+/CD3+ T cells. The concentrations of collagen type II-specific IgG2a, IgG1, and total IgG was obtained via ELISA. Finally, leukocyte infiltration into synovial tissue was imaged via confocal microscopy and compared across treatment groups on day 14 and day 28. The results indicate that there may be a potential role for BBR as a prophylactic supplement, although further examination into mechanism of action must be elucidated.
Two morphological changes of cancer cells under natural killer cell killing
Authors: CHOI YEO JIN , Ahn Gi Beom , Kim Hyung Sook , Lee Hong Kyung , Kim Youngsoo , Hong Jin Tae , Han Sang- Bae
Published: January 2023

Natural killer (NK) cells use granule exocytosis to kill cancer cells, which involves perforin-mediated delivery of granzymes from NK cells to cancer cells. Once inside the cancer cells, granzyme B, a member of serine protease family, activates caspase-3, which activates the serine/threonine kinase Rho-associated coiled coil–containing protein kinase (ROCK) I, an effector of the small GTPase Rho. Activated ROCK I increases the phosphorylation of myosin regulatory light chain and thus stimulates actomyosin contraction, required for apoptotic membrane blebbing. Ultimately, cancer cells under NK cell attack show rounding, shrinkage, and membrane rupture, which are classical morphological changes observed at the onset of apoptosis. Although much has been learned about these morphological changes, a fundamental question of how melanoma cell death is induced by NK cells remains unanswered. Do all melanoma cell lines undergo the classical dying process? If not, is there another dying process? To address this simple issue, we examined the dying of B16 melanoma cell lines under NK cell attack by using time-lapse imaging. Upon contact with NK cells, B16-F10 cells rounded and most of them showed membrane rupture (98 min); however, B16 parent cells showed writhing and delayed membrane rupture (235 min). This morphological difference depended on the expression levels of myosin regulatory light chain 9 (MYL9) but not activating ligands (CD112, CD155, Rae-1, and MULT-1), SPI, FasL, or PD-L1. Taken together, our data show that melanoma cells show two distinct types of morphological changes upon contact with NK cells and suggest that a strategy to decrease MYL9 expression by melanoma cells may improve the efficacy of NK cell–based immunotherapy.
Knockout immune regulator FGL2 in tumor cells impairs tumor progression in the CNS by facilitating CD103+ dendritic cell differentiation
Authors: Yan Jun , Zhao Qingnan , Gabrusiewicz Konrad , Kong Ling-Yuan , Xia Xueqing , Wang Jian , Ott Martina , Xu Jingda , Davis Eric Richard , Huo Longfei , Rao Ganesh , Sun Shao-Cong , Watowich Stephanie S , Heimberger Amy B. , Li Shulin
Published: January 2023

Although many studies link brain tumor progression to oncogene activation and tumor-suppressor gene inactivation in tumor cells, few studies implicate immune regulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout (FGL2KO) in GL261, DBT, and LLC tumor cells did not affect tumor cell proliferation in vitro or tumor progression in immunodeficient NSG mice, but completely impaired GBM progression in immune-competent C57bl/6 mice. This impairment was reversed in mice with a defect in Batf3 (a key transcription factor for CD103+ DCs differentiation). Mechanistic studies revealed that FGL2KO in tumor cells induces CD103+ DCs differentiation in both the central nervous system (CNS) and in tumor draining lymph nodes (TDLN). The increased CD103+ DCs population in the CNS and TDLNs induce CD8+ T cells priming and activation and thereby gliomas regress. More specifically, the presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.
Dendritic cells present cancer-derived p:MHCI molecules to prime anti-tumor CD8 T cells
Authors: MacNabb Brendan , Kline Douglas , Chen Xiufen , Tumuluru Sravya , Kline Justin
Published: January 2023

Migratory CD103+ dendritic cells (DCs) initiate anti-tumor CD8+ T cell responses by presenting tumor antigens on class I major histocompatibility complex (MHCI) molecules. Given that CD103+ DCs are adept at cross-presenting antigens, the current paradigm is that they present tumor antigens via classical TAP-dependent cross-presentation. However, recent studies have shown that DCs can present cellular antigens by other means, including by acquiring and presenting intact peptide:MHC (p:MHC) complexes from other cells, a mechanism known as cross-dressing. To assess the role of antigen cross-dressing in priming spontaneous anti-tumor CD8+ T cell responses in vivo, the murine MHCI molecule H2-Kb was knocked out in B16 melanoma and C1498 leukemia cell lines. Engraftment of these syngeneic tumors expressing Kb-restricted model antigens into C57BL/6 mice revealed a defect in antigen-specific CD8+ T cell priming when the cancer cells lacked Kb. While the absence of Kb expression by the tumor did not alter the level of costimulatory molecules on DCs or the cytokine milieu of the draining lymph node, it was correlated with a reduction in p:MHCI molecules for a model tumor antigen, SIINFEKL, presented by CD103+ DCs. Further, tumor-derived GFP-tagged Kb molecules were readily observed on the surface of DCs isolated from the tumor, and the presentation of acquired tumor-derived p:MHCI complexes by migratory DCs from tumor-draining lymph nodes of Kb/Db−/− and Tap1−/− mice was sufficient for ex vivo CD8+ T cell priming. Together, these data address a critical knowledge gap in tumor antigen presentation, and highlight a previously under-appreciated pathway as a crucial and non-redundant mechanism of CD8+ T cell activation by DCs.
Effector T cell stimulation using Staphylococcal enterotoxin G and I loaded HLA transgenic dendritic cells
Authors: Knopick Peter Leo , Riha Nathan , Biswas Nivedita , Nilles Matthew , Bradley David S
Published: January 2023

Staphylococcal enterotoxins (SE) induce powerful T cell stimulation with specificity for the variable beta (Vβ) region of the T cell receptor (TCR). Additionally, MHC in mice (H2) and humans (human leukocyte antigen; HLA) differ in binding superantigens and, in turn, provide different T cell activating potentials. We have previously demonstrated that SEG and SEI, administered together, elicited progression free survival of B16–F10 challenged HLA-DQ8 (DQ8; DQA1*0301 and DQB1*0302) transgenic mice. C57BL/6 (B6) mice did not benefit from SEG/SEI given post B16–F10 challenge, as expected, given a markedly reduced T cell proliferation in the B6 mice compared to the DQ8 mice. We provide evidence here of the potential of dendritic cells (DCs) to be the vector for a SEG and SEI immunotherapy. Primary antigen presenting cells, DCs and monocytes, were isolated from the bone marrow of DQ8 or HLA-DR3 (DRB1*0301) transgenic mice. SEG/SEI loaded DQ8 and DR3 DCs drove a strong B6 CD8 T cell response at low T cell:DC ratios; whereas both DQ8 and DR3 DCs at a high T cell:DC ratios activated CD4 T cells. Collectively, these data support incorporating SEG and SEI into the current DC based immunotherapeutic strategies to induce a stronger and direct anti-cancer T cell response.
Inhibition of SHP-1 expands the repertoire of anti-tumor T cells available to respond to checkpoint blockade therapy
Authors: Snook Jeremy , Soedel Ashleigh , Williams Matthew A
Published: January 2023

The presence and activity of CD8+T cells within the tumor microenvironment is essential for the control of tumor growth and impediment of cancer progression. Utilizing B16 F10 melanoma tumors that express altered peptide ligands of OVA257–264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-1 CD8+T cell into tumor-bearing mice. As expected, we found that TCR affinity positively correlated with the frequency of OT-1 tumor infiltrating lymphocytes (TILs). The differences in TCR affinity inversely corresponded to in vivo tumor growth rate. We next sought to determine whether checkpoint blockade therapy (CB: anti-PD-1; anti-CTLA-4) could enhance the recruitment of low affinity T cells. However, CB only increased the frequency of OT-1 TILs responding to high affinity antigens, while failing to alter the response or protective capacity of intermediate to low affinity T cell responses. To explore whether lowering TCR activation threshold could enhance the breadth and magnitude of the anti-tumor T cell response, we used shRNA to knock down Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-1 T cells prior to tumor antigen exposure. Unlike CB, SHP-1 knockdown increased the magnitude and effector function of both low affinity and high affinity OT-1 T cells. When SHP-1 knockdown was combined with immunotherapy, we observed an additional boost in the OT-1 response, as well as enhanced tumor control by intermediate to low-affinity OT-1s. We conclude that targeting TCR activation threshold expands the repertoire of T cells available to respond to conventional CB, leading to enhanced control of tumor growth.
Guadecitabine, in combination with a TLR9 agonist, promotes anti-cancer immunity in BALB/c mice bearing 4T1 mouse mammary carcinoma
Authors: Smith Timothy M , Martin Rebecca
Published: January 2023

The classical approach to vaccine development establishes an effective, lasting memory response by challenging an organism with an antigen/adjuvant combination to prime the immune system against the antigen challenge. However, with respect to cancer vaccine development, this approach has often failed to elicit positive outcomes. This is, in part, due to the ability of tumor cells to downregulate specific antigens and forego immune surveillance. The objective of this study is to develop a treatment strategy that induces expression of cancer testis antigens (CTAs) on tumor cells while eliciting a strong and lasting immune response against these CTAs. CTAs are derived from proteins only expressed in immune-privileged sites under normal conditions, but de novo expression can be induced following treatment with a DNA methyltransferase inhibitor, such as guadecitabine (GUA). Previous publications have shown that intratumoral injections of a TLR9 agonist, CpG, results in an immune response against antigens expressed within the treated tumor. Our experiments have shown that 4T1 cells, a mouse mammary carcinoma, treated in vitro with GUA resulted in increased expression of CTAs as measured by flow cytometry. In addition, BALB/c mice challenged with 4T1 cells then treated with GUA + CpG resulted in depletion of myeloid derived suppressor cells and significantly reduced tumor burden. Tumor-bearing mice that received GUA treatment followed by adoptive T-cell therapy (ATC) also had significantly smaller tumors compared to the ATC only group. These findings warrant further investigation into whether treatment with GUA prior to treatment with CpG results in a lasting memory response against CTAs.
Glycogen synthase kinase 3 (GSK-3) synergizes with immune-checkpoint blockade (ICB) to overcome tumor resistance due to unique tumor infiltrating T-cell subsets as revealed by mass cytometry (CyTOF)
Authors: Krueger Janna , Taylor Alison , Motta Vinicius , Stagg John , Watson Ian , Rudd Christopher E
Published: January 2023

We have shown that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a major regulator of programmed cell death-1 (PD-1) transcription and expression in T-cells (Taylor et al., 2016, Immunity). Further, small molecule inhibitors (SMIs) increased Tbx21 transcription for enhanced Tbet expression which suppressed Pdcd1 transcription in CD8+cytolytic T-cells. SMIs against GSK-3 are as effective as anti-PD-1 blocking antibody in the control of spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells (Taylor et al., 2018, Cancer Research). This introduced a new chemical approach to down-regulate PD-1 for the treatment of cancer. In addition, GSK-3 effects in Tbet expression can induce the expression of interferon-γ1 (IFNγ1) and granzyme B (GZMB) for enhanced cytolytic T-cell (CTL) killing of tumors suggesting that GSK-3 SMIs might synergize with check-point blockade.

We now show that GSK-3α/β inhibition can cooperate with anti-PD-1 blockade to overcome tumor resistance. Using high dimensional profiling of tumor infiltrated lymphocytes (TILs) with mass cytometry (CyTOF), we show that the synergy of GSK-3 SMI and anti-PD-1 is accompanied by 2 specific findings: (i) an increase in the presence of a unique subset of CD8+ TIL effector-memory T-cells beyond that observed with anti-PD-1 alone, and (ii) the significant decrease of a subset of CD4+ suppressor regulatory T-cells (Tregs) not observed with anti-PD-1 alone. Our findings offer a small molecule approach for synergy with anti-PD-1 that leads to an increase in CD8+ T-cells and the marked decrease of Tregs in tumors.
Splenic CD169+ marginal zone macrophages directly cross-prime CD8+ T cells in vivo using a vacuolar processing pathway
Authors: Van Endert Peter M , Mauvais François-Xavier , Hamel Yamina , Diana Julien , Garfa Meriem
Published: January 2023

Cross-priming of CD8+ T cells is of critical importance in triggering CD8+ T cell responses against pathogens and tumors. It is commonly thought that conventional type 1 dendritic cells (cDC1) cross-prime with unequalled efficacy, conferred to them by a proteasome and TAP-dependent pathway. Here we present data challenging the unique cross-priming efficacy of both the cDC1 subset and the proteasome/TAP-dependent pathway. Splenic CD169 macrophages are strategically placed in the marginal zone for capture of blood-borne pathogens. However, they are considered unable to directly cross-present and to depend on antigen transfer to cDC1 for cross-priming. We have developed a protocol for purification of CD169 macrophages. Extensive characterization shows that these cells display morphology, phenotype and gene expression distinguishing them clearly from cDC1, cDC2, plasmacytoid DC and red pulp macrophages. In vitro, CD169 cells cross-present soluble, receptor-targeted and phagocytosed antigen with equal or better efficiency than cDC1. Remarkably, this is achieved through a vacuolar, BFA and epoxomicin-independent pathway. Using intravital biphoton microscopy, 3D light sheet imaging and antigen targeting to CD169, we show that CD169 macrophages establish long-lasting antigen-dependent contacts with cognate CD8+ T cells that result in T cell activation in situ and generation of triple-positive effector as well as memory cells. Therefore, splenic CD169 macrophages represent a second set of myeloid cells equipped for highly efficient cross-priming. The biological role of cross-priming by these cells as well as their cooperation with cDC1 cells are under investigation.
Wdfy4-deficiency reveals a critical role for cross-presentation in anti-viral and anti-tumor responses
Authors: Theisen Derek J , Davidson Jesse T , Briseño Carlos G , Gargaro Marco , Lauron Elvin J , Wang Qiuling , Desai Pritesh , Durai Vivek , Bagadia Prachi , Brickner Joshua R , Beatty Wandy L , Virgin Herbert W , Gillanders William E , Mossamaparast Nima , Diamond Michael S , Sibley L David , Yokoyama Wayne , Schreiber Robert D , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.
High GILT expression and an active and intact MHC class II antigen presentation pathway are associated with improved survival in melanoma
Authors: Hastings Karen , Meador Lydia , Menon Hari , Lu Yih-Kaung , Brill Jacob , Cui Haiyan , Roe Denise J. , DiCaudo David J. , Buetow Kenneth H.
Published: January 2023

The MHC class I antigen presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of tumors. However, the clinical significance of the MHC class II antigen presentation pathway in melanoma cells is less clear. In antigen presenting cells, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens. While not expressed in benign melanocytes of nevi, GILT and MHC class II expression is induced in malignant melanocytes in a portion of melanoma specimens. Analysis of The Cancer Genome Atlas (TCGA) cutaneous melanoma dataset showed that high GILT mRNA expression was associated with improved overall survival. Expression of IFN-γ, TNF-α, and IL-1β was positively associated with GILT expression in the TCGA melanoma specimens. These cytokines were capable of inducing GILT expression in melanoma cells in vitro. GILT protein expression in melanocytes was induced in halo nevi, which are nevi undergoing immune-mediated regression, and is consistent with the association of GILT expression with improved survival in melanoma. To explore potential mechanisms of GILT’s association with patient outcome, we analyzed the mRNA expression pattern in the MHC class II antigen presentation pathway. In contrast to healthy skin where the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway of the TCGA melanoma dataset. Both an active and intact MHC class II pathway were associated with improved overall survival in melanoma. These studies support a role for GILT and the MHC class II antigen presentation pathway in melanoma outcome.
Comprehensive Immunopeptidome Analysis of I-Ab-bound peptides from Thymus and Splenic B cells in C57BL/6 mice
Authors: Nanaware Padma Pandurang , Stern Lawrence J
Published: January 2023

Antigen presentation in the thymus is central to selection of CD4 and CD8 T cells. Despite postulated roles for differential central and peripheral antigen presentation pathways for MHC-I and MHC-II, comprehensive immunopeptidome analysis of the thymus has not been reported. Advances in mass spectrometry (MS) technology now allow analysis of the MHC-presented peptidomes of tissue samples containing small numbers of antigen-presenting cells. To map the immunopeptidome in the thymus of C57/BL6 mice presented by I-Ab, we used immunoaffinity coupled to MS. We identified >1000 peptides using a conventional data-dependent acquisition strategy (DDA). We compared the immunopeptidome of thymus to that of the splenic B cells. Some of the core epitopes were unique to thymus, and some were unique to splenic B cells, although most were shared. We observed differences in the general characteristics of the peptidomes presented by thymus and splenic B cells, including length distribution and hydrophobicity. To help understand the impact of the hydrophobicity differences, we selected 57 peptides representative of thymic-derived, splenic B-derived, or shared peptidomes, and studied their binding affinity to IAb. Results suggest that the pool of peptides presented by I-Ab in thymus has lower MHC binding than does the pool presented by splenic B cells. We are exploring a data-independent acquisition strategy (DIA) for quantitative comparison of peptides identified in thymus and splenic B cells to further characterize antigen presentation differences in thymus and periphery.
VSV-primed CD8+ T cells suppress autoreactive germinal centers
Authors: Kole Hemanta K , Scott Bethany , Crampton Steve , Sule Gautam , Deane Jonathan , Bolland Silvia
Published: January 2023

Pathogenic infections are important environmental modifiers of autoimmune disease as they can alter the immune system in a way that either promotes or reduces autoimmune responses. We tested the effect of infection with vesicular stomatitis virus (VSV) on the FcγR2B−/− (R2) mouse strain that spontaneously develops lupus disease. Infecting FcγR2B−/− mice with live VSV reduces autoantibody levels and inflammatory pathology and prolongs survival. We showed that VSV-mediated protection can be transferred in a CD8 cell adoptive transfer experiment. After further testing the transfer of various CD8 T cell subsets, we have identified CD8+CD122+NKG2D+ cells as mediator in VSV-induced Lupus amelioration. RNA Sequencing data for gene expression profiles among various CD8 subsets, indicate that the protective cells express higher level of genes linked to memory phenotype. Our working hypothesis is that the viral infection may induce the expansion of long lived CD8 populations that regulate the antibody response.

In support of the idea we initiated to identify the target of the VSV-primed CD8 T cells in vivo. We examined the effect of VSV-primed CD8 T cells on the immune phenotypes of recipient R2 mice at the initial stages, e.g. after 1, 2 or 3 weeks of transfer. Interestingly, we found that Tfh and germinal center B cells are significantly reduced after three weeks of VSV-CD8 transfer. Thus, the protective CD8 T cells from VSV-infected mice might target autoreactive germinal center to regulate the antibody response. How VSV-CD8 T cells might control germinal center & why it takes about 3 weeks for controlling is under investigation.
Induction of Regulatory B Cells by Bacterial DNA in the Gut Microbiota at Early Age is Beneficial in Lupus-prone Mice
Authors: Mu Qinghui , Swartwout Brianna , Cabana-Puig Xavier , Grieco Joe , Cecere Thomas E , Luo Xin M
Published: January 2023

SLE is a complex autoimmune disorder with no known cure. We previously reported that oral vancomycin treatment of female MRL/lpr mice during active disease (9 to 15 weeks of age) could significantly ameliorate disease symptoms. However, when vancomycin treatment was initiated from an earlier age (from 3 to 15 weeks), the beneficial effect was not observed. Strikingly, mice given vancomycin during the pre-disease stage (from 3 to 8 weeks) exhibited exacerbated lupus disease. As vancomycin works by removing parts of the gut microbiota, we hypothesized that the regulatory immunity induced by gut microbiota at early age is essential in hammering lupus disease development in MRL/lpr mice. To test the hypothesis, we analyzed diverse regulatory cell types from the mice receiving vancomycin from 3 to 8 weeks of age. Regulatory B (Breg) cells, in particular, were found to be reduced in both the percentage and absolute number in multiple lymphoid organs. Importantly, adoptive transfer of Breg cells at 6–7 weeks of age to long-term vancomycin-treated mice improved lupus-like symptoms. This clearly indicates that Breg cells, inducible by vancomycin-sensitive gut microbiota, plays an important role in suppressing lupus disease initiation and progression. We next sought to determine Breg inducers in the gut microbiota. The serum level of bacterial DNA was found to be significantly lower in mice treated with vancomycin. More importantly, early administration of bacterial DNA reproduced the beneficial effect seen in the Breg adoptive transfer experiment. Together, these results suggest an important protective mechanism against lupus initiation that involves bacterial DNA in the gut microbiota and the induction of Breg cells.
Linking diet, gut microbiota and autoimmunity: A phytoestrogen diet alters the gut microbiota and influences Experimental Autoimmune Encephalomyelitis (EAE)
Authors: Freedman Samantha N , Shahi Shailesh , Zarei Kasra , Gupta Arnav , Guseva Natalya , Bossler Aaron , Legge Kevin L , Mangalam Ashutosh K
Published: January 2023

The etiology of multiple sclerosis (MS) involves genetic predisposition and an unknown environmental trigger. The gut microbiome may be an environmental contributing factor. Previously, we have shown that certain gut bacteria especially those with ability to metabolize phytoestrogen are less abundant in MS patients compared to healthy individuals. We hypothesize that phytoestrogen metabolism via microbial flora induces an anti-inflammatory state in the intestine. Therefore their reduced levels might predispose to disease development and/or severity. To test this, we placed C57Bl/6 mice on a diet with or without phytoestrogens and induced Experimental Autoimmune Encephalomyelitis (EAE), an experimental model of MS. We observed that mice on a phytoestrogen diet developed milder disease compared to those on a phytoestrogen free diet; a phenomenon dependent on the presence of phytoestrogen-metabolizing bacteria in the gut. We also found that a phytoestrogen diet alters the gut microbiota compared to a phytoestrogen-free diet with the gut microbiota of phytoestrogen group closely resembling the gut microbiota of healthy individuals. The gut microbiota of mice on a phytoestrogen-free diet closely resembles the gut microbiota of MS patients. Furthermore, mice on a phytoestrogen-diet exhibit an altered immune profile in the colonic lamina propria and mesenteric lymph nodes. Finally, we found that phytoestrogen-metabolites altered the cytokine-producing function of human colonic epithelial cells in vitro. Our results suggest that dietary phytoestrogen metabolites produced by commensal bacteria can alter the phenotype and function of the intestinal immune system, which may influence central nervous system autoimmunity.
Dysbiosis reprograms the differentiation of thymus-derived regulatory T cells
Authors: Wang Ke , Yang Bi-Huei , Fu Wenxian
Published: January 2023

Foxp3+ regulatory T (Treg) cells can be developed from the thymus (tTreg) or induced in the peripheral tissues (pTreg). While abundant literature has reported that gut microbiota and derived metabolites profoundly influence the generation and abundance of pTreg cells, whether these environmental cues also affect tTreg cells remains poorly understood. Our previous work has defined tTreg cells in peripheral organs into three distinct subsets, exhibiting different profiles of differentiations and functions. We here report that intestinal tTreg cells exhibited a "reversed” phenotype of differentiation in antibiotic (Abx) treated mice. This "dedifferentiation” was mediated by gut microbiota because cohousing Abx treated mice with untreated mice rectified this abnormal phenotype. In addition, we found that dysbiosis altered Treg's cytokine dependence for their maintenance and survival. Thus, gut microbiota have a critical role in shaping the differentiation and functional programs of tTreg cells.

This work is supported by NIH DK114427, AI139753 (to W.F.)
Heightened levels of antimicrobial response factors in patients with systemic lupus erythematosus
Authors: Ayyappan Prathapan , Harms Robert Z , Buckner Jane H , Sarvetnick Nora E
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by immune system abnormalities and severe inflammation leading to multi-organ damage. Compromised gut permeability and microbial translocation is suggested to be associated with SLE. The objective of this study was to assess whether SLE subjects have increased systemic exposure to bacteria. Plasma from 30 SLE patients and 30 age and sex matched healthy controls were assessed for a series of clinically relevant antimicrobial response factors (ARFs) including sCD14, EndoCAbs, LBP, lysozyme, CXCL16, FABP2, galectin-3, and LL-37. The levels of sCD14 (P<0.0001), lysozyme (P<0.0001) and CXCL16 (P=0.001) were significantly elevated in SLE subjects compared to healthy controls. sCD14 exhibited a positive correlation with SELENA-SLEDAI score. EndoCAbs are antibodies directed against the endotoxin core of LPS. Interestingly, the ratio of EndoCAb IgM:total IgM was significantly decreased in SLE (P=0.006) and this ratio was negatively correlated with sCD14 levels (P=0.030, r=-0.396). We observed significant positive correlations between lysozyme levels and sCD14 (r=0.499, P=0.005), LBP (r=0.434, P=0.016), and FABP2 (r=0.396, P=0.030). Levels of CXCL16 was also positively correlated with the levels of sCD14 in SLE cohort (r=0.551, P=0.001). Moreover, Galectin-3 levels also positively correlated with lysozyme (r=0.447, P=0.013), sCD14 (r=0.425, P=0.019), and LBP (r=0.416, P=0.022). Overall, these changes in the levels and relationships between ARFs link microbial exposure and SLE. Thus, our study results are relevant to clinical SLE that will help to define better therapeutic strategies for SLE patients.
Aberrant MDA5 signaling and IFN-β synthesis elicit protection from Coxsackievirus-accelerated Type 1 diabetes
Authors: Blum Samuel I , Burg Ashley R , Chen Yi-Guang , Tse Hubert Ming
Published: January 2023

Viral infections can trigger the synthesis of proinflammatory cytokines and free radicals that contribute to pancreatic β-cell destruction leading to Type 1 diabetes (T1D). We previously demonstrated that Coxsackievirus B3 (CB3) infection of Non-Obese Diabetic (NOD) mice can accelerate T1D, due to the induction of antiviral signaling pathways including melanoma differentiation-associated protein 5 (MDA5). SNPs within Ifih1, the gene encoding for MDA5, is associated with T1D pathogenesis, but the molecular mechanism contributing to innate immune dysregulation is not known. We hypothesized that attenuated MDA5 expression and signaling can delay diabetogenic viral-accelerated T1D. We generated NOD mice mutated in Ifih1 that displayed a loss in MDA5 expression (NOD.Ifih1-m1) or an in-frame deletion in the helicase domain that decreased MDA5 synthesis (NOD.Ifih1-m4). Interestingly, CB3-infected NOD and NOD.Ifih1-m1 mice displayed virus-accelerated T1D, but NOD.Ifih1-m4 (n=9 per group) mice were significantly delayed in both spontaneous and CB3-accelerated T1D (p=0.0036 and p=0.0004, respectively). CB3-infected NOD.Ifih1-m4 mice exhibited a 4-fold decrease in pancreas IFN-β levels and may partly explain the delay in CB3-induction of T1D. The Ifih1-m4 mutation did not compromise pancreatic viral clearance, and this was complemented by a ~34% and ~32% reduction in pancreas-infiltrating CD8 T cells and activated macrophages (F4/80+, I-Ag7+), respectively, in comparison to CB3-infected NOD mice. The protective Ifih1-m4 mutation may establish an acute level of IFN-β and islet-infiltrating lymphocytes to inhibit CB3 replication without mediating bystander activation and virus-accelerated autoimmune diabetes.