bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased lymphatic circulation decreases CD4 counts in the lymph nodes of rats in adjuvant-induced arthritis
Authors: Swanson-Mungerson Michelle Anne , Magnuson Michael , Chang Min-Kyung , Hager Denver , Sperrazza Stephanie , Zanotti Brian , Incrocci Ryan , Volin Michael V
Published: January 2023

Rheumatoid arthritis (RA) is a disorder characterized by chronic inflammation. Rat adjuvant-induced arthritis (AIA) is a RA model with dysfunctional lymphatic circulation. We hypothesized that increasing lymphatic circulation would decrease arthritic inflammation. In order to determine if increasing lymphatic flow would decrease arthritic inflammation, we utilized osteopathic lymphatic manipulation (Lymphatic pump treatment (LPT)) during either the onset or during arthritis. LPT was performed by gradually increasing compression on the rat chest wall over 5 seconds followed by rapid release for 5 seconds. This technique was repeated for 1 minute 3X/day for 7 consecutive days either before the onset of inflammation (preventative treatment) or after measurable inflammation (therapeutic treatment). Articular index scoring, ankle caliper measurements, and peripheral blood, spleens, and popliteal lymph nodes were collected. In both the preventative and therapeutic studies, the LPT-treated rats had reduced inflammation. Therapeutically treated animals also had significantly decreased CD4+ lymphocytes in the draining popliteal lymph nodes, with increases in CD4+ cells in the spleen. These results suggest that LPT can reduce arthritis and alter lymphocyte trafficking in secondary lymphoid organs during inflammation. These data suggest that forced lymphatic circulation through osteopathic manipulation may serve to enhance current treatments to decrease inflammatory diseases.
Cancer stem cells inhibit NK cell effector function via PCNA-NKp44 interaction
Authors: Malaer Joseph D. , Mathew Porunelloor A
Published: January 2023

NK cells participate in the innate immune response against infection and cancer without prior sensitization. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of cells. Cancer cells may evade NK-mediated killing by expressing ligands for inhibitory receptors. Proliferating cell nuclear antigen (PCNA) associates with MHC I and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. Pancreatic and colon CSC can be identified by co-expression of surface markers CD44 and CD133. In both cell lines, Panc-1 and HCT 116, cell surface PCNA is associated with co-expression of CD44 and CD133 as well as increased CSC transcription factor expression (NANOG, SOX2, and Oct-4). Blocking the interaction of NKp44 and PCNA enhanced the specific lysis of cells by NK cells. Collectively these data demonstrate that surface PCNA, CD44, and CD133 co-expression is a marker of pancreatic and colon CSC. Our research implicates that blocking NKp44-PCNA interaction may provide a novel immunotherapeutic target for pancreatic and colon cancer stem cells and prevent metastasis.
Pulsatile MEK inhibition improves anti-tumor immunity and T cell function in Kras mutant lung cancer
Authors: Choi Hyejin , Deng Jiehui , Li Shuai , Silk Tarik , Dong Lauren , Brea Eliott J , Redmond David C , Houghton Sean , Falik Nouraiz , Boiarsky Jonathan , Zhong Hong , Akbay Esra A , Smith Paul D , Merghoub Taha , Wong Kwok-Kin , Wolchok Jedd D
Published: January 2023

KRAS is one of the most commonly identified driver oncogene in non-small cell lung cancer (NSCLC) and is commonly associated with disease refractory to current available modalities of treatment. Targeted therapy to inhibit MEK has shown promising tumor growth control in preclinical models, but is followed by quick rebound of tumor growth. Recently, immune checkpoint blockade has shown clinical activity by dis-inhibiting T cells that are suppressed in the tumor microenvironment. We sought to identify the most effective therapy for the treatment of KRAS mutant NSCLC patients by targeting cancer cells and activating infiltrating immune cells concurrently. We focused on studying the impact of pulsatile MEK inhibition on the immune microenvironment. We found that pulsatile MEK inhibition maintained T cell activation better than continuous treatment ex vivo and in vivo. Treatment with two different MEK inhibitors (selumetinib and trametinib) each resulted in higher CTLA-4 and PD-1 expression in tumor-infiltrating T cells using pulsatile treatment, compared to continuous treatment in Kras mutant lung cancer mice. In addition, the pulsatile schedule showed a superior anti-tumor effect and delayed drug resistance compared to the continuous schedule. Further, combination of pulsatile MEK inhibitor treatment and CTLA-4 blockade resulted in the longest survival of Kras tumor-bearing mice. Similar experiments in immune deficient mice confirmed that this prolonged survival is conferred by adaptive immunity. Our findings set the foundation for a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy in patients to optimally enhance tumor apoptosis and promote immune response simultaneously.
REAlease® Immunomagnetic Separation Technology with reversible labeling for tumor infiltrating T cells and sub-populations
Authors: Evaristo Cesar , Siemer Ramona , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Richter Anne
Published: January 2023

Immunotherapies engaging T cells have proven clinical efficacy and tremendous potential. However, responses are often suboptimal. Further research is required to understand tumor-infiltrating leukocytes (TILs) biology and enhance outcomes. TIL analysis is technically challenging and labor intensive. Their number can be very low and small subpopulations may escape analysis as they get lost in the background noise. Importantly, tumor-infiltrating (TI) T cells are embedded in a highly immunomodulatory environment such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping TILs by flow cytometry is time consuming and data processing is laborious. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time and generate high quality and reliable data.

We developed state-of-the-art CD4, CD8, CD3 and CD4/CD8 T cell specific enrichment reagents for magnetic cell sorting incorporating novel technology enabling the removal of both superparamagnetic beads and antibody fragments (REAlease®). Single cell suspension of human ovarian, pancreas or colon tumors was obtained by automated dissociation. TI T cell frequencies ranged from 0,5–14%, were isolated to high purities and with high yield. CD69 was not upregulated after 18h culture, and cells were functional for downstream applications. Importantly, REAlease technology allowed secondary magnetic isolation of sub-populations of T cells, such as gamma-delta T cells.

Our data demonstrates that magnetic enrichment using REAlease technology reduces time and increases quality of analysis, and is essential for the isolation of important T cell sub-populations within the tumor microenvironment.
Harnessing the anti-tumor activity of invariant natural killer T cells for neuroblastoma immunotherapy
Authors: Khurana Priya , Ferry Gabrielle , Karageorgos Spyridon , Vu Annette , McNerney Kevin , Hogarty Michael , Bassiri Hamid
Published: January 2023

Background

Invariant natural killer T (iNKT) cells are innate-like CD1d-restricted T cells that nucleate many anti-tumor immune pathways. In neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, intratumoral iNKT cell presence predicts better 5-year survival and prognosis. For optimal iNKT cell immunotherapy, an understanding of how they sustain effector function long-term within the tumor microenvironment (TME) is needed.

Results

Immunophenotyping of tumors from a mouse model of high-risk NB revealed a significant frequency of iNKT cells relative to conventional T cells (TCONV). To specifically redirect iNKT cell activity toward NB tumors, we developed a conjugate molecule (“CAb”) fusing a glycolipid antigen-loaded CD1d to an antibody directed against an NB-specific antigen. Acute CAb stimulation increased expression of cytokines and activation and degranulation markers specifically on intratumoral iNKT cells. Moreover, these expression levels were maintained upon chronic CAb stimulation. We postulate that iNKT cells can metabolically adapt to the nutrient-poor TME to withstand exhaustion. Transcriptional profiling of in vitro-stimulated iNKT cells and TCONV revealed differential expression patterns of genes in several metabolic pathways. We are now further defining the metabolic profile of iNKT cells and its link to anti-tumor function, and determining whether CAb activation of iNKT cells controls NB growth to improve survival.

Conclusions

iNKT cells within NB tumors can be activated and may possess unique metabolic and functional features from TCONV that confer sustained anti-tumor activity. Defining these properties will better inform the use of iNKT cells for solid tumor immunotherapy.
CD28 homolog is a strong activator of natural killer cells for lysis of B7H7-positive tumor cells
Authors: Zhuang Xiaoxuan , Long Eric O
Published: January 2023

The CD28–B7 family of receptor–ligand pairs regulate lymphocyte responses through costimulation and coinhibition. It includes checkpoint inhibitors, such as PD-1, which limit anti-tumor and anti-virus T cell responses. CD28 homolog (CD28H) and B7H7 have been identified as a new receptor–ligand pair in this family, which has costimulatory activity in T cells. Here we show that CD28H is expressed in primary natural killer (NK) cells and that it is a strong activator of NK cells through selective synergy with receptors NKp46 and 2B4 to induce degranulation, lysis of target cells, and production of pro-inflammatory cytokines. Expression of B7H7 on target cells enhanced both natural and antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Mutation of tyrosine 192 on the CD28H cytoplasmic tail abolished NK cell activation through CD28H. As B7H7 is broadly expressed in tumor tissues, we engineered a CD28H chimeric antigen receptor (CD28H-CAR) consisting of full-length CD28H fused to the cytoplasmic domain of T cell receptor zeta chain. NK cell activation is controlled by dominant “self-inhibition” mediated by inhibitory receptors for MHC class I, which has been a constraint for NK cellular cancer therapy. Remarkably, expression of CD28H-CAR in NK cells triggered lysis of tumor cells expressing HLA-E and B7H7 by overriding inhibition mediated by the HLA-E receptor NKG2A. The cytoplasmic domains of both CD28H and TCR zeta chain were required for this activity. Thus, CD28H is a powerful activation receptor of NK cells that broadens their anti-tumor activity and holds promise as a component of NK-based CARs for cancer immunotherapy.
Monocyte-lineage dendritic cell (DC) precursors are pre-positioned in normal lymph nodes and rapidly differentiate into Batf3+ cross-presenting APCs during inflammation
Authors: Sharma Madhav D , Munn David H.
Published: January 2023

We have shown (Immunity 48:91–106, 2018) that a subset of murine monocytic precursor cells are plastic, and can up-regulate a Batf3-driven differentiation program resembling classical cDC1 cells. The resulting inflammatory DCs are important in tumor immunity, but it has been unclear whether they played a role in normal immune responses. We now identify the precursor cells for these inflammatory DCs as a population of Ly6c+ ckit+ c− fms+ CD11b-neg cells. These are constitutively present in all resting LNs (comprising up to half of the Ly6c+ cells). In response to a standard vaccination model of cross-presented antigen (whole OVA protein plus CpG adjuvant in IFA), the precursor cells rapidly up-regulated Batf3 and differentiated into CD103+ IRF8+ DCs. Just as in tumors, maturation was dependent on intracellular p53 signaling in the precursor cells. Targeted disruption of p53 (LysMcre/p53-KO) had no effect on the number of precursors, but abrogated their ability to mature into Batf3+ DCs. In KO mice, the loss of this single monocytic DC population entirely abrogated the ability to create an inflammatory milieu in the vaccine-draining LN, and abolished cross-presentation of protein antigen to T cells. In contrast, direct presentation (soluble siinfekl peptide) remained intact. We hypothesize that the ability of monocyte-lineage cells to differentiate into Batf3+ APCs during inflammation is an important component of the normal cross-presentation process.
Cannabinoids decrease intestinal permeability and induce colonic CD103+ dendritic cells to increase T regulatory cells leading to decreased murine colitis-associated colon cancer
Authors: Becker William J , Alrafas Haider , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: January 2023

Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two abundant phytocannabinoids found in Cannabis sativa. Cannabinoids bind to cannabinoid receptors 1 and 2 (CB1 and CB2), located primarily on the neurons and immune cells, respectively. Cannabis and synthetic THC and CBD are quickly gaining attention for their therapeutic potential in diseases ranging from epilepsy to multiple sclerosis, and cancer pain.

Colon cancer is the third leading cause of cancer-related deaths, and treatment options are limited to surgery, chemotherapy or radiation. Many physicians are now moving towards a strategy focused on prevention. Chronic inflammation is a cause of many cancers including colon cancer. Thus, we used a murine model of colitis induced colon cancer, wherein BL6 mice were given a single administration of carcinogen (azoxymethane) followed by DSS (1%) in the drinking water daily for 1 week followed by 2 weeks without DSS. This cycle, along with bi-weekly treatment with cannabinoids (CBD, THC, THC+CBD, or vehicle; all 10mg/kg; by oral gavage) continued for 10 weeks, as the progression of cancer was monitored.

Our data revealed that treatment with THC and THC+CBD resulted in decreased polyp formation in mice with colon cancer. We also showed that cannabinoids increased CD103+ dendritic cells in the colonic lamina propria that led to an increase in the CD4+Nrp1+FoxP3+ T regulatory cells (nTregs), which was dependent on CB2. Cannabinoids also increased the colonic barrier integrity as assessed by increased levels of ZO-1, through CB1, and increased mucus production. These changes collectively demonstrated cannabinoid treatment reduced the incidence of colitis-associated colon cancer by inducing an anti-inflammatory response.
A novel CSF-1R neutralizing antibody, DCB-AB21, inhibits colony stimulating factor 1 receptor signaling activity
Authors: Chen-Hsuan HO , Liao Chu-Bin , Chen Yu-Kai , Huang Chen-Wei , Yang Tze-Pin
Published: January 2023

Tumor-associated macrophages (TAMs) represent the major cell components of immune infiltrate in solid tumors, and it has been reported that high TAMs density is associated with poor clinical prognosis. Within the tumor microenvironment, TAMs rely on signaling through colony stimulating factor 1 receptor (CSF-1R)/CSF-1 axis for various tumor promoting processes such as tumor growth, angiogenesis, and metastasis. Therefore, decrease of TAMs density in tumors through CSF-1R inhibition is considered an attractive therapeutic target for drug development. In this study, we report that a novel anti-CSF-1R monoclonal antibody DCB-AB21 which specifically binds to CSF-1R and inhibits CSF-1R downstream signaling. Anti-proliferative activity was measured on murine Ba/F3 cells engineered to be dependent on CSF-1R, and our results showed DCB-AB21 can blocks the CSF-1 and IL-34-induced cell proliferation. Potent inhibition of CSF-1R-dependent signaling was observed in the Ba/F3 CSF-1R driven model cells, and in human THP-1 leukemia monocytic cell lines. Humanization engineering further improves this antibody’s affinity and anti-proliferative efficacy. The discovery of novel binding epitope of DCB-AB21 differentiates itself from the current CSF-1R targeting antibodies testing in clinical trials. In vivo efficacy of DCB-AB21 is now in progress.

Together, these data demonstrate that DCB-AB21 can potently inhibit CSF-1R in cellular contexts and that this activity has the potential to induce a phenotypic effect on macrophages. These data suggest that DCB-AB21 is a promising new agent with possibility to combine with immune checkpoint inhibitors to relief macrophage-dependent immune suppression and would yield clinical benefit.
MIF functional polymorphisms (−794 CATT5–8 and −173 G>C) in Basal Cell Carcinoma in western Mexican population
Authors: Alvarado Emmanuel Valdés , Gutierrez Jorge Ramón Padilla , Valle Yeminia , González Mariana Pedroza , Martinez-Fernandez Diana Emilia , Valle Jose Francisco Muñoz , Guevera Elizabeth , Tlacuilo Alberto
Published: January 2023

Basal cell carcinoma (BCC) is the dermatological neoplasm more prevalent worldwide, in recent years there has been an increase in the number of cases becoming a health problem. Despite its high incidence, there is little information about genetic factors that lead to the carcinogenesis of this neoplasms. In the last years, macrophage migration inhibitory factor (MIF) has received particular attention, its expression has been linked to the development of different types of cancer. Therefore, the aim of this study was to investigate the genetic association of −794 CATT5–8 and −173 G>C MIF polymorphisms with BCC.

A total of 150 BCC patients and 150 individuals with similar age than cases as a control group were recruited. Total genomic DNA was extracted from peripheral blood leukocytes by the salting out method. The −794 CATT5–8 MIF polymorphism was analyzed by conventional PCR and polyacrylamide gel electrophoresis, the −173 G>C MIF polymorphism was genotyped by PCR-RFLP as was done in previous studies by our research group.

It was found that BCC incidence is higher in women than men additionally the localization of this neoplasia are more prevalent in head and neck. Also, it was found that the 5/5 genotype of the −794 CATT5–8 MIF polymorphisms of confers a risk to BCC development (OR=4.55, p=0.04). Respect to the −173 G>C MIF polymorphism we did not find a statistical difference.

In conclusion, our findings suggest that the 5/5 genotyope of −794 CATT5–8 MIF polymorphism is associated with the development of BCC in patients from western Mexico. Further studies are needed due to the relation of the functions of MIF and de development of cancer.
Mesenchymal stem cells educate breast tumor associated macrophages to acquire increased immunosuppressive features
Authors: BISWAS SUBIR , Mandal Gunjan , Chowdhury Sougata Roy , Purohit Suman , Payne Kyle K. , Galindo Carmen Maria Anadon , Gupta Arnab , Yu Xiaoqing , Conejo-Garcia Jose R. , Bhattacharyya Arindam
Published: January 2023

Breast cancer metastasis is known to be extensively promoted by immunosuppression. Here we describe a novel mechanism by which mesenchymal stem cell (MSC)-derived exosomes drive an immunosuppressive program within the breast tumor microenvironment. Initial q-PCR and immunophenotyping of human breast tumor samples, and TCGA data analysis confirmed a positive correlation between infiltrations of MSC and M2 macrophage phenotype, therefore we hypothesized that MSC promote M2 macrophage differentiation in breast tumors. We then performed a series of experiments in vitro and in vivo, and found that MSC-derived exosomes significantly promote differentiation of macrophages into PD-L1 expressing ‘M2-like’ phenotype, while also enhancing malignant progression of breast tumors, in vivo. Additionally, we observed increased invasive potential of tumor cells with higher expression of mesenchymal markers when induced with MSC-derived exosomes. Importantly, our observation of a significantly higher TGF-β production by tumor associated macrophages in exosome-induced tumors, with stronger PD-1 expression by intra-tumoral T cells, suggests TGF-β driven PD-1 upregulation. Blocking of PD-L1 abrogates exosome-induced tumor growth signifying the PD-L1/PD-1 checkpoint pathway is vital to MSC-induced tumor progression. Together, infiltration of MSCs within breast tumors drives the increased invasive potential as well as immune-checkpoint-mediated immunosuppression through inducing PD-L1 expression by tumor associated macrophages and PD-1 expression by T cells. Targeting infiltration of MSCs into the breast tumor therefore is a potential approach to reduce breast tumor metastases and improve efficacy of immunotherapies.
IL-15 enhances anti-PD-1-induced reinvigoration of tumor-infiltrating CD8+ T cells
Authors: Leem Galam , Kim Kun Woo , Lee Jea-Ik , Ha Seung Yeon , Park Su-Hyung , Kang Shin Myung , Shin Eui-Cheol
Published: January 2023

IL-15 is a homeostatic cytokine that induces proliferation of NK cells and memory CD8+ T cells. Recently, clinical trials using various forms of IL-15 in combination with immune checkpoint inhibitors in multiple intractable cancers are on progress. In the present study, we examined effects of IL-15 on tumor-infiltrating CD8+ T cells obtained from human non-small cell lung cancer (NSCLC; n = 20) and synergic effects of IL-15 in combination with anti- PD-1 blocking antibodies. More than 80% of tumor-infiltrating CD8+ T cells expressed PD-1, indicating that they are exhausted. We stimulated tumor-infiltrating CD8+ T cells with anti-CD3 antibodies in the absence or presence of anti-PD-1 blocking antibodies and IL-15 and found that anti-PD-1 treatment increased anti-CD3-stimulated proliferation of tumor-infiltrating CD8+ T cells, indicating that tumor-infiltrating CD8+ T cells were reinvigorated by anti-PD-1. Moreover, IL-15 further enhances anti-PD-1-induced reinvigoration of tumor-infiltrating CD8+ T cells in a dose-dependent manner. These findings indicate that IL-15 might increase therapeutic effects of anti-PD-1 by synergistically enhancing anti-PD-1-induced reinvigoration of tumor-infiltrating CD8+ T cells.
Soluble FasL deficiency promotes CD8 T cell dependent elimination of primary intraocular tumors and liver metastases
Authors: McKenna Kyle C , McCullough Peter , Durbin Drew , O’Brien Grace , McDermott Meghan , Rothstein Ann Marshak
Published: January 2023

CD95/FasL is a membrane bound molecule that can be cleaved by metalloproteinases to produce a soluble form. The purpose of these experiments was to determine the influence of soluble CD95/FasL on intraocular tumor growth and liver metastases. P815 tumors transplanted into the anterior chamber of the eye of Balb/C J mice formed intraocular tumors that metastasized to the liver in 33% of mice. In contrast, ΔCS mice, engineered with a point mutation that prevents cleavage of membrane CD95/FasL to a soluble form, rejected intraocular tumors (ΔCS ocular tumor weight = 20+/− 15 mg; Balb/C J ocular tumor weight = 99+/−34) and did not develop liver metastases. Anti-tumor antibody responses were equivalent in Balb/C J and ΔCS mice with intraocular tumors. ΔCS mice depleted of CD8+ T Cells uniformly developed fatal liver metastases. These data indicate that soluble FasL normally inhibits tumor-specific CD8+ T cell responses to promote growth of primary intraocular tumors that metastasize to the liver.
Synergistic activity between IL-15 signaling and checkpoint blockade drives enhanced NK cell expansion and tumor control in ovarian cancer
Authors: Felices Martin , Bendzick Laura , Kodal Behiye , Hinderlie Peter , Miller Jeffrey S , Geller Melissa
Published: January 2023

Survival in ovarian cancer patients has not improved in the past 40 years, stressing a need for new lines of therapy. NK cell immunotherapy in ovarian cancer has not been extensively explored due to limited data showing defective NK cell function in these patients. Our previous work shows that an IL-15 super-agonist (NANT N-803 (formerly ALT-803)) can bypass some of the deficiencies. However, we show here that though N-803 enhances NK cell activation, it also induces NK PD-1 expression and inflammatory cytokines secreted by the NK cells induce PD-L1 expression on ovarian tumors. To bypass this negative feedback we combined N-803 with an anti-PD-L1 antibody (Avelumab) and show a potent increase in NK cell activation and long-term real-time in vitro tumor killing. Furthermore, combined N-803/Avelumab induces robust killing of ovarian cancer tumor spheroids, not seen with individual treatments. To evaluate the benefits of this combined treatment in vivo we tested them in a xenogeneic model of ovarian cancer containing human ovarian cancer cells (MA148lucs or OVCAR8lucs) and human NK cells. Our data clearly shows reduced tumor burden, by bioluminescent imaging, and potently increased NK cell expansion with the combined treatment when compared to individual treatments. Since Avelumab is capable of driving antibody dependent cell-mediated cytotoxicity (ADCC) we wanted to evaluate the impact of checkpoint signals in the synergy excluding ADCC using an anti-PD-1 antibody (Pembrolizumab). Combination of N-803 with Pembrolizumab yielded similar tumor control and NK cell expansion in the in vivo model, indicating that the synergy lies in checkpoint signals and IL-15 signaling, and not just in amplification of ADCC via N-803.
Pathology of B cell and neutrophil infiltration in the YUMMER1.7 mouse model of spontaneous melanoma tumor regression
Authors: Blenman Kim RM , Wang Jake , Cowper Shawn , Bosenberg Marcus
Published: January 2023

We evaluated the spontaneous and immunotherapy-induced histological changes in the tumor microenvironment of a mouse melanoma regression model consisting of C57BL/6J mice implanted with syngeneic YUMMER1.7 melanoma cells. We found common themes to the host response to cancer irrespective of mode of regression. Theme 1: Three regression phenotypes were identified: (1) nonregression, (2) regression, and (3) mixed regression. In nonregression, melanoma cells were epithelioid shaped and tightly packed. In regression, melanoma cells were spindle shaped and discohesive. Theme 2: In nonregression, B220+ B cells were small and located peritumor. In regression, B220+ B cells were intratumor and were plasmablasts/plasma cells. B cell numbers and distribution increased over time in spontaneous regression and was enhanced with immunotherapy. Theme 3: Ly6B.2+ neutrophils were in direct contact with dead/dying melanoma cells. The cytoplasm of the contacted melanoma cells displayed low/intermediate levels of Ly6B.2 protein, which suggested a role in melanoma cell death. Theme 4: At the peak of the adaptive immune response, in the regression tumors, most of the tumor mass were immune cells. Differentiators: The most striking difference between the two modes of regression was the presence of neutrophil extracellular traps (NETs)-like formations and geographic necrosis after immunotherapy treatment. Our preliminary data from patients with metastatic melanoma treated with anti-PD-1 therapy show that higher numbers of tumor-infiltrating B cells corresponded with longer progression-free survival. Beyond tumor regression, the increase in the B cell and neutrophil response could play a role in immunotherapy-induced adverse reactions.
The Human NK Cell Receptor KIR2DS4 Detects a Conserved Bacterial Epitope Presented by HLA-C
Authors: Long Eric O , Sim Malcolm J. W.
Published: January 2023

Natural killer (NK) cells are innate immune effector cells regulated by many germline-encoded activating and inhibitory receptors. The DAP12-associated activating receptor KIR2DS4 has been linked with multiple disease processes including cancer, disorders of pregnancy, and resistance to HIV. However, the precise ligands for KIR2DS4 remain poorly defined and the role of this receptor in immune responses is unclear. Here we show that human KIR2DS4 is a highly peptide-specific receptor for the human MHC-I molecule HLA-C*05:01. Of over 60 different peptides tested, only two conferred strong binding to KIR2DS4. Recognition of these peptides presented by HLA-C*05:01 potently activated KIR2DS4+ NK cells to degranulate and produce IFN-gamma and TNF-alpha. A recombinant peptide:HLA-C complex was sufficient to activate KIR2DS4+ NK cells. An alignment search of the KIR2DS4 binding peptides identified an epitope in recombinase A (RecA), a highly conserved bacterial protein. RecA epitopes from Chlamydia, Campylobacter, Brucella and Helicobacter pathogens were presented by HLA-C* 05:01, bound KIR2DS4 and activated KIR2DS4+ NK cells. By sequence alignment we predict that hundreds of species of bacteria contain RecA epitopes that can be presented by HLA-C*05:01 and bind KIR2DS4. These data provide clear evidence that KIR2DS4 is a highly peptide specific activating receptor and suggest that KIR2DS4 evolved to play a role in immune defense to bacteria.
Investigating a role for HLA-DO in the development of CD4 memory cells
Authors: Song Nianbin , Welsh Robin A , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO) is a MHC class II-encoded protein in humans (known as H2-O in mice). It is expressed only in thymic medulla, B cells and some subsets of dendritic cells. Although DO is found to form a stable complex with HLA—DM, its biological functions remain poorly understood. Based on biochemical experiments, our lab has previously proposed a model suggesting that DO enhances binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules in vitro. We also have established that a reduced density of antigen presentation by B cells during the contraction phase of an infection, induces of anergy in CD4 memory T cells, a condition that helps memory CD4 T cell survival and effective memory response to a second challenge. Based on our model, it would be expected that in immunized DO-KO mice, presentation of altered densities of DM-resistant pMHC by B cells would fail to induce long-lived, multifunctional memory CD4 T cells. We tracked the development of DR1 restricted flu specific CD4 memory T cells three months post flu vaccine immunization in DR1+/DO-KO mice. We found that the DR1+/DO-KO mice had fewer flu specific CD4 memory T cells than DR1+/DO-WT mice. By in vitro and in vivo recall experiments, we also found that the secondary immunological responses in DR1+/DO-KO mice were compromised. In addition, we recovered fewer memory B cells from immunized DR1+/DO-KO mice. These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can potentially hamper the proper development of memory cells after the resolution of the infection. These findings support the idea that the expression of DO might ensure the proper development of memory cells.
Parkinson’s disease related proteins PINK1 and Parkin are major regulators of the immune system
Authors: Matheoud Diana , Cannon Tyler , Voisin Aurore , Penttinen Anna-Maija , Ramet Lauriane , Fahmy Ahmed , Ducrot Charles , Laplante Annie , Bourque Marie-Josée , Zhu Lei , Le Campion Armelle , McBride Heidi , Gruenheid Samantha , Trudeau Louis-Eric , Desjardins Michel
Published: January 2023

Parkinson’s disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons (DN) in the substantia nigra. Although the mechanisms triggering the loss of DN are unclear, mitochondrial dysfunction and inflammation are viewed as playing a key role. PINK1 and Parkin are major regulators of mitophagy and failure in this pathway in DNs is hypothesized to enhanced oxidative stress and cause cell death. However, we showed that PINK1 and Parkin play also a role in adaptive immunity by repressing mitochondrial antigen presentation (MitAP) (Matheoud et al., Cell 2016), suggesting that autoimmune mechanisms participate in the aetiology of PD. Here, following on the finding that LPS triggers MitAP in vitro and in vivo, we present evidence that intestinal infection with Gram - bacteria in Pink1 KO mice increases the release of pro-inflammatory cytokines, activates MitAP and induces autoimmune mechanisms eliciting the activation of cytotoxic mitochondria-specific CD8+ T cells. Remarkably, infection in these mice also leads to the emergence of severe motor impairment, reversed by L-DOPA treatment, accompanied by a sharp decrease in the density of dopaminergic axonal varicosities in the striatum. These data support the role of PINK1 as a modulator of the immune system and provide a new pathophysiological model where intestinal infection acts as a triggering event in PD, highlighting the relevance of the gut-brain axis in the disease.
Polymorphism in chicken MHC-Y class I molecules that bind lipid ligands
Authors: Goto Ronald , Gugiu Gabriel , Zhang Jibin , Stadtmueller Beth , Bjorkman Pamela J , Miller Marcia M
Published: January 2023

Polymorphism is a major structural feature considered to set peptide-binding MHC class I molecules apart from other types of MHC class I molecules that bind other types of ligands. Most polymorphic residues in peptide-binding MHC class I molecules point into the ligand binding groove and delimit the types of peptides bound. For illustration, in an analysis of the α1 and α2 domains of 25 HLA class I sequences, most of the highly polymorphic positions (12 out of 15 positions with variability indices greater than six) have amino acids with side-chains pointing into or toward the ligand binding groove. In contrast, distribution of polymorphic positions in chicken MHC-Y class I molecules is strikingly different. An analysis of 25 MHC-Y class I molecules revealed 16 highly polymorphic positions (variability indices greater than six) in α1 and α2 domain sequences. Only seven of these have amino acids with side chains pointing into or toward the binding groove. The remaining polymorphic positions are located mostly along the α1 helix, pointing up and away from the binding groove. These findings are consistent with the MHC-Y class I binding groove being narrow and binding simple lipid ligands through hydrophobic interactions. It may be that the remaining surface polymorphism is the basis for specificity in the interactions of MHC-Y class I molecules in immune responses that remain to be defined.

Support - USDA NIFA 2016-10247 & Caltech-City of Hope Biomedical Research Initiative
Androgens may influence lupus development via an effect on the composition and metabolic activities of intestinal microbiota in BWF1 mice
Authors: Harder James W , Ma Jing , Chhabra Anita , Alard Pascale , Zhang Xiang , Yuan Fang , Ferrill Rachel , Hua Yuan , Kosiewicz Michele M
Published: January 2023

Like many autoimmune diseases, systemic lupus erythematosus (SLE) occurs much more often in females than males in humans and spontaneous mouse models of lupus, such as NZBxNZWF1 (BWF1) mice. Castration increases disease incidence, severity, and mortality in BWF1 males, and BWF1 females are protected from disease by androgen treatment, suggesting that male sex steroids, androgens, play a protective role in lupus. Accumulating evidence suggests that microbiota dysbiosis is associated with development of autoimmune diseases, and the microbiota influences and is influenced by androgens. Our previous research has linked sex differences in microbiota to the sex-bias of SLE and differences in immunoregulation in BWF1 mice. To investigate the role of androgen in this sex-bias, we have studied the effects of androgen depletion via castration on male BWF1 mice. Fecal samples were collected from castrated and control male and female BWF1 mice, and analyzed for microbiota composition, function, and metabolites. We found that androgen-depletion induced a significant shift in the overall microbiota profile away from that found in control male mice, and toward one that was more similar to BWF1 female mice. Androgen-depletion also altered the fecal metabolite profile; 72 metabolites were altered in castrated compared to control male mice, and of these, 34 were also differentially produced in control female vs male mice. These changes in microbiota and metabolite profiles correlated with increased disease/mortality and altered immunoregulation in castrated male mice. Taken together, these data indicate that androgens have a dramatic effect on the microbiota, and it may be through the microbiota that androgens affect SLE development.
Systemic activation of B and CD4 but not CD8 cells drives early disease in lupus prone mice, while macrophages, CD4 and CD8 cells infiltrate the kidney with the exclusion of B cells
Authors: Wu Juan , Wang Hongsheng , Amo Laura , Scott Bethany , Kole Hemanta K , Bolland Silvia
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies in serum and multi-organ inflammation. Immune complex deposition in kidney can lead to lupus nephritis, which is a major risk factor for morbidity and mortality in SLE. Using FcγRIIB−/− mice as a lupus animal model, we performed a detailed time course analysis of the extent of the spontaneous leukocyte activation and expansion in spleen and in renal, inguinal, mesenteric lymph nodes. We then correlated the data with the onset of pathology and leukocyte infiltration in the kidney. In the FcγRIIB−/− mouse model, effector-memory CD4 cells and germinal centers appeared first in the spleen and renal lymph nodes, but not in other immune organs such as inguinal or mesenteric lymph nodes. As the disease progresses in these mice, lymphocytes undergo an extensive expansion that involves all immune organs in a systemic manner. At the same moment we can detect the first signs of kidney pathology, measured by histopathology and by the levels of creatinine and albumin in urine. In FcγRIIB−/− mice, the onset of kidney pathology correlates with leukocyte infiltration in kidney, detected both by parenchyma-restricted flow cytometry and histopathology. Macrophages, CD4 and CD8 cells are found both in glomerular and interstitial spaces with the exclusion of B cells, most likely suggesting a role for T cells and/or macrophage specific chemokine signals in lupus nephritic kidney.
Divergent roles for TLR7 and TLR9 in modulating the NFκB pathway in B cells
Authors: Nundel Kerstin (Kiefer) , Moses Stephanie , Shim JaeHyuck , Marshak-Rothstein Ann
Published: January 2023

BCR signal strength plays a key role in the fate decision of B cells with strong BCR signals promoting plasma cell differentiation and weaker BCR signals driving germinal center formation. Signals through toll-like receptors (TLRs) also influence B cell differentiation. It is still unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and B cell activation depends on TLR7 and/or TLR9. ICs formed using the auto-antibody (autoAb) PL2-3 can activate AM14 B cells through either BCR/TLR9 or BCR/TLR7, allowing us to use TLR knockout lines to dissect signaling pathways unique to each TLR. Using this system, we reported earlier that BCR/TLR7-activated AM14 B cells differentiate into short-lived plasmablasts while plasma cell development does not occur in BCR/TLR9-activated AM14 B cells. Intriguingly, we now show that BCR/TLR9 activation of AM14 B cells leads to sustained activation of the NFκB pathway. BCR/TLR7-activated AM14 B cells are unable to sustain the initial NFκB response and fail to express the non-classical IκB member, IκBζ. IκBζ is known to be important in regulating cytokine responses in T cells and macrophages. However, its role in B cells is not well defined. We find that several IκBζ regulated cytokines are expressed after BCR/TLR9 dependent signaling but not after BCR/TLR7 dependent signaling. Our results point to divergent roles for TLR7 and TLR9 in modulating the NFκB signaling cascades, leading to differential expression of NFκB target genes which may influence B cell fate decisions.
Characterization of CD11c+ B-cells from human blood
Authors: Calbo Sébastien F , Demeules Mélanie , Derambure Céline , Riou Gaetan , Vaillant Maud , Boyer Olivier , Joly Pascal , Golinski Marie-Laure
Published: January 2023

Different B cell subpopulations with immuno-regulatory properties have been recently identified. We previously reported a CD11c+ B-cell subset that was not able to produce IL-10 even under optimal conditions (1). Interestingly, despite the fact that CD11c+ B-cells are detected in the blood of healthy individuals, they have been reported to be increased in autoimmune diseases. We aimed to characterize CD11c+ B cells from healthy humans by flow cytometry, microarray analysis, in vitro functional assays, and by measuring their frequency in patients with autoimmune diseases. Here we report that CD11c+ B cells are a distinct subpopulation of B cells, even if their phenotype is heterogeneous, with overexpression of genes involved in B cell activation and antigen presentation. Moreover, upon activation, CD11c+ B cells can differentiate into plasma cells and CD11c could be up-regulated in CD11c− B cells. Finally, we showed that patients with pemphigus, an autoimmune disease mediated by B cells, have a decreased frequency of CD11c+ B-cell after treatment, relative to baseline. Our findings could help to identify a B-cell population that may represent novel targets for immunotherapy.
Analysis of age-associated B cells (ABCs) in Systemic Lupus Erythematosus (SLE) patients
Authors: HERNÁNDEZ VÍCTOR ANDRÉS SOSA , Navarro-Hernandez Itze Cecilia , Torres-Ruiz Jiram José , Whitall Laura Patricia , Gómez-Martín Diana , Santos-Argumedo Leopoldo , Maravillas-Montero José Luis
Published: January 2023

SLE is an autoimmune disease which is characterized by the formation of auto-antibodies and loss of tolerance. In addition to the production of auto-antibodies, certain sub-populations of B cells may play a critical role in the resolution or severity of the disease.

Recently, a novel CD11c+, CD21low, and T-bet+ expressing B-cell subset has been identified as critical for the development of autoimmune diseases; the transcriptional program that develops this phenotype is induced in response to signaling by IFN-γ, TLR-9 and the costimulatory molecule CD40 and is linked to the induction of IgG2a, IgG2b, IgG3 and repression of IgG1 and IgE. However, the relationship between the presence of these cells and autoimmunity development is not yet well understood.

In this study, we analyzed blood samples from a cohort of 15 patients with SLE through flow cytometry. The frequency of this ABCs sub-population was correlated with clinical and laboratory variables such as C3, C4, antidsDNA antibodies, anti-nucleosomes antibodies, serum creatinine and total urine proteins. These data were subjected to Spearman’s correlation analyses. Interestingly, we found a positive correlation with ABCs percentages and anti-dsDNA antibodies (r=0.604, p=0.028).

These results suggest that circulating ABCs number can be potentially used as a diagnostic/prognostic marker for this autoimmune disease.
Peptides of endogenous retrovirus Gag gene variants are not agonistic ligands for autoreactive T cells in non-obese diabetic mice
Authors: Dai Yang D , Bashratyan Roman , Marquardt Kristi , Hu Wen-Yuan , Evans Leonard , Haskins Kathryn
Published: January 2023

Endogenous retroviruses (ERV) have been isolated from the pancreatic islets of non-obese diabetic (NOD) mice. We previously demonstrated that a murine leukemia retrovirus–like ERV Gag protein was secreted together with exosomes by an insulinoma and by primary islet mesenchymal stem cells. Here, we further demonstrated that Gag is an effective antigen to stimulate IFN-gamma production by autoreactive T cells using recombinant Gag protein or virus-like particles (VLPs) expressing the Gag gene. However, candidate peptides of this Gag protein were not effective in stimulating the T cells. Upon analyses of ERV Gag genes expressed in the islets of prediabetic NOD mice, we were surprised to find multiple Gag gene variants with complete open-reading frames. These results suggested the expression of different endogenous proviruses, or alternatively, the detection of point mutations incurred during viral replication. A new batch of altered peptides was synthesized reflecting the variations among the Gag gene variants, and a few of them induced detectable amount of cytokine secretion from the T cells. But, the levels of IFN-gamma induced by the peptides were much lower than that induced by the Gag protein and the VLPs, suggesting a low affinity activation. This weak T-cell-stimulating activity of the Gag peptides was further confirmed by testing diabetogenic T-cell clones, as well as by using MHC tetramers loaded with candidate peptides. The data suggest that ERV Gag or its gene variants may trigger autoimmunity via a pathway that could be independent from direct presentation of viral peptides to autoreactive T cells.
Molecular Targets of inflammation in pancreatic endocrine cells in Type 1 Diabetes (T1D)
Authors: Mora Conchi , Sala Ester , Saavedra-Ávila Noemí Alejandra , Sengupta Upasana , Vived Celia , Verdaguer Joan , Stratmann Thomas , Lahti Jill
Published: January 2023

T1D (Type 1 Diabetes) is an autoimmune disorder caused by the immune-mediated destruction of insulin-producing beta cells in the pancreas. There are direct mechanisms that have been demonstrated to cause final beta cell demise, such as the pathways triggered by Fas engagement and Perforin release. However, focus should be placed on other indirect mechanisms, such as pro-inflammatory cytokines present in the beta cell niche early on, that progressively debilitate and alter beta cells from earlier phases of T1D progression, rendering them as helpless targets of the immune system at the end. Though beta cells may resist adverse stimuli in early phases of the autoimmune process, the complicity of both, direct and indirect deleterious mechanisms, leads to beta cell death. In an effort to identify those indirect mechanisms that chronically besiege beta cells from the T1D-prone NOD mouse model, we used the microarray technology. We have identified several candidate genes the expression of which is altered in the NOD islet prior to T1D onset. Several of these genes are related to cell cycle progression and are downregulated due to the insulitic attack to the islet. One of these genes is cyclin D-3, a Dtype cyclin that interacts with either Cdk4 or Cdk6 to promote G1/S cycle progression. We have reported that cyclin D3 protects beta cells against cytokine-induced apoptosis and is required for proper beta cell function in a cell-cycle independent fashion. Cdk11 is also affected by inflammation in islet cells and is a cyclin-dependent kinase which is involved in transcription, mitosis and apoptosis. The natural partners of Cdk11 are L-type cyclins. We have examined whether the immune function is affected in the relative absence of Cdk11.