bluuebunny/crossref_metadata_2025 huggingface dataset:
IgA B cell receptor signaling protects from FasL counterselection during germinal center reaction in Peyer’s patches and shapes humoral mucosal response.
Authors: Reboldi Andrea , Raso Fiona , Berthellete Alyssa , Sagadiev Sara , Moses Stephanie , Acharya Mridu , Barton Gregory , Muppidi Jagan , Marshak-Rothstein Ann
Published: January 2023

Immunoglobulin A (IgA) is the main antibody isotype found in the intestine: deficiency in IgA alters microbiome composition, leads to increased susceptibility to enteric pathogens and reduces tissue response to oral vaccine. IgA can bind multiple unrelated bacterial taxa, but mechanistic insights into the generation of such cross-species reactivity are lacking. IgA+ B cells arise during germinal center (GC) reaction in Peyer’s patches: however, the role of IgA B cell receptor (BCR) in shaping the humoral response at the intestinal interface remains unknown.

Here we showed that GC B cells lacking IgA are rapidly outcompeted by IgA+ B cells during GC reaction in Peyer’s patches and they are unable to contribute to memory B cell and intestinal-homing plasma cell compartments. IgA BCR did not impact cell proliferation in dark zone, and ectopic Bcl-2 expression was unable to rescue IgA-deficient GC B cells in mixed bone marrow chimeras. In contrast, IgA-deficient GC B cells underwent increased apoptosis in the light zone. In line with these findings, we observed that IgA BCR mediated faster and stronger intracellular Ca2+ signaling and led to increase BCR-dependent phosphorylation events. Mechanistically, IgA BCR signaling conferred resistance to Fas ligand (FasL)-dependent cell death in vitro, and genetic inhibition of Fas-FasL pathway restored IgA-deficient B cell fitness during GC reaction in vivo.

Thus, our results showed that IgA-expressing GC B cells are protected from FasL counterselection in the light zone via enhanced BCR intracellular signaling. This process might allow low affinity B cell clones to participate to the GC reaction and assure a comprehensive humoral response to poorly immunogenic commensal species.

Supported by grants from NIH (T32 AI132152, T32 AI007349 and R01 AI155727-02)
MyD88-mediated signaling in myo-/fibroblasts is required for control of macrophage maturation under mucosal tolerance in the gut
Authors: Chulkina Marina , Uribe Gabriela , He Bing , McAninch Steven , Khanipov Kamil , Golovko George , Markov Nikolay S , Powell Don W , Beswick Ellen , Pinchuk Iryna V
Published: January 2023

Introduction

Colonic myo-/fibroblasts (MFs) are cells of mesenchymal origin in a healthy gut are among the key players in the mucosal tolerance that involve MF-restricted MyD88 signaling. MFs and macrophages interact in colonic tissue. However, the mechanisms of MF mediated regulation of macrophages under intestinal mucosal tolerance has emerging evidence. We hypothesized that MF-restricted MyD88 signaling regulates macrophage maturation under colonic mucosal tolerance.

Methods

RNAseq, qRT-PCR, H&E, and flow cytometry was used for analysis of colonic tissue/cell derived from B6ACTA2CreMyD88fl/fl, B6Col1a2CreMyD88fl/fl and B6RagCol1a2CreMyD88fl/fl MF-specific conditional KO mice.

Results

MF-specific deletion of MyD88 in vivo resulted in the development of microcolitis and was associated with dysbiotic decrease in Firmicutes to Bacteroidetes bacterial ratio, a hallmark of intestinal inflammation. This process was associated with the increase in colonic expression of genes linked to the inflammatory activity of myeloid cells and accumulation of immature monocyte-derived macrophages. In vitro deletion of MyD88 from MFs (isolated from MF-specific MyD88 conditional KO mice) resulted in changes of basal and TLR4 mediated pathways involved in myeloid cell migration/maturation and inflammation. Using MF-macrophage co-cultures we demonstrated the MF-restricted MyD88 signaling suppresses myeloid cell inflammatory activity.

Conclusion

Intrinsic MyD88 signaling within colonic mesenchymal cells is critical to control myeloid cell differentiation and inflammatory activity under mucosal tolerance in the gut.

Supported by CTSA/TL1 5TL1TR001440-04 UTMB Institute for Translational Science and R01 DK103150 National Institute of Diabetes and Digestive and Kidney Diseases.
The role of progesterone in barrier responses to intravaginal viral infection.
Authors: Dulson Sarah J , Lopez Cesar A , Lazear Helen M
Published: January 2023

The female reproductive tract (FRT) is a robust physical and immunological barrier tasked with balancing tolerogenic immunity to non-self antigens such as commensal bacteria, sperm, and fetal tissues, with protection against sexually transmitted pathogens such as herpes simplex virus (HSV) and Zika virus (ZIKV). The FRT is dynamically regulated by hormones, but the mechanisms by which hormones affect antiviral responses in immune and non-immune cells are incompletely understood. We found that a high progesterone state, such as pregnancy or treatment with Depo-medroxyprogesterone acetate (DMPA, Depo-provera), was necessary to render mice susceptible to vaginal infection with HSV-1 or ZIKV, and that this was true for both wild-type and immunocompromised Ifnar1−/− mice. The effect of progesterone was specific to vaginal infection as treatment with DMPA did not enhance susceptibility to HSV or ZIKV infection by dermal or subcutaneous inoculation, respectively. Importantly, we found that only DMPA-treated mice were protected against secondary vaginal infection, suggesting that productive viral replication is necessary for the establishment of protective memory responses in the vagina. Although we found that DMPA increased dye permeability of the vagina, mechanical abrasion without DMPA treatment was not sufficient to induce susceptibility, implicating other roles of progesterone in modulating epithelial antiviral responses. Together, these data underscore a crucial endocrine-immune axis involved in early antiviral responses in the vagina. Ongoing studies seek to identify progesterone-responsive cells in the vagina and to define the mechanism by which progesterone renders these cells permissive to viral infection.

Supported by grants from NIH K12-GM000678 (SJD), NIH F31 AI143237 (CAL), NIH R01 AI139512 (HML), and NIH R21 AI44631 (HML).
Enzymatic/mechanical digestion outperforms explant culture for isolation of tissue resident memory t cells from human lungs
Authors: Geber Adam B , Sportiello Michael John , Hyuck Heidie , Pryhuber Gloria , Topham David J
Published: January 2023

Tissue resident memory T cells (TRM) comprise a distinct population which persists in the tissue after infection. Lung TRM help to coordinate early immune responses to infections of the respiratory tract, but sampling of the relevant anatomical sites in humans is logistically difficult and not easily standardized. We used fresh human tissues to compare methods for organ dissociation and lymphocyte enrichment with regard to T cell phenotype and viability. The two main approaches to tissue processing were explant culture and combined enzymatic/mechanical digestion. While cytokine supplementation was able to induce T cell egress from intact respiratory samples, the majority of TRM remained in situ and the viability of all cells was compromised after three days of ex vivo culture. More T cells were recovered after IL-2 supplementation than with IL-7, but it remains unclear whether IL-2 is inducing proliferation in vitro. Enzymatic and mechanical digestion did not appear to alter the antigenic phenotype of the lymphocyte population and yielded enriched and purified cellular samples that were suitable for downstream flow cytometric or transcriptomic assays. Results suggest that minimizing processing time and using a targeted enzymatic cocktail are important considerations for working with human lung TRM.

Supported by NIH grant GR531572.
Bystander CD8 T cell memory responses partially protect mice against lethal vaginal HSV-2 challenge
Authors: Arkatkar Tanvi , Dave Veronica , Talavera Irene , Prlic Martin , Lund Jennifer M
Published: January 2023

Herpes Simplex Virus type-2 (HSV-2) infection is one of the most prevalent sexually transmitted infections, yet no vaccine is currently available. Detailed analysis of HSV-infected human tissue revealed tissue-resident CD8 T cells (TRM) limit the duration and severity of HSV-2 episodes. Interestingly, recent studies indicate that the sensing and alarming function of CD8 TRMs is not restricted to cognate antigen interaction, but CD8 TRM can mediate protection against antigenically unrelated pathogens, termed “bystander activation.” Here, we investigated if antigen non-specific CD8 T cells could provide some degree of protection in a bystander fashion in the context of HSV infection. To test this, we created antigen non-specific memory compartments through immunization of mice with Listeria expressing ovalbumin (OVA) (LM-OVA). Mice were then challenged with wild-type HSV-2 to assess the degree of bystander-mediated protection. Immunization with LM-OVA-delayed disease progression from lethal viral challenge, suggesting that bystander CD8 T cells may mediate protection despite the lack of antigen-specificity. Furthermore, we found early infiltration of antigen-non-specific CD8 T cells (OVA-specific) in the vaginal tract in the infection setting. Additionally, assessment of the memory CD8 compartment in human vaginal tissue upon in-vitro treatment with cytokines caused bystander activation of memory CD8 T cells. Finally, we found that in-vitro treatment of CD8 T cells with cytokines delayed disease progression and reduced viral burden upon adoptive transfer. Altogether, our findings suggest that local bystander-activation of CD8 memory T cells can participate in protection from HSV-2.
Intranasal, not parenteral, vaccination induces the formation of tissue-resident memory CD8 T cells in nasal mucosa that rapidly clear influenza virus infection
Authors: Matysiak Colette , Kazer Samuel , Ordovas-Montanes Jose , von Andrian Ulrich H
Published: January 2023

Respiratory tract infections are among the leading causes of death. While current vaccines reduce severe disease, they provide suboptimal mucosal protection (e.g. against influenza virus and SARS-CoV-2). Nasal vaccines offer an advantage by quickly limiting viral shedding, in part by generating tissue-resident memory (Trm) CD8 T cells in the nasal mucosa. But little is known about the molecular requirements for this Trm formation. The common paradigm is that lymphocytes home to tissues after having been imprinted within draining lymph nodes to express a tissue-specific combination of trafficking molecules, and Trm cells form rapidly from this pool. To probe the paradigm of tissue homing to the nasal mucosa, we compared CD8 T cell trafficking following intranasal and intramuscular vaccination. To investigate the trafficking molecules expressed by CD8 T cells and nasal cells we generated a single-cell RNA sequencing profile of the nasal mucosa over the course of an acute influenza infection. We found significantly more CD8 Trm cells in the nasal mucosa following intranasal vaccination that rapidly cleared influenza infection. Notably, CD8 T cells could be “pulled” into the nasal mucosa with an inflammatory stimulus following intramuscular vaccination, suggesting a new strategy for generating nasal CD8 Trm cells. Unlike canonical T cell homing to the gut and skin, nasal CD8 T cell trafficking depended on a4/VCAM1 (not a4b7/MADCAM1 or P and E-Selectin), and CD8 T cells expressed high levels of CXCR3 and CXCR6. This combination of trafficking molecules suggests a distinct multi-step adhesion cascade for CD8 T cell recruitment to the nasal mucosa and provides critical insights to rationally design vaccines for respiratory tract protection.

Supported by grants from NIH (R01 AR068383-01, P01 AI 112521) and HMS-AbbVie Alliance
Defining the transcriptional signature and heterogeneity of lung resident memory B cells
Authors: Padilla-Quirarte Herbey Oswaldo , Williams Elliot , Scharer Christopher
Published: January 2023

Lung resident memory cells have been shown to drive protection against viruses and bacteria, and recently resident memory B cells (BRM) have been also recognized in humans and animal models as critical immune mediators of protection from pathogens. Compared to naïve B cells, circulating influenza-specific memory B cells harbor a unique epigenetic and transcriptional landscape. Although lung-resident memory T cells have distinct molecular programming, it is not known if lung BRM are similarly programmed or distinct. Recent work has defined the emergence and requirement for T cell help in the formation of BRM, suggesting a distinct set of signals are required to promote lung residency compared to circulating memory. Here we investigated the unique programming of lung BRM and plasma cells to determine how their unique transcriptional, metabolic, and epigenetic properties. We used single-cell RNA sequencing (sc-RNA-seq) on sorted lung resident and mediastinal LN (mLN) memory B cells and antibody secreting cells from influenza-infected C57BL/6 mice 35 days post-infection. As a comparison, circulating naïve B cells were also sorted. Here, we investigated BRM heterogeneity and transcriptional programming in the lung tissue. Comparisons to tissue resident memory T cells define metabolic characteristics that promote survival in the lung microenvironment. These data defined the BRM molecular properties compared to circulating B cells and their unique relationship to plasma cells in the lung.

Supported by grant 5R01AI148471-02 from NIH
Human Defensin-5 (HD-5) and HD-6 mRNA Produced by Intestinal Crypt Paneth Cells are Decreased in Fecal Samples of Patients Positive for SARS-CoV2
Authors: Ryan Lisa K , Robinson Tanya O , Figgins Erika H , Do Uyen ‘Emily’ , Wilson Kenneth J , Glover Sarah C , Diamond Gill
Published: January 2023

Alpha-defensins (AD) are antimicrobial peptides (AMP) that are found on mucosal surfaces, providing innate immune defense against pathogens. Human defensin-5 (HD-5) and HD-6 are AD produced only by Paneth cells of small intestinal crypts. SARS-CoV2 virus has been shown to infect the small intestinal cells via the ACE2 receptor and suppress inflammation in the gut. HD-5 and HD-6 bind ACE2 to prevent entry to intestinal cells. Based on the observation that human beta defensin-1 (hBD-1) is downregulated by influenza virus in airway epithelial cells, we hypothesized that HD-5 and HD-6 could be similarly downregulated by SARS-CoV2. To examine this in situ, we sampled patient cohorts with similar co-morbidities, with and without SARS-CoV2 positivity, for HD-5 and HD-6 gene expression via qRT-PCR. Stool samples were collected from all patients, diluted in Qiagen RNA Later and frozen at −80°C prior to total RNA extraction. Results revealed a significant decrease in both HD-5 (p=0.000006) and HD-6 (p=0.002) of SARS-CoV2 positive patients compared with non-Covid19 patients negative for SARS-CoV2. The results were consistent in fecal swabs or fecal streams. Gene expression in other significant markers of inflammation were also blunted: sPLA-2 (p=0.006), alpha-1-anti-trypsin (p=0.0001) and trypsin 2 (p=0.005). Other gut inflammatory markers PSTI, HIP/PAP, trypsin 3 and IL-8 were unchanged. There was a trend in IL-8 upregulation in Covid19 patients. This is the first demonstration of defensin mRNA detection in stool samples. The results suggest that decreasing AMPs may be a mechanism by which the virus increases its chance for survival.

Supported by R21DE028378
Nasopharyngeal inflammatory lymphocytes perturbation in COVID-19 severity prediction
Authors: Ayass Mohamad Ammar , Tripathi Trivendra , Griko Natalya , Zhang Jin , Nair Ramya Ramankutty , Dai Jun , Zhu Kevin , Cao Wanying , Pashkov Victor , Singh Sharda Kalla , Mosleh Lina Abi
Published: January 2023

SARS-CoV-2 causes severe respiratory infections in some patients due to an uncontrolled immune response. The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19, however, the immune-responses at the site of viral entry remain poorly understood. We used flow cytometry to decipher nasopharyngeal immune profiling of COVID-19 positive/negative patients; viral detection and load were performed by RT-PCR. Analysis of viral-induced immune-inflammatory responses, lymphocytes population/activation lymphocytes markers, T cell subtypes and associated inflammatory cytokines. Furthermore, we performed in vitro assays using cultured monocytes-derived-macrophages trained with/without SARS-CoV-2 spike protein and co-cultured with/without autologous T cells. Results showed that positive patients had high inflammatory CD45+ and CD8+ T cells including elevated % of Th1/Th17.1, associated with a worse outcome in a subgroup of COVID-19 infected patients. Positive patients showed increased % of cytokines, IFN-γ+, IL-17+, and IL-10+ T cells suggesting a crucial role of these cytokines in recruiting and activating neutrophils that can migrate to target organs (e.g., lungs) and potentially play an important role in the disease severity. Moreover, upregulated Tregs along with monocytic-induced inflammatory T-cell responses and increased TH1/TH17 cytokines in positive patients as compared to negative, potentially having a nefarious effect on disease progression. Our findings indicate a distinct signature of inflammatory lymphocytes/cytokines responses within the nasopharynx of COVID-19 patients that might predict the clinical outcome/progression of the disease.

This work is supported by Dr. Mohamad Ayass, CEO of Ayass Bioscience, LLC
Changes in the structure of lipid A in glycosylated outer membrane vesicles (glycOMVs) suppress the activation of glycan-specific B1 cells in vivo
Authors: Lopez Natalia , Tyagi Srishti , DeLisa Matthew P
Published: January 2023

B1 cells differ from B2 cells in their developmental origin, phenotype, and function. They are anatomically located in various tissues of the immune system, and contribute to around 90% of the circulating antibodies in blood. B1 cells are often activated in a T-cell independent manner promoted by either antigens that contain a B-cell mitogen such as LPS, or by molecules that have highly repetitive structures such as bacterial capsular polysaccharides, with both pathways having the potential to undergo class-switch recombination and somatic hypermutation. Previously, we have shown that immunization with bacterial outer membrane vesicles engineered to display defined glycan structures on their surface (glycOMVs) promotes a strong glycan-specific IgG response for a wide range of bacterial and mammalian glycans. Here, we show that the structure of lipid A in these (glycOMVs) displaying the oncofetal antigen polysialic acid (polySia), influences the activation and class-switching of polySia-specific B1 cells from the spleen and peritoneal cavity in mice. We found that structural remodeling of lipid A from its normal hexa-acylated structure to a penta-acylated variant prevents the activation of polySia-specific B1 cells in the spleen and enhances the activation of polySia-specific B2 cells from the peritoneal cavity. Interestingly, we observed no changes in the glycan-specific IgG titers following immunization of wildtype B6 mice with either construct, even though there was no B cell activation in the spleen of mice receiving penta-acylated glycOMVs. However, these same glycOMVs failed to promote a glycan-specific IgG response in T-deficient mice, suggesting that this response was entirely T-cell dependent.

This work was supported by Bill and Melinda Gates Foundation grant OPP1217652 (to MPD), NSF grants CBET-1159581, CBET-1264701 and CBET-1936823 (to MPD), the Defense Threat Reduction Agency (DTRA) grants HDTRA1-15-10052 and HDTRA1-20-10004 (to MPD), NIH grants 1R01GM137314 and 1R01GM127578 (to MPD) and the Fleming Scholars Fellowship (to NLB).
Heightened levels of circulating cell-free DNA predict aggressive onset of experimental glioblastoma
Authors: Ayasoufi Katayoun , Wolf Delaney , Tritz Zachariah Peter , Jin Fang , Gukbicki Lauren , Hansen Michael , Fain Cori E , Khadka Roman , Johnson Aaron J
Published: January 2023

Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including spleen atrophy, T cell lymphopenia, and T cell sequestration within the bone marrow. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel circulating soluble factors that are found in the serum of glioma-bearing mice. This high molecular weight species potently inhibits T cell proliferation in vitro. Cell-free extracellular DNA has been recently identified as an immune suppressive factor in circulation. We, therefore, assessed cell-free DNA levels in the GL261 model. We determined that serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Serial assessment of DNA concentrations in serum of glioma-bearing mice indicated that the most prominent increase was concurrent with end-stage disease. These findings provide a tractable approach to investigate the cellular source and immune suppressive effects of cell-free DNA in the leading murine model of GBM.

Supported by K99NS117799
IFNγ mediated PD-L1 expression mitigate the immunomodulatory effect of irreversible electroporation on pancreatic cancer
Authors: Imran Khan Mohammad , Brock Rebecca , Alinezhadbalalami Nastaran , Aycock Kenneth N , Morrison Holly A , Davalos Rafael V , Allen Irving Coy
Published: January 2023

Pancreatic cancer is one of the deadliest diagnoses leaving patients with few therapeutic options. Irreversible electroporation (IRE), a novel non-thermal ablation system, utilizes very short, high voltage electrical pulses to form micropores in cell membranes and stimulate cell death. IRE has been tested in human clinical trials for pancreatic cancer treatment with a significant improvement in progression-free survival, but not much is understood about the immunomodulatory effects of IRE and the reasons for tumor relapse. We hypothesized that, we could identify a molecular rationale for the tumor recurrence and potential co-therapy targets for IRE treatment. We have utilized Pan02 cells, and a subcutaneous flank model of pancreatic cancer. Here, we show that IRE can significantly delay cancer progression by shifting the tumor microenvironment to a pro-inflammatory state. In doing so, IRE treatment recruits cytotoxic CD8+ T-cells to the tumor sites, reduces contralateral tumor burden, and increases interferon-gamma (IFNγ) levels in the serum. Within two weeks after treatment, immune cells in the tumor site reverts to the pre-treatment condition, tumor relapses, and tumor cells express programmed-cell death-ligand 1 (PD-L1). Our in vitro findings of IFNγ dependent PD-L1 expression and ineffective in vivo blocking of IFNγ hints feedback loop pathway involving IFNγ and PD-L1. Since other findings have shown blocking PD-L1 is not always very effective, our findings indicate another compensatory bypass immune checkpoint mechanism. The effects of IRE can therefore be extended by additional combination therapy methods, including PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors.

This work was supported, in part, by the Virginia Maryland College of Veterinary Medicine; The Virginia Tech Institute for Critical Technology and Applied Sciences Center for Engineered Health; and The National Institutes of Health (R03AI151494 (ICA); R21EB028429 (ICA). The contents solely the responsibility of the authors and does not necessarily represent the official views of the NIH or any other funding agency.
Triple synergy between vaccine and checkpoint inhibitors in a pre-clinical tumor model
Authors: Becker William J , Olkhanud Purevdorj B , Berzofsky Jay A
Published: January 2023

Despite advances in checkpoint inhibitor (CPI) therapy for cancer treatment, many malignancies remain resistant. Tumors deemed ‘cold’ based on lack of T cell infiltration into the stroma of the tumor show reduced potential for CPI therapy and finding the right combination to balance safety and efficacy is arduous. To study ways to circumvent these limitations, we used the preclinical mouse model of TC1 tumor cells resistant to conventional CPI therapy. The TC1 cells are transfected with oncogenes E6 and E7 of HPV16, and we designed a tumor-vaccine specific for an E7(43–77) peptide. We show the synergy between the tumor-antigen specific vaccine and the combination of two CPIs, anti-TIGIT and anti-PD-L1. The synergistic effect of the triple combination provides more protection against tumor growth than either treatment alone or any pairwise combination and significantly improves survival in a CD8+ T cell dependent manner. Combining the tumor-specific vaccine with CPIs induces tumor-specific CD8+ T cells that infiltrate the tumor in young and aged mice, although aged mice show less protection than their younger counterparts. These data show proof-of-concept for a novel combination of a vaccine designed to elicit de novo anti-tumor T cell responses that can be amplified by synergistic CPIs that lead to greater survival.

Supported by Intramural NCI funding: ZIA-C-004020
Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immune tolerance
Authors: Reticker-Flynn Nathan E , Zhang Weiruo , Belk Julia Ann , Basto Pamela Antonia , Satpathy Ansuman , Plevritis Sylvia Katina , Engleman Edgar G
Published: January 2023

The majority of cancer deaths result from distant organ metastasis. Lymph nodes (LNs) are major sites of anti-tumor lymphocyte education, yet LN metastasis frequently precedes distant metastasis. Here, we find that LN metastasis represents a critical step in tumor progression by inducing tumor-specific immune tolerance, thus enabling further dissemination of tumors to distant organs. Using an in vivo passaging approach, we generated 300 cell lines exhibiting varying degrees of LN metastatic capacity. We show that the LN metastases promote distant organ metastasis in a manner that is tumor specific. Through organism-wide immune profiling by single cell sequencing, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs). Adoptive transfer of Tregs from the LNs of mice bearing LN metastasis to naïve mice facilitates metastasis in a manner that Tregs from mice without LN metastases cannot. Additionally, these Tregs are induced in an antigen-specific manner. Using whole exome sequencing, we show LN metastases do not evolve through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I, or decreases in melanoma antigens. Rather, by RNA-seq and ATAC-seq, we show that a conserved interferon signaling axis is upregulated in LN metastases and is rendered stable through epigenetic regulation of chromatin accessibility. Knockout studies reveal that these pathways are required for LN metastatic seeding, and we validate their significance in additional mouse models and patients. These findings demonstrate a critical role for LN metastasis in promoting tumor-specific immunosuppression.

This work was supported by NIH grants U54 CA209971 and F32 CA189408.
Response to anti-PD-1 and anti-LAG-3 immune checkpoint blockade is associated with induction of pro-inflammatory Tregs
Authors: Rolig Annah S , Sturgill Elizabeth R , Mick Courtney , Rose Daniel , Kaufmann Johanna , Yanamandra Niranjan , Griffin Sue , Smothers James , Redmond William L
Published: January 2023

Only a subset of patients durable clinical responses to aPD-1 and/or aCTLA-4 immunotherapies, thus, developing new therapeutic agents to increase the proportion of responding patients is a priority. Combining aPD-1 with aLAG-3 has shown promising results; however, lack of mechanistic understanding of aPD-1/aLAG-3 synergy remains a barrier for its optimal clinical use. Here, we examined the mechanism of aPD-1/aLAG-3 synergy in multiple mouse models using flow cytometry and single cell RNA sequencing. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma) and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. Regulatory T cells (Tregs) suppressed response to this therapy, as in the absence of CD4+ T cells, 100% of mice responded. To understand how responders overcome Treg suppression, we performed an in-depth analysis of tumor-infiltrating lymphocytes (TIL) comparing mice that responded to treatment (decreased tumor size post-treatment) to non-responders (same tumor growth trajectory as control). Responders had reduced Foxp3+ CD4+ Tregs in comparison to non-responders and, in addition, those Tregs had a ‘fragile’ phenotype, including a pro-inflammatory cytokine profile (TNF-a; IFN-g), increased LAG-3, and decreased NRP1 expression. Within responders, CD8+ TIL exhibited increased frequency, effector cytokine production (TNF-a; IFN-g), and LAG-3 expression as compared to non-responders. Together, these data suggest that aPD-1/aLAG-3 can reduce Treg frequency and function leading to expansion of active tumor-specific CD8+ T cells capable of supporting tumor regression and improved survival.

Supported by the Providence Portland Medical Foundation and GlaxoSmithKline
TCF-1 controls Treg functions that regulate inflammation, CD8 T-cell cytotoxicity, and severity of colon cancer
Authors: Khazaie Khashayarsha , Osman Abu , Yan Bingyu , Kazemian Majid , Gounari Fotini
Published: January 2023

The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome.

Supported by NIH R01 AI 108682 (FG & KK), NIH RO1 AI 147652 (FG), NIH R35GM138283 (MK), and Praespero Innovation Award Alberta, Canada (FG & KK)
Tumor-intrinsic factors dictate beneficial effect of microbiota-based therapies
Authors: Lam Khiem C , Huang April , Araya Romina E , Chen Quanyi , Goldszmid Romina S
Published: January 2023

Gut microbiota impacts antitumor immunity and recent studies have shown that manipulating microbiota via fecal transplant can rescue immune checkpoint blockade (ICB) response in refractory melanoma patients. However, not all patients benefited from this approach. We hypothesize that tumor-intrinsic factors (e.g. neoantigen load and immune microenvironment) contribute to the microbiota effect on anticancer response. To test this, we performed preclinical microbiota manipulation studies using dietary intervention. Mice fed high-fiber diet (FD) had improved spontaneous tumor control and response to ICB. Mechanistically, FD-induced changes in microbiota composition skewed the tumor innate immune repertoire towards an antitumor profile marked by increased dendritic cell infiltration. Downstream of the innate compartment, FD reduced the proportion of intratumoral Tregs and exhausted CD8+ T cells. We further assessed the effects of FD across 8 tumor models including EL4 lymphoma, MC38 colon carcinoma, and 6 melanomas (M1–M6) that mimic the variety of human melanoma subtypes. Although FD had a beneficial effect overall, not all models responded to the same extent, and were accompanied by different changes in the tumor immune profile. FD delayed tumor initiation for EL4 and M4, reduced growth rate for M3 and M5, both delayed initiation and reduced growth rate for MC38, M2, and M6, but had no significant effects for M1. Additionally, we found that increasing tumor immunogenicity resulted in a synergistic effect with FD. Our data suggests that tumor-intrinsic factors influence the beneficial effect of microbiota on antitumor immunity. Identifying these factors will help refine the use of microbiota-based therapies in the clinic.

This research was supported by the Intramural Research Program of the NIH (CCR-NCI).

This research was supported by the Intramural Research Program of the NIH (CCR-NCI).
Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism
Authors: Fisher Emilie L , Madden Matthew Z , Chi Channing , Suguira Ayaka , Rathmell Jeffrey
Published: January 2023

Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve complete and durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are therefore crucial in the effort to improve immunotherapies. One avenue for manipulation of CTLs is through alterations to cellular metabolic programs. T cells radically alter their metabolism upon activation. Prior studies have shown pan-inhibition of glutaminolysis using the inhibitor 6-Diazo-5-oxo-l-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. Herein, I utilize a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – the enzyme responsible for catalyzing the conversion of glutamate to glutamine – conferred a fitness advantage to CTLs. In future experiments, inhibition and genetic deletion of GS will be utilized to investigate the role of GS in CTL activation, proliferation, effector cytokine production, and memory formation.
T-cells resist CD5 CAR mediated fratricide by continuously degrading CD5 protein
Authors: Ma Royce , Popat Divya , Chaumette Alex , Carisey Alexandre , Brenner Malcolm K , Mamonkin Maksim
Published: January 2023

Developing chimeric antigen receptor (CAR) T cells for patients with T cell malignancies remains challenging as most CARs targeting T-lineage antigens induce T cell fratricide. A notable exception is a CD5-specific CAR, which does not produce extensive fratricide, coinciding with a loss of detectable surface CD5 by an unknown mechanism.

In this study, we used live imaging fluorescent microscopy, Western Blot, and flow cytometry to show CD5 protein is rapidly internalized and degraded in transduced T cells following CD5 CAR expression. Experiments with C-and N-tagged CD5 confirmed complete degradation of the entire protein in CD5 CAR T cells. Physical ligation of CD5 antigen by the CAR in cis is sufficient to drive loss of CD5 protein, independently of either CAR or CD5 signaling, resulting in full protection from CD5-directed fratricide.

In an ongoing Phase I clinical trial (NCT03081910), CD5 CAR T cells expand and produce robust anti-tumor activity in patients with T cell malignancies. However, patients exhibit incomplete aplasia of non-transduced normal T-cells and a population of circulating T cells remain resistant to CD5-directed elimination and persistalongside CAR T cells. These resistant T cells are surface CD5-negative suggesting that in the presence of CD5 CAR T cells there can be removal of the target antigen as a means of evading killing. Indeed, in vitro experiments showed normal T cells degrade CD5 protein upon coculture with CD5 CAR T cells indicating antigen down modulation occurs on target cells not only in cis but also in trans. These data identify a possible mechanism of resistance of normal T cells to CD5-directed CAR T-cell elimination.

Supported by CAGT T32 Training Grant NIH NCI SPORE P2
Glycolipid-loaded nanoparticle immunotherapy cooperates with checkpoint inhibitors to harness iNKT cells for tumor control
Authors: Shute Travis J , Dudley Elizabeth A , Nash Kelly , Leadbetter Elizabeth A
Published: January 2023

Invariant natural killer T cells (iNKT) have a well-documented role in direct and indirect killing of tumor cells through their release of proinflammatory cytokines and cytotoxic compounds. To improve outcomes in poorly immunogenic cancers while avoiding NKT cell anergy, we induced anti-tumor responses by activating NKT cells with polymeric nanoparticles containing a low dose of a glycolipid adjuvant, α-Galactosyl-Ceramide (aGalCer), and a model tumor neoantigen, ovalbumin (OVA). Prophylactic vaccination of mice with aGalCer-OVA nanoP before inoculation with B16F10-OVA melanoma cells reduced tumor growth rate and increased overall survival compared to mice immunized with control nanoparticles. In mice with an established B16 tumor, therapeutic administration of aGalCer-OVA nanoP provided significantly greater anti-tumor effects, reduced tumor growth rate and increased survival, compared to control nanoparticles. Therapeutic treatment with aGalCer-OVA nanoP, but not control nanoPs, also induced significant infiltration of iNKT, NK, and antigen-specific CD8 T cells in the tumor, and enhanced production of granzyme B and perforin by CD8 T cells. Furthermore, immune checkpoint blockade treatment (anti-CTLA-4 + anti-PD-1) enhanced the effectiveness of aGalCer-OVA nanoP, leading to clearance of established tumors nearly 50% of the time. These results provide strong evidence that activation of iNKT cells via nanoparticles containing a glycolipid adjuvant and tumor antigen is a viable anti-cancer strategy that partners well with other clinically used immunotherapies.

Supported by grants from NIH (T32 AI138944) and the CTSA (TL1 TR002467)
A tumor-targeted cytokine/antibody fusion protein to stimulate anti-cancer immunity
Authors: Silver Aliyah Brianne , Spangler Jamie
Published: January 2023

Interleukin-2 (IL-2) is a pleiotropic cytokine that has clinical utility in stimulating anti-cancer immunity. However, the clinical efficacy of IL-2 is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory natural killer (NK) and effector T (Teff) cells and immunosuppressive regulatory T (Treg) cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor beta (IL-2Rβ) and common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Tregs express high levels of IL-2Rα, making them much more responsive to IL-2 than naïve NK and Teff cells. To extend the serum half-life and selectively bias the immunostimulatory functions of IL-2 in a stable unimolecular format, we created a single-chain fusion protein, denoted F10 immunocytokine (F10 IC), that intramolecularly fuses human IL-2 to an anti-IL-2 antibody that sterically blocks the IL-2/IL-2Rα interaction and allosterically enhances the IL-2/IL-2Rβ interaction. To exploit the abundance and accessibility of collagen in the tumor microenvironment for targeting, we fused collagen binding domains to F10 IC in various topographies. These novel fusion proteins occlude IL-2Rα binding and enhance IL-2Rβ binding, thereby eliminating the Treg advantage conferred by IL-2Rα and preferentially activating immune effector cells. Furthermore, these proteins selectively bind purified collagen and are capable of homing to, and being retained in, the tumor in vivo. Building on these promising results, we are assessing the in vivo therapeutic efficacy of these fusion proteins in various murine tumor models to illustrate the potential of collagen binding F10 IC as a powerful anti-cancer therapeutic.

Supported by a Sanofi Innovation Award and NIH grants (R01 EB029341, T32 AI007417)
Resveratrol, Modafinil and Caffeine’s modulation of B cell’s CD19 activation receptor
Authors: Valenzuela Hector F , Samayoa Giovanni , Martinez Kassandra , Salvador Samantha
Published: January 2023

Resveratrol a natural compound with anti-oxidant properties shown to inhibit cell proliferation and induces cellular apoptosis in Jurkat cell lines. Mechanistically, it was observed that interleukin-2 expression was blocked in T cells, suggesting resveratrol’s anti-inflammatory mechanism may involve downregulation of activation molecules. In this study, we investigated the effects of resveratrol or in combination with Modafinil or caffeine on a B cell line, Nor20. Modafinil is a compound shown to directly act on enzymes in the brain’s free-radical scavenging system and therefore directly reduce free radical levels. Caffeine has been shown to abolish the anti-inflammatory effects of resveratrol in astrocytes. Here we show that Nor20 treated with resveratrol can have up to 47.3% reduction in viability, consistent with previous observations. To determine a mechanism to explain these results, we used flow cytometry to measure the expression of CD19, the dominant receptor for all forms of B cell activation and modulator of B cell activation threshold combined with Modafinil or caffeine. We observed that resveratrol reduced the expression of CD19 up to 51%, Modafinil, inhibited up to 65%, but when resveratrol and Modafinil were combined the effect was not additive remaining at 53% inhibition when compare to the controls. In contrast, caffeine did not affect CD19 expression but when combined with resveratrol, caffeine abolished the downregulation of CD19 by resveratrol returning to the same levels as the untreated controls. Taken together, these results may indicate that resveratrol’s anti-inflammatory effects may initiate at the lymphocyte’s early recognition signaling receptors as a mechanisms to control cell growth.

Supported by RevGenetics and Whittier College’s Faculty Research Grant.
Demonstration of regulatory CD8 T cell prevalence, phenotype, and functions in autoimmune patients treated with a tolerizing peptide vaccine.
Authors: Maurer Meghan , Bowser Justin , Fasnacht Rachael , Gardell Jennifer L , Julien Susan , Templeton Megan , Therriault Jon , Yang Su J , Swiderek Kristine , Crane Courtney
Published: January 2023

INTRODUCTION

Others have described a subset of CD8 T cells with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation, through a canonical T cell receptor, results in oligoclonal expansion and perforin dependent elimination of pathogenic CD4 T cells. We have demonstrated the CD8 Treg network in Celiac patients, and that CD8 Tregs can be modulated to enhance their cytolytic elimination of pathogenic CD4 T cells. Here, we evaluate culture conditions to enhance CD8 Treg functions, and describe the impact of a gluten tolerizing peptide vaccine on CD8 Treg in Celiac disease patients.

METHODS

We examined the phenotype and function of Celiac patient-derived peripheral blood CD8 Tregs using flow cytometry, multicolor immunohistochemical staining, supernatant analysis, and TCR sequencing. We then evaluated the impact of a gluten tolerizing peptide vaccine on the CD8 Treg network in Celiac patient derived peripheral blood and tissues.

RESULTS

Using our previously reported phenotypic and functional readouts to define CD8 Treg activity, we describe culture conditions that support oligoclonal CD8 Treg cell expansion, plasticity, and cytolytic function. In patients treated with a gluten tolerizing vaccine, we found evidence for CD8 Treg expansion and preferential recruitment to duodenal tissues; however, pathogenic CD4 T cells were still detectable, suggesting insufficient CD8 Treg functional activation.

CONCLUSION

We describe parameters that support cytolytic CD8 Treg function, as well as the impact of a gluten tolerizing vaccine on the mobilization of CD8 Treg. Collectively, our data support the potential for CD8 Treg directed therapies to eliminate pathogenic CD4 T cells in autoimmune diseases.
Development of no-wash, rapid Fcγ Receptor Binding Immunoassays using NanoBiT® Technology
Authors: Swanson Brad , Nath Nidhi , Godat Becky , Flemming Rod , Urh Marjeta
Published: January 2023

Antibody driven therapies including monoclonal antibodies (mAbs), intravenous IgG (IVIG), subcutaneous IgG (SCIG), and vaccines have made a significant impact in the treatment of many diseases including multiple cancers and autoimmune, metabolic, viral, and infectious diseases. A key mechanism of action (MOA) that makes antibody-based therapies so effective is the ability of the Fc domain of the antibody to recruit immune effector cells through their interaction with a variety of Fc receptors (FcγR). However, assessing the combined impact of interaction of the Fc domain of various antibody isotypes with diverse FcγRs (Fc/FcγR interaction) on the efficacy of the drug is challenging, primarily due to the lack of simple to use and reproducible Fc/FcγR binding assays. Furthermore, existing technologies like SPR can introduce artifacts based on assay format, sensor chip characteristics, and immobilization methods.

To meet the need for reliable, simple to use assays, we have developed a suite of bioluminescent biochemical assays for the following receptors: Lumit™ FcγRI Binding Immunoassay Lumit™ FcγRIIA (H131) Binding Immunoassay Lumit™ FcγRIIA (R131) Binding Immunoassay Lumit™ FcγRIIIA (V158) Binding Immunoassay Lumit™ FcγRIIIA (F158) Binding Immunoassay

These assays are homogeneous, meaning no immobilization or washing steps are required. In addition, they are rapid, easy to use in a 96-well format, and require a simple luminometer. These assays can enable better understanding of antibody MOA and hence better therapies.