bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody-mediated Recruitment of Viral-specific Effector Cells via Recombinant Antibody-MHC T cells Engagers
Authors: Cohen Maya , Efimov Vera , Reiter Yoram
Published: January 2023

Our lab has developed a novel T cell engager (TCE) whereby a recombinant antibody fragment specific for a tumor-associated antigen (TAA) of choice is genetically fused to HLA-A2/peptide complex that presents a viral T cell epitope. The HLA-A2/peptide complex is designed to engage the TCR of a CD8+ T cell while the scFV or Fab redirects specificity towards a TAA, thus recruiting potent antiviral memory CTLs to attack tumor cells. We used the highly immunogenic cytomegalovirus (CMV) pp65-derived peptide as a target peptide for CTLs, taking into consideration the CMV prevalence of seropositive humans. Mesothelin was used as a TAA.

We monitored the binding of TCEs to target cells demonstrating that their binding is dependent exclusively on the interaction of the targeting domain (scFv/Fab) of the fusion molecule with mesothelin, and is dose-dependent. Potency of TCEs was determined in vitro using co-cultures of CMV-specific CTLs and mesothelin-expressing human tumor cell lines. The TCEs specifically and potently mediated the killing of mesothelin-positive cells in TCE-dose-dependent matter. Moreover, a significant increase in CD25 expression and dose-dependent secretion of IL2 and IFNγ were observed in CTLs exposed to mesothelin-positive cells.

The results represent a powerful new approach for immunotherapy. The MHC-based TCEs can mediate specific and efficient recruitment of viral-specific CTLs to kill tumor cells, adding an approach by which specific population of T cells can be engaged and recruited to the tumor site in a controllable manner in contrast to current CD3-based T cells engagers that cannot selectively target the specificity of interest. Such treatments can be very potent but with decreased toxicities involved.
Metronomic low dose cyclophosphamide in combination with a DNA methyltransferase inhibitor limits tumor growth in murine triple negative breast cancer
Authors: Camarena Carmen , Smith Timothy M , Bear Harry , Martin Rebecca
Published: January 2023

Many breast cancers are treated with therapies that target herceptin, estrogen, or progesterone receptors; however, for triple-negative breast cancers (TNBC) that lack these receptors, treatment options are limited, and prognosis is often unfavorable. The goal of this study is to design a therapeutic intervention that can elicit an effective immune response against the tumor and instill immunological memory to eradicate primary and metastatic lesions. Guadecitabine (Guad) is a second-generation DNA methyltransferase inhibitor (DMNTi) that has been reported to have several antitumor properties such as increased antigenicity and depletion of myeloid-derived suppressor cells (MDSC’s). Cyclophosphamide is a FDA approved chemotherapy that has been shown, when given as a low-dose treatment, to selectively deplete regulatory T-cells (T-regs). Both MDSCs and T-regs suppress antitumor immunity. We hypothesize that the combination of Guad and Cyp will synergize and promote anticancer immunity through increased expression of de novo tumor antigens and depletion of MDSCs and T-regs in a low-dose setting. To test this hypothesis, BALB/c mice were challenged with murine TNBC 4T1 tumor cells and the 4T1-bearing mice were administered low-dose Guad and Cyp daily for ten consecutive days. This experiment showed a degree of synergy between Guad and Cyp with the dual therapy reducing tumor burden to a greater extent than either monotherapy.
All-trans Retinoic Acid (atRA) promotes tumor resistence to NK cells by downregulating tumor B7-H6 expression
Authors: Cao Guoshuai , Sun Rui , Tian Zhigang
Published: January 2023

The interaction between the activating receptor NKp30 and its major tumor ligand B7-H6 is important for NK cell mediated tumor rejection while the regulation of B7-H6 by tumor therapeutics remains largely unknown. In this study, we investigated the regulation of B7-H6 by all-trans Retinoic acid (atRA), which is a terminal differentiation inducer of tumor cells and in use for tumor therapy. We found that B7-H6 expression on tumor cell suface was downregulated upon atRA treatment. We further found that atRA downregulated B7-H6 at the mRNA level. Epgenetic modifications such as DNA methylation and HDAC was not responsible as inhibitors of these pathways did not restore the B7-H6 mRNA expression. Instead, atRA downregulated c-myc and inhibited the transcription of B7-H6. NK92 and NKG cells mediated U937 cell killing was mainly dependent on the NKp30-B7-H6 interaction. Thus atRA treatment dampened U937 lysis by NK92 and NKG cells. Together, our data showed that atRA treatment downregulated B7-H6 expression and promoted tumor cell resistence to NK cells.
Paradoxical Role of MLK3-CTLA-4 axis in Triple-negative Breast Cancer and CD8+ T cells
Authors: Kumar Sandeep , Singh Sunil Kumar , Viswakarma Navin , Sondarva Gautam , Nair Rakesh Sathish , Thatcher Gregory , Rana Basabi , Rana Ajay
Published: January 2023

Mixed Lineage Kinase 3 (MLK3), a MAP3K family member, is an upstream regulator of MAP kinases and reported to mediate cytotoxic drug-induced breast cancer cell death, suggesting that MLK3 could serve as a therapeutic target in breast cancer. However, the function of MLK3 in host immunity and Triple-negative Breast Cancer (TNBC) are not known. The small-molecule inhibitor of MLK3, URMC-099, was able to induce caspase activity and promote apoptosis in TNBC cells, both in vivo and in vitro settings. The cytokine levels in TNBC xenograft tumors were either decreased or unchanged except for a significant increase in IL-1β level upon MLK3 inhibition. However, most of the Th1/Th2/Th17 cytokines were found to be up-regulated in the serum of tumor-bearing mice. The expression of T cell activation marker, CD69 on splenic, and tumor-infiltrating CD8+ T cells was increased upon inhibition of MLK3. Furthermore, the number of effector CD8+ T cells was also increased upon inhibition of MLK3 in tumor-bearing mice. We also observed that proteins, implicated in CD8+ T cell exhaustion, TIM3, LAG3, NFATc1, and CTLA-4 were up-regulated on CD8+ T cells upon MLK3 inhibition. Additionally, genetic loss/pharmacological inhibition of MLK3 increased expression of CTLA-4 on TNBC and CD8+ T cells via NFATc1. Paradoxically, the cellular proliferation of TNBC cells, expressing CTLA-4 was up-regulated, however it was opposite in CD8+ T cells. Most importantly, the combined blockade of MLK3 and CTLA-4 increased tumor infiltration of cytotoxic CD8+ T cells and correspondingly decreased the cellular proliferation and survival of tumor cells. Collectively, these results suggest that combined blocked of MLK3 and CTLA-4 could serve as a novel immunotherapy approach for TNBC.
Utilization of genetically diverse mouse model identifies genetic loci that correlate to response to immune checkpoint inhibitors to B16F0 melanoma
Authors: Hackett Justin , McCasland Jennifer , Wiesend Wendy , Dyson Greg , Muñiz Maria , Gibson Heather
Published: January 2023

Historically metastatic melanoma has been associated with poor prognosis. The advent of immune checkpoint inhibitors targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. To explore the effect of genetic background on immune checkpoint inhibitor efficacy, we have developed a tumor immunotherapy model using genetically heterogeneous Diversity Outbred (DO) mice and the C57BL/6 syngeneic B16F0 melanoma model. To implement genetic diversity, DO mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice. Untreated (C57BL/6xDO)F1 mice (n=34) reliably develop B16F0 tumors after subcutaneous inoculation, with some variation in tumor latency. To test the role of genetics in response to ICI, (C57BL/6xDO)F1 mice (n=142) were treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after inoculation with 2×105 B16F0 cells. Mice receiving therapy show wide variation in tumor development of up to 65 days (mean 20.86 +/− 11.04, CV 52.94%), with 19 never developing tumor by day 88. Re-challenge of tumor free mice confirmed adaptive response to tumor. Preliminary Genome Wide Association shows suggestive associations on chromosomes 8, 12, and 13. The locus on chromosome 13 contains a family of related genes with known function in autoimmune conditions and expression in several immune cell types. QTL effects show the NZO genotype is a positive driver at this locus and C57BL/6 is a negative driver, reflecting known ICI resistance in the C57BL/6 strain. These results demonstrate host genetic background significantly contributes to response to ICI and identifies genomic loci associating with primary resistance. NIH R37 CA220482.
Redirecting Immune Activation in the Tumor Microenvironment of Estrogen Receptor Negative Breast Cancer
Authors: Pioli Patricia Ann , Ball Michael S. , Bhandari Rajan , Santiesteban Gretel Maria Torres , Martyanov Viktor , Eltanbouly Mohamed , Whitfield Michael L , Liby Karen
Published: January 2023

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immune suppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from tumor-promoting to tumor-inhibiting in vitro. We show now for the first time that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of tumor CD4+Foxp3+ regulatory T cells. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.
Fine Needle Aspiration Based Immune Organoids Recapitulate TME in clear cell Renal Cell Carcinoma (ccRCC)
Authors: Wolf Melissa , Madden Matthew Z , Hongo Rachel , Rathmell W. Kimryn , Weiss Vivian L
Published: January 2023

Replication of the tumor microenvironment (TME) is a major limitation in organoid development as a model system for cancer research. Studies aimed at understanding the TME are typically performed in an oversimplified 2D cell culture system, or utilizing complex mouse models. Not only are in vivo mouse studies time consuming and costly, significant differences in the mouse immune system and TME may hinder applicability of the results in a translational paradigm. Here, we show that human specimens obtained via fine needle aspiration (FNA) from cancerous clear cell renal cell (ccRCC) tumors can grow and be maintained as immune organoids harboring a diverse lymphoid and myeloid population in a matrigel-based culture model. This system provides several advantages over cocultured cell lines with primary immune cells. In our FNA based method, the immune cell population is obtained directly from the TME, reflecting a realistic representation of tumor-associated exhaustive and/or regulatory immune cells. In addition, these studies provide a framework for drug development and allow for the evaluation of a 3D organization of tumor cells with immune cells. Ultimately, there is a translational gap between 2D cell culture and animal model testing; therefore, evaluation of the 3D organization of tumor cells within an ideal TME could recapitulate physiological cancer processes.
IL-6 Promotes Cell Growth and Is Associated With Poor Prognosis In Patients With Oral Cancer
Authors: Chia Jean-San , Wei Ling-Ying
Published: January 2023

Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine that regulates growth and differentiation of various types of malignant tumors, including oral cancers. Compared to healthy controls, the levels of IL-6 are elevated in sera of patients with oral cancer. In this study, the role of IL-6 in the growth regulation of oral cancer cells was evaluated.

The effects of IL-6 on cell growth were evaluated by expressing IL-6 cDNA into the human tongue cancer cell line (SAS), resulting in high-producer clones. In vivo, the effects of tumor-derived IL-6 on growth were studied by using an experimental vaccine model. Two stable clones, consisting of low-producer (IL-6LE), and high-producer (IL-6OE), were subcutaneously inoculated on the flank of each severe combined immune-deficient (SCID) mice. A total of 111 surgically treated HNSCC patients were studied. IL-6 and STAT3 levels were measured by immunohistochemical staining and correlated with clinicopathological variables and patient survival.

Treatment of recombinant IL-6 and overexpression of IL-6 by intorducing IL-6 into SAS cells significantly increased clonogenic ability and cell proliferation in vitro compared to the parental SAS cells and the vector controls. In animal model, a statistically significant difference of tumor weight was obtained between the IL-6LE and IL-6OE (*P=0.024) groups in experiment. Clinical data revealed significant relationships between IL-6 and stage*, lymph node metastasis*, recurrence**, disease-free survival* and overall survival* (P < 0.01; *P < 0.05).

These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo and in vivo oral cancers.
Role of the receptor tyrosine kinase AXL in regulating antitumor response and relationship with hypoxic stress
Authors: Mami-Chouaib Fathia , Terry Stephane , Buart Stephanie , Gausdal Gro , Angelsen Agnete , Lorens James , Chouaib Salem
Published: January 2023

Tumor hypoxia has long been considered as a driving force of tumor progression and to play a key role in remodeling the tumor stroma and favoring the emergence of tolerance. The receptor tyrosine kinase AXL is associated with drug resistance in several solid tumors and AXL-targeting agents are currently in clinical trials. We investigated the relationship between tumor hypoxia, AXL expression and the emergence of tumor resistant variants. Our data indicate that hypoxia promotes EMT, AXL expression and resistance to CTLs and NK in NSCLC. We have next investigated the molecular basis of AXL-induced resistance to cell-mediated cytotoxicity and demonstrated that AXL targeting resulted in an increase in target susceptibility to CTLs and NK mediated cell lysis by a mechanism involving ICAM and the NKG2D ligand (ULBP1).

We next investigated the effect of pseudo hypoxia (hypoxic stress in presence of oxygen) in clear cell renal cell carcinoma (ccRCC). In fact, in this type of cancer, Von Hippel-Lindau (VHL) mutations induce a hypoxic microenvironment under normoxic conditions. We showed that pseudo-hypoxia induced by VHL dysfunctions, promotes ccRCC resistance to immune cell-mediated cytotoxicity by a mechanism involving HIF-2α.

We then sked wheter the pseudo-hypoxia associated with VHL mutation in RCC could impact AXL expression. We obtained in vtro evidence indicating that both HIF2α and EMT associated transcription factors (ZEB1, ZEB2) play a role in AXL expression regulation. Using a cohort of 324 metastatic RCC patients treated with anti-PD1, we could demonstrate that AXL expression may interfere with the clinical outcome. The relationship between VHL dysfunction, AXL expression and patient response to anti-PD1 will be discussed.
Tumor growth following genetic modifications of mouse prostate tumor cells
Authors: Aguilar Roberto , Zhang Carl Tinghao , Ma Tengchuan , Feng Chongyu
Published: January 2023

Objectives

1) To determine the growth features of MyC-CaP mouse prostate tumors induced in immunodeficient nude mice and in immunocompetent FVB mice; and 2) To assess growth characteristics of MyC-CaP-p53 null tumor cells in vitro.

Methods

MyC-CaP cells were transfected with enhanced green fluorescent protein (eGFP), and the MyC-CaP-eGFP cells were injected intraperitoneally into naive immunodeficient nude recipients and into naive immunocompetetent FVB recipients. The transplanted tumor cells were subsequently tracked by fluorescent stereo microscopy. MyC-CaP-p53 null tumor cells were generated by inducible CRISPR/Caspase-9 gene deletion targeted to the native mutated p53 tumor suppressor gene.

Results

1) Increased growth of MyC-CaP-eGFP tumors occurred in immunodeficient nude mice compared to growth in immunocompetent FVB mice; 2) decreased metastasis of MyC-CaP-eGFP tumors compared to MyC-CaP tumors occurred following injection into immunocompetent FVB mice; and 3) MyC-CaP-p53 null cells showed reduced proliferation and growth in vitro compared to non-modified MyC-CaP tumor cells.

Conclusions

Three conclusions can be drawn from these studies: 1) Injection of MyC-CaP-eGFP tumor cells into naive immunodeficient nude recipients allowed for adequate examination of initial tumor viability and subsequent tumor growth and metastasis; 2) Decreased MyC-CaP-eGFP tumor growth in immunocompetent FVB mice compared to immunodeficient nude mice indicates that an immune response to the eGFP protein inhibited growth of the MyC-CaP-eGFP tumor; and 3) The growth inhibition following deletion of the mutated p53 tumor suppressor gene in MyC-CaP tumor cells indicates that the mutated allele helps drive the malignancy.
TGF-β signature is a potential biomarker for identifying individualized immunotherapeutic approaches in hepatocellular carcinoma
Authors: Chen Jian , Su Xiaoping
Published: January 2023

Background

Several immune checkpoint inhibitors have recently shown promising results in patients with advanced hepatocellular carcinoma (HCC). However, tumor response rates ranged from 14% to 17%. Thus, there remains a large unmet medical need for novel and effective immunotherapeutic strategies against HCC. Dysregulated signaling in the transforming growth factor (TGF)-β pathway plays a central role in immunomodulation, inflammation, and fibrogenesis in the HCC microenvironment. We therefore used The Cancer Genome Atlas database to analyze potential therapeutic approaches combining TGF-β inhibition and immunotherapies for HCC.

Methods

Transcriptomic analyses were performed in 193 HCC cases. Pathway analyses were performed by correlating 70 published signatures with TGF-β signatures.

Results

1. Unsupervised hierarchical clustering of TGF-β genes in HCC revealed four distinct clusters with unique TGF-β signatures: highly activated (14.5%), activated (45.0%), normal (30.6%), and inactivated (9.9%). 2. Highly activated TGF-β signatures were significantly associated with fibrosis and activated stromal signatures. 3. TGF-β signature subtypes were significantly associated with immune cell infiltration, T cell exhaustion, and checkpoint blockade response subclasses. 4. Potentially targetable genes were associated with specific TGF-β signatures. 5. TGF-β signatures provide potential combination therapeutic options for HCC.

Conclusion

We demonstrated a specific immunosuppressive role of TGF-β in mediating immunotherapy resistance in HCC, highlighting the TGF-β signature as a potential biomarker in individualized immunotherapeutic approaches in HCC.
Conventional type 1 dendritic cells and natural killer cells demonstrate strong correlation to cytotoxic T lymphocyte infiltration in cervical cancer tumors
Authors: Dikshit Anushka , Anderson Courtney M , Zhang Bingqing , Ma Xiao-Jun
Published: January 2023

Professional antigen presenting cells such as dendritic cells (DCs) are vital for priming naive CD8+ T cells and developing a sustainable anti-tumor immune response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s). However, these cells are low in abundance making their detection in the tissue context challenging.

To interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship to each other we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. NK cells and cDC1 cells were identified by using cell specific marker probes in 4 cervical cancer samples. Similarly, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and infiltration of CTLs within the cervical cancer tumors.

Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells in the TME. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration.

In conclusion, by utilizing the RNAscope Multiplex ISH assay we identified and visualized the spatial relationship between NK cells, CTLs, and cDC1 cells, highlighting the strength of this technology to spatially interrogate the TME.
Heat shock protein gp96 controls regulatory T cell lineage stability via IL-2/pSTAT5 signaling
Authors: Zhou Lei , Kwon Hyunwoo , Rubinstein Mark P , Li Zihai
Published: January 2023

Gp96 is an essential endoplasmic reticulum chaperone for various proteins including GARP, TLRs, integrins, Wnt co-receptor LRP6 and the platelet glycoprotein Ib/IX/V complex. Our laboratory has previously shown that loss of gp96 in murine Foxp3+ regulatory T cells (Tregs) results in their instability as determined by FOXP3 downregulation, impaired immunosuppressive function and production of effector cytokines (Zhang, et al., J Clin Invest., 2015, 125: 859–869). However, the mechanism underlying Treg dysfunction upon gp96 deletion remains unknown. Using tamoxifen-inducible Treg-specific gp96 gene (encoded by Hsp90b1) knockout (KO) mice (Foxp3-ERT2creHsp90b1f/f), we first phenotypically characterized gp96-deleted Tregs in mouse spleen and peripheral lymph nodes (pLNs). Gp96 deficient Tregs showed decreased frequency of TCF1−CD62L− effector population, but were enriched with short-lived KLRG1+ and Helios+ subsets, in both spleen and pLNs. We also confirmed that the expression of Foxp3 was decreased in splenic gp96-deleted Tregs, which correlated with a reduction of cell surface CD25. Intriguingly, both Foxp3 and surface CD25 levels stayed high in gp96-null Tregs in pLNs. In addition, gp96-null Tregs in mouse spleen, but not in pLNs, demonstrated compromised suppressive function. We further found that IL-2/CD25 signaling, as assessed by STAT5 phosphorylation, was more activated in gp96-null Tregs in pLNs, compared to that in spleen. In vitro IL-2 treatment was able to rescue the attenuated Foxp3 expression in gp96-null Tregs. Our work indicates that IL-2/pSTAT-5 signaling may be a key factor in maintaining Treg Foxp3 expression and its lineage stability upon gp96 deletion.
Protein kinase C-eta is required for Treg-mediated suppression of anti-tumor and viral immunity
Authors: Liu Hsin-Yu , Pedros Christophe , Balancio Ann , Kong Kok-Fai , Altman Amnon
Published: January 2023

We previously reported that protein kinase C-eta plays an important role in the contact-dependent suppressive activity of Tregs via its association with CTLA4, and that PKC-eta-deficient (Prkch−/−) Tregs fail to suppress anti-melanoma tumor immunity. Here we extend this study to a genetically engineered mouse model of HCC driven by CRISPR-Cas9-driven deletion of Pten and p53. In the Pten-p53 HCC model, Prkch Treg-specific conditional knockout (cKO) mice developed a lower tumor incidence, fewer tumors, lower degree of steatosis phenotype, and showed higher intratumoral CD4+ T cells than WT mice. In addition, B cells and resident DCs displayed higher levels of the costimulatory ligand CD86 in dLNs of cKO mice. Increased CD4+ T cells, memory T lymphocytes, and cytokines production were observed in spleen of cKO mice. These results indicate that Treg-expressed PKC-eta is required for Treg-mediated suppression of anti-HCC tumor immunity. To further explore the importance of PKC-eta in Teff cells, the LCMVArm acute infection model, as well as the in vitro activation of murine or human CD8+ T cells were used. We found that purified Prkch−/− mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact T cell activation in vitro as measured by proliferation and expression of GzmB and IFNg. Interestingly, Treg-specific cKO mice showed improved viral clearance and displayed enhanced expression of GzmB and IFNg by virus-specific CD8+ T cells. Thus, global PKC-eta deletion does not impair overall CD8+ T cell-mediated immunity implying that selective pharmacological PKC-eta inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor- and viral-specific immunity.
Foxp3+ regulatory T cells suppress T cell immunity in inflamed and non-inflamed tumors by distinct mechanisms
Authors: Delfino Teresita , Wang Yingyun , Muller Soren , Iftikar Hina , Modrusan Zora , Schartner Jill , Rutz Sascha
Published: January 2023

Foxp3+ regulatory T cells (Treg) are essential to prevent autoimmunity, but may also suppress anti-tumor immune responses. The relevance of Treg cell-mediated suppression in inflamed and non-inflamed (immune excluded or immune desert) tumors is not well understood. Here, we used Foxp3-DTR transgenic mice to selectively ablate Treg cells in mice with established tumors. We observed robust and rapid tumor clearance in a number of models, independent of the immune infiltration at baseline. These responses were driven by the induction of infiltration, proliferation, and cytokine production by CD8 T cells in tumor tissues. Importantly, pairing single cell TCR sequencing with transcriptome analysis revealed that CD8 T cells in the tumor acquired a progenitor exhausted phenotype and a clonally diverse TCR repertoire following Treg cell depletion. Pharmacological blockade of lymph node egress demonstrated that local Treg cell depletion and the resulting activation of tumor-infiltrating CD8 T cells was sufficient for tumor rejection in inflamed tumors. In contrast, short-term Treg cell depletion outside of the tumor and the resulting de novo generation of CD8 T cell responses facilitated the rejection of immune desert tumors. These data suggest that Treg cells play a critical role in suppressing both priming of novel anti-tumor CD8 T cell responses as well as limiting effector functionality of infiltrating CD8 T cells within the tumor microenvironment. More importantly, our data suggest that transient systemic Treg cell depletion can convert non-inflamed tumors into inflamed tumors.
Identifying a uniform T cell signature associated with protective anti-tumor immunity and autoimmune systemic sclerosis
Authors: Lust Hannah E , Whitfield Michael , Huang Yina H
Published: January 2023

Precise tug-of-war between immunosuppressive regulatory T (Treg) cells and inflammatory IL-17-producing (Th17) cells ensures appropriate reactivity to pathogenic challenge. While an imbalance towards Tregs leads to suboptimal responses as observed in cancer, unchecked Th17 activity leads to autoimmunity. Though numerous mechanisms have been identified for how Tregs suppress effector cells, productive effector T cell responses are believed to result from passive escape of Treg suppression. Here we report a novel active role for Th17 cells in dampening Tregs to promote effector activity in the context of melanoma. We hypothesize that Th17 cells can physically impede Treg suppression resulting in enrichment of CD8 T cell prevalence and effector function. Using a melanoma mouse model to characterize Th17 effectors responsible for inhibiting Tregs, we found elimination of IL-17-producing cells prevents vaccine-induced anti-melanoma immune responses. This suggests Th17 cells are important for induction of CD8-mediated anti-tumor immunity. Using RNA-Seq analysis, we generated a gene expression (GE) signature to characterize Th17 effectors responsible for inhibiting Tregs in the context of tumor vaccination. Interestingly, increased ratio of Th17 to Treg cells may contribute to pathogenesis of localized and systemic scleroderma. Diminished Treg- and enhanced Th17-associated genes are consistently found in scleroderma patient skin, leading us to hypothesize that scleroderma pathogenesis has a similar Th17 GE signature to that associated with enhanced tumor clearance. Using the tumor-associated Th17 GE signature to examine scleroderma patient samples we determined transcriptional overlap between the Th17 cell subsets.
Regulatory T cell control of systemic tumor-specific immunity and response to checkpoint immunotherapy response in liver metastasis
Authors: Lee James , Mehdizadeh Sadaf , Bridge Jennifer Anne , Young Arabella , Mufazalov Ilgiz A. , Daud Adil , Bluestone Jeffrey A
Published: January 2023

A significant number of metastatic melanoma patients have disease progression despite checkpoint inhibitor therapy. We lack salvage strategies for this population and a mechanistic understanding of checkpoint inhibitor refractory disease. Previously, we reported that patients with liver metastasis have worse response rate and survival than those without liver metastasis despite treatment with checkpoint immunotherapy, but how liver metastasis can influence the antitumor immune response in this context is unclear. We hypothesize that tumor antigen-specific tolerance can be induced in the context of liver metastasis, resulting in therapy resistance.

In this study, we developed a syngeneic two-site tumor model in which tumor cells are injected simultaneously into a subcutaneous (SQ) site and into the liver or other organ sites of B6 mice to study the effects of the intrahepatic tumor on antitumor immunity at a distant site. We found that the presence of tumor or tumor antigen within the liver reduced systemic antigen-specific effector T cell function. Tetramer and sc-RNAseq studies suggest a liver and antigen-specific immunoregulatory process associated with the activation of regulatory T cells and the remodeling of innate immune cells. Importantly, this systemic dysfunctional immune state was not reversed by anti-PD-1 monotherapy but rather by deleting (anti-CTLA-4) or destabilizing (EZH2 inhibitor) intratumoral Tregs, suggesting a distinct regulatory mechanism contributing to anti-PD-1 resistance that could be overcome to achieve complete tumor regression at all sites.

Our results reveal a novel mechanism of checkpoint inhibitor resistance and suggest a strategy to improve the outcome for patients with liver metastasis.
Sex differences in immune responses to influenza vaccine in healthcare workers during the 2019–2020 influenza season
Authors: Park Han-Sol , Kuo Helen , Dhakal Santosh , Ursin Rebecca , Shaw-Saliba Kathryn , Fenstermacher Katherine , Gearhart Patricia J , Rothman Richard , Pekosz Andrew , Morgan Rosemary , Klein Sabra L
Published: January 2023

Seasonal influenza epidemics affect 5–15% of the world population with an estimated 290,000–650,000 annual respiratory deaths due to influenza. The Center for Disease Control recommends annual influenza vaccination for healthcare workers (HCWs) because not only does their occupation increase their risk of contracting influenza, but HCWs care for people at higher risk for developing influenza-related complications. Studies have shown immune responses, such as seroconversion, differ between male and female vaccine recipients, but few have focused on HCWs and their influenza vaccine responses by sex. To understand sex differences in immune responses to the influenza vaccine in the HCW population, 50 male and 50 female were recruited during the 2019–2020 influenza season as part of the Johns Hopkins Center for Excellence in Influenza Research and Surveillance (JH-CEIRS) annual serological surveys among Johns Hopkins Hospital HCWs that takes part in the annual hospital-wide vaccination campaigns. Participants were recruited just prior to receiving their annual quadrivalent influenza vaccine (QIV), which targets influenza strains A/Brisbane/H1N1, A/Kansas/H3N2, B/Colorado (Victoria lineage), and B/Phuket (Yamagata lineage). Whole blood was collected at 0, 7 and 28 days post-vaccination. Neutralizing antibody assays will be used to measure pre and post antibody titers against the H1N1 and H3N2 vaccine components. We will also analyze vaccine-specific B cell responses from peripheral blood mononuclear cells (PBMCs) at 7 and 28 days post-vaccination via flow-cytometry. This study will provide extensive data on the effects of patient sex on influenza vaccine-induced antibody responses and B cell phenotypes in the HCW.
Molecular mechanisms underlying T cell co-potentiation by anti-CD3 Fab fragments
Authors: Huynh Hien T. , Nelson Alfreda D , Hoffmann Michele , Abergel Megan , Hu Shuang , Alarcon Balbino , Schrum Adam G , Gil Diana
Published: January 2023

Cognate antigen binding to the T cell receptor (TCR) induces a conformational change of the CD3 complex (CD3Δc), which is required but not sufficient to trigger productive signaling from the TCR/CD3. CD3Δc fails to occur when the TCR ligates poorly immunogenic antigens. In the absence of CD3Δc, T cells do not display productive immune responses. CD3Δc involves the exposure of a conserved proline-rich-sequence (PRS) in CD3ɛ cytoplasmic tail that functions as a docking site for cytoplasmic adaptor proteins like Nck, which participate in the CD3 signaling cascade. The opening of the PRS is controlled by a CXXC motif in CD3ɛ located adjacent to the plasma membrane. We previously described it is possible to mimic CD3Δc on T cells when adding Fab fragments capable to bind to the CD3 complex. When combining TCR engagement to weak antigens with binding of Fabs to the CD3 complex that not interfere with antigen binding, T cell co-potentiation occurs. In this instance, T cells become capable to display responses to poorly immunogenic stimulation. As an example of the consequences of T cell co-potentiation in vivo, we studied anti-mouse CD3 Fabs as a novel immunotherapeutic against poorly immunogenic types of cancer. Anti-CD3 Fabs reduced melanoma burden in B6 mice bearing B16-F10 tumor in lungs. Now, we have explored the molecular mechanisms supporting T cell co-potentiation in monoclonal and polyclonal mouse models. In B6 mice expressing null mutations for either the CXXC or the PRS motifs, anti-CD3 Fabs fail to induce CD3Δc, as well as to co-potentiate murine T cells in vitro and in vivo. These data suggest PRS opening by anti-CD3 Fabs may be linked to their capacity to co-potentiate T cells responses to poorly immunogenic antigens.
Querying resources in the tumor microenvironment using bystander memory T cells
Authors: Sarkar Surojit , Sullivan Peter , Yuzefpolskiy Yevgeniy , Kalia Vandana
Published: January 2023

The general paradigm is that the tumor microenvironment is non-conducive to sustaining productive cytotoxic T cell responses due to suppressive and counter-immunity T cell extrinsic factors. It has been shown that the tumor microenvironment can harbor large numbers of bystander memory cells. However, it is not very clear if given the right stimulation these bystander memory cells can be commandeered to utilize the available resources of the tumor microenvironment, get optimally activated and elaborate productive immune responses. This has wide-ranging implications in hijacking the tumor-resident bystander memory cells for efficacious tumor control. In this study we show that the tumor microenvironment is capable of harboring ultra-functional bystander memory cells, which display complete panoply of memory markers. These tumor–resident bystander memory cells are not only capable of elaborating robust ex vivo and transplantable memory T cell functions, but they can also efficiently utilize the resources within the tumor microenvironment to elaborate in situ recall effector functions. We further show that these memory T cells utilize CXCR3 as a key chemokine receptor for trafficking to the tumor sites. Our data identify bystander tumor-resident memory cells as Spartan warriors to mediate enhanced tumor control during adoptive T cell immunotherapy.
Characterization of serological response to dengue and Zika viruses in pregnant women during the Zika outbreak in Brazil
Authors: Driesse Kaitlin , Tsai Wen-Yang , Brites Carlos , Pedroso Celia , Wang Wei-Kung
Published: January 2023

The outbreak of Zika virus (ZIKV) and associated congenital Zika syndrome in dengue virus (DENV)-endemic regions raised questions about the effects of ZIKV infection on DENV immunity and disease outcome and vice versa, which highlighted the need of serological tests that can discriminate between DENV and ZIKV infections. Previously we developed combined DENV and ZIKV non-structural protein 1 (NS1) enzyme-linked immunosorbent assays (ELISAs) to overcome the cross-reactivity of traditional envelope protein-based serological tests (Tsai et al. Clin Infect Dis 2017;65:1829). In this study, we used this assay to screen 120 serum samples collected from asymptomatic parturient women during the ZIKV outbreak in Salvador, Brazil and identified four serostatus groups. We further characterized antibody responses in 8 participants including 2 primary DENV (pDENV), 2 secondary DENV (sDENV), 1 primary ZIKV (pZIKV), and 3 ZIKV with previous DENV (ZIKVwprDENV) infections. Using depletion experiments with inactivated ZIKV and urea tests, we determined the IgG avidity to DENV. Using depletion experiments with inactivated DENV and endpoint ELISA titers, we determined ZIKV type-specific and cross-reactive antibody and found pZIKV had higher proportion of ZIKV type-specific antibody (82%) than ZIKVwprDENV (10-16%). These results were supported by microneutralization tests. Our findings suggest that combined DENV and ZIKV NS1 IgG ELISAs plus depletion experiments can delineate past and present DENV and ZIKV infections and are a potential tool to further our understanding of pathogenesis and epidemiology of DENV and ZIKV in endemic regions.

Research supported by R01 AI11076901, R21AI135292-01A1 from NIAID, and P30GM114737 from NIGMS, NIH.
Mixed origins: HIV vaccine induced Env-specific antibodies arise from naive and cross-reactive memory B cells
Authors: Kobie James , Piepenbrink Michael , Fucile Christopher , Rosenberg Alexander F. , Basu Madhubanti
Published: January 2023

The most potent and broad HIV envelope (Env)-specific antibodies (Abs) often when reverted to their inferred germline versions representing the naïve B cell receptor, usually fail to bind Env. This may suggest that the primary B cell population that responds to the initial Env exposure is not exclusively comprised of a naïve population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated 66 gp120 reactive mAbs from participants from HVTN105, a phase I trial testing AIDSVAX B/E (gp120 protein) combined with a DNA immunogen. In order to determine the origin of these mAb producing B cells, we used Miseq-based VH lineage tracking approach and identified several of these mAb lineages in peripheral blood prior to vaccination (pre-immune). Further, several of these pre-immune lineages also persist in the bone marrow ~8 months after the final vaccination, including within in the CD138+ long-lived plasma cell compartment. When tested by ELISA, pre-immune mAbs bound to HIV Env, with relatively lower ability than their post-vaccination forms, but showed higher reactivity than their germline form. The majority of the pre-immune lineage members were IgM, however IgG and IgA members were predominant prior to vaccination and the majority of the pre-immune lineage members exhibited somatic hypermutation. These results may indicate that the vaccine-induced Env-specific antibody lineages originated from a mixed ancestor population of naïve and cross-reactive memory cells.
A CXCR5−PD1+ T helper subset triggers excessive plasma cell differentiation and antibody responses in dengue
Authors: Gupta Nimesh , Ansari Asgar , Sachan Shilpa , Kumar Vinod , Coshic Poonam , Sood Rita , Sette Alessandro , Wig Naveet , Weiskopf Daniela , Soneja Manish , Gupta Nimesh
Published: January 2023

Understanding the underlying determinants of cross-reactive and weakly neutralizing antibody response in dengue virus infection will facilitate the rational development of vaccines and therapeutics. Accordingly, we investigated CD4 T cell subsets in promoting the antibody development in dengue. In longitudinal and antigen-specific analysis we demonstrate the existence of a CXCR5−PD1+ T helper (Th)-subset that co-expresses PD1/ICOS and expands robustly in dengue with warning sign and severe dengue. The magnitude of CXCR5− PD1+ Th-subset is significantly higher than the frequency of activated Tfh cells in dengue. Interestingly, this PD1+/ICOS+ phenotype is exclusively enriched in the DENV-specific CXCR3 expressing Th population. Moreover, the CXCR5−PD1+ Th-subset harbors the potential of B-cell help in terms of expressing key help factors; IL21, CD40L, IL10 and IFNγ. We further demonstrate that IgG responses to NS1 are independent of germinal center (GC) reaction and that CXCR5−PD1+ Th-subset is superior to Tfh subset in inducing plasma cell differentiation and NS1-specific IgG in autologous T-B co-cultures. The targeted gene expression analysis and antigen stimulation assay confirms unique traits in CXCR5−PD1+ Th-subset over the conventional Tfh cells in dengue. Targeting this unique CXCR5−PD1+ Th-subset might limit the excessive differentiation of plasma cells and shape the optimal antibody responses in dengue virus infection.
The Pro-autoimmune allele of tyrosine phosphatase PTPN22 enhances anti-viral and anti-tumor immunity
Authors: Orozco Robin C , Marquardt Kristi , Mowen Kerri A , Pratumchai Isaraphorn , Teijaro John R , Sherman Linda A
Published: January 2023

The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W) is present in 5–10% of the North American population and is strongly associated with numerous autoimmune diseases. Specifically, this alternative allele affects lymphocyte activation, toll-like receptor signaling, and cytokine production in various autoimmune contexts. Despite the importance of these inflammatory networks to clear pathogens and tumors, the influence of PTPN22, and its pro-autoimmune allele, have on viral and tumorigenic conditions is still being defined. To interrogate these roles, we used CRISPR/Cas9 to generate mice expressing the Ptpn22 R619W molecule, which is the ortholog of R620W in humans. Using LCMV-cl13 infection and B16 tumor models, we tested the hypothesis that pleiotropic effects of Ptpn22 R619W enhances anti-viral and anti-tumor immunity. Importantly, R619W-bearing mice have less tumor burden and clear chronic viral infection faster than wild-type mice. Consistent with these results, R619W mice have enhanced antigen-specific T cell activation during viral infection and increased tumor-infiltrating Th1 CD4 T cells. Additionally, we define an underlying mechanism by which Ptpn22 promotes PD-L1 expression on conventional dendritic cells (cDCs) in wild-type mice, but is abrogated in R619W mice during disease. Lastly, R619W expression in lymphocytes alone can enhance T cell activation; however, the combination of R619W expressing lymphocytes and DCs enhances T cell activation even more. Our results suggest that a pro-autoimmune allele can be beneficial by promoting anti-viral and anti-tumor immune responses through multiple immune cell types, and may have a protective effect in these diseases.
Chronic viral infection promotes efficient germinal center B cell responses
Authors: Pinschewer Daniel D. , Fallet Bénédict , Hao Yi , Florova Marianna , Cornille Karen , de los Aires Alba Verge , Zubani Giulia Girelli , Ertuna Yusuf , Greiff Victor , Menzel Ulrike , Hammad Karim , Merkler Doron , Reddy Sai T. , Weill Jean-Claude , Reynaud Claude-Agnès
Published: January 2023

Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infection, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID-mutant mice, chronic infection selects for GC B cells with hypermutated BCRs and neutralizing antibody formation. These findings demonstrate that, unlike for CD8+ T cells, chronic viral infection drives a functional, productive and protective GC B cell response.
T cell-derived XCL1 induces protective immunity against West Nile virus within the CNS
Authors: Durrant Douglas M. , Cortes Alejandro , Alvarado Marina
Published: January 2023

West Nile Virus (WNV) is a leading cause of mosquito-borne neuroinvasive disease in the US. Following infection, WNV can cause neuronal injury and disease. Previous studies have shown that the proper activation of virus-specific T cells results in protection from WNV infection and any associated disease. Dendritic cells (DCs) are often defined as pivotal inducers of immunity due to their role in activating immune defense mechanisms against invading pathogens. XCR1, a chemokine receptor expressed predominantly by a subset of DCs, has been shown to be essential in cross-presenting antigen and in activating anti-viral immunity. However, DCs are not readily present within the CNS and it remains unknown how DCs are recruited to the CNS during WNV encephalitis to fully activate antiviral immunity. XCL1, the ligand for XCR1, is expressed by activated T cells, DCs, macrophages, and NK cells. We hypothesize that following WNV infection, virus-specific T cells within the CNS produce XCL1 in order to recruit and stabilize their interaction with XCR1+ DCs thus ensuring their proper activation and subsequent protection against WNV encephalitis. Here we demonstrate that mice lacking XCR1+ DCs are more susceptible to WNV encephalitis, display increased neuronal cell death, as well as increased viral burden compared to control animals. In addition, in the absence of XCR1+ DCs, WNV-infected animals displayed decreased parenchymal localization of T cells within the brain compared to controls. These data indicate that XCL1-XCR1 interactions are crucial in the development of efficient anti-viral immunity. The knowledge gained from this work may then be applicable to other viral diseases of the CNS.