bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary resident effector CD4+ T cells fail to effectively utilize glycolysis
Authors: Roberts Lydia M , Evans Tyler J , Bosio Catharine M
Published: January 2023

The transition of T cells from naïve to effector cells has been reported to require a metabolic shift from oxidative phosphorylation to glycolysis for the production of effector molecules such as IFN-γ. This phenomenon is primarily restricted to study of splenic T cells and it is largely unknown if T cells in other organs undergo the same metabolic shift. Intranasal vaccination establishes a pool of lung tissue-resident, effector memory T cells (trTem) that are typically sufficient to control a secondary infection via production of cytokines. Therefore, we hypothesized trTem would be highly glycolytic upon activation. To test this hypothesis, we compared the metabolic activity of both trTem and circulating Tem (cTem) isolated from mice vaccinated with either Bordetella pertussis or Francisella tularensis. As expected, regardless of the vaccinating pathogen, CD4+ cTem readily shifted to glycolysis following activation with anti-CD3/CD28 beads. In contrast, CD4+ trTem cells did not shift to glycolysis following the same activation scheme, nor did they increase mitochondrial respiration as a mechanism to compensate for the absence of glycolysis. Consistent with an inability to shift to glycolysis, ex vivo stimulation of trTem with antigen also resulted in limited uptake of glucose as compared to cTem. These data suggested that, unlike previous reports, pulmonary CD4+ trTem do not rapidly shift to glycolysis even in the presence of antigen. Together these data highlight the unique environment of the pulmonary compartment and present critical information that will influence our understanding of pulmonary T cell responsiveness during infection which may shape future vaccination strategies.
Distinct Roles of TβRIII and LRP-1 in Lactoferrin-mediated Foxp3+ Inducible Tregs
Authors: Kim Sun-Myoung , Jang Young-Saeng , Kim Pyeung-Hyeun
Published: January 2023

We recently found that lactoferrin (LF) stimulated mouse naïve CD4+T cells to differentiate into inducible Foxp3+Tregs (iTregs). However, LF decreased cell proliferation, CD62L shedding, and CD44 expression. In the present study, we characterized these phenomena at the receptor level. Addition of excess amount of soluble TβRIII (betaglycan) virtually abrogated the LF-induced Foxp3 expression but little affected LF-suppressed cell proliferation/CD62L shedding/CD44 expression. On the other hand, treatment of anti-LRP-1 Ab slightly enhanced LF-induced Foxp3 expression while substantially restored LF-suppressed cell proliferation/CD62L shedding/CD44 expression. These results indicate that LF enhances Foxp3 expression mainly through TβRIII and simultaneously suppresses cell proliferation/CD62L shedding/CD44 expression mainly through LRP-1. Thus, it is assumed that LF renders activated CD4+T cells to become central memory-like Tregs (CD4+Foxp3+CD62L+CD44lo).
BATF is an essential regulator of migratory ILC2 cell fate and function
Authors: Miller Mindy M , Patel Preeyam , Bao Katherine , Danhorn Thomas , O’Connor Brian , Reinhardt R Lee
Published: January 2023

A recently identified subset of group-2 innate lymphoid cells (ILC2s) migrates to mucosal tissues early during type-2 inflammation and is characterized by responsiveness to interleukin-25 and high expression of KLRG1. This study identifies the major transcriptional regulator required for their generation and function. RNA-sequencing and unbiased pathway analysis revealed the AP-1 transcription factor BATF as a potential modulator of ILC2 cell fate. Indeed, infection of BATF-deficient mice with the helminth N. brasiliensis showed a selective defect in IL-25-induced, ILC2-mediated worm clearance as well as a corresponding loss of IL17RB+ (IL-25 receptor) KLRG1high ILC2s in the lung. Moreover, these BATF-dependent migratory ILC2s serve as the major early source of IL-4 and IL-13 after infection. Interestingly, while the presence of KLRG1high ILC2s in the small intestine are unaffected by BATF-deficiency, their ability to produce IL-13 is significantly impacted. Defects in IL-13 expression by intestinal KLRG1high ILC2s corresponded with decreased goblet and tuft cell hyperplasia in BATF-deficient mice, supporting the critical role of these migratory ILC2s as early sentinels of mucosal barrier integrity.
Bivalent anti-CD3 Fab dimers drive T cells into a highly efficient fratricide with potential clinical application
Authors: Pages Diana Gil , Nelson Alfreda D , White Tommi A , Schrum Adam G
Published: January 2023

Non-covalent anti-CD3 Fab dimers (Bi-Fabs) bind to the TCR/CD3 complex and activate T cells. In the presence of proper co-stimulation, anti-CD3 Bi-Fabs promote T cell cycling and acquisition of effector functions on CD4 and CD8 T cells more robustly than other bivalent antibody species like F(ab′)2 and mAb with the same specificity. Surprisingly, the net effect of anti-CD3 Bi-Fab treatment on T cells in vitro is contraction of CD4 and CD8 T cells, via AICD. This is in contrast with the net T cell proliferation observed with soluble anti-CD3 mAbs that are stimulatory in the presence of proper co-stimulation. Bi-Fab binding to the TCR/CD3 complex, and direct cellular contact with Bi-Fab engaged T cells, are required to observe the net contraction of treated T cells. Using perforin-deficient T cells, and Fas-FasL blockade, we show this net contraction results from T cell apoptosis via Fas-FasL and CTL degranulation. Structural studies on Bi-Fabs suggest a rigid rod like structure. We hypothesize that the rigid rod allows for a potent crosslinking structure with both paratopes from the anti-CD3 Fabs capable to engage effector T cells on fratricide. In vivo, mice injected with anti-CD3 Bi-Fabs present depletion of T cells from blood, and absence of any adverse effects derived from T cell activation like cytokine storm. We have begun exploring the therapeutic potential of T cell depletion in mouse models of autoimmune disease with promising results.
Genetic and pharmacological validation of TAK1 inhibition in macrophages as a therapeutic strategy to effectively inhibit TNF secretion
Authors: Scarneo Scott A , Eibschutz Liesl , Hughes Phillip , Haystead Timothy
Published: January 2023

Immune challenge of invading macrophages at sites of infection is associated with release of TNF, which triggers a local cytokine storm as part of the normal inflammatory response. Whereas this response maybe beneficial in fighting off infections, similar responses triggered in autoimmune diseases contribute significantly to the underlying damaging pathology associated with these diseases. Here we show that Takinib, a highly discriminatory inhibitor of transforming growth factor Beta-activated kinase 1 (TAK1), selectively and potently reduces TNF production in pro-inflammatory THP-1 macrophages. A complete survey of 110 cytokines, showed robust loss of proinflammatory cytokine responsiveness to lipopolysaccharide (LPS) and interferon gamma (IFNg) challenge in response to Takinib. The mechanisms of action of Takinib was recapitulated in TAK1 KO macrophages. TAK1 KO cells showed significant loss of TNF production as well as release of IL-6 in response to LPS challenge. Furthermore, Takinib blocked the ability of exogenously added LPS to promote phosphorylation of, c-Jun, p38 protein kinases as well as downstream transcription factors regulated by nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). In a mouse LPS challenge model, Takinib significantly reduced TNF serum levels. Our findings demonstrate that Takinib has utility in the treatment inflammatory disease by locally suppressing TNF production from invading macrophages.
Controlling the Residual Inflammatory Risk in Type 1 Diabetic Atherosclerosis
Authors: Yussman Martin , Wagner David H , Vaitaitis Gisela , Waid Dan
Published: January 2023

Prevention trials with statins reduce the relative risk of cardiovascular (CV) events by 10 to 40%. This leaves a ‘residual risk’ of 60–90% for which the CANTOS trial (IL1β inhibition) provided proof of concept that targeting inflammation reduces CV event rates; unfortunately, it did not change rates of type 1 diabetes (T1D) and infections in T1D patients were higher. A major molecular driver of auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad with unique T cell subset, CD3+CD4+CD40+ (TH40 cell) known to be significantly expanded in T1D subjects. Normalization of the aberrant contact dependent interaction of this dyad has shown efficacy in animal models using antibody to CD154, reversing T1D and rate of atherosclerosis, but unfortunately has deleterious side effects when given in humans.

We measured TH40 cells in T1D subjects with high calcium scores (a measure of atherosclerotic burden) and those with low calcium scores. Additional to the higher percentage of TH40 cells compared to controls, we have found a statistically significant elevation in in INFg production in the high calcium score cohort, indicating a more pathogenic state of these cells.

We designed peptides capable of binding directly to the CD40 receptor, which have been shown to reverse T1D in animals. Peptide was administered to ApoE−/− mice, a model of atherosclerosis. Immunohistochemical staining demonstrated significant reduction in plaque as well as smooth muscle and collagen content. In-vitro analysis of TH40 mouse splenic cells showed modulation in IL2, INFγ and IL17, all potent cytokines of atherosclerosis.

This new information relays promise for a more directed diagnostic and therapeutic target for T1D related atherosclerosis.
Modeling graft-vs-host disease in humanized mice using different hematopoietic stem cells sources reveals inherent differences in Tc and myeloid cell biology
Authors: Hess Nicholas J , Hematti Peiman , Gumperz Jenny E.
Published: January 2023

Cancers and autoimmune diseases, which affect 1.6% and 4.9% of the U.S. population respectively, are often treated with chemotherapy and immunosuppressive drugs with variable response rates. One alternative treatment option are hematopoietic stem cell (HSC) transplantations. Unfortunately, the biology behind these transplants remains unclear despite being in practice for over 60 years. Our research aims to use a humanized mouse model to understand the different molecular mechanisms that lead to acute GVHD, hematopoietic recovery/rejection and GVL. Here, we will focus on our data recapitulating clinical GVHD in different graft sources. Injecting equivalent numbers (2E7) of bone marrow (BM) mononuclear cells and G-CSF mobilized (MB) mononuclear cells into non-irradiated NSG mice results in 50% & 0% penetrance of GVHD respectively at 12 weeks. Concurrently, we have measured the Tc proliferation and IFNγ secretion in these mice over the course of 12 weeks that reveals an elevated activation status in BM Tc’s from an early stage. Interestingly, stimulating the Tc’s directly in vitro results in equivalent activation potentials suggesting a prominent role of myeloid cells in the induction of GVHD and not Tc’s. The following investigations have shown an increased expression of the B7 complex on BM myeloid cells and an increase in their IL-6 secretion. These early observations suggest that the “accessory cells” injected from the different HSC grafts are vastly important for driving clinical outcomes. Understanding the molecular cross-talk between the “accessory” cells and the underlying cellular differences attributed to each HSC source will better inform clinicians on the best treatment practices for patients.
Defining the role of graft-infiltrating B cells in the acute rejection of kidney allografts in mice
Authors: JAIN DHARMENDRA , Yin Dengping , Chong Anita S
Published: January 2023

B-cell infiltrates has been observed in kidney biopsies undergoing clinical cellular or mixed rejection. Some studies suggest poorer graft survival is associated with the presence of B cell infiltrates in biopsies diagnosed with cellular rejection, whereas others have not. We have developed a mouse kidney transplant model to study the role of donor-specific B cells infiltrate kidney allografts undergoing acute rejection. To this end, C57BL/6 female mice were subjected to left nephrectomy and were transplanted with kidneys from BALB/c mice expressing 2W-OVA, transgene. Donor-specific B and T cells were identified with donor MHC Class I & II tetramers. All graft were rejected by D28 post-transplant and showed an average 36-fold increase in donor specific antibodies (DSA). At D14, ~5% of the donor specific B cells had a Fas+GL7+ germinal center phenotype in both SLO’s (pooled spleen and peripheral lymphnodes) and kidney allografts. In addition, about 47% of donor-specific T cells in both the SLO and allograft had a CD4+ICOS+ expression, suggesting Tfh phenotype. ~4000 and ~900 Anti Kd-IgG secreting B cells (ASC’s) were detected in both SLO’s and in kidney allografts, respectively. Our data raise the possibility that in situ donor-specific T and B cell interactions are occurring in SLOs and also in the rejecting allografts. We plan to use this transplant model to further investigate whether graft infiltrating B cells serve as antigen-presenting cells to alloreactive T cells mediating acute rejection, whether these B cells differentiate into ASC in situ, and to define the relationship between the intrarenal B cells and ASCs in the allograft and those expanded in the SLO.
The Th17 lineage in heart transplant rejection: Examining plasticity and pathogenicity
Authors: Williams Linus Clark , Iacomini John
Published: January 2023

Heart transplants are the only treatment for end-stage heart failure. Over 3000 heart transplants were performed in the United States in 2016, making heart transplant the most common cadaveric organ transplant. Long-term survival of heart transplants remains a significant clinical concern, and median survival rates after the first year have not significantly improved in 25 years. We have previously shown that pro-inflammatory co-morbidities such as hyperlipidemia contribute to accelerated IL-17 dependent rejection of the transplanted heart. Hyperlipidemia, an increased level of triglycerides and lipids in the blood, is extremely prevalent in the transplant patient population, with nearly 90% of heart transplant recipients becoming hyperlipidemic after their transplant, despite universal use of statins in the transplant patient population. The work described in this poster aims to further explore the mechanisms by which hyperlipidemia promotes Th17-mediated graft rejection, especially the potential interplay between the hyperlipidemic state and CD4 T cell plasticity. By using a MHC-II mismatched heart graft model, immunocytochemistry staining, a Th17 lineage trace, and genetic and chemical ablation of inflammatory cytokines and lineages, we aim to elucidate the differences in inflammatory T-cell infiltrate between graft-destructive accelerated hyperlipidemic rejection and chronic non-hyperlipidemic transplant rejection. This work may lead to more precise targeting of immunosuppression in heart transplant patients and improved survival and quality of life in this patient population
The enigmatic impact of donor T cell subsets on the therapeutic efficacy of tolerogenic protocols for transplant rejection
Authors: Lozano Marcos Iglesias , Bibicheff Darrel , Chicco Maria , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Costimulation blockade (CoB) regimens are a promising immunomodulatory strategy to prevent transplant rejection, especially when combined with antigen specific therapy. However their efficacy is affected by multiple factors. Recent reports have highlighted the unexpected capacity of passenger donor lymphocytes to enhance the recipient’s anti-graft response.

In this study we assess if T lymphocytes contained in the donor specific infusion (DSI) used in combination with anti-CD154 (MR1), a powerful CoB regimen, impact the regulation of mouse skin transplant rejection. Our data indicated donor T cells persist for at least 7d after infusion. When T cells were depleted from the DSI, transplant survival was significantly increased (MST=103 vs 68). Unexpectedly, the removal of CD4 T cells did not improve survival (MST=66). Concordantly, the addition of CD8+CD44hi memory T cells to T-depleted DSI restored the limited survival to that of full DSI (MST=71). ELISPot and flow cytometry results showed a greater anti-donor memory response in post-rejection spleen samples. Surprisingly, the removal of donor CD8 T cells in the DSI inoculum induced a survival even greater than that achieved with total T cell depletion (MST=148). The addition of CD4+CD44hi memory T cells to T-depleted DSI (inoculum still depleted in CD8 T cells), matched the survival of T-depleted DSI (MST=113) and was not associated to any increment in anti-donor memory response post-rejection.

Overall, these data reveal a novel role for donor lymphocytes in the modulation of recipient’s alloimmunity by DST+MR1. Identification of the mechanisms and specificities underlying this effect will be pivotal for the optimization of clinically effective tolerogenic therapies.
Type-1 Interferons impair the immunoregulatory activity of IL-10: understanding novel mechanisms of abrogation of transplant tolerance
Authors: Lozano Marcos Iglesias , Bibicheff Darrel , Chicco Maria , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. Type-1 interferons (TI-IFNs) are recognized mediators of this effect in multiple models, but their target population and specific mechanism used remain undefined. To better understand how TI-IFN could interfere with transplant tolerance, we studied their impact on the immunomodulatory properties of IL-10.

Using a murine full-mismatch skin transplant model we showed that IL-10 has a fundamental role in the protective effect of CoB. Bone marrow chimeras suggested this effect is partially dependent on IL-10 signaling in T cells. We then studied the impact of T cell exposure to TI-IFN on IL-10 signaling. Following in vitro exposure, T cells produced significantly less phospho-STAT3 in response to IL-10. This effect was very selective - IL-6 signaling remained unaltered - and resulted in the inhibition of specific IL-10 induced genes. Encouragingly, this effect was slowly reversible with removal of TI-IFN, suggesting a “druggable” pathway. This unresponsiveness was not associated with reduced IL-10-receptor expression or increased SOCS1_3 levels. Instead, microarray analysis of exposed cells suggested a novel role for STAT1 in dampening IL-10 signaling. Preliminary experiments with STAT1-KO cells supported this hypothesis.

Overall, these results highlight the importance of IL-10 in the efficacy of CoB regimens and reveal a new molecular mechanism whereby TI-IFNs can interfere with IL-10 signaling, and ultimately with the regulation of alloreactivity. A strategy to selectively target this mechanism could significantly enhance the clinical efficacy of CoB-based immunomodulatory strategies.
Inhibiting EZH2 function prevents and treats chronic graft vs host disease (cGVHD) by disrupting germinal center formation and plasma cell maturation
Authors: Zaiken Michael C , Flynn Ryan , Paz Katelyn , Rhee Stephanie , Park Paul , Hemming Matthew , Sage Peter , Sharpe Arlene , Luznik Leonard , Mailard Ivan , Hill Geoffrey R. , MacDonald Kelli , Serody Jonathon , Cutler Corey , Koreth John , Miklos David , Antin Joseph , Soiffer Robert , Ritz Jerome , Bradner Jay , Qi Jun , Blazar Bruce R.
Published: January 2023

To determine whether targeting EZH2, a histone methyltransferase necessary for germinal center (GC) formation, could reduce murine cGVHD (cGVHD), we first used a clinically relevant murine model of cGVHD, which recapitulates multi-organ cGVHD with bronchiolitis obliterans (BOS). Bone marrow (BM) from EZH2 fl/fl CD19 Cre B6 mice and WT T cells, or WT B6 BM and EZH2 fl/fl CD4 Cre B6 T cells were given to conditioned B10.BR mice. EZH2 expression in BM and T cells was obligatory for cGVHD. To determine whether EZH2 function could be targeted to treat cGVHD, we tested a novel EZH2 small molecule inhibitor, JQ5 with a favorable toxicity and efficacy profile to other inhibitors. Mice with established cGVHD given JQ5 had dramatically improved cGVHD. Similar results were seen in a sclerodermatous cGVHD model. To assess possible causes of this improvement we performed RNA-seq and ATAC-seq data of MACS purified CD19+ B cells obtained from control and JQ5 treated BOS cGVHD mice. Unsupervised gene set enrichment analysis and weighted gene correlation network analysis showed that among gene clusters associated with JQ5 treatment there was an enrichment for genes expressed in naïve B cells compared to plasma cells (GSEA4142) and an impairment of genes associated with late stage GC B cells compared to early follicular B cells (GSEA11961). This suggested that JQ5 impaired GC B cell and plasma cell maturation. Consistent with these data, splenic follicular cells, GC size and number, and lung resident PCs were reduced compared to cGVHD controls. Taken together these results show that EZH2 is necessary for the initiation of cGVHD and show for the first time that EZH2 inhibition using a novel inhibitor JQ5 can reverse established cGVHD in mice with BOS or scleroderma.
Donor Vγ4+T cells promote graft-versus-leukemia effect and ameliorate acute graftversus- host disease in allogeneic hematopoietic stem cell transplantation
Authors: Song Yuan , Zhu Ying , Liu Haiyan
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most effective ways of treating leukemia and other malignant diseases. However, disease recurrence and GVHD are still the major causes of death after allo-HSCT. Previous studies showed that immune cells involved in graft versus leukemia (GVL) effect traditionally include CD8+ T, CD4+ T and NK cells. Unfortunately, some of these T-cell subsets also cause graft-versus- host disease (GVHD). The role of donor γδT cells after transplantation is less well defined. We detected direct anti-leukemia cytotoxicity of γδT cells in vitro and the cytotoxicity of Vγ1 subset was stronger than the Vγ4 subset. To evaluate the role of donor γδT cells in aGVHD and GVL, we established the murine HLA fully mismatched allo-HSCT GVL/GVHD model. Our results showed that TCRδ−/− donor bone marrow cells caused enhanced disease progression and aggravated aGVHD compared with wildtype donors. Furthermore, flow cytometry analysis demonstrated γδT cells mediated anti-leukemia effects through promoting IFN-γ production by CD8+ T cells while suppressed aGVHD by inhibiting the activation of CD4+ T cells in liver, lung and IEL. The experiments of adoptive transfer of in vitro expanded γδT/Vγ1/Vγ4 cells and the in vivo depletion of Vγ1/Vγ4 cells indicated that the subset of donor γδT cells to alleviate murine aGVHD and enhance GVL effect might be Vγ4 γδT cells. The GVL effect of Vγ4 cells in vivo could be mediated by the production of IL-17A and regulation of CD8+ T cell function. These findings suggest donor γδT cells infusion could be a novel strategy to improve patients’ overall survival after allo-HSCT.
Bcl-2 Inhibition with Venetoclax Promotes Induction of Mixed Chimerism and Renal Allograft Tolerance Without Severe Myelosuppression in Non-human Primates
Authors: Kawai Tatsuo , Sasaki Hajime , Ma David , Dehnadi Abbas , Cosimi A Benedict , Cippa Pietro , Fehr Thomas
Published: January 2023

Background

Specific Bcl-2 inhibition has been shown to promote mixed chimerism and allograft tolerance without myelosuppressive conditioning in murine models. We extended this approach to nonhuman primates in combined kidney and bone marrow transplantation (CKBMT).

Methods

In Group A, recipients received CKBMT with the regimen including total body irradiation (TBI,3 Gy) thymic irradiation (TI, 7Gy) and ATG, followed by a short course of anti-CD154 antibody and cyclosporine. In Group B, TBI was reduced to 1.5Gy. In Group C, peri-transplant Bcl-2 inhibitor (Venetoclax) was added to Group B regimen. In Group D, Group C regimen without TBI. In Group E, Group C regimen without TI.

Results

7/8 recipients in Group A developed transient chimerism and achieved long-term renal allograft survival without immunosuppression. However, these recipients developed transient but severe pancytopenia between days 10–17 post CKBMT. Both recipients in Group B failed to develop chimerism. By adding Venetoclax to Group B regimen, 3/3 Group C recipients developed significantly higher and longer mixed chimerism without severe myelosuppression. These three recipients also achieved long-term immunosuppression free renal allograft survival without rejection (>316, >575, >239 days). In Group D, both recipients failed to develop chimerism and rejected their allografts on days 140 and 120. Although three Group E recipients successfully developed chimerism, all rejected their renal allografts on days 100, 97, 163 with early recovery of CD31+ naive T cells.

Conclusion

Enhancement of intrinsic apoptosis with Bcl-2 inhibition is a promising strategy to achieve robust mixed chimerism and allograft tolerance with reduced myelosuppressive conditioning.
Enhancing Antigen Delivery Using Spherical Nucleic Acidsfor Optimal Prostate Cancer Immunotherapy
Authors: Qin Lei , Wang Shuya , Dominguez Donye , Long Alan , Chen Siqi , Fan Jie , Ahn Ji Hae , Skakuj Kacper , Lee Andrew , Mirkin Chad , Zhang Bin
Published: January 2023

Prostate cancer ranks as the second leading cause of cancer-related death in the United States in men. For patients with metastatic or recurrent disease, cancer vaccines remain an attractive treatment approach while therapeutic efficacy is rather limited. Here, we developed Spherical Nucleic Acid nanostructures (SNAs) to deliver adjuvant CpG and prostate cancer peptide antigens to potentiate antitumor responses. We show that SNAs that differ in the positioning of the antigen within the nanostructure lead to differences in the quality of immune responses generated by vaccination with SNAs. SNAs conjugated with both antigen and adjuvant CpG on the surface (HM SNAs)induce stronger cytotoxic T lymphocyte (CTL) mediated antigen-specific killing of target cells than that for SNAs with CpG on the surface and antigen encapsulated within the core (EM SNAs). Mechanistically, HM SNAs increase co-delivery of CpG and antigen, thereby promoting dendritic cells-mediated cross-priming compared to EM SNAs, or admix of free peptide and linear CpG. Furthermore, vaccination with HM SNAs led to significantly inhibits tumor growth in two syngeneic mouse models of prostate cancer. These data suggest that the positioning of peptide antigens within SNA structures can be exploited to enhance the therapeutic performance of SNAs, representing a novel approach to vaccine design for cancer immunotherapy and potentially other diseases that can be addressed through modulating the immune system.
Assessing the impact of neoantigen load on checkpoint blockade efficacy
Authors: Yalcin Zeynep Kosaloglu , Frentzen Angela , Premlal Ashmitaa Logandha Ramamoorthy , Sidney John , Greenbaum Jason A , Sette Alessandro , Peters Bjoern
Published: January 2023

Neoantigens were suggested to be crucial for the outcome of immune checkpoint blockade (ICB) therapies and accordingly, the neoantigen load of a tumor is often being used to predict therapy outcome. Current approaches utilize HLA binding predictions to rank peptides, and the neoantigen load of a given tumor is then usually defined as the number of peptides binding above a certain threshold. Using this approach some reports found a strong correlation of neoantigen load and response to ICB while others found a better correlation when mutational load was used instead. However, comparing results between studies is hard because they used different tools and thresholds to identify mutations. We sought to overcome this lack of uniformity in re-analyzing data from several published studies to consistently define the mutational burden and neoantigen load, and correlate them with therapy outcomes.

We obtained sequencing data from several ICB studies and run all datasets through our in-house mapping and variant calling pipelines. We applied different scoring schemes to define the neoantigen load which were based on HLA binding predictions with varying thresholds and different metrics used. We found that the exact definition of neoantigen load has a major impact on how well this metric performs in predicting checkpoint blockade therapy outcome. We also implemented a likelihood score which is based on data of known immunogenic neoantigens and describes the likelihood of a predicted neoantigen to be immunogenic. Based on our preliminary analysis to date, we found that the cumulative likelihood score of a patient is a good predictor of ICB outcome, outperforming tumor mutational burden and predicted neoantigen load in several cohorts.
Adjuvant screen identifies DC modifying growth factor Flt3 ligand as candidate for prostate cancer DNA vaccine
Authors: Malo Kirsten , Wong Ashley J. , Thorne Amy H. , Humeau Laurent , Masteller Emma
Published: January 2023

Prostate cancer is the most common type of cancer in men. Immunotherapies such as Sipuleucel-T have shown that stimulating the immune system to target the prostate is a viable therapeutic option. Inovio is a clinical-stage biotechnology company that uses DNA vaccines as a novel immunotherapy strategy. An advantage to plasmid DNA vaccine therapy is the ability to encode adjuvants within the vaccine in order to increase immunogenicity and efficacy.

We conducted an adjuvant screen in BALB/c mice with 25 candidate plasmid encoded genetic adjuvants in combination with the prostate cancer specific tumor-associated antigen STEAP1. Antigen-specific T cell responses were measured by IFNg ELISpot. The screen revealed that the addition of a plasmid encoding dendritic cell (DC)-activating Fms-like tyrosine kinase 3 ligand (Flt3L) fused to an Fc domain, significantly increased antigen-specific T cells (p<0.001, 2.8 fold). Mice treated with Flt3L-Fc had an enhanced immune response to STEAP1 vaccination as early as 7 days post dose 1 (p<0.01, 2.4 fold), which developed into an enhanced memory response measured 12 weeks later (p<0.001, 8.5 fold). Flow cytometry showed that Flt3L-Fc increased DC populations at the site of injection and at the draining lymph node 8 days following the initial vaccination. DCs are considered the most potent antigen-presenting cells in the immune system and DCs at the tumor site have been shown to be critical for T cell immunity. Our data shows that enhancing DC populations and function through the use of a genetic adjuvant can enhance the immunogenicity of a DNA cancer vaccine. Future studies will assess the efficacy of Flt3L-Fc in combination with STEAP1 in a mouse tumor model.
Regional pressure-enabled drug delivery of anti-PD-1 agent for colorectal liver metastases improves anti-tumor activity without increased hepatic toxicity
Authors: Chai Louis F. , Hardaway John C. , Lopes Mikayla C. , Rabinowitz Benjamin A. , Guha Prajna , Jaroch David , Cox Bryan , Espat N. Joseph , Katz Steven C.
Published: January 2023

Introduction

Checkpoint inhibitors (CI) have greatly impacted the treatment of solid tumors. Success in the liver has been more limited. When intravenously infused, the CI is delivered predominantly to extrahepatic sites, which results in systemic side effects. Previously, we reported on regional delivery of cellular immunotherapy for liver tumors using pressure-enabled drug delivery (PEDD™, TriSalus™ Life Sciences, Inc.). We hypothesized that PEDD of CI would improve the therapeutic index (TI).

Methods

In a murine model of colorectal liver metastases (CRLM), we infused high (HP) or low pressure (LP) anti-PD-1 antibodies (RMP1-14) via the portal vein and compared to systemic tail vein delivery (SD) at 0, 0.3, 1.0 and 3.0 mg/kg. Tumor bioluminescence (TB), histopathology, and serologic analysis informed treatment effects and toxicity.

Results

At a dose of 3mg/kg, LP resulted in similar IVIS TB growth kinetics compared to SD 3 mg/kg at 7 days post-treatment (geometric mean, p = 0.94). In animals treated with a 10-fold lower dose (0.3 mg/kg), tumor growth was significantly lower with HP vs. LP (7.5-fold improvement, p = 0.04). Circulating serum anti-PD-1 levels were similar when we compared SD and LP at 1 and 3 mg/kg (Mean 2512.0 vs. 2250.2 ng/mL, p = 0.25). Comparatively, systemic exposure was significantly lower at 0.3 mg/kg LP (Mean 508.1 ng/mL, p < 0.0001). No increases in hepatotoxicity were seen with PEDD (AST p = 0.57, ALT p = 0.43).

Conclusion

PEDD of anti-PD-1 CI improved liver tumor control while also limiting systemic exposure. These findings support PEDD for anti-PD-1 therapy to improve TI. Phase 1 exploration of anti-PD-1 PEDD for patients with hepatic malignancy is thus warranted.
Interleukin-15 in cancer immunotherapy: IL-15 receptor complex versus soluble IL-15 in a cancer cell-delivered murine leukemia model
Authors: Berger Alexandra , Colpitts Sarah , Seabrook Melanie , Furlonger Caren , Bendix Maura , Moreau Joshua , McKillop William , Medin Jeffrey , Paige Christopher J
Published: January 2023

Cancer immunotherapy endeavors to stimulate the immune system in order to recognize, reject and destroy tumor cells. Previously we have demonstrated that IL-12 is an ideal candidate for leukemia immunotherapy and there is a Phase 1 human trial underway at the Princess Margaret Cancer Center to test this approach in AML patients. In order to further our knowledge on immunomodulators we have expanded our studies to other cytokines such as IL-15. IL-15, either in its soluble form (IL-15sol) or complexed with IL-15Ra (IL-15Rc), has been shown to exhibit potent anti-tumor activities in various cancer studies.

In this study we describe the impact of intraperitoneal IL-15 in a cancer cell delivered IL-15 immunotherapy approach using the 70Z/3-L leukemia mouse model. Whereas both forms of IL-15 led to significantly improved survival rates compared to the parent cell line, there were striking differences in the extent of the improved survival: mice receiving cancer cells secreting IL-15sol showed near 100% survival for up to 250 days, whereas mice receiving cancer cells secreting IL-15Rc started to perish around day 50 post-injection, with few survivors left on day 250.

Interestingly, cell-secreted IL-15sol and IL-15Rc activated different cell types in the peritoneum, leading to different immune responses. IL-15Rc resulted in an influx and/or expansion of NK1.1+ natural killer cells in the peritoneum, while injection of cells secreting IL-15sol lead to an increased expansion of CD4+ and CD8+ T-cells. To our knowledge, this is the first study detailing a significantly different biological effect of cell-delivered IL-15Rc vs IL-15sol in a mouse cancer immunotherapy study, suggesting a different mechanism of action of the two IL-15 forms.
Lenolidamide and IL-15 enhance natural killer cell-mediated immunity and augments the anti-tumor effect of an anti-CD30/CD16A bispecific antibody against Hodgkin’s lymphoma B-cells
Authors: Manna Alak , Aulakh Sonikpreet , Ailawadhi Sikander , Chanan-Khan Asher , Sher Taimur , Paulus Aneel
Published: January 2023

Hodgkin’s lymphoma (HL) is characterized by malignant CD30+ Reed-Sternberg (HRS) B-cells surrounded by an ineffective infiltrate of inflammatory immune cells. T-helper and T-regulatory cells presumably provide survival signals for HRS cells; rescuing them from immune-surveillance by CD8+ cytotoxic T lymphocytes and natural killer (NK) cells, which are also functionally anergic. Clinically, ≈10–30% of HL patients resulted in relapse after front line therapy; highlighting the need for development of new treatments. We investigated a novel bispecific CD16axCD30 antibody, AFM13 (Affimed Therapeutics, AG), which engages NK cells and recruits them towards CD30+ tumor cells. Safety of single-agent AFM13 has been demonstrated in a phase I clinical study, however, disease remission was observed in only 23% of patients. We hypothesized that combining AFM13 with another agent whose safety profile is already been established in HL and also activates NK cells (lenalidomide) could potentially increase anti-tumor efficacy of AFM13. We observed increased expression of CD107a, CD69, CD25 and IFNγ (activation markers) in NK cells isolated from HL patients (HL-NK), that were stimulated ex vivo with IL- 15 + lenalidomide. Pre-treatment with IL-2/IL-15 antibody has been shown to activate and restore T/NK cell cytolytic function. In line with this, IL-15+lenalidomide-stimulated HL-NK cells showed functional restoration by inducing specific lysis of human CD30+ lymphoma cells (30.55±1.49%) in a co-culture assay. While, AFM13 is being investigated in combination with anti-PD1 antibody therapy (NCT02665650), our preclinical data provides the rationale for an AFM13 + Len combination regimen for HL and potentially other CD30+ malignancies.
Towards identification of a lipid metabolic checkpoint for immuno-oncology
Authors: Hsu Ku-Lung , Borne Adam L. , Brulet Jeffrey W. , Campbell Sean T. , Franks Caroline E. , Huang Tao , McCloud Rebecca L. , Shin Myungsun , Ware Timothy B.
Published: January 2023

Lipid phosphorylation is an understudied regulatory mechanism for T cell metabolism and signaling. Specifically, diacylglycerol kinases (DGKs) modulate intracellular levels of the secondary messengers diacylglycerol and phosphatidic acid, which are implicated in regulation of T cell activation and anergy. Development of isoform-selective DGK inhibitors is challenging but needed to understand specificity of DGK biology in vivo. Towards this goal, we use ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, the DGK ATP-binding site (DAGKc/DAGKa subdomains) and propose a potential role for C1 domains in DGK catalytic function. We discovered fragments from old drugs (ritanserin) that reveal pharmacophores required for selective binding to DGK active sites that are not in the canonical ATP binding pocket. We provide evidence of a single contiguous inhibitor binding site composed of DAGKc, DAGKa, and C1 sites by demonstrating equipotent inhibition using a fragment inhibitor. Our current working model is that C1 domains of DGKs are involved in substrate and inhibitor recognition of type 1, 4, and 5 DGKs and is a site for pursuing development of isoform-selective inhibitors. We describe new lead compounds that will be optimized into the first selective and in vivoactive DGK-alpha inhibitor with potential applications for immuno-oncology. We also describe our efforts to use mass spectrometry-metabolomics to annotate substrate specificity of all 10 mammalian DGKs in live cells to identify biomarkers for future applications in studying T cell biology.
Recombinant human IL-15 (rhIL-15) in combination with anti-PD-L1 (Avelumab) in SIV infected rhesus macaques leads to the expansion of a subset of CXCR3+PD1−/low CD8 T cells
Authors: MOUSSA Maha , Cheng Jie , Chen Ping , Chen Hui , Li Tong , Qin Jing , Lifson Jeffrey D. , Sneller Michael C. , Krymskaya Ludmila , Godin Steven , Lane Clifford , Catalfamo Marta
Published: January 2023

The PD1/PD-L1 pathway contributes to the pathogenesis of HIV/SIV infection. Expression of PD1 by HIV/SIV-specific CD8 T cells can limit antiviral responses and expression of checkpoint receptors including PD1 by HIV infected cells can contribute to maintenance of viral reservoirs. In addition, reports have shown that IL-15 induces the expansion of effector memory CD8 T and NK cells. In this study, we hypothesized that expansion of memory CD8 T cells in the context of PD1/PD-L1 blockade may lead to enhanced anti-SIV responses. We design a study in which we evaluated the effect of rhIL-15 administered in cycles of ten days by continuous infusion in combination with weekly administration of anti-PD-L1 (Avelumab) over 24 weeks. After seven doses of anti-PD-L1 and completion of two cycles of rhIL15, cART therapy was interrupted and anti-PD-L1 administration continued to complete 24 weeks treatment. We found that administration of rhIL-15 in combination with anti-PD-L1 treatment was well tolerated. rhIL-15 infusions were associated with transient increases in proliferation of NK cells and effector memory CD8 T cells. rhIL-15/anti-PD-L1 treatment led to a sustained expression of CXCR3+ with low to undetectable expression of PD1on CD8 T cells, suggesting potential of increased tissue trafficking. Multidimensional single cell analysis showed that CXCR3+PD1−/low CD8 T cells secrete cytokine in response to viral antigens. Despite these clear biological effects, no impact was observed on rebound plasma viremia after cART discontinuation and during anti-PD-L1 administration. Futures studies should characterize the CXCR3+PD1−/low CD8 T cell subset expanded by this treatment to understand the potential impact of this subset in viral control.
A replicating Adenovirus-SIV recombinant prime/protein boost regimen induces SIVspecific T and B cell responses in lymph nodes associated with humoral immunity in the genital mucosa of female Rhesus macaques
Authors: Hait Sabrina Helmold , Hogge Christopher James , Mushtaq Zuena , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

The efficacy of HIV vaccines relies on potent antibody (Ab) responses at major transmission sites such as the female genital tract (FGT). CD4+ T follicular helper (TFH) cells, specialized in providing B cell help in germinal centers (GCs) of secondary lymphoid organs, play an important role in initial stages of HIV-specific Ab responses. Here we investigated SIV-specific T and B cell responses and FGT immunity in Rhesus macaques following mucosal priming with Ad5hr-SIV(Env/gag/nef) vectors at wks 0 & 13 and intramuscular boosting with SIV gp120 in alum at wks 26 & 38. Lymph nodes (LNs) were collected pre-vaccination and 3–4 days post-2nd prime and post-2nd boost. Vaginal washes were obtained prior to and 3 wks post-immunizations. GC TFH and B cell frequencies positively correlated post-2nd prime (p=0.04; r=0.6) suggesting an effect of early TFH cells in GCs. GC SIV-specific TFH cells were significantly enhanced post-2nd boost (p=0.01). BCL6 upregulation was also seen in GC-TFH cells at this time, confirming the GC phenotype of TFH cells. GC SIV-specific memory B cell levels were higher post-2nd boost compared to post-prime (p=0.03) indicating efficient SIV-specific B cell generation. GC B cells post-2nd prime and boost strongly associated with SIV-specific IgG levels in vaginal washes (p=0.03, r=0.7; p=0.002, r=0.8). GC centrocytes are critical for long-lasting Ab secreting cells. GC centrocytes post 2nd prime and boost tended to correlate with vaginal wash SIV-specific IgG levels (p=0.06, p=0.05; r=0.6 for both). Overall, our vaccine regimen elicited GC T and B cell responses that supported SIV-specific humoral immunity in the FGT suggesting further development of replicating mucosal Ad-vaccines is warranted for females as well as males.
Weaning and postnatal age influence the early time course and nature of intestinal mast cell activation and mucosal inflammation in a porcine model of early life adversity
Authors: Wilson Neco , Li Yihang , Rajput Mrigendra , Thelen Kyan , Kerr Katie , Moeser Adam J.
Published: January 2023

Early life adversity (ELA) is a risk factor for later life emergence of functional and inflammatory GI disorders in people and animals. In this study, we compared the early GI immune responses in male piglets exposed to early weaning (EW) at 16–18 d of age, a form of ELA we’ve previously shown result in altered GI developmental and health trajectories similar to ELA in humans, with that of late weaned piglets (LW: 28 d of wean age). RNA transcriptome analysis of ileal mucosa at 24h post-weaning revealed that EW pigs exhibited a greater number of differentially expressed genes (765 vs 110 genes, for EW and LW piglets, respectively), characterized by a large number of upregulated genes associated with immune cell trafficking and inflammation. Mast cells (MC), stress-sensitive innate immune cells which orchestrate the immune response, were acutely activated in EW and LW pigs but in a differential manner. Compared with LW piglets, EW piglets exhibited higher levels of serum histamine which coincided with a downregulation in the gene expression of histamine degrading enzymes, DAO and HNMT in intestinal mucosa. Mast cell tryptase (MCT7) and chymase (CMA1) gene expression were upregulated in ileal and colonic mucosa (within 3h post-weaning) in both EW and LW piglets; however, this response was greater in LW pigs. Together, these data show that weaning stress in piglets induces rapid GI MC activation which precedes immune cell recruitment and intestinal inflammation and that postnatal age has a significant influence on the level and nature of this response. Further investigation of how EW and LW piglets differentially regulate early GI immune responses is expected to provide new insight into the mechanisms of GI and immune disorders associated with ELA.
Characterization of bovine γδ T cells phenotype following Mycobacterium bovis vaccination or virulent infection
Authors: Guerra-Maupome Mariana , Palmer Mitchell V , Waters Ray , McGill Jodi L.
Published: January 2023

Bovine tuberculosis caused by Mycobacterium bovis is a significant veterinary health problem. γδ T cells participate in the control of mycobacterial infections. Accumulating data suggest that mycobacterial infection regulates memory/effector phenotype and immune functions of mycobacterium-responsive γδ T cells. To date, the impact of both virulent M. bovis infection and Bacille Calmette-Guerin (BCG) vaccination on bovine γδ T cells memory/effector phenotype remains unknown. In this study, we addressed the functional and phenotypical differences in memory/activation marker, and chemokine receptor expression of M. bovis-specific γδ T cells following BCG vaccination or virulent M. bovis infection. Analysis of γδ T cells in blood, bronchoalveolar lavage fluid, and regional lymph nodes indicated that γδ T cells have phenotypic differences based on the expression of CD27 that suggest distinct functional properties. Here, we show that most peripheral γδ T cells display a CD27+ phenotype. Antigen recall responses showed that expression of CD27 correlates with the expansion of M. bovis-specific γδ T-cells. Thus, the CD27+ subset may comprise the adaptive γδ T cell compartment responding to virulent M. bovis infection and vaccination. Furthermore, we show that γδ T cells from neonatal calves are activated by aerosol BCG vaccination and adopt a lung-homing phenotype that is similar to that observed during virulent M. bovis infection, suggesting an important role for this T cell subset in the response to mucosal vaccination. The ability of γδ T cells to mount a robust local immune response supports the hypothesis that vaccine-elicited γδ T cell immunity might prove beneficial.