Chromosomally-Encoded Yersinia pestis Type III Secretion Effector Proteins Promote Infection in Cells and in Mice

Yersinia pestis, the causative agent of plague, possesses a number of virulence mechanisms that allows it to survive and proliferate during its interaction with the host. To discover additional infection-specific Y. pestis factors, a transposon site hybridization (TraSH)-based genome-wide screen was employed to identify genomic regions required for its survival during cellular infection. In addition to several well-characterized infection-specific genes, this screen identified three chromosomal genes (y3397, y3399, and y3400), located in an apparent operon, that promoted successful infection. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. These genes were designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB), and C (ylrC). Engineered strains with deletions of y3397 (ylrC), y3399 (ylrB), or y3400 (ylrA), exhibited infection defects both in cultured cells and in the mouse. C-terminal FLAG-tagged YlrA, YlrB, and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium and deletions of the DNA sequences encoding the predicted N-terminal type III secretion signals of YlrA, YlrB, and YlrC prevented their secretion, indicating that these proteins are substrates of the type III secretion system (T3SS). Further strengthening the connection with the T3SS, YlrB was readily translocated into HeLa cells and expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins may function as anti-host T3S effector proteins

Enhancing Antitumor Immunity by Selectively Targeting the High-Affinity IL-2 Receptor on Neoantigen-Specific T Cells

Tumor-specific T cells are central mediators of the antitumor response&nbsp;but their low frequency is one major reason for failed immunotherapy. IL-2 is an agent that boosts&nbsp;tumor-reactive T cells but its benefit is limited due to its short half life which necessitates high dosing that contributes to&nbsp;toxicity and potential off-target effects. We developed a mouse (m)IL-2/CD25 fusion protein which exhibits more favorable pharmacokinetics and pharmacodynamics when compared to IL-2. A single administration of high dose mIL-2/CD25 following vaccination with&nbsp;cognate peptide and LPS amplified tumor-specific Pmel-1 CD8+&nbsp;and TRP-1 CD4+&nbsp;Teff cells and Pmel-1 memory cells while only transiently increasing Tregs. This approach effectively controlled pre-established&nbsp;B16.F10 melanoma in vivo. This scheme was modified to amplify endogenous polyclonal melanoma-reactive T cells, which is translationally more relevant. Development of tumor-specific T cells required priming and boosting with a mixture of B16.F10 neoantigens and poly (I:C) followed by application of IL-2/CD25 only after the boost. This resulted in largely a CD4+&nbsp;anti-neoantigen specific T cell response. This immunization scheme delayed tumor growth in&nbsp;mice with pre-established B16.F10 tumors. The immune response within the tumor microenvironment was accompanied by increased frequencies of granzyme B-expressing CD4+&nbsp;and CD8+&nbsp;T cells. Together, our data show that limited application of high-dose IL-2/CD25 in the context of neoantigen vaccines promotes effective antitumor immunity and&nbsp;has potential clinical translation.</p

Abstract 1725: Short-term administration of high dose mIL-2/CD25 fusion protein elicits CD8+ T cell-driven anti-tumor immunity and memory

Abstract Recombinant IL-2 (rIL-2) promotes the differentiation, proliferation, and survival of antigen-activated T cells and induces potent anti-tumor activity. High dose (HD) rIL-2 broadly targets T effector (Teff) and NK cells. However, rIL-2 cancer therapy requires long-term infusions of high doses due to its short half-life and is associated with severe toxicities which limit efficacy. To circumvent these limitations, our lab has developed and is testing a new IL-2-based biologic, mIL-2/CD25, which can be used at high doses to promote anti-tumor immunity. mIL-2/CD25 is a fusion protein (FP) composed of mouse IL-2 fused to the mouse CD25 extracellular domain via a non-cleavable glycine-serine linker with selectivity toward the high affinity IL-2R. This FP has a much longer half-life (16 hr) than rIL-2 (15 min). At a low dose, mIL-2/CD25 selectively expands Tregs, but at a HD, Teff cells are also engaged. These properties allow for HD FP to readily expand activated endogenous tumor-reactive Teff cells, but not memory-phenotypic CD8+ T cells and NK cells. In the current study, we tested the extent short-term HD FP therapy activated tumor-reactive Teff cells to elicit efficacious anti-tumor immunity responses despite Treg stimulation. HD FP prolonged survival in mice bearing B16.F10 melanoma, MC-38 colon adenocarcinoma, or CT26 colon carcinoma. In the CT26 model, a significant increase in both the number and the frequency of CD8+ T cells was detected in the tumor microenvironment (TME) of treated mice. FP-dependent Treg expansion was observed in secondary lymphoid tissues but not the TME. This therapeutic approach, therefore, led to a higher CD8+ Teff to Treg ratio specifically in the TME. After FP treatment, CD8+ T cells isolated from the TME expressed lower levels of TOX, PD-1, and LAG-3, suggesting that they were less exhausted. T cell depletion studies using anti-CD8 revealed that CD8+ T cells are the major driver of the FP-induced anti-tumor response. Rechallenge of animals that had previously cleared CT26 due to FP treatment led to successful tumor rejection, showing that the FP supported anti-tumor memory. These findings demonstrate that mIL-2/CD25 amplifies recently activated tumor-reactive CD8+ Teff cells to generate efficacious anti-tumor responses that limit Treg-dependent negative regulation in the TME and lead to anti-tumor memory. Citation Format: Kathryn LaPorte, Rosmely Hernandez, Thomas Malek. Short-term administration of high dose mIL-2/CD25 fusion protein elicits CD8+ T cell-driven anti-tumor immunity and memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1725

Fueling Cancer Vaccines to Improve T Cell-Mediated Antitumor Immunity

Cancer vaccines offer the potential to enhance T cell-mediated antitumor immunity by expanding and increasing the function of tumor-specific T cells and shaping the recall response against recurring tumors. While the use of cancer vaccines is not a new immunotherapeutic approach, the cancer vaccine field continues to evolve as new antigen types emerge and vaccine formulations and delivery strategies are developed. As monotherapies, cancer vaccines have not been very efficacious in part due to pre-existing peripheral- and tumor-mediated tolerance mechanisms that limit T cell function. Over the years, various agents including Toll-like receptor agonists, cytokines, and checkpoint inhibitors have been employed as vaccine adjuvants and immune modulators to increase antigen-mediated activation, expansion, memory formation, and T effector cell function. A renewed interest in this approach has emerged as better neoepitope discovery tools are being developed and our understanding of what constitutes an effective cancer vaccine is improved. In the coming years, cancer vaccines will likely be vital to enhance the response to current immunotherapies. In this review, we discuss the various types of therapeutic cancer vaccines, including types of antigens and approaches used to enhance cancer vaccine responses such as TLR agonists, recombinant interleukin-2 and interleukin-2 derivatives, and checkpoint inhibitors

Engineering IL-2 for immunotherapy of autoimmunity and cancer

Preclinical studies of the T cell growth factor activity of IL-2 resulted in this cytokine becoming the first immunotherapy to be approved nearly 30 years ago by the US Food and Drug Administration for the treatment of cancer. Since then, we have learnt the important role of IL-2 in regulating tolerance through regulatory T cells (T cells) besides promoting immunity through its action on effector T cells and memory T cells. Another pivotal event in the history of IL-2 research was solving the crystal structure of IL-2 bound to its tripartite receptor, which spurred the development of cell type-selective engineered IL-2 products. These new IL-2 analogues target T cells to counteract the dysregulated immune system in the context of autoimmunity and inflammatory disorders or target effector T cells, memory T cells and natural killer cells to enhance their antitumour responses. IL-2 biologics have proven to be effective in preclinical studies and clinical assessment of some is now underway. These studies will soon reveal whether engineered IL-2 biologics are truly capable of harnessing the IL-2-IL-2 receptor pathway as effective monotherapies or combination therapies for autoimmunity and cancer

Robust IL-2-dependent antitumor immunotherapy requires targeting the high-affinity IL-2R on tumor-specific CD8+ T cells

Background Development of interleukin (IL)-2-dependent antitumor responses focus on targeting the intermediate affinity IL-2R to stimulate memory-phenotypic CD8+ T and natural killer (NK) cells while minimizing regulatory T cell (Treg) expansion. However, this approach may not effectively engage tumor-specific T effector cells. Since tumor-antigen specific T cells upregulate the high-affinity IL-2R, we tested an IL-2 biologic, mouse IL-2/CD25, with selectivity toward the high-affinity IL-2R to support antitumor responses to tumors that vary in their immunogenicity.Methods Mice were first implanted with either CT26, MC38, B16.F10, or 4T1 and after a tumor mass developed, they were treated with high-dose (HD) mouse (m)IL-2/CD25 alone or in combination with anti-programmed cell death protein-1 (PD-1) checkpoint blockade. Tumor growth was monitored and in parallel the immune signature in the tumor microenvironment (TME) was determined by a combination of multiparameter flow cytometry, functional assays, and enumeration of tumor-reactive T cells.Results We show that HD mIL-2/CD25, which preferentially stimulates the high-affinity IL-2R, but not IL-2/anti-IL-2 complexes with preferential activity toward the intermediate-affinity IL-2R, supports vigorous antitumor responses to immunogenic tumors as a monotherapy that were enhanced when combined with anti-PD-1. Treatment of CT26-bearing mice with HD mIL-2/CD25 led to a high CD8+:Treg ratio in the TME, increased frequency and function of tumor-specific CD8+ T effector cells with a less exhausted phenotype, and antitumor memory responses.Conclusions Targeting the high-affinity IL-2R on tumor-specific T cells with HD mIL-2/CD25 alone or with PD-1 blockade supports antitumor responses, where the resulting memory response may afford long-term protection against tumor re-emergence

High-dose IL-2/CD25 fusion protein amplifies vaccine-induced CD4+ and CD8+ neoantigen-specific T cells to promote antitumor immunity

BackgroundImmunization with tumor neoantigens is a promising vaccine approach to promote antitumor immunity due to their high immunogenicity, lack of expression in normal tissue, and preferential induction of tumor neoantigen-specific T cells, which are central mediators of the anti-cancer response. A drawback to targeting tumor neoantigen-specific T cells is that these cells are found at a low frequency in patients with cancer, limiting their therapeutic benefit. Interleukin-2 (IL-2) promotes expansion and persistence of tumor-reactive T cells. However, its clinical use has been hampered by toxicities arising from its multiple cellular targets. Thus, new engineered IL-2 receptor (IL-2R) agonists with distinctive cell type selectivity have been designed to harness the potential of IL-2 for tumor immunotherapy.MethodsWe investigated the potential to amplify neoantigen-specific CD4+ and CD8+ T cell immune responses to promote antitumor immunity through vaccination with tumor neoantigens. Following T cell receptor (TCR)-mediated induction of the high-affinity IL-2R on these T cells, amplification of the neoantigen-specific T cell response was achieved using a high dose of the mouse IL-2/CD25 (mIL-2/CD25) fusion protein, an IL-2R agonist with more favorable pharmacokinetics and pharmacodynamics than IL-2 and selectivity toward the high-affinity IL-2R.ResultsAdministration of a high dose of mIL-2/CD25 shortly after antigen-dependent induction of the high-affinity IL-2R amplified the numbers and function of TCR transgenic tumor-reactive tyrosinase-related protein-1 (TRP-1) CD4+ T cells, leading to antitumor immunity to B16-F10 melanoma. This approach was adapted to amplify endogenous polyclonal B16-F10 neoantigen-specific T cells. Maximal expansion of these cells required prime/boost neoantigen vaccinations, where mIL-2/CD25 was optimal when administered only after the boosting steps. The ensuing mIL-2/CD25-driven immune response supported antitumor immunity to B16-F10 and was more effective than treatment with a similar amount of IL-2. Optimal antitumor effects required amplification of CD4+ and CD8+ neoantigen-specific T cells. High-dose mIL-2/CD25 supported a tumor microenvironment with higher numbers of CD4+ and CD8+ T effectors cells with increased granzyme B expression and importantly a more robust expansion of neoantigen-specific T cells.ConclusionThese results indicate that neoantigen-based vaccines are optimized by potentiating IL-2R signaling in CD4+ and CD8+ neoantigen-reactive T cells by using high-dose mIL-2/CD25, leading to more effective tumor clearance

Sustained IL-2R signaling of limited duration by high-dose mIL-2/mCD25 fusion protein amplifies tumor-reactive CD8.sup.+ T cells to enhance antitumor immunity

Keywords: IL-2; Tumor vaccine; Melanoma; T effector cells; CD8.sup.+ T cell memory High-dose IL-2 induces cancer regression but its therapeutic use is limited due to high toxicities resulting from its broad cell targeting. In one strategy to overcome this limitation, IL-2 has been modified to selectively target the intermediate affinity IL-2R that broadly activates memory-phenotypic CD8.sup.+ T and NK cells, while minimizing Treg-associated tolerance. In this study, we modeled an alternative strategy to amplify tumor antigen-specific TCR transgenic CD8.sup.+ T cells through limited application of a long-acting IL-2 fusion protein, mIL-2/mCD25, which selectively targets the high-affinity IL-2R. Here, mice were vaccinated with a tumor antigen and high-dose mIL-2/mCD25 was applied to coincide with the induction of the high affinity IL-2R on tumor-specific T cells. A single high dose of mIL-2/mCD25, but not an equivalent amount of IL-2, amplified the frequency and function of tumor-reactive CD8.sup.+ T effector (Teff) and memory cells. These mIL-2/mCD25-dependent effects relied on distinctive requirements for TLR signals during priming of CD8.sup.+ tumor-specific T cells. The mIL-2/mCD25-amplified tumor-reactive effector and memory T cells supported long-lasting antitumor responses to B16-F10 melanoma. This regimen only transiently increased Tregs, yielding a favorable Teff--Treg ratio within the tumor microenvironment. Notably, mIL-2/mCD25 did not increase non-tumor-specific Teff or NK cells within tumors, further substantiating the specificity of mIL-2/mCD25 for tumor antigen-activated T cells. Thus, the selectivity and persistence of mIL-2/mCD25 in conjunction with a tumor vaccine supports antitumor immunity through a mechanism that is distinct from recombinant IL-2 or IL-2-based biologics that target the intermediate affinity IL-2R. Author Affiliation: (1) Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, 33136, Miami, FL, USA (f) [email protected] Article History: Registration Date: 09/09/2020 Received Date: 06/01/2020 Accepted Date: 09/09/2020 Online Date: 10/10/2020 Byline:Academi

Acute Lipopolysaccharide-Induced Inflammation Lowers IL-2R Signaling and the Proliferative Potential of Regulatory T Cells

IL-2R signaling is essential for the development and homeostasis of CD4 Foxp3 regulatory T cells (Tregs). Low-dose IL-2 is being advanced as a therapy for autoimmune diseases because of its ability to expand Tregs. Although Treg stability and function is diminished by chronic inflammation, the impact of inflammation on proximal IL-2R signaling and/or responsiveness to low-dose IL-2 is poorly understood. In this study, we show that acute inflammation induced by LPS, analogous to responses to acute bacterial infection, led to decreased endogenous STAT5 signaling and proliferative potential as measured by Ki67 in mouse Tregs. This impaired Treg activity was transient, did not lead to a reduction in Treg numbers or function, and was due to TLR signaling by non-Tregs. Although acute LPS induced high levels of IL-1 and IL-6, these cytokines did not solely mediate dysregulated Treg activity. Global gene expression analyses demonstrated that acute LPS-induced inflammation substantially and rapidly altered the Treg transcriptome. In the presence of an IL-2R agonist, the mouse IL-2/CD25 fusion protein (mIL-2/CD25), this type of inflammatory response tempered the transcription of IL-2R-dependent genes in vivo. Gene enrichment and pathway analyses are consistent with LPS attenuating mIL-2/CD25-dependent genes related to the cell cycle, DNA replication, and cholesterol biosynthesis while enhancing mRNAs that mediated Treg suppression in vivo. Acute LPS-induced inflammation diminished some responses by Tregs to mIL-2/CD25 treatment in vivo. Together, these results suggest a role for persistent IL-2R signaling in mitigating some but not all of the deleterious effects of inflammation on Treg proliferation while supporting their function

Perforin-2 limits pathogen proliferation at the maternal-fetal interface

Placental immune responses are highly regulated to strike a balance between protection and tolerance. For relatively mild infections, protection encompasses both the mother and fetus; however, during worsening conditions, protection becomes exclusively reserved for the mother. Previously, we and others have shown that the host factor Perforin-2 plays a central role in protecting mice and cells against infection. Here, we analyzed Perforin-2 activity in the mouse placenta to determine whether Perforin-2 plays a similarly protective role. We show that Perforin-2 is critical for inhibiting Listeria monocytogenes colonization of the placenta and fetus and that this protection is due to both maternal and fetal-encoded Perforin-2. Perforin-2 mRNA is readily detectable in individual immune cells of the decidua and these levels are further enhanced specifically in decidual macrophages during high-dose infections that result in fetal expulsion. Unexpectedly, inductive Perforin-2 expression in decidual macrophages did not occur during milder infections in which fetal viability remained intact. This pattern of expression significantly differed from that observed in splenic macrophages in which inductive Perforin-2 expression was observed in both high and mild infection conditions. In the placenta, inductive Perforin-2 expression in decidual macrophages was co-incident with their polarization from a M2 to M1 phenotype that normally occurs in the placenta during high-burden infections. Our results suggest that Perforin-2 is part of a host response that is protective either for both the mother and fetus in milder infections or exclusively for the mother during high-dose infections. Competing Interest Statement The authors have declared no competing interest