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Background: TIGIT is a co-inhibitory receptor and immune checkpoint associated with T cell and natural killer (NK) cell dysfunction in cancer. Tiragolumab is an anti-TIGIT antibody with an active IgG1/kappa Fc. In a randomized double-blind phase 2 clinical trial in non-small cell lung cancer (NSCLC), tiragolumab + atezolizumab (anti-PD-L1) combination treatment demonstrated significant improvement relative to atezolizumab alone. However, the mechanisms underlying the efficacy of this combination are not well understood. Methods: In CITYSCAPE (phase 2, NCT03563716), chemotherapy-naïve patients with locally advanced/metastatic NSCLC received either placebo + 1200 mg atezolizumab or 600 mg tiragolumab + 1200 mg atezolizumab q3w IV. We collected tumor pretreatment samples and serum samples (baseline and on-treatment) from patients enrolled in the trial, which were subject to bulk RNA-seq and proteomics, respectively. In the mouse tumor model, BALB/c mice were implanted with syngeneic CT26 tumors. After tumor establishment, mice were randomized by tumor volume and then treated with control IgG2a, anti-PD-L1, anti-TIGIT IgG2a-LALAPG (Fc-inactive) ± anti-PD-L1, anti-TIGIT IgG2b ± anti-PD-L1, or anti-TIGIT IgG2a ± anti-PD-L1. Three days after treatment, CD45+ immune cells were collected from the peripheral blood and tumor from selected treatment groups and underwent single cell RNA sequencing or flow cytometry profiling. Tumor growth was also monitored to determine efficacy. Results: Here, we show that tiragolumab functions as both a conventional checkpoint inhibitor and, via Fc gamma receptor engagement, as a modulator of immunosuppressive myeloid cells and T regulatory (Treg) cells. Gene expression analysis of patient tumor samples revealed high levels of these cell subsets, were instead associated with treatment benefit in the tiragolumab + atezolizumab arm but not atezolizumab arm. Analysis of patient serum proteins suggested an association of myeloid cell activation with clinical benefit mediated by the combination therapy. In mouse tumor models, treatment with anti-PD-L1 + anti-TIGIT IgG2a (but not anti-TIGIT IgG2b or Fc-silent anti-TIGIT) led to effective tumor control in mice, suggesting a pivotal role for activating Fc receptors. Phenotypic profiling by single cell RNAseq and flow cytometry revealed that Fc-active anti-TIGIT remodels the tumor microenvironment, most prominently by inducing antigen presentation machinery in myeloid cells, counteracting of anti-PDL1-enforced CD8+ T cell exhaustion, and reducing Treg suppressive capacity. Conclusions: These findings reveal that FcR engagement is one of several distinct mechanisms by which tiragolumab unleashes antitumor immune responses, and inform further clinical development of anti-TIGIT therapies. Citation Format: Namrata S. Patil, Shyam Srivats, Yoonha Choi, Xiangnan Guan, Barzin Nabet, Lisa McGinnis, Eugene Chiang, Alexis Dunkle, Bill O’Gorman, Patrick S. Chang, Ruozhen Hu, John Silva, Joy Han, Amelia Au-Yeung, Chikara Takahashi, Nandini Molden, Pallavi Daggumati, Wendy Connolly, Melissa Johnson, Delvys Rodriguez Abreu, Byoung Chul Cho, Antoine Italiano, Ignacio Gil-Bazo, Enriqueta Felip, Ira Mellman, Raymond Meng, Sanjeev Mariathasan, Robert Johnston, David S. Shames. Anti-TIGIT antibody tiragolumab leverages myeloid cells and regulatory T cells to improve PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5712.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.

This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore’s interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis., Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis. We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.

Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.

The processes that regulate T cell memory generation are important for therapeutic design and the immune response to disease. However, what allows a subset of effector T cells to survive the contraction period to become memory cells is incompletely understood. The Bcl-2 family is critical for T cell survival, and Bcl-2 has been proposed to be important for the survival of memory cells. However, previous studies have relied on double-knockout models, potentially skewing the role of Bcl-2, and the use of Bcl-2 as a marker in adoptive transfer experiments, a method required to confirm the memory potential of cell subsets, has not been possible because of the intracellular localization of the protein. In this study, we present a novel Bcl-2 reporter mouse model and, to our knowledge, show for the first time that a distinct subset of effector T cells, and also a subset within the CD127hiKLRG1lo memory precursor effector cell population, retains high Bcl-2 expression at the peak of the CD8+ T cell response to Listeria monocytogenes. Furthermore, we show that Bcl-2 correlates with memory potential in adoptive transfer experiments using both total responding CD8+ T cells and memory precursor effector cells. These results show that even within the memory precursor effector cell population, Bcl-2 confers a survival advantage in a subset of effector CD8+ T cells that allows differentiation into memory cells and cement Bcl-2 as a critical factor for T cell memory.

Background Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses. Methodology/Principal Findings When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life. Conclusions/Significance We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15–mediated thymic and peripheral NKT cell survival critically depended on Bcl-xL expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-xL, and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Myeloid cell leukemia sequence-1 (Mcl-1) is a critical anti-apoptotic factor in T lymphocytes. However, in spite of the many pro-apoptotic proteins with proposed binding to Mcl-1, the specific interactions by which Mcl-1 regulates primary T-cell survival under different conditions have not been fully explored. Further, how different trophic cytokines modulate the specific role(s) of Mcl-1 is unknown. Here, we use genetic mouse models to dissect the roles of Mcl-1 in primary T lymphocytes. Using the inducible Mcl-1-floxed estrogen receptor-Cre fusion protein (Mcl-1(f/f)ERCre) deletion system in combination with genetic modification of other B-cell lymphoma 2 (Bcl-2) family members, we show that loss of pro-apoptotic Bcl-2 homologous antagonist/killer (Bak) rescues the survival of Mcl-1-deficient T cells in the presence of IL-7. Without IL-7, the survival of Mcl-1-deficient cells cannot be rescued by loss of Bak, but is partially rescued by overexpression of Bcl-2 or loss of Bcl-2-interacting mediator of cell death (Bim). Thus, Mcl-1 and Bcl-2 have a shared role, the inhibition of Bim, in promoting T-cell survival during cytokine withdrawal. Finally, we show that other common gamma-chain (γc) cytokines differentially modulate the roles of Mcl-1. IL-15 has effects similar to those of IL-7 in memory T cells and naïve CD8(+) cells, but not naïve CD4(+) cells. However, IL-4 maintains Mcl-1 and Bcl-2 but also upregulates Bim and Bcl-2-associated X protein (Bax), thus altering the cell's dependence on Mcl-1.

During the development and normal function of T lymphocytes, the cells are subject to several checkpoints at which they must “decide” to live or die. At these critical times and during homeostasis, the molecules that regulate the classical apoptotic pathways and survival pathways such as autophagy have critical roles in controlling this decision. Our laboratory has focused on the roles of apoptotic and autophagic proteins in T lymphocyte development and function. Using genetic models in mice and in vitro analyses of T cell functions, we have outlined critical roles for the Bcl-2 family (regulators of the intrinsic pathway of apoptosis), c-FLIP (an anti-apoptotic protein in the extrinsic pathway of apoptosis), and autophagy in T lymphocytes.

The antiapoptotic proteins Mcl-1 and Bcl-2 have been shown to be critical in T-cell development and homeostasis, but the precise mechanism by which these proteins function in T cells and other cells of the body is unclear. Potential mechanisms have allowed both for overlapping and unique roles for these proteins because of their abilities to bind different proapoptotic Bcl-2 family members, but it is unclear which of these mechanisms are important in an in vivo context. By generation of various genetic mouse models, we found that Mcl-1-deficient thymocytes die largely by a Bak-specific mechanism. In vivo deletion of Bak rescued the survival and developmental blocks of Mcl-1-deficient thymocytes at the double-negative and single-positive stages. Transgenic overexpression of Bcl-2 and in vivo deletion of Bax or Bim were unable to rescue Mcl-1-deficient thymocytes. Thus, Mcl-1 functions in a unique pathway from Bcl-2 in T lymphocytes, likely because of its specific ability to bind and sequester proapoptotic Bak. Together, these data provide an in vivo model for Mcl-1 activity and present us with a greater understanding of the pathways that promote thymocyte survival.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs vs. delivery with free MPLA induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the species-matched NLP scaffold protein were detected. Using the ovalbumin/TCR transgenic model, MPLA-NLPs enhanced antigen-specific T cell expansion in lung-draining lymph nodes. In summary, the NLP platform enhances cellular, humoral, and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Commentary to:T351 protein, A Novel Tumor Associated Antigen and Potential Target for ImmunotherapyJunhai Lu, Ning Kang, Lianxian Cui, Denian Ba, Shujun Cheng, Wei He