Effector T Cell Egress via Afferent Lymph Modulates Local Tissue Inflammation.

Memory/effector T cells recirculate through extralymphoid tissues by entering from blood and egressing via afferent lymph. Although T cell entry into effector sites is key to inflammation, the relevance of T cell egress to this process is unknown. In this study, we found that Ag recognition at the effector site reduced the tissue egress of proinflammatory Th1 cells in a mouse model of delayed hypersensitivity. Transgenic expression of "tissue exit receptor" CCR7 enhanced lymphatic egress of Ag-sequestered Th1 cells from the inflamed site and alleviated inflammation. In contrast, lack of CCR7 on Th1 cells diminished their tissue egress while enhancing inflammation. Lymph-borne Th1 and Th17 cells draining the inflamed skin of sheep migrated toward the CCR7 ligand CCL21, suggesting the CCR7-CCL21 axis as a physiological target in regulating inflammation. In conclusion, exit receptors can be targeted to modulate T cell dwell time and inflammation at effector sites, revealing T cell tissue egress as a novel control point of inflammation.

Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major.

Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections.

Engagement of NKG2D on bystander memory CD8 T cells promotes increased immunopathology following Leishmania major infection.

One of the hallmarks of adaptive immunity is the development of a long-term pathogen specific memory response. While persistent memory T cells certainly impact the immune response during a secondary challenge, their role in unrelated infections is less clear. To address this issue, we utilized lymphocytic choriomeningitis virus (LCMV) and Listeria monocytogenes immune mice to investigate whether bystander memory T cells influence Leishmania major infection. Despite similar parasite burdens, LCMV and Listeria immune mice exhibited a significant increase in leishmanial lesion size compared to mice infected with L. major alone. This increased lesion size was due to a severe inflammatory response, consisting not only of monocytes and neutrophils, but also significantly more CD8 T cells. Many of the CD8 T cells were LCMV specific and expressed gzmB and NKG2D, but unexpectedly expressed very little IFN-γ. Moreover, if CD8 T cells were depleted in LCMV immune mice prior to challenge with L. major, the increase in lesion size was lost. Strikingly, treating with NKG2D blocking antibodies abrogated the increased immunopathology observed in LCMV immune mice, showing that NKG2D engagement on LCMV specific memory CD8 T cells was required for the observed phenotype. These results indicate that bystander memory CD8 T cells can participate in an unrelated immune response and induce immunopathology through an NKG2D dependent mechanism without providing increased protection.

Vaccines targeting ESR1 activating mutations elicit anti-tumor immune responses and suppress estrogen signaling in therapy resistant ER+ breast cancer.

ER+ breast cancers (BC) are characterized by the elevated expression and signaling of estrogen receptor alpha (ESR1), which renders them sensitive to anti-endocrine therapy. While these therapies are clinically effective, prolonged treatment inevitably results in therapeutic resistance, which can occur through the emergence of gain-of-function mutations in ESR1. The central importance of ESR1 and development of mutated forms of ESR1 suggest that vaccines targeting these proteins could potentially be effective in preventing or treating endocrine resistance. To explore the potential of this approach, we developed several recombinant vaccines encoding different mutant forms of ESR1 (ESR1mut) and validated their ability to elicit ESR1-specific T cell responses. We then developed novel ESR1mut-expressing murine mammary cancer models to test the anti-tumor potential of ESR1mut vaccines. We found that these vaccines could suppress tumor growth, ESR1mut expression and estrogen signaling in vivo. To illustrate the applicability of these findings, we utilize HPLC to demonstrate the presentation of ESR1 and ESR1mut peptides on human ER+ BC cell MHC complexes. We then show the presence of human T cells reactive to ESR1mut epitopes in an ER+ BC patient. These findings support the development of ESR1mut vaccines, which we are testing in a Phase I clinical trial.

Beyond Neoantigens: Antigens Derived from Tumor Drivers as Cancer Vaccine Targets.

A vaccine targeting HER2, a nonmutated but overexpressed tumor antigen, readily primed T cells for ex vivo expansion and adoptive transfer with minimal toxicity. This regimen led to intramolecular epitope spreading in a majority of patients and offers a treatment modality that may improve outcomes for patients with metastatic breast cancer expressing HER2. See related article by Disis et al., p. 3362.

Cancer vaccine strategies using self-replicating RNA viral platforms.

The development and success of RNA-based vaccines targeting SARS-CoV-2 has awakened new interest in utilizing RNA vaccines against cancer, particularly in the emerging use of self-replicating RNA (srRNA) viral vaccine platforms. These vaccines are based on different single-stranded RNA viruses, which encode RNA for target antigens in addition to replication genes that are capable of massively amplifying RNA messages after infection. The encoded replicase genes also stimulate innate immunity, making srRNA vectors ideal candidates for anti-tumor vaccination. In this review, we summarize different types of srRNA platforms that have emerged and review evidence for their efficacy in provoking anti-tumor immunity to different antigens. These srRNA platforms encompass the use of naked RNA, DNA-launched replicons, viral replicon particles (VRP), and most recently, synthetic srRNA replicon particles. Across these platforms, studies have demonstrated srRNA vaccine platforms to be potent inducers of anti-tumor immunity, which can be enhanced by homologous vaccine boosting and combining with chemotherapies, radiation, and immune checkpoint inhibition. As such, while this remains an active area of research, the past and present trajectory of srRNA vaccine development suggests immense potential for this platform in producing effective cancer vaccines.

Clinical trials of self-replicating RNA-based cancer vaccines.

Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.

Key takeaways for knowledge expansion of early-career scientists conducting Transdisciplinary Research in Energetics and Cancer (TREC): a report from the TREC Training Workshop 2022.

The overall goal of the annual Transdisciplinary Research in Energetics and Cancer (TREC) Training Workshop is to provide transdisciplinary training for scientists in energetics and cancer and clinical care. The 2022 Workshop included 27 early-to-mid career investigators (trainees) pursuing diverse TREC research areas in basic, clinical, and population sciences. The 2022 trainees participated in a gallery walk, an interactive qualitative program evaluation method, to summarize key takeaways related to program objectives. Writing groups were formed and collaborated on this summary of the 5 key takeaways from the TREC Workshop. The 2022 TREC Workshop provided a targeted and unique networking opportunity that facilitated meaningful collaborative work addressing research and clinical needs in energetics and cancer. This report summarizes the 2022 TREC Workshop's key takeaways and future directions for innovative transdisciplinary energetics and cancer research.

Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis.

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity.

BACKGROUND: Despite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment systemic antitumor immunity, in part by appropriately presenting tumor antigens, but also by modulating the immunosuppressive tumor microenvironment (TME). We previously validated this concept in models of murine carcinoma treated with a novel predominately microcavitating version of high-intensity focused ultrasound (HIFU), mechanical high-intensity focused ultrasound (M-HIFU). Here we elucidated the mechanisms of enhanced antitumor immunity by M-HIFU over conventional thermal high-intensity focused ultrasound (T-HIFU) and investigated the potential of the combinatorial strategy with an immune checkpoint inhibitor, anti-PD-L1 antibody. METHODS: The antitumor efficacy of treatments was investigated in syngeneic murine breast cancer models using triple-negative (E0771) or human ErbB-2 (HER2) expressing (MM3MG-HER2) tumors in C57BL/6 or BALB/c mice, respectively. Induction of systemic antitumor immunity by the treatments was tested using bilateral tumor implantation models. Flow cytometry, immunohistochemistry, and single-cell RNA sequencing were performed to elucidate detailed effects of HIFU treatments or combination treatment on TME, including the activation status of CD8 T cells and polarization of tumor-associated macrophages (TAMs). RESULTS: More potent systemic antitumor immunity and tumor growth suppression were induced by M-HIFU compared with T-HIFU. Molecular characterization of the TME after M-HIFU by single-cell RNA sequencing demonstrated repolarization of TAM to the immunostimulatory M1 subtype compared with TME post-T-HIFU. Concurrent anti-PD-L1 antibody administration or depletion of CD4+ T cells containing a population of regulatory T cells markedly increased T cell-mediated antitumor immunity and tumor growth suppression at distant, untreated tumor sites in M-HIFU treated mice compared with M-HIFU monotherapy. CD8 T and natural killer cells played major roles as effector cells in the combination treatment. CONCLUSIONS: Physical disruption of the TME by M-HIFU repolarizes TAM, enhances T-cell infiltration, and, when combined with anti-PD-L1 antibody, mediates superior systemic antitumor immune responses and distant tumor growth suppression. These findings suggest M-HIFU combined with anti-PD-L1 may be useful in reducing late recurrence or metastasis when applied to primary tumors.