Corrigendum: Children and Adults With Mild COVID-19: Dynamics of the Memory T Cell Response Up to 10 Months.

[This corrects the article DOI: 10.3389/fimmu.2022.817876.].

Releasing the brakes of tumor immunity with anti-PD-L1 and pushing its accelerator with L19-IL2 cures poorly immunogenic tumors when combined with radiotherapy.

BACKGROUND: Poorly immunogenic tumors are hardly responsive to immunotherapies such as immune checkpoint blockade (ICB) and are, therefore, a therapeutic challenge. Combination with other immunotherapies and/or immunogenic therapies, such as radiotherapy (RT), could make these tumors more immune responsive. We have previously shown that the immunocytokine L19-IL2 combined with single-dose RT resulted in 75% tumor remission and a 20% curative abscopal effect in the T cell-inflamed C51 colon carcinoma model. This treatment schedule was associated with the upregulation of inhibitory immune checkpoint (IC) molecules on tumor-infiltrating T cells, leading to only tumor growth delay in the poorly immunogenic Lewis lung carcinoma (LLC) model. METHODS: We aimed to trigger curative therapeutic responses in three tumor models (LLC, C51 and CT26) by "pushing the accelerator" of tumor immunity with L19-IL2 and/or "releasing the brakes" with ICB, such as antibodies directed against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) or its ligand (PD-L1), combined with single-dose RT (10 Gy or 5 Gy). Primary tumor endpoint was defined as time to reach four times the size of tumor volume at start of treatment (4T×SV). Multivariate analysis of 4T×SV was performed using the Cox proportional hazards model comparing each treatment group with controls. Causal involvement of T and natural killer (NK) cells in the anti-tumor effect was assessed by in vivo depletion of T, NK or both cell populations. Immune profiling was performed using flow cytometry on single cell suspensions from spleens, bone marrow, tumors and blood. RESULTS: Combining RT, anti-PD-L1 and L19-IL2 cured 38% of LLC tumors, which was both CD8+ T and NK cell dependent. LLC tumors were resistant to RT +anti-PD-L1 likely explained by the upregulation of other IC molecules and increased T regulatory cell tumor infiltration. RT+L19-IL2 outperformed RT+ICB in C51 tumors; effects were comparable in CT26 tumors. Triple combinations were not superior to RT+L19-IL2 in both these models. CONCLUSIONS: This study demonstrated that combinatorial strategies rationally designed on biological effects can turn immunotherapy-resistant tumors into immunologically responsive tumors. This hypothesis is currently being tested in the international multicentric randomized phase 2 trial: ImmunoSABR (NCT03705403).

A novel co-culture assay to assess anti-tumor CD8+ T cell cytotoxicity via luminescence and multicolor flow cytometry.

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8+ T cells evidenced by in vitro CD8+ T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFNγ, and TNFα by CD8+ T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8+ T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.

HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells.

The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Critical Contribution of NK Group 2 Member D Expressed on Invariant Natural Killer T Cells in Concanavalin A-Induced Liver Hepatitis in Mice.

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (Klrk1-/-) mice, we found that the absence of NKG2D reduced the hepatic injury upon Con A administration. This was not due to an intrinsic functional defect of NKG2D-deficient iNKT cells as mice missing NKG2D have normal distribution and function of iNKT cells. Furthermore, increased resistance to Con A-induced hepatitis was confirmed using neutralizing anti-NKG2D antibodies. The reduced pathogenic effect of Con A in the absence of NKG2D correlates with a reduction in pathogenic cytokine production and FAS-Ligand (FAS-L) expression by iNKT cells. We also found that Con A administration led to an increase in the retinoic acid early inducible (RAE-1) surface expression on wild-type hepatocytes. Finally, we found that Con A has no direct action on FAS-L expression or cytokine production by iNKT cells and thus propose that NKG2D-L expression on stressed hepatocytes promote cytotoxic activity of iNKT cells via its interaction with NKG2D contributing to hepatic injury. In conclusion, our results highlight NKG2D as an essential receptor required for the activation of iNKT cells in Con A-induced hepatitis and indicate that it represents a potential drug target for prevention of autoimmune hepatitis.

The immunocytokine L19-IL2: An interplay between radiotherapy and long-lasting systemic anti-tumour immune responses.

Recently, we have shown that the administration of the tumour-targeted antibody-based immunocytokine L19-IL2 after radiotherapy (RT) resulted in synergistic anti-tumour effect. Here we show that RT and L19-IL2 can activate a curative abscopal effect, with a long-lasting immunological memory. Ionizing radiation (single dose of 15Gy, 5 × 2Gy or 5 × 5Gy) was delivered to primary C51 colon tumour-bearing immunocompetent mice in combination with L19-IL2 and response of secondary non-irradiated C51 or CT26 colon tumours was evaluated. 15Gy + L19-IL2 triggered a curative (20%) abscopal effect, which was T cell dependent. Moreover, 10Gy + L19-IL2 treated and cured mice were re-injected after 150 days with C51 tumour cells and tumour uptake was assessed. Age-matched controls (matrigel injected mice treated with 10Gy + L19-IL2, mice cured after treatment with surgery + L19-IL2 and mice cured after high dose RT 40Gy + vehicle) were included. Several immunological parameters in blood, tumours, lymph nodes and spleens were investigated. Treatment with 10Gy + L19-IL2 resulted in long-lasting immunological memory, associated with CD44+CD127+ expression on circulating T cells. This combination treatment can induce long-lasting curative abscopal responses, and therefore it has also great potential for treatment of metastatic disease. Preclinical findings have led to the initiation of a phase I clinical trial (NCT02086721) in our institute investigating stereotactic ablative radiotherapy with L19-IL2 in patients with oligometastatic solid tumours.

Combining radiotherapy with immunotherapy: the past, the present and the future.

The advent of immunotherapy is currently revolutionizing the field of oncology, where different drugs are used to stimulate different steps in a failing cancer immune response chain. This review gives a basic overview of the immune response against cancer, as well as the historical and current evidence on the interaction of radiotherapy with the immune system and the different forms of immunotherapy. Furthermore the review elaborates on the many open questions on how to exploit this interaction to the full extent in clinical practice.