PD-1/PD-L1 checkpoint therapy for cancer is commonly considered to act by reactivating T cells in the tumor microenvironment. Here, we present data from 2 mouse tumor models demonstrating an essential involvement of tumor-draining lymph nodes in PD-1 and PD-L1 therapeutic efficacy. Immune activation induced by checkpoint treatment was predominantly observed in the tumor-draining, but not nondraining, lymph nodes and was reflected in local accumulation of CD8 ⁺ T cells. Surgical resection of these lymph nodes, but not contralateral lymph nodes, abolished therapy-induced tumor regressions and was associated with decreased immune infiltrate in the tumor microenvironment. Moreover, inhibitor FTY720, which locks lymphocytes in lymph organs, also abrogated checkpoint therapy, suggesting that the tumor-draining lymph nodes function as sites of T cell invigoration required for checkpoint blockade therapy. Now that PD-1/PD-L1 checkpoint treatment is applied in earlier clinical stages of cancer, our preclinical data advocate for enrolling patients with their tumor-draining lymph nodes still in place, to optimally engage the antitumor immune response and thereby enhance clinical benefit.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ^−/− mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods: We developed protocols that minimize the inhibitory effects of ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (⁶⁴Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ–producing CD4⁺ T (Th1) cells with 0.7–2.2 MBq of ⁶⁴Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular ⁶⁴Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10^{7 64}Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of ⁶⁴Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10^{7 64}Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, ⁶⁴Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered ⁶⁴Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of ⁶⁴Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm³]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity–diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm³). As expected, ⁶⁴Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm³) when compared with phosphate-buffered saline–challenged animals (4.6 ± 0.5 %ID/cm³). Conclusion: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for ⁶⁴Cu-PTSM–labeled T cells in clinical trials and novel therapy concepts focusing on T-cell–based immunotherapies of autoimmune diseases or cancer.

Purpose: Cancer immunotherapy depends on a systemic immune response, but the basic underlying mechanisms are still largely unknown. Despite the very successful and widespread use of checkpoint inhibitors in the clinic, the majority of cancer patients do not benefit from this type of treatment. In this translational study, we investigated whether noninvasive in vivo positron emission tomography (PET) imaging using 2-[¹⁸F]fluoro-2-deoxy-D-glucose (¹⁸F-FDG) is capable of detecting immunotherapy-associated metabolic changes in the primary and secondary lymphoid organs and whether this detection enables the prediction of a successful anti-cancer immune response.

Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production.

Hypoxia is essential for the development of autoimmune diseases such as rheumatoid arthritis (RA) and is associated with the expression of reactive oxygen species (ROS), because of the enhanced infiltration of immune cells. The aim of this study was to demonstrate the feasibility of measuring hypoxia noninvasively in vivo in arthritic ankles with PET/MRI using the hypoxia tracers ¹⁸F-fluoromisonidazole (¹⁸F-FMISO) and ¹⁸F-fluoroazomycinarabinoside (¹⁸F-FAZA). Additionally, we quantified the temporal dynamics of hypoxia and ROS stress using L-012, an ROS-sensitive chemiluminescence optical imaging probe, and analyzed the expression of hypoxia-inducible factors (HIFs). Methods: Mice underwent noninvasive in vivo PET/MRI to measure hypoxia or optical imaging to analyze ROS expression. Additionally, we performed ex vivo pimonidazole-/HIF-1α immunohistochemistry and HIF-1α/2α Western blot/messenger RNA analysis of inflamed and healthy ankles to confirm our in vivo results. Results: Mice diseased from experimental RA exhibited a 3-fold enhancement in hypoxia tracer uptake, even in the early disease stages, and a 45-fold elevation in ROS expression in inflamed ankles compared with the ankles of healthy controls. We further found strong correlations of our noninvasive in vivo hypoxia PET data with pimonidazole and expression of HIF-1α in arthritic ankles. The strongest hypoxia tracer uptake was observed as soon as day 3, whereas the most pronounced ROS stress was evident on day 6 after the onset of experimental RA, indicating that tissue hypoxia can precede ROS stress in RA. Conclusion: Collectively, for the first time to our knowledge, we have demonstrated that the noninvasive measurement of hypoxia in inflammation using ¹⁸F-FAZA and ¹⁸F-FMISO PET imaging represents a promising new tool for uncovering and monitoring rheumatic inflammation in vivo. Further, because hypoxic inflamed tissues are associated with the overexpression of HIFs, specific inhibition of HIFs might represent a new powerful treatment strategy.