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Noninvasive and quantitative tracking of CD8 T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. Radiolabeling of an anti-CD8 cys-diabody (169cDb) with Cu was developed. The accumulation ofCu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains ( = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8 T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped ( = 6-8/group studied with imaging and IHC or flow cytometry). We demonstrate the ability of immunoPET to detect small differences in CD8 T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic -driven model of mammary adenocarcinoma (NDL),Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8 T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols. Cu-169cDb imaging can spatially map the distribution of CD8 T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8 T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. .

Background and purpose COVID-19 pandemic led to wide-spread use of face-masks, respirators and other personal protective equipment (PPE) by healthcare workers. Various symptoms attributed to the use of PPE are believed to be, at least in part, due to elevated carbon-dioxide (CO2) levels. We evaluated concentrations of CO2 under various PPE. Methods In a prospective observational study on healthy volunteers, CO2 levels were measured during regular breathing while donning 1) no mask, 2) JustAir® powered air purifying respirator (PAPR), 3) KN95 respirator, and 4) valved-respirator. Serial CO2 measurements were taken with a nasal canula at a frequency of 1-Hz for 15-min for each PPE configuration to evaluate whether National Institute for Occupational Safety and Health (NIOSH) limits were breached. Results The study included 11 healthy volunteers, median age 32 years (range 16–54) and 6 (55%) men. Percent mean (SD) changes in CO2 values for no mask, JustAir® PAPR, KN95 respirator and valve respirator were 0.26 (0.12), 0.59 (0.097), 2.6 (0.14) and 2.4 (0.59), respectively. Use of face masks (KN95 and valved-respirator) resulted in significant increases in CO2 concentrations, which exceeded the 8-h NIOSH exposure threshold limit value-weighted average (TLV-TWA). However, the increases in CO2 concentrations did not breach short-term (15-min) limits. Importantly, these levels were considerably lower than the long-term (8-h) NIOSH limits during donning JustAir® PAPR. There was a statistically significant difference between all pairs (p < 0.0001, except KN95 and valved-respirator (p = 0.25). However, whether increase in CO2 levels are clinically significant remains debatable. Conclusion Although, significant increase in CO2 concentrations are noted with routinely used face-masks, the levels still remain within the NIOSH limits for short-term use. Therefore, there should not be a concern in their regular day-to-day use for healthcare providers. The clinical implications of elevated CO2 levels with long-term use of face masks needs further studies. Use of PAPR prevents relative hypercapnoea. However, whether PAPR should be advocated for healthcare workers requiring PPE for extended hours needs to evaluated in further studies.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments.Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components.Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation.Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.