Characterizing the Dysfunctional NK Cell: Assessing the Clinical Relevance of Exhaustion, Anergy, and Senescence.

There is a growing body of literature demonstrating the importance of T cell exhaustion in regulating and shaping immune responses to pathogens and cancer. Simultaneously, the parallel development of therapeutic antibodies targeting inhibitory molecules associated with immune exhaustion (such as PD-1, but also TIGIT, and LAG-3) has led to a revolution in oncology with dramatic benefits in a growing list of solid and hematologic malignancies. Given this success in reinvigorating exhausted T cells and the related anti-tumor effects, there are increasing efforts to apply immune checkpoint blockade to other exhausted immune cells beyond T cells. One approach involves the reinvigoration of "exhausted" NK cells, a non-T, non-B lymphoid cell of the innate immune system. However, in contrast to the more well-defined and established molecular, genetic, and immunophenotypic characteristics of T cell exhaustion, a consensus on the defining functional and phenotypic features of NK "exhaustion" is less clear. As is well-known from T cell biology, separate and distinct molecular and cellular processes including senescence, anergy and exhaustion can lead to diminished immune effector function with different implications for immune regulation and recovery. For NK cells, it is unclear if exhaustion, anergy, and senescence entail separate and distinct entities of dysfunction, though all are typically characterized by decreased effector function or proliferation. In this review, we seek to define these distinct spheres of NK cell dysfunction, analyzing how they have been shown to impact NK biology and clinical applications, and ultimately highlight key characteristics in NK cell function, particularly in relation to the role of "exhaustion.

Memory in the time of COVID

This dissertation examines six characteristics of memory in accordance with the COVID-19 pandemic. COVID-19 is a major event that has changed how society operates. With the conjunction of digital archives, memory performances have changed. In light of a major world pandemic and the shift to the digital a reassessment of memory is needed. 1) With many museums physically closed, the context of the pandemic with the affordances of technology allowed for memory to symbolically be performed through the digital archives. The future of memory studies must contend with digital archives as memory performances. 2) Computer assisted analysis may help with decoding a broad set, but the human aspect cannot be overlooked. Examining the digital archived COVID-19 memory showcases the importance of human analysis when understanding trauma and trauma recovery. 3) The rapid speed of memory making today and the reduced distance between event and recollection requires memory studies to evaluate the role of time and distance. The expedited memory making creates more references for others to use and abuse increasing the importance of rhetoric to understand all the available means of persuasion. 4) While the digital opens up room for multiple voices, it also allows individuals to be selective. Future memory studies must be aware of how memory performances are used to create echo chambers in order to attract likeminded newcomers and establish a group identity. 5) From the partisan and contested voices, COVID-19 and the digital have created new ways to forget the other. Memory studies must take on the mantle of exposing the silencing of inequalities. 6) Evaluating the characteristics of collective memory after COVID-19 and the increase of digital archives demonstrates the importance of rhetoric. With the trouble of truth and the disconnectedness between institutions and the people, rhetoric becomes integral in solving the issues of society

Bortezomib Augments Natural Killer Cell Targeting of Stem-Like Tumor Cells.

Tumor cells harboring stem-like/cancer stem cell (CSC) properties have been identified and isolated from numerous hematological and solid malignancies. These stem-like tumor cells can persist following conventional cytoreductive therapies, such as chemotherapy and radiotherapy, thereby repopulating the tumor and seeding relapse and/or metastasis. We have previously shown that natural killer (NK) cells preferentially target stem-like tumor cells via non- major histocompatibility complex (MHC) restricted mechanisms. Here, we demonstrated that the proteasome inhibitor, bortezomib, augments NK cell targeting of stem cell-like tumor cells against multiple solid human tumor-derived cancer lines and primary tissue samples. Mechanistically, this was mediated by the upregulation of cell surface NK ligands MHC class I chain-related protein A and B (MICA and MICB) on aldehyde dehydrogenases (ALDH)-positive CSCs. The increased expression of MICA and MICB on CSC targets thereby enhanced NK cell mediated killing in vitro and ex vivo from both human primary tumor and patient-derived xenograft samples. In vivo, the combination of bortezomib and allogeneic NK cell adoptive transfer in immunodeficient mice led to increased elimination of CSCs as well as tumor growth delay of orthotopic glioblastoma tumors. Taken together, our data support the combination bortezomib and NK transfer as a strategy for both CSC targeting and potentially improved outcomes in clinical cancer patients

Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma.

BackgroundIncreasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic).MethodsWe exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate.ResultsAfter functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05).ConclusionsAnti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations

Blood and tissue biomarker analysis in dogs with osteosarcoma treated with palliative radiation and intra-tumoral autologous natural killer cell transfer.

We have previously reported radiation-induced sensitization of canine osteosarcoma (OSA) to natural killer (NK) therapy, including results from a first-in-dog clinical trial. Here, we report correlative analyses of blood and tissue specimens for signals of immune activation in trial subjects. Among 10 dogs treated with palliative radiotherapy (RT) and intra-tumoral adoptive NK transfer, we performed ELISA on serum cytokines, flow cytometry for immune phenotype of PBMCs, and PCR on tumor tissue for immune-related gene expression. We then queried The Cancer Genome Atlas (TCGA) to evaluate the association of cytotoxic/immune-related gene expression with human sarcoma survival. Updated survival analysis revealed five 6-month survivors, including one dog who lived 17.9 months. Using feeder line co-culture for NK expansion, we observed maximal activation of dog NK cells on day 17-19 post isolation with near 100% expression of granzyme B and NKp46 and high cytotoxic function in the injected NK product. Among dogs on trial, we observed a trend for higher baseline serum IL-6 to predict worse lung metastasis-free and overall survival (P = 0.08). PCR analysis revealed low absolute gene expression of CD3, CD8, and NKG2D in untreated OSA. Among treated dogs, there was marked heterogeneity in the expression of immune-related genes pre- and post-treatment, but increases in CD3 and CD8 gene expression were higher among dogs that lived > 6 months compared to those who did not. Analysis of the TCGA confirmed significant differences in survival among human sarcoma patients with high and low expression of genes associated with greater immune activation and cytotoxicity (CD3e, CD8a, IFN-γ, perforin, and CD122/IL-2 receptor beta). Updated results from a first-in-dog clinical trial of palliative RT and autologous NK cell immunotherapy for OSA illustrate the translational relevance of companion dogs for novel cancer therapies. Similar to human studies, analyses of immune markers from canine serum, PBMCs, and tumor tissue are feasible and provide insight into potential biomarkers of response and resistance

Anti-proliferative but not anti-angiogenic tyrosine kinase inhibitors enrich for cancer stem cells in soft tissue sarcoma

The templated zeolite-analogue GaPO-34 (CHA structure type) crystallises from a gel precursor Ga2O3:2H3PO4:1HF:1.7SDA:70H2O (where SDA = structure directing agent), treated hydrothermally for 24 hours at 170 °C using either pyridine or 1-methylimizadole as SDA and one of either poorly crystalline ε-Ga2O3 or γ-Ga2O3 as gallium precursor. If the same gels are stirred for periods shorter than 2 hours but treated under identical hydrothermal conditions, then a second phase crystallises, free of GaPO-34. If β-Ga2O3 is used as a reagent only the second phase is found to crystallise, irrespective of gel aging time. The competing phase, which we denote GaPO-34A, has been structurally characterised using synchrotron powder X-ray diffraction for the pyridine material, GaPO-34A(pyr), and using single-crystal X-ray diffraction for the 1-methylimiazole material, GaPO-34A(mim). The structure of GaPO-34A(pyr), P1 @#x0305;, a = 10.22682(6) Å, b = 12.09585(7) Å, c = 13.86713(8) Å, α = 104.6531(4) °, β = 100.8111(6) °, γ = 102.5228(6) °, contains 7 unique gallium sites and 6 phosphorus sites, with empirical formula [Ga7P6O24(OH)2F3(H2O)2].2(C5NH6). GaPO-34A(mim) is isostructural but is modelled as a half volume unit cell, P1 @#x0305;, a = 5.0991(2) Å, b = 12.0631(6) Å, c = 13.8405(9) Å, α = 104.626(5) °, β = 100.346(5) °, γ = 101.936(4) °, with a gallium and a bridging fluoride partially occupied and two partially occupied SDA sites. Solid-state 31P and 71Ga NMR spectroscopy confirms the structural complexity of GaPO-34A with signals resulting from overlapping lineshapes from multiple Ga and P sites, while 1H and 13C solid-state NMR spectra confirm the presence of the protonated SDA and provide evidence for disorder in the SDA. The protonated SDA is located in 14-ring one-dimensional channels with hydrogen bonding deduced from the SDA nitrogens to framework oxygen distances. Upon thermal treatment to investigate SDA removal, structure collapse occurs, which may be due the large number of bridging hydroxides and fluorides in the as-made material, and the unequal amounts of gallium and phosphorus present

Aging augments obesity-induced thymic involution and peripheral T cell exhaustion altering the “obesity paradox”

IntroductionThe incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the “obesity paradox” which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment.MethodsThe role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice.ResultsWe observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts.DiscussionThese results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses

Low-Dose Sorafenib Promotes Cancer Stem Cell Expansion and Accelerated Tumor Progression in Soft Tissue Sarcomas

The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients