Disease: correlates of protection, determinants of outcome and clinical management' amended to incorporate urgent COVID-19 studies and contract number 75F40120C00085 'Characterization of severe coronavirus infection in humans and model systems for medical countermeasure development and evaluation' awarded to

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Key Points• CML LSC demonstrate increased IL-1 receptor expression and enhanced signaling response.• Inhibition of IL-1 signaling using the antagonist IL-1RA enhances targeting of CML LSC in combination with TKI.Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-kB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML. (Blood. 2016;128(23):2671-2682

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34+ CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34+ CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34+ CP-CML cell engraftment in NSG mice and, upon administration to EGFP+ /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Object Transpedicular instrumentation at C-7 has been well accepted, but salvage techniques are limited. Lamina screws have been shown to be a biomechanically sound salvage technique in the proximal thoracic spine, but have not been evaluated in the lower cervical spine. The following study evaluates the anatomical feasibility of lamina screws at C-7 as well as their bone-screw interface strength as a salvage technique. Methods Nine fresh-frozen C-7 cadaveric specimens were scanned for bone mineral density using dual energy x-ray absorptiometry. Prior to testing, all specimens were imaged using CT to obtain 1-mm axial sections. Caliper measurements of both pedicle width and laminar thickness were obtained. On the right side, pedicle screws were first inserted and then pulled out. Salvage intralaminar screws were inserted into the left lamina from the right spinous process/lamina junction and then pulled out. All screws were placed by experienced cervical spine surgeons under direct fluoroscopic visualization. Pedicle and lamina screws were 4.35- and 3.5-mm in diameter, respectively. Screws sizes were chosen based on direct and radiographic measurements of the respective anatomical regions. Insertional torque (IT) was measured in pounds per inch. Tensile loading to failure was performed in-line with the screw axis at a rate of 0.25 mm/sec using a MiniBionix II system with data recorded in Newtons. Results Using lamina screws as a salvage technique generated mean pullout forces (778.9 ± 161.4 N) similar to that of the index pedicle screws (805.3 ± 261.7 N; p = 0.796). However, mean lamina screw peak IT (5.2 ± 2.0 lbs/in) was significantly lower than mean index pedicle screw peak IT (9.1 ± 3.6 lbs/in; p = 0.012). Bone mineral density was strongly correlated with pedicle screw pullout strength (r = 0.95) but less with lamina screw pullout strength (r = 0.04). The mean lamina width measured using calipers (5.7 ± 1.0 mm) was significantly different from the CTmeasured mean lamina width (5.1 ± 0.8 mm; p = 0.003). Similarly, the mean pedicle width recorded with calipers (6.6 ± 1.1 mm) was significantly different from the CT-measured mean pedicle width (6.2 ± 1.3 mm; p = 0.014). The mean laminar width measured on CT at the thinnest point ranged from 3.8 to 6.8 mm, allowing a 3.5-mm screw to be placed without difficulty. Conclusions These results suggest that using lamina screws as a salvage technique at C-7 provides similar fixation strength as the index pedicle screw. The C-7 lamina appears to have an ideal anatomical width for the insertion of 3.5-mm screws commonly used for cervical fusions. Therefore, if the transpedicular screw fails, using intralaminar screws appear to be a biomechanically sound salvage technique.

The late effects of chemotherapy on immunologic parameters in AIDS-related non-Hodgkin lymphoma (NHL) have not been described.

Although CD4 + T cells likely play key roles in antitumor immune responses, most immunooncology studies have been limited to CD8 + T-cell responses due to multiple technical barriers and a lack of shared antigens across patients. Merkel cell carcinoma (MCC) is an aggressive skin cancer caused by Merkel cell polyomavirus (MCPyV) oncoproteins in 80% of cases. Because MCPyV oncoproteins are shared across most patients with MCC, it is unusually feasible to identify, characterize, and potentially augment tumor-specific CD4 + T cells. Here, we report the identification of CD4 + T-cell responses against six MCPyV epitopes, one of which included a conserved, essential viral oncogenic domain that binds/disables the cellular retinoblastoma (Rb) tumor suppressor. We found that this epitope (WED LT209-228 ) could be presented by three population-prevalent HLA class II alleles, making it a relevant target in 64% of virus-positive

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are populationprevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detected a low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of open reading frame (ORF) products recognized was nine per person. Overall, the data expand the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides, and HLA restriction was defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population prevalent, including U73, U72, U95, and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens. RESULTS Peripheral blood of healthy donors contains HHV-6B-specific CD4 T cells that can be enriched in vitro.The initial cohort of 53 healthy donors was 34% male, and the median age at the time of blood draw was 40 years (range, 21 to 68 years). To ascertain the frequency of HHV-6B-specific CD4 T cells in peripheral blood, peripheral blood mononuclear cells (PBMCs) were assayed by an interleukin-2 (IL-2)/interferon gamma (IFN-␥) intracellular cytokine cytometry (ICC) assay (Fig. 1A). Donors had virus-specific cell populations that were of low abundance but clearly discernible in most subjects. Responses were typically less than 0.1% of total CD4 T cells, with an overall median of 0.048% (Fig. 1B).Given the low abundance of HHV-6-reactive CD4 T cells in PBMCs, we enriched these cells from 42 donors prior to interrogation of the HHV-6B protein library. These donors were chosen based on PBMC availability and a detectable response to HHV-6B ex vivo.