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ABSTRACTIntroduction: Therapeutic options for mycosis fungoides and Sézary syndrome include a variety of immunomodulatory, epigenetic, and cytotoxic options; however, none has been demonstrated to be efficacious for all patients, or to deliver deep and durable responses to the majority of patients. In this review, we examine the monoclonal antibody, IPH4102, a novel agent for the treatment of cutaneous T-cell lymphoma.Areas covered: In this review, we examine data demonstrating the tissue specificity of KIR3DL2 receptor, which is highly expressed on the malignant cells in cutaneous T-cell lymphoma, including mycosis fungoides and Sézary syndrome. This specificity has led to the development of the agent IPH4102. Preclinical data showing efficacy of IPH4102 in vivoare outlined, as well as the results from Phase I clinical trials, which suggest that the agent is both efficacious and well-tolerated. Larger scale clinical trials are to follow.Expert Opinion: We examine the putative benefit of IPH4102 in comparison to established agents already in the clinic, highlighting its efficacy and relative safety. We also examine possible directions that may better define the role of IPH4102 in the treatment of T-cell lymphoma in the future.

Surgery remains the standard of care for localised renal cell carcinoma (RCC). Nevertheless, nearly 50% of patients with high-risk disease experience relapse after surgery, with distant sites being common. Considering improved outcomes in terms of disease-free survival with adjuvant immunotherapy with pembrolizumab, we hypothesise that neoadjuvant SABR with or without the addition of pembrolizumab before nephrectomy will lead to improved disease outcomes by evoking better immune response in the presence of an extensive reserve of tumor-associated antigens.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR–Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

Elotuzumab, a humanized monoclonal antibody that binds human signaling lymphocytic activation molecule F7 (hSLAMF7) on myeloma cells, was developed to treat patients with multiple myeloma (MM). Elotuzumab has a dual mechanism of action that includes the direct activation of natural killer (NK) cells and the induction of NK cell–mediated antibody-dependent cellular cytotoxicity. This study aimed to characterize the effects of elotuzumab on NK cells in vitro and in patients with MM and to determine whether elotuzumab antitumor activity was improved by programmed death receptor-1 (PD-1) blockade. Elotuzumab promoted NK cell activation when added to a coculture of human NK cells and SLAMF7-expressing myeloma cells. An increased frequency of activated NK cells was observed in bone marrow aspirates from elotuzumab-treated patients. In mouse tumor models expressing hSLAMF7, maximal antitumor efficacy of a murine immunoglobulin G2a version of elotuzumab (elotuzumab-g2a) required both Fcγ receptor–expressing NK cells and CD8+T cells and was significantly enhanced by coadministration of anti–PD-1 antibody. In these mouse models, elotuzumab-g2a and anti–PD-1 combination treatment promoted tumor-infiltrating NK and CD8+T-cell activation, as well as increased intratumoral cytokine and chemokine release. These observations support the rationale for clinical investigation of elotuzumab/anti–PD-1 combination therapy in patients with MM.

Structures that block movement of fish through river networks are built to serve a variety of societal needs, including transportation, hydroelectric power, and exclusion of exotic species. Due to their abundance, road crossings and dams reduce the amount of habitat available to fish that migrate from the sea or lakes into rivers to breed. The benefits to fish of removing any particular barrier depends on its location within the river network, its passability to fish, and the relative position of other barriers within the network. Balancing the trade-offs between ecological and societal values makes choosing among potential removal projects difficult. To facilitate prioritization of barrier removals, we developed an online decision support tool (DST) with three functions: (1) view existing barriers at various spatial scales; (2) modify information about barriers, including removal costs; and (3) run optimization models to identify portfolios of removals that provide the greatest amount of habitat access for a given budget. A survey of available DSTs addressing barrier removal prioritization indicates that barrier visualization is becoming widespread but few tools allow dynamic calculation of connectivity metrics, scenario analysis, or optimization. Having these additional functions, our DST enables organizations to develop barrier removal priorities based on cost-effectiveness in restoring aquatic connectivity.

Patients who receive chimeric antigen receptor (CAR)–T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEMcells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti–programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

N-methyl-2-pyrrolidone (NMP) is a common solvent and drug vehicle. We discovered unexpected antineoplastic and immunomodulatory activity of NMP in a cMYC-driven myeloma model. Coincident to this, NMP was identified as an acetyllysine mimetic and candidate bromodomain ligand. Accordingly, NMP-treated cells demonstrated transcriptional overlap with BET-bromodomain inhibition, including downregulation of cMYCand IRF4. NMP’s immunomodulatory activity occurred at sub-BET inhibitory concentrations, and, despite phenotypic similarities to lenalidomide, its antimyeloma activity was independent of the IMiD targets cereblon and Ikaros-1/3. Thus, low-affinity yet broad-spectrum bromodomain inhibition by NMP mediates biologically potent, cereblon-independent immunomodulation and at higher doses targets malignant cells directly via BET antagonism. These data reveal that NMP is a functional acetyllysine mimetic with pleotropic antimyeloma and immunomodulatory activities. Our studies highlight the potential therapeutic benefits of NMP, the consequences of current human NMP exposures, and the need for reassessment of scientific literature where NMP was used as an “inert” drug-delivery vehicle.

We report results from a study exploring the combination of romidepsin, bortezomib, and dexamethasone for the treatment of patients with multiple myeloma (MM) previously treated with > 1 prior therapy. The primary objective was to determine the maximum tolerated dose (MTD) of the combination using a novel accelerated dose-escalation schedule in patients with relapsed or refractory MM. The secondary objective was to determine overall response (OR), time to progression (TTP), and overall survival (OS). The MTD identified was bortezomib 1.3 mg/m2(days 1, 4, 8, and 11), dexamethasone 20 mg (days 1, 2, 4, 5, 8, 9, 11, and 12), and romidepsin 10 mg/m2(days 1, 8, and 15) every 28 days. Thrombocytopenia (64%) was the most common ≥ grade 3 hematologic toxicity. Peripheral neuropathy occurred in 76% of patients (n = 19) (≥ grade 3, 8%; 95% confidence interval [CI] 1%-26%). Maintenance romidepsin 10 mg/m2(on days 1 and 8 of a 28-day cycle) proved feasible, with 12 patients receiving a median of 7.5 cycles (range: 1-29). An OR (M-protein) of > minor response (MR) was seen in 18 of 25 patients (72%); 2 (8%) had complete remissions (CRs) and 13 (52%) had partial responses (PRs), including 7 (28%) with very good PRs (VGPRs). The median TTP was 7.2 (95% CI: 5.5-19.6) months, and the median OS was > 36 months. This regimen shows activity with manageable toxicity and warrants further evaluation. This trial was registered at www.clinicaltrials.govas NCT00431990.

Lenalidomide combined with dexamethasone is an effective treatment for refractory/relapsed multiple myeloma (MM). Lenalidomide stimulates natural killer (NK) cells and enhances antitumor responses. We assessed NK cell number and function in 25 patients with MM participating in a clinical trial of lenalidomide and dexamethasone. NK cell numbers increased from a mean of 2.20 ± 0.05 × 105/mL (baseline) to a mean of 3.90 ± 0.03 × 105/mL (cycle 6; P = .05); however, in vitro NK-cell–mediated cytotoxicity decreased from 48.9% ± 6.8% to 27.6% ± 5.1% (P = .0028) and could not be rescued by lenalidomide retreatment. Lenalidomide increased normal donor NK-cell cytotoxicity in vitro from 38.5% to 53.3%, but this was completely abrogated by dexamethasone. Dexamethasone suppression of NK cell–mediated cytotoxicity was partially reversed by a 3-day washout, but these cells remained refractory to lenalidomide-induced enhanced function. Lymphocyte subset depletion experiments revealed that lenalidomide's enhancement of NK cell–mediated cytotoxicity was mediated by CD4+ T-cell production of interleukin 2 and that dexamethasone acted by suppressing interleukin-2 production. Similarly, the reduced ability of NK cells from patients with MM to respond to lenalidomide was also due to impaired CD4 T-cell function. Our findings indicate that lenalidomide immunostimulatory effects on patient NK cells are severely blunted by concurrent dexamethasone administration.

Simple colormetric assay to measure glycolysis in AML blasts at diagnosis can distinguish two distinct populations with significantly different prognoses. This research investigated the level of glycolytic metabolism in leukemic blasts as a prognostic marker in AML. Using an in vitro dye‐reduction assay, we determined the level of glycolytic metabolism in 26 BM samples taken from 23 adult patients with newly diagnosed (n=19) or relapsed (n=4) AML, and AML blasts stratified into two distinct cohorts of moderate (<70%) or high (>80%) levels of glycolytic metabolism. All samples taken at relapse were moderately glycolytic. However, nine of the 19 samples taken at diagnosis were highly glycolytic, and 10 were moderately glycolytic. Three patients had paired samples taken at diagnosis and relapse, and the glycolytic metabolism of these samples did not alter between the two time‐points. The level of glycolytic metabolism did not correlate with the percentage of marrow blasts, patient age, or CG/molecular risk group. Highly glycolytic AML blasts were more resistant to apoptosis induced by ATRA and/or ATO in vitro, suggesting potential resistance to induction chemotherapy, as has been observed in solid tumors. Despite this, high levels of glycolytic metabolism at diagnosis were predictive of a significantly improved duration of CR1 and OS following AML remission induction chemotherapy. In conclusion, we found that the extent of myeloblast glycolysis may be an effective and easily applied method to determine the pretreatment prognosis of AML.

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 105CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

ABSTRACTBacillus anthracisis the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracisprotective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella entericaserovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. entericaserovar Typhi ClyA and under the control of the ompCpromoter. Oral immunization of A/J mice with Salmonellaexpressing full-length PA protected five of six mice against a challenge with 105CFU of aerosolized B. anthracisSTI spores, whereas Salmonellaexpressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonellaexpressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracisspores.

Lymphocytes transiently express an active form of the β−2integrin LFA-1 (LFA-1Af) which has conformational changes in extracellular domains enabling higher affinity binding to the ligand ICAM-1. In this study, we investigated the role of lymphocytes bearing LFA-1Afas potential mediators of binding of 1CAM-1-positive tumour cells to endothelium.

ABSTRACTWe describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensisto type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F. tularensisstrains into five main groups corresponding to strains of F. tularensissubsp. tularensis, F. tularensisstrain ATCC 6223, strains of F. tularensissubsp. holarctica, strains of F. tularensissubsp. holarcticafrom Japan, and F. tularensissubsp. mediaasiatica. The results confirm the genetic identities of these subspecies and also support the suggestion that strains of F. tularensissubsp. holarcticafrom Japan should be considered members of a separate biovar. These findings should support future studies to determine the genetic differences between strains of F. tularensisat the whole-genome level.

1In the CA1 region of hippocampal slices prepared from juvenile (12‐ to 18‐day‐old) rats, activation of group I metabotropic l‐glutamate (mGlu) receptors by the specific agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG) induces a form of long‐term depression (LTD) of excitatory synaptic transmission.2We have used a variety of electrophysiological techniques applied to CA1 neurones in hippocampal slices and from pyramidal cells in dissociated hippocampal cultures to investigate the Ca2+dependence and locus of expression of DHPG‐induced LTD.3In patch‐clamp experiments from hippocampal slices, bath application of DHPG induced a depression of synaptically evoked responses that persisted for the duration of the recording (up to 2 h after commencing washout of DHPG) in 27 of 29 neurones investigated.4DHPG‐induced LTD was associated with an increase in both the paired‐pulse facilitation ratio and the coefficient of variation of EPSCs.5Using dendritic recording, there was a decrease in EPSC success rate (number of trials that elicited a detectable response) but no change in potency (mean EPSC amplitude excluding failures) associated with DHPG‐induced LTD.6In experiments using dissociated hippocampal cultures, application of DHPG elicited a persistent decrease in the frequency of tetrodotoxin‐resistant miniature EPSCs but no change in the amplitude of such events.7DHPG‐induced LTD was not blocked by intracellular application of the calcium chelator BAPTA. It was also unaffected when intracellular calcium stores were depleted by perfusion with thapsigargin. Furthermore, when synaptic transmission was blocked by perfusing with Ca2+‐free medium, DHPG application reliably induced LTD.8These data suggest that DHPG‐induced LTD is Ca2+independent and is expressed presynaptically.

This study demonstrates the variable expression of ICAM‐1 and leukocyte function antigen‐3 (LFA‐3) on four tumor cell lines (COLO526, K562, Daudi, and HT‐29). In addition, phorbol ester (PMA) activation of lymphocytes modulated LFA‐1 from a uniform to a clustered surface distribution; whereas after treatment with high levels of Mg2+ions, the unique epitope for high‐affinity LFA‐1 was identified using clone Mab24. Using a flow cytometric adhesion assay it was demonstrated that PMA‐activated lymphocytes formed conjugates with COLO526 and Daudi, and that these conjugates were inhibited by anti‐CD2 with varying inhibition by LFA‐1 clones MHM24 and 25.3.1. When lymphocytes were induced to express the high‐affinity form of LFA‐1, conjugates were identified with COLO526, K562, and Daudi and these conjugates were sensitive to the presence of both CD2 and LFA‐1 antibodies. Further studies using confocal microscopy confirmed significant adhesion between peripheral blood lymphocytes pre‐treated with either PMA or high levels of Mg2+and the adherent cell line COLO526. In conclusion, this unique study has demonstrated for the first time the important role of the active form of LFA‐1 on the lymphocyte cell surface for conjugate formation with an ICAM‐1‐expressing tumor cell; also, two pathways of cell signaling were identified for conjugate formation to occur. J. Leukoc. Biol.67: 847–855; 2000.

Background:Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies have demonstrated promising, though limited, efficacy against melanoma. Methods:We designed a model system to explore the efficacy of dual specific T cells derived from melanoma patient TILs by transduction with a Her2-specific CAR. Results:Metastatic melanoma cells in our biobank constitutively expressed Her2 antigen. CAR-TIL produced greater amounts of IFN compared with parental TIL, when co-cultured with Her2 expressing tumor lines, including autologous melanoma tumor lines, although no consistent increase in cytotoxicity by TIL was afforded by expression of a CAR. Results of an in vivostudy in NSG mice demonstrated tumor shrinkage when CAR-TILs were used in an adoptive cell therapy protocol. Conclusion:Potential limitations of transduced TIL in our study included limited proliferative potential and a terminally differentiated phenotype, which would need addressing in further work before consideration of clinical translation.

Objective: To test the hypothesis that, controlling for socio‐demographic factors, destructive behaviour among Aboriginal and non‐Aboriginal female sole parents will not be significantly different. Method: This study took place among an urban population of sole parents in Adelaide, South Australia, living in government housing. Two sample subsets were made up of 52 Aboriginal and 45 non‐Aboriginal mothers from similar postcodes. Trained interviewers administered a questionnaire which, in addition to basic demographic data, elicited information concerning finance, housing, upbringing, experience of abuse and police interaction. The major issue of concern in the study was suicide attempt. Results: 1‐in‐3 of the whole sample, 2‐in‐5 of the non‐Aboriginal and 1 ‐in‐4 of the Aboriginal subset had attempted suicide at least once and half more than once. Statistical differences among ‘attempters’ vs. ‘non‐attempters’, irrespective of ethnicity, included increased familial alcohol abuse, physical and sexual abuse, economic difficulty, poor self esteem and perceived discriminatory treatment by welfare agencies and, in the case of Aboriginals, by police. Conclusion: The social environment is critical to understanding destructive behaviour, including self‐harm, regardless of culture or ethnicity. The data show that suicide attempts among female sole parents in State‐housing is one of the few health indices for which Aboriginal statistics are less than for non‐Aboriginals. Implications: It is evident that class, rather than ethnicity, better explains self‐harm in this urban population. It is suggested that reluctance to access services, especially in times of crisis, relates in part to perceptions of care services and that, for Aboriginals, the value of culturally appropriate community‐run services have specific public health and policy implications.