Novel vaccination strategies for CD4+ T cell immunotherapy of melanoma

Immunotherapy has emerged as a standard treatment modality in melanoma and many other cancers. While a lot is known about the anti-tumoral effector functions of CD8+ T cells, CD4+ T cells remain less well understood in cancer immunotherapy. In the current work, it was hypothesized that melanocyte antigen-specific CD4+ T cells can control the growth of melanomas as efficiently as corresponding CD8+ T cells but differ in the way they recognize antigen and exert their effector functions against tumor cells in the tissue microenvironment. It has been previously shown by the Tüting lab that a single administration of an adenovirus vector expressing the melanocytic antigen gp100 can promote effective expansion of adoptively transferred gp100-specific Pmel-1 TCR transgenic CD8+ T cells and cause regression of established melanomas in syngeneic mice. Here, a similar therapy protocol was established for Trp1-specific TCR transgenic CD4+ T cells. For this, the adenoviral vaccine vector Ad-GTY expressing both gp100 and Trp1 epitopes was first generated. Ad-GTY could expand adoptively transferred Trp1 CD4+ T cells in vivo, albeit less efficiently when compared to Pmel-1 CD8+ T cells. Nevertheless, a Trp1 CD4+ T cell ACT protocol with Ad-GTY showed significant anti-tumor efficacy and could control the growth of HCmel12 melanomas. The recombinant MVA virus vector MVA-PMTP that also expressed both the gp100 and Trp1 epitopes was generated to evaluate prime-boost vaccine strategies. However, MVA-PMTP was only able to re-expand CD8+ T cells but not CD4+ T cells. Moreover, the Ad-MVA prime-boost vaccination strategy did not significantly increase the therapeutic efficacy of the ACT protocols. Following Trp1 CD4+ ACT escaping melanoma cells frequently down-regulated melanocytic antigen expression and acquired a dedifferentiated phenotype presumably due to therapy-induced inflammation. As shown previously with CD8+ T cells, this also represented a major limitation of targeting melanocytic antigens with antigen-specific CD4+ T cells. Experiments using HCmel12 Trp1 antigen loss variants generated with CRISPR-Cas9 genome editing techniques revealed that the control of tumor growth by Trp1 CD4+ T cells is antigen-specific. Experiments with mixtures of HCmel12 control and Trp1 knockout cells demonstrated that Trp1 CD4+ cells can exert significant bystander killing and that immunoselection for irreversible genetic antigen loss is dominant over reversible phenotypic antigen loss for immune escape of melanoma cells. HCmel12 Ciita loss variants were also generated with CRISPR-Cas9 genome editing techniques. Unlike unmodified HCmel12 cells they fail to upregulate MHC class II and therefore cannot be directly recognized by Trp1 CD4+ T cells. Experiments revealed that direct MHC class II restricted recognition of melanoma cells by Trp1 CD4+ T cells was not required for tumor growth control in vivo. This suggested an important role for indirect stimulation of Trp1 CD4+ T cells by APC in the tumor microenvironment. Likely, Trp1 CD4+ T cells indirectly control melanoma growth in the tumor microenvironment through Th1 associated cytokines such as IFN-γ and TNF-α. Future studies will have to address the spatial location of Trp1 CD4+ T cells in the tumor microenvironment, their interaction with other immune cells and the role of Th1-associated cytokines for their anti-tumor efficacy. Combining T cell therapies with signal transduction inhibitors or checkpoint inhibitors to counteract mechanisms of therapy resistance and immune escape in mouse models will help to delineate strategies for more effective treatment of melanoma patients that should be tested in the clinic

Multicomponent analysis of the tumour microenvironment reveals low CD8 T cell number, low stromal caveolin-1 and high tenascin-C and their combination as significant prognostic markers in non-small cell lung cancer

The complex interplay of the tumour microenvironment (TME) and its role in disease progression and response to therapy is poorly understood. The majority of studies to date focus on individual components or molecules within the TME and so lack the power correlative analysis. Here we have performed a multi-parameter analysis of the TME in 62 resectable non-small cell lung cancer (NSCLC) specimens detailing number and location of immune infiltrate, assessing markers of cancer-associated fibroblasts, caveolin-1 and tenascin-C, and correlating with clinicopathological details, as well as markers of disease progression such as epithelial-to-mesenchymal transition (EMT). The influence of individual parameters on overall survival was determined in univariate and multivariate analysis and the combination of risk factors and interplay between components analysed. Low numbers of CD8 T cells, low stromal levels of caveolin-1 or high levels of tenascin-C were significant prognostic markers of decreased overall survival in both univariate and multivariate analysis. Patients with two or more risk factors had dramatically reduced overall survival and those with all three a median survival of just 7.5 months. In addition, low levels of tumour E-cadherin correlated with reduced immune infiltrate into the tumour nests, possibly linking EMT to the avoidance of CD8 T cell control. The multicomponent approach has allowed identification of the dominant influences on overall survival, and exploration of the interplay between different components of the TME in NSCLC

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

Experimental Study on Thickness and Surface Roughness of Co-Sputtered Titanium and Alumina Films

Titanium (Ti) and Alumina (Al2O3) thin films have been coated using co-sputtering technique on SS304, Copper substrates. Magnetron sputtering technique has been employed, in this Direct current and Radio Frequency powers have been used in the presence of argon gas to sputter Ti and Al2O3 respectively. Experimental runs are planned by Design of Experiments (DOE) approach. Investigations have been conducted to determine optimize conditions for thickness and surface roughness of the thin films. It is vital to have a precise knowledge of film thickness and its roughness. Both of these have been measured in terms of nanometer using surface profilometer and Atomic Force Microscope (AFM) respectivel

Endovascular Management in a Case of Recurrent Bleed Following Transurethral Resection of the Prostate

Transurethral resection of prostate (TURP) remains the gold standard surgical therapy for symptomatic benign prostatic enlargement, with reported International Prostate Symptom Score reduction of up to 70%. However, as many as 20% of patients can have significant complications including sexual dysfunction, perioperative bleeding requiring blood transfusion, and incontinence. Intractable hematuria from the prostate can be life-threatening, and its management remains a difficult clinical problem. Prostate artery embolization (PAE) is occasionally indicated in such patients when all other measures have failed. PAE has been used to treat benign prostatic hyperplasia; however, literature related to its use for bleeding following TURP remains limited. We report a case of an elderly male who presented with recurrent episodes of hematuria following TURP and was successfully treated by endovascular management

Use of strain, strain rate, tissue velocity imaging, and endothelial function for early detection of cardiovascular involvement in patients with beta-thalassemia

Background: Global ventricular function often remains normal in patients with beta-thalassemia major. (β-TM) until late. Tissue Doppler and strain imaging may be useful to assess regional myocardial function abnormalities in these patients. Methods: Systolic (Sm), early diastolic (Em), and late diastolic (Am) (Em/Am) myocardial velocities at basal lateral and septal left ventricular (LV) segments, strain (S), and strain rate (SR) in basal and mid LV, right ventricular (RV) and septum were measured in 30 patients (β-TM, 12.4 ± 5.2 years, serum ferritin 2603.1 μg/L) and twenty controls (12.5 ± 5.2 years). Flow-mediated dilatation (FMD) vasodilatation as a measure of endothelial function was also assessed. Results: Patients had significantly higher LV mass index (169.45 ± 61.14 vs. 104.66 ± 24.42; P = 0.009) while global LV Sm and diastolic function was similar to controls. Patients had significantly lower lateral Em velocity, Em (10.12 ± 1.16 vs. 17.9 ± 2.11; P = 0.002), Em/Am ratio (0.811 ± 0.192 vs. 2.06 ± 0.62; P = 0.001) at the basal lateral LV, lower strain values at the basal lateral LV (19.5 ± 4.17 vs. 24.196 ± 1.81; P = 0.002), mid lateral LV (19.07 ± 3.98 vs. 25.56 ± 2.62; P = 0.042), basal septum (17.04 ± 3.44 vs. 25.43 ± 2.53; P = 0.001), and mid septum (20.49 ± 5.34 vs. 24.45 ± 2.20; P = 0.001) as compared to controls. SR at the basal and mid segment of the lateral LV wall and at the basal and mid septum was also significantly lower in patients. SR in basal and mid RV although lower was not significantly different from controls. Patients also had significantly lower FMD (7.57 ± 3.16 vs. 18.08 ± 1.9, P = 0.018) implying endothelial dysfunction. Conclusions: Tissue Doppler, strain and SR imaging are useful to quantify regional myocardial function in asymptomatic α-TM patients with preserved global Sm and diastolic function

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

<div><p>One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. <i>Mycobacterium tuberculosis</i> (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro <i>vs</i>. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of <i>rpoB>aroK>ppk>rpoC>ilvB</i>. <i>RpoB</i> was used as the cidality control. In-vitro and in-vivo studies feature <i>aroK</i> (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute infection phase for the high fidelity translation, the compound efficacy may also be evaluated in the low pH, in addition to the standard replication condition.</p></div

AS-silencing of Mtb targets under replicating in-vitro growth conditions.

<p>The survival kinetics of target AS-recombinants of Mtb enumerated up to 63-days = almost ~70 generations; are shown here as log₁₀ cfu/ml <i>vs</i>. the vector control. Under replicating growth condition, <i>ilvB</i> demonstrated the maximum AS-repression among different magnitude of cidality in comparison to <i>ppk</i>. The order of cidality (log₁₀ cfu/ml) was <i>ilvB</i> (5.4)<i>> ppk</i>(4.8)<i>> rpoC</i>(3.5)<i>> rpoB</i>(2.5)<i>> aroK</i>(2.2).</p

Cidality SCORE of Mtb targets by in-vitro AS-silencing.

<p>The normal or stringent physiological conditions used are: Msx = Nitrogen depletion, LpH = low pH, NO2 = Nitric oxide, NSM = Nutrient Starvation Model, Hpx = Hypoxia, REP = logarithmically replicating condition. The graph depicts the net compounded effect, the Cidality SCORE, of respective genes under various physiological conditions (a total of) as the inhibition on the upper scale, and growth on the lower scale. It represents the behaviour of the respective target under a diseased situation. WT = Wild-type Mtb strain, V = WT strain of Mtb containing the blank vector. The rest are all the gene-specific recombinants of Mtb. The blue colour boxes show the cidality SCORE representing the overall cidality potential of a target based on the AS-RNA gene silencing magnitudes as <i>ppk</i>(7.5)><i>ilvB</i>(7.3)><i>rpoB</i>(7.1)><i>rpoC</i>(5.2)><i>pyrH</i>(3.6)><i>aroK</i>(3.2). Statistically significant (***), the error bars (SEM) represent the robustness of data from the triplicates.</p

Maximum fold target repression during the course of infection.

<p>The data validates IPTG-inducible in-vivo AS-repression of Mtb targets. The net target transcript levels, as evaluated by RTPCR of Mtb from lung homogenates; showed a variable -fold down regulation (13- to 103-fold), during the entire course of in-vivo studies. The maximum fold repression of targets equated that the in-vivo transcript translation into cidality is target-vulnerability-dependent. Target <i>rpoB</i> translated into maximum cidality of 3.9 log₁₀ cfu reduction with mere 13-fold transcript level repression; whereas, in the case of <i>ppk</i>, only 1.3 log₁₀ cfu reduction could be achieved in-vivo despite a maximum of 103-fold transcript repression (as in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154513#pone.0154513.s002" target="_blank">S1 File</a>).</p