Overcoming triple-negative breast cancer (TNBC) resistance to oncolytic virotherapy by histone deacetylase inhibitor, trichostatin A

"Triple-negative breast cancer (TNBC) is a tumor classification that lack receptors for the hormones estrogen, progesterone and HER2 protein. These malignancies are characterized to be of poor prognosis, refractoriness to conventional therapy and high rates of recurrence. Virotherapy with oncolytic adenovirus (OAd) consists of cancer selective viruses that replicate, spread, and kill cancer cells by oncolysis, without affecting the normal cells."--Introduction

Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-), FoxP3(-)) and suppressor (CD25(+), FoxP3(+)) CD4(+) T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4(+) subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+)CD25(-) T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis

4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression.

Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4+ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4+CD25+FoxP3+ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role

CD4+ T Cells Play a Critical Role in the Generation of Primary and Memory Antitumor Immune Responses Elicited by SA-4-1BBL and TAA-Based Vaccines in Mouse Tumor Models

The role of CD4+ T cells in the generation of therapeutic primary and memory immune responses in cancer diverse immunotherapy settings remains ambiguous. We herein investigated this issue using two vaccine formulations containing a novel costimulatory molecule, SA-4-1BBL, as adjuvant and HPV E7 or survivin (SVN) as tumor associated antigens (TAAs) in two mouse transplantable tumor models; the TC-1 cervical cancer expressing xenogeneic HPV E7 and 3LL lung carcinoma overexpressing autologous SVN. Single vaccination with optimized SA-4-1BBL/TAA formulations resulted in the eradication of 6-day established TC-1 and 3LL tumors in.70 % of mice in both models. The in vivo depletion of CD4+ T cells one day before tumor challenge resulted in compromised vaccine efficacy in both TC-1 (25%) and 3LL (12.5%) tumor models. In marked contrast, depletion of CD4+ T cells 5 days post-tumor challenge and one day prior to vaccination did not significantly alter the therapeutic efficacy of these vaccines. However, long-term immunological memory was compromised in the 3LL, but not in TC-1 model as a significant number (85.7%) of tumor free-mice succumbed to tumor growth when rechallenged with 3LL cells 60 days after the initial tumor inoculation. Collectively, these results demonstrate the indispensable role CD4+ T cells play in the generation of therapeutic primary immune responses elicited by SA-4-1BBL/TAA-based vaccines irrespective of the nature of TAAs and establish the importance of CD4+ T cells for long-term immun

4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4⁺CD25⁺FoxP3⁺ T Regulatory Cell Suppression

<div><p>Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4⁺ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.</p></div

Costimulation by SA-4-1BBL overcomes suppression of Tconvs by Tregs independent of APCs.

<p><b>(A)</b> Flow-sorted Tconvs were cocultured with freshly sorted or expanded Tregs at 1:1 ratio in the presence of irradiated splenocytes from wild type or 4-1BB^-/- mice as APCs and anti-CD3 Ab (0.5 μg/ml). SA-4-1BBL (1 μg/ml) was added to cultures where indicated. Cultures were incubated for 48 h, pulsed with [³H]-thymidine for an additional 16 h, and proliferation was measured and graphed as cpm. <b>(B)</b> Suppression assay was conducted with sorted Tconvs and Tregs without APCs using plate bound anti-CD3 Ab (5 μg/ml) and soluble SA-4-1BBL (1 μg/ml) where indicated. Each data point is indicative of mean ± SEM of triplicate wells and representative of 2 separate experiments. Student’s t-test (two-tailed) was performed for statistical analysis with <i>*p</i> ≤ 0.05 being significant.</p

IL-2 is the predominant cytokine upregulated in Tconv and Tregs cocultures costimulated with SA-4-1BBL.

<p>Sorted Tconvs and Tregs were cocultured at 1:1 ratio and 48 h later supernatants were collected and subjected to cytometric bead array analysis. <b>(A)</b> Tconv:Treg cocultures containing irradiated APCs, SA-4-1BBL (1 μg/ml) as shown, and soluble anti-CD3 Ab (0.5 μg/ml). <b>(B)</b> Tconv:Treg cocultures without APCs, but including plate bound anti-CD3 Ab (5 μg/ml) and SA-4-1BBL (1 μg/ml) as indicated. Data shown in (<b>A</b> and <b>B</b>) is combination of two independent experiments with mean ± SEM reported. <b>(C, D)</b> RT-PCR ΔΔCT values for relative IL-2 mRNA expression with respect to GAPDH gene in indicated cultures stimulated with SA-4-1BBL (1 μg/ml) and plate-bound anti-CD3 Ab (5 μg/ml). IL-2 expression was assessed at 24 <b>(C)</b> and 48 <b>(D)</b> hours post-stimulation. Each data point in (<b>C</b> and <b>D</b>) represents the mean of triplicate wells ± SD, with *<i>p</i> ≤ 0.05 being significant.</p

Removal of SA-4-1BBL and IL-2 from Tconv culture supernatants.

<p><b>(A)</b> Supernatant (sup) of Tconvs stimulated with an agonistic anti-CD3 Ab (5 μg/ml) and SA-4-1BBL (1 μg/ml) were incubated with CuSO₄-charged sepharose beads to remove SA-4-1BBL carrying a 6xHis tag. Regular sup (batch 1) and SA-4-1BBL-depleted sup (batch 2) were analyzed using anti-SA antibodies for detection in Western blots. The indicated amounts of SA-4-1BBL protein were used as detection controls. <b>(B)</b> Batch 2 supernatant described in (A) was incubated with an anti-IL-2 Ab (20 μg/ml) followed by the removal of Ab/IL-2 complexes using immobilized protein G beads to generate batch 3. Batch 2 as well as batch 3 supernatants (50 μl/each) were tested on the IL-2-dependent CTTL-2 cell line with the indicated commercial recombinant IL-2 doses (IU/ml) as positive and PBS as negative controls. Cultures were incubated for 28 h with [³H]-thymidine added for the last 8 h. Proliferation was measured and graphed as cpm. Each data point is indicative of mean ± SEM of triplicate wells. Student’s t-test (two-tailed) was performed for statistical analysis with *<i>p</i> ≤ 0.05 being significant. The data is representative of two independent experiments.</p

SA-4-1BBL costimulation-mediated IL-2 production by Tconvs is both necessary and sufficient in overcoming Treg suppression.

<p>Sorted Tconvs and Tregs were cocultured at 1:1 ratio in the presence <b>(A)</b> or absence <b>(B)</b> of irradiated APCs and anti-CD3 Ab. Cultures were also supplemented with SA-4-1BBL (1 μg/ml), IL-2 (3 IU/ml), SA-4-1BBL-depleted supernatants (100 μl/well), or SA-4-1BBL- and IL-2-depleted supernatants (100 μl/well) where indicated. <b>(C)</b> Tconvs sorted from WT C57BL/6 or Tregs sorted from C57BL/6.Foxp3^hCD2 mice were labeled with 2.5μM CFSE and used in coculture suppression assays with irradiated APCs. Cultures were incubated for 64 h and cells were analyzed using flow cytometry for proliferation. <b>(D)</b> Tregs were irradiated at 2000 cGy (Tregs*) and used in coculture experiments mimicking panel <b>A</b>. Experiments were repeated twice with similar patterns. Student’s t-test (two-tailed) was performed for statistical analysis with *<i>p</i> ≤ 0.05 being significant.</p

SA-4-1BBL/MPL as a novel immune adjuvant platform to combat cancer

WOS: 000373383600041Practical experience with cancer vaccines combined with accumulated knowledge of the complex interactions between cancer and immune system rationalize the combinatorial use of immune adjuvants for better efficacy. We recently described a novel adjuvant system based on the costimulatory SA-4-1BBL and TLR4 agonist MPL that has desired therapeutic and safety profiles