Domain-swapped T cell receptors improve the safety of TCR gene therapy

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy

Osteoclast Activated FoxP3+ CD8+ T-Cells Suppress Bone Resorption in vitro

BACKGROUND: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption. PRINCIPAL FINDINGS: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts. SIGNIFICANCE: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology

The role of testosterone, the androgen receptor, and hypothalamic-pituitary–gonadal axis in depression in ageing Men

Considerable research has shown that testosterone regulates many physiological systems, modulates clinical disorders, and contributes to health outcome. However, studies on the interaction of testosterone levels with depression and the antidepressant effect of testosterone replacement therapy in hypogonadal men with depression have been inconclusive. Current findings indicate that low circulating levels of total testosterone meeting stringent clinical criteria for hypogonadism and testosterone deficiency induced by androgen deprivation therapy are associated with increased risk for depression and current depressive symptoms. The benefits of testosterone replacement therapy in men with major depressive disorder and low testosterone levels in the clinically defined hypogonadal range remain uncertain and require further investigation. Important considerations going forward are that major depressive disorder is a heterogeneous phenotype with depressed individuals differing in inherited polygenic determinants, onset and clinical course, symptom complexes, and comorbidities that contribute to potential multifactorial differences in pathophysiology. Furthermore, polygenic mechanisms are likely to be critical to the biological heterogeneity that influences testosterone-depression interactions. A genetically informed precision medicine approach using genes regulating testosterone levels and androgen receptor sensitivity will likely be essential in gaining critical insight into the role of testosterone in depression

Germline DNA damage response gene mutations as predictive biomarkers of immune checkpoint inhibitor efficacy

BackgroundImpaired DNA damage response (DDR) can affect immune checkpoint inhibitors (ICI) efficacy and lead to heightened immune activation. We assessed the impact of pathogenic or likely pathogenic (P/LP) germline DDR mutations on ICI response and toxicity.Materials and methodsA retrospective analysis of 131 cancer patients with germline DNA testing and ICI treatment was performed.ResultsNinety-two patients were DDR-negative (DDR-), and 39 had ≥1 DDR mutation (DDR+). DDR+ patients showed higher objective response rates (ORRs) compared to DDR- in univariate and multivariable analyses, adjusting for age and metastatic disease (62% vs. 23%, unadjusted OR = 5.41; 95% CI, 2.41-12.14; adjusted OR 5.94; 95% CI, 2.35-15.06). Similar results were seen in mismatch repair (MMR), DDR pathways with intact MMR (DDR+MMRi), and homologous recombination (HR) subgroups versus DDR- (adjusted OR MMR = 24.52; 95% CI 2.72-221.38, DDR+MMRi = 4.26; 95% CI, 1.57-11.59, HR = 4.74; 95% CI, 1.49-15.11). DDR+ patients also had higher ORRs with concurrent chemotherapy (82% vs. 39% DDR-, p=0.03) or concurrent tyrosine kinase inhibitors (50% vs. 5% DDR-, p=0.03). No significant differences in immune-related adverse events were observed between DDR+ and DDR- cohorts.ConclusionP/LP germline DDR mutations may enhance ICI response without significant additional toxicity

Tc_REG suppress mature osteoclasts by effecting the cytoskeletal reorganization:

<p>Osteoclasts were cultured either alone or with Tc_REG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel <b>A</b>. Quantitation of three independent experiments is shown in panel <b>B</b>. Panel <b>C</b> is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Tc_REG inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.

<p><b>A</b>. Osteoclasts were seeded on hydroxyapatite-coated plates and allowed to adhere overnight. The osteoclasts were pulsed with SIINFEKL peptide (+Antigen) or a control FLAG peptide (-Antigen). OT-I Tc_REG or naïve CD8 T-cells were added and co-cultured for 7 days. Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days. The plates were then treated with bleach solution, washed, dried and photographed. Representative photomicrographs are shown on the left. Quantitation from four experiments of three wells each is shown on the left. <b>B</b>. Tc_REG were added to top-insert of transwell (0.45-µ membrane) separated from the osteoclast plated on 24-well Corning Osteo-Assay plate. Osteoclast activity was determined by quantifying total pit area resorbed following 10 days of co-incubation. <b>C</b>. In a culture of both osteoclast and Tc_REG on Corning Osteo-Assay plates, neutralizing antibodies against IL-10 (25 µg/ml), IFN-γ (50 µg/ml), IL-6 (20 µg/ml) or CTLA-4 (10 µg/ml) were added to determine their impact on osteoclast activity. The cells were co-cultured for 10 additional days, and re-fed with media containing M-CSF, RANKL every three days. Re-feed media with antibodies were added on days 3 and 6. <b>D</b>. Pitting assay as described in Panel C were conducted in parallel using osteoclasts from either wild type (WT) or IFNγR1^−/− mice. Statistical significance of area resorbed was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01 and ***: P<0.001 in comparison to osteoclast alone wells.</p

TcREG inhibit osteoclast resorption.

<p>A. Osteoclasts (day 3) lifted and plated on hydroxyapatite-coated plates. OT-I Tc_REG or OT-II TGFβ-induced FoxP3⁺ CD4 T-cells (iT_REG) generated in separate dishes were added next day. The co-cultures were re-fed every two days. After seven days, the wells were treated with bleach, photographed, and total pit area was quantified (results in panel B). Representative images from five replicates are shown in panel <b>A</b>. No pitting was observed in the presence of Tc_REG (top right). Larger pits were observed in the presence of iT_REG (bottom left), but Tc_REG were dominant suppressors (bottom right). <b>B</b>. Tc_REG suppressed pitting on hydroxyapatite plates without re-stimulation. In contrast, iT_REG could only suppress after re-stimulation (see methods for details). IFN-γ producing OT-I T-cells activated by anti-CD3 and anti-CD28 in the presence of IL-2 could partially suppress pitting by osteoclasts. Activated GFP⁺ CD8 T-cells purified by cell sorting from FoxP3^eGFP reporter mice could also suppress osteoclast pitting, while conventional (GFP⁻) CD8 had no affect on pitting. Statistical significance of area resorbed was assessed by Wilcoxon test: *: P<0.05, **: P<0.01 and ***: P<0.001 relative to osteoclast alone wells.</p