Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

Background: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). Methodology/Principal Findings In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4+ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G+ FcγRIIIa(CD16A)+ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4+ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A+ CD56+ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. Conclusions/Significance: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A+ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration

CHS response is significantly reduced in dermal γδ T cell-deficient chimeric mice.

<p>A, Generation of chimeric mice. 5–10 x 10⁶ neonatal thymocytes from newborn C57BL/6 mice (day 0–1 after birth) were transferred to half of the irradiated mice. 24 hours later, 5 x 10⁶ bone marrow (BM) cells from adult naïve C57BL/6 mice were intravenously transferred to all irradiated mice. At least 12 weeks later, these BM or BM + thymocytes chimeric mice were sensitized with 0.25% DNFB on left ears at first two days. 5 days later, the right ears were challenged with one dose of 0.25% DNFB. The ear thickness was measured at 0–120 hours after challenge (B). *: P<0.05. 5 mice for each group were tested. Results are representative of two independent experiments.</p

Dermal γδ T cells rapidly increase in situ and produce large amounts of IL-17 after DNFB challenge.

<p>The DNFB-induced CHS model using wild type C57BL/6 mice was established as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a>. 24 hours after DNFB challenge, right ears were collected and the frequency and number of dermal γδ T cells were analyzed by flow cytometry (A: of total skin cells; and B: per ear). In parallel, parts of skin cells were incubated in RPMI 1640 media supplemented with 5% FCS, 1% P/S, 0.25% DNFB and Brefeldin A at 37°C. 6–7 hours later, IL-17 production of total dermal γδ T cells and the proportion of IL-17-producing Vγ4⁺ cells were analyzed by intracellular staining (C). D, Vγ4⁺ γδ T cells were depleted by i.v. injection of anti-Vγ4 antibody or isotype antibody for 3 consecutive days. DNFB-induced CHS model was created as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a>. Ear swelling was measured during 0–120 hours after challenge. E: Vγ4⁺ cells were depleted in LN and skin after 3 consecutive days of anti-Vγ4 injection. F: The increase of ear thickness after DNFB challenge. *: P<0.05. **: P<0.01. 5 mice for each group were tested. Results are representative of three independent experiments.</p

CHS response is reduced significantly after depletion of Gr-1⁺ neutrophils in vivo.

<p>Naive C57BL/6 mice were treated with DNFB as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a> to generate DNFB-CHS model. 1 day prior to DNFB challenge, mice were i.p. injected with 100 μg anti-Gr-1 or 100 μg isotype antibodies (rat-IgG2b). Neutrophils (CD11b⁺ Gr-1⁺) in blood, spleen, and challenged skin site at 48 hours after DNFB challenge were analyzed by FACS (A). The ear thickness was measured at 0–120 hours after challenge (B). *: P<0.05. 5 mice for each group were tested. Results are representative of three independent experiments.</p

H&E staining of DNFB-challenged skin.

<p>Naive C57BL/6 mice were treated with DNFB as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169397#pone.0169397.g002" target="_blank">Fig 2</a> to generate DNFB-CHS model. 1 day prior to DNFB challenge, mice were i.p. injected with 100 μg anti-Gr-1, 100 μg anti-IL-17, or 100 μg isotype antibodies. 48 hours after DNFB challenge, the challenged skin site was harvested for H&E staining. 5 mice for each group were tested. Results are representative of two independent experiments. The bar represents 100 μm.</p

The sensitized CD4⁺ or CD8⁺ T cells infiltrate into DNFB-treated skin normally after intravenous transfer while Gr-1⁺ CD11b⁺ neutrophils are greatly reduced in dermal γδ T cell-deficient chimeric mice.

<p>A, Wild type CD45.1⁺ C57BL/6 mice were sensitized with DNFB at first 2 days. 5 days later, dLNs were collected to prepare cell suspension. After washed with PBS, 2 x 10⁷ CD45.1⁺ sensitized leukocytes were intravenously transferred to CD45.2⁺ BM or BM+thymocytes chimeric mice. 1 day after transfer, the ears of chimeric mice were treated with 0.25% DNFB. 48 hours later, the treated ears were collected for flow cytometry analysis. B, The frequency of total or donor T cells. C and D are the numbers of total or donor T (CD4⁺ or CD8⁺) cells in the skin, respectively. E and F are the frequency and number of total or donor Gr-1⁺CD11b⁺ neutrophils in the skin, respectively. NS: no significant. **: P<0.01. Results are representative of two independent experiments.</p

CHS response is remarkably reduced in TCR δ^-/- mice.

<p>WT or TCR δ^-/- mice were sensitized with 0.25% DNFB on left ears for 2 consecutive days. 5 days later, the right ears were challenged with one dose of 0.25% DNFB. The ear thickness was measured at 0–120 hours after challenge (A and B). *: P<0.05. 5 mice for each group were tested. Results are representative of two independent experiments.</p