Objective To describe the ophthalmic and systemic autoimmune findings after successful adoptive cell transfer of ex vivo expanded, autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) for metastatic melanoma. Design Retrospective, interventional case report. Participant A 35-year-old man who underwent immunotherapy for metastatic melanoma with adoptive cell transfer of tumor-reactive TIL. Methods A 35-year-old man with metastatic melanoma was treated with TIL plus interleukin-2 (IL-2) therapy after a lymphodepleting regimen of cyclophosphamide and fludarabine for metastatic melanoma, which led to a complete and durable remission. Bilateral panuveitis, hearing loss, vitiligo, poliosis, and alopecia developed in the patient, requiring local ophthalmic immunosuppressive therapy. The clinical course, diagnostic testing, and therapeutic interventions over a 2-year period are reviewed. Main Outcome Measures Visual acuity, anterior chamber and vitreous inflammation, optical coherence tomography findings, serial electro-oculograms (EOGs), microperimetry (MP-1) testing, flow cytometric analysis of cells derived from the aqueous humor, and aqueous humor cytokine profiles were evaluated. Results After melanoma immunotherapy, complete tumor regression was achieved at 5 months after treatment with a durable, ongoing, complete remission at 24 months. Early in the treatment course, a high fever, a diffuse rash, hearing loss, and bilateral anterior uveitis developed acutely in the patient. Late autoimmune sequelae included the development of alopecia, vitiligo, poliosis, and bilateral panuveitis with diffuse retinal pigment epithelium (RPE) hypopigmentation, reminiscent of Vogt-Koyanagi-Harada (VKH) syndrome. Bilateral cystoid macular edema also developed that was responsive to acetazolamide. Serial EOGs showed alterations in RPE standing potentials in dark conditions, and MP-1 testing revealed diminished foveal and perifoveal sensitivity. An aqueous humor aspirate revealed a high concentration of melanoma tumor antigen-reactive T cells compared with that of peripheral blood samples, as well as a proinflammatory aqueous cytokine profile. At the time of cataract surgery 22 months after immunotherapy, a repeat aqueous humor sample showed the disappearance of the previously seen melanoma differentiation antigen-reactive lymphocytes, but the proinflammatory cytokine profile persisted. Conclusions Ocular and systemic autoimmune sequelae resembling VKH may develop after successful melanoma immunotherapy. This report provides insight into the pathogenesis of VKH disease. The patient’s clinical course illustrates the fine balance between tumor-specific immunity and loss of self-tolerance.

Usually mast cells (MCs) modulate other cellular activities through the release of their cytoplasmic granules. Recently, gap junctional intercellular communication (GJIC) between an established human MC cell line (HMC-1) co-cultured with human dermal fibroblasts in fibroblast populated collagen lattices (FPCLs), enhanced the rate and degree of FPCL contraction. However, HMC-1 cells were unable to generate GJIC with human neonatal fibroblasts in monolayer culture. Here freshly isolated rat peritoneal MCs are co-cultured with fibroblasts in collagen lattices and in monolayer culture in vitro and introduced into rat polyvinyl alcohol (PVA) sponge implants in vivo. Co-cultured MC-FPCL contracted faster and to a greater degree. Loading Calcein AM green fluorescent dye into red fluorescent Dil tagged MC generates MC-paratroopers. When MC-paratroopers form GJIC with fibroblasts, some green dye is passed into the fibroblast, while the MC-paratrooper retains both its red and green fluorescence. MC-paratroopers passed green fluorescent dye into both human and rat dermal fibroblasts in monolayer culture. In rats 7-day-old subcutaneous PVA sponge implants, which received an injection of MC-paratroopers, exhibited auto-fluorescent green fibroblasts, when harvested 24 h later. MC-paratroopers pretreated with a long-acting GJIC inhibitor prior to their introduction into PVA sponge implants, failed to pass dye into fibroblasts. It is proposed that GJIC between granulation tissue fibroblasts and MCs can modulate some aspects of wound repair and fibrosis.

Human ex vivo gene therapy protocols have been used successfully to treat a variety of genetic disorders, infectious diseases, and cancer. Murine oncoretroviruses (specifically, gammaretroviruses) have served as the primary gene delivery vehicles for these trials. However, in some cases, such vectors have been associated with insertional mutagenesis. As a result, alternative vector platforms such as lentiviral vectors (LVVs) are being developed. LVVs may provide advantages compared with gammaretroviral vectors, including the ability to transduce large numbers of nondividing cells, resistance to gene silencing, and a potentially safer integration profile. The aim of this study was to develop a simplified process for the rapid production of clinical-grade LVVs. To that end, we used a self-inactivating bicistronic LVV encoding an MART (melanoma antigen recognized by T cells)-1-reactive T cell receptor containing oPRE, an optimized and truncated version of woodchuck hepatitis virus posttranslational regulatory element (wPRE). Using our simplified clinical production process, 293T cells were transiently transfected in roller bottles. The LVV supernatant was collected, treated with Benzonase, and clarified by modified step filtration. LVV produced in this manner exhibited titers and a biosafety profile similar to those of cGMP (current Good Manufacturing Practices) LVVs previously manufactured at the Indiana University Vector Production Facility in support of a phase I/II clinical trial. We describe a simple, efficient, and low-cost method for the production of clinical-grade LVV for ex vivo gene therapy protocols.