Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy

<div><p>Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with <i>in vitro</i> scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the <i>in vitro</i> scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.</p></div

Scratch closure is faster in fetal fibroblast and keratinocyte co-cultures than in fibroblast or keratinocyte mono-cultures.

<p><b>A</b>, Representative images of the progression of the scratch closure at T0, 5 h and 20 h for keratinocytes (upper panel), fibroblasts (middle panel) and both in co-culture (lower panel). <b>B</b>, The extent of the scratch closure in scratch assays performed on fetal fibroblasts, keratinocytes or both in co-culture at 5 h and 20 h. Data shown are the means ± SD (n = 4). Asterisks denote significant differences in the extent of the scratch closure (*, p<0.05; **, p<0.01).</p

Multiplex cytokine analysis of cell culture supernatants from fetal and adult fibroblasts and keratinocytes mono-cultures or fibroblasts and keratinocytes co-cultures.

<p>Supernatants from fibroblast and keratinocyte mono-cultures or both cell types in co-culture (ratio 1∶1) were collected to detect soluble HGF, GM-CSF, IL-1α, VEGF-A and IL-8 by multiplex immunoassay (see materials and methods). For adult skin cells, values are the means of three samples (see table 1). Fib., fibroblasts mono-culture; Ker., keratinocytes mono-culture; Fib./Ker. (1∶1), fibroblasts and keratinocytes co-culture at the ratio 1∶1.</p

Immunogenicity and Immunosuppressive activity of fetal cells.

<p><b>A,</b> MHC class I and class II expression in fetal cells. Flow cytometry analysis of MHC class I and class II expression in fetal fibroblasts and kerationcytes treated or not with 1 µg/ml IFN-γ for 48 hours. Full histograms represent test samples, solid lines represent isotypic controls. Percentage of positive cells and mean fluorescence intensity values (MFI) are indicated. Cytograms are typical of two independent experiments. <b>B,</b> Proliferation of allogeneic PBMC in co-culture with fetal fibroblasts, keratinocytes or both cell types in co-culture. PBMC were initially plated with various fetal cell numbers corresponding to four different ratios between fetal cells and PMBC, 1∶4, 1∶20, 1∶100 and 1∶200. PBMC were collected from the co-cultures after 5 days and treated with ³H-thymidine for 16 hours before radioactive counting. Data represent the means ± SD (n = 3) of ³H-thymidine incorporation by proliferative PBMC. Asterisks denote significant differences in PBMC proliferation compared to the positive control (column 2) (*, p<0.05; **, p<0.01; ***, p<0.001). Data are representative of three independent experiments performed with PBMC obtained from different healthy donors.</p

Characterization of the fetal fibroblasts and keratinocytes manufactured of the clinical batches.

<p><b>A</b>, Morphological and molecular characterization. Left panel, microphotography of the fetal fibroblasts and keratinocytes. Middle panel, immunofluorescence of cultured fetal fibroblasts and kerationcytes from the clinical batches, stained for nuclei (blue), prolyl-4-hydroxylase (green) and keratin 14 (red). Right panel, cytometry histograms showing the percentage of prolyl-4-hydroxylase positive cells and the percentage of keratin14 positive cells in the fetal fibroblasts and keratinocytes, respectively. Full histograms represent test samples, solid lines represent isotypic controls. <b>B</b>, Integrin expression profiles. Cytometry histograms showing β1-, α2- and α3-integrin expression in fetal fibroblasts (upper panel) and keratinocytes (lower panel). Cytograms are typical of at least three independent experiments.</p

Phase I/II clinical trial of adoptive cell transfer of sorted specific T cells for metastatic melanoma patients

International audienceAdoptive cell transfer (ACT) of tumor-specific T lymphocytes represents a relevant therapeutic strategy to treat metastatic melanoma patients. Ideal T-cells should combine tumor specificity and reactivity with survival in vivo, while avoiding autoimmune side effects. Here we report results from a Phase I/II clinical trial (NCT02424916, performed between 2015 and 2018) in which 6 metastatic HLA-A2 melanoma patients received autologous antigen-specific T-cells produced from PBMC, after peptide stimulation in vitro, followed by sorting with HLA-peptide multimers and amplification. Each patient received a combination of Melan-A and MELOE-1 polyclonal specific T-cells, whose specificity and anti-tumor reactivity were checked prior to injection, with subcutaneous IL-2. Transferred T-cells were also characterized in terms of functional avidity, diversity and phenotype and their blood persistence was evaluated. An increase in specific T-cells was detected in the blood of all patients at day 1 and progressively disappeared from day 7 onwards. No serious adverse events occurred after this ACT. Clinically, five patients progressed and one patient experienced a partial response following therapy. Melan-A and MELOE-1 specific T-cells infused to this patient were diverse, of high avidity, with a high proportion of T lymphocytes coexpressing PD-1 and TIGIT but few other exhaustion markers. In conclusion, we demonstrated the feasibility and safety of ACT with multimer-sorted Melan-A and MELOE-1 specific T cells to metastatic melanoma patients. The clinical efficacy of such therapeutic strategy could be further enhanced by the selection of highly reactive T-cells, based on PD-1 and TIGIT co-expression, and a combination with ICI, such as anti-PD-1

Tissue biomarkers in melanoma patients treated with TIL.

While treating stage III melanoma patients with autologous therapeutic TIL in an adjuvant setting, we previously reported a significant benefit of treatment on both progression-free survival and overall survival in patients with only one invaded lymph node (early stage III) compared to patients with more than one invaded lymph nodes (advanced stage III). In this context, in order to understand the difference of activity of TIL therapy according to the progression of the illness at stage III, the first objective of the present study was to determine potential differences in the characteristics of TIL populations obtained from an early stage III and a more advanced stage III when tumor burden is more important. The second objective was to determine possible differences in tissue expression level of several molecules involved in interactions between tumor cells and T cells between early and advanced stage III considering that the tumor microenvironment of invaded lymph nodes could become more tolerant with the progression of the disease. A total of 47 samples of melanoma invaded LN from stage IIIb (AJCC 2007) melanoma patients treated with TIL plus IL-2 were included in this study. We confirmed that both PFS and OS were significantly associated to the presence of tumor-reactive T-cells among TIL injected to the patients and that these tumor reactive T cells were more frequently observed at the early stage III. Moreover, while analyzing the expression of 17 markers on 34/47 tumor specimens using immunohistochemistry, we identified that 3 tissue markers involved in interactions between melanoma cells and T cells have a significant difference of expression between early and advanced stage III: MHC class I, adhesion molecule ICAM-1 and the co-stimulation molecule LFA-3 had a significantly weaker expression in melanoma tissue specimens from advanced stage III. In addition, the expression of the alpha chain of the IL-2 receptor (CD25) and the nuclear transcription factor Foxp3 was significantly increased in the melanoma tissue specimens from advanced stage III. Our results suggest differences in the immunological status of the tumor microenvironment between early and advanced stage III, which could explain the difference in clinical response to TIL infusion in an adjuvant setting between early and advanced stage III

A phase I/II feasibility vaccine study by autologous leukemic apoptotic corpse-pulsed dendritic cells for elderly AML patients

International audienceThis was a phase I/II study testing the feasibility of a vaccine by autologous leukemic apoptotic corpse-pulsed dendritic cells (DC) in elderly acute myeloid leukemia (AML) patients in first or second complete remission. Pulsed DC were administered at doses of 9 × 106 subcutaneously (1 mL) and 1 × 106 intra-dermally (0.1 mL). Five doses of vaccine were planned on days +1 + 7 + 14 + 21 and +35. Five DC-vaccines were produced and injected for all five patients included in the study. No severe adverse event was documented. Larger Phase 2 studies are now required to precise the role of DC-vaccines with leukemic apoptotic bodies in older as well as younger AML populations

Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes in Advanced Melanoma Patients

Immunotherapy for melanoma includes adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TILs). This monocenter retrospective study was undertaken to evaluate the efficacy and safety of this treatment of patients with advanced melanoma. All advanced melanoma patients treated with TILs using the same TIL expansion methodology and same treatment interleukin-2 (IL-2) regimen between 2009 and 2012 were included. After sterile intralesional excision of a cutaneous or subcutaneous metastasis, TILs were produced according to a previously described method and then infused into the patient who also received a complementary subcutaneous IL-2 regimen. Nine women and 1 man were treated for unresectable stage IIIC (n=4) or IV (n=6) melanoma. All but 1 patient with unresectable stage III melanoma (1st line) had received at least 2 previous treatments, including anti-CTLA-4 antibody for 4. The number of TILs infused ranged from 0.23 × 109 to 22.9 × 109. Regarding safety, no serious adverse effect was reported. Therapeutic responses included a complete remission, a partial remission, 2 stabilizations, and 6 progressions. Among these 4 patients with clinical benefit, 1 is still alive with 9 years of follow-up and 1 died from another cause after 8 years of follow-up. Notably, patients treated with high percentages of CD4 + CD25 + CD127lowFoxp3+ T cells among their TILs had significantly shorter OS. The therapeutic effect of combining TILs with new immunotherapies needs further investigation