Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

Whilst the importance of keratinocytes as a first-line defense has been widely investigated, little is known about their interactions with non-resident immune cells. In this study, the impact of human keratinocytes on T cell effector functions was analyzed in an antigen-specific in vitro model of allergic contact dermatitis (ACD) to nickel sulfate. Keratinocytes partially inhibited T cell proliferation and cytokine production. This effect was dependent on the keratinocyte/T cell ratio and was partially reversible by increasing the number of autologous dendritic cells. The inhibition of T cell proliferation by keratinocytes was independent of the T cell subtype and antigen presentation by different professional antigen-presenting cells. Autologous and heterologous keratinocytes showed comparable effects, while the fixation of keratinocytes with paraformaldehyde abrogated the immunosuppressive effect. The separation of keratinocytes and T cells by a transwell chamber, as well as a cell-free keratinocyte supernatant, inhibited T cell effector functions to the same amount as directly co-cultured keratinocytes, thus proving that soluble factor/s account for the observed suppressive effects. In conclusion, keratinocytes critically control the threshold of inflammatory processes in the skin by inhibiting T cell proliferation and cytokine production.

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Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

Frontiers in Immunology ◽

10.3389/fimmu.2020.592553 ◽

2020 ◽

Vol 11 ◽

Author(s):

Christian Binder ◽

Felix Sellberg ◽

Filip Cvetkovski ◽

Erik Berglund ◽

David Berglund

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Mixed Lymphocyte Reaction ◽

T Cell Proliferation ◽

Allogeneic Mixed Lymphocyte Reaction ◽

Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

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