Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

Ilona Hromadnikova,Katerina Kotlabova,Anne M Dickinson,Shuang Li,Alan Graham Pockley

doi:10.1371/journal.pone.0151535

Abstract

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.

Highlights

Previous studies from Multhoff and colleagues have reported the selective expression of membrane form of the 70 kDa heat shock protein Hsp70 on tumour cells, including leukaemia blasts, but not on corresponding non-malignant tissues or non-transformed cells [1, 2, 3]
We have recently reported the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70 derived 14-mer peptide (TKD) on cell surface expression of NK activatory and inhibitory receptors in CD3-CD56+ cellular population within peripheral blood mononuclear cells of healthy individuals [12]
The major NK activatory receptor NKG2D was highly expressed mainly on NKT cells, NKG2D was found on the cell surface of CD8+ T cells and B cells

Summary

Introduction

Previous studies from Multhoff and colleagues have reported the selective expression of membrane form of the 70 kDa heat shock protein Hsp on tumour cells, including leukaemia blasts, but not on corresponding non-malignant tissues or non-transformed cells [1, 2, 3]. A membrane Hsp positive tumor phenotype has been found to be associated with a significantly decreased overall survival in patients with lower rectal and lung carcinomas [4]. Plasma membrane Hsp was demonstrated to act as a tumor-specific recognition structure for pre-activated NK cells expressing high amounts of CD94. Multhoff et al [8] demonstrated that similar to full-length Hsp protein, the N-terminalextended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450–463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp protein. Gastpar et al [9] previously shown that the incubation of lymphocytes with Hsp70-derived peptide TKD in the presence of a low dose of IL-2 results in an enhanced cytolytic and migratory capacity of NK cells toward Hsp membrane-positive tumor cells in vitro and in a xenograft tumor mouse model [10]. In a clinical phase I trial, the tolerability, feasibility, and safety of adoptively transferred, autologous IL-2/TKD-activated NK cells have been shown in patients having colorectal and lung carcinoma [4, 11]

Methods

Results

Conclusion