IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: “S2A”. Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.
The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A * 0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A * 0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.
Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38+CD138+ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.
Lung cancer cells in the tumor microenvironment facilitate immune evasion that leads to failure of conventional chemotherapies, despite provisionally decided on the genetic diagnosis of patients in a clinical setup. The current study follows three lung cancer patients who underwent “personalized” chemotherapeutic intervention. Patient-derived xenografts (PDXs) were subjected to tumor microarray and treatment screening with chemotherapies, either individually or in combination with the peptide R11-NLS-pep8; this peptide targets both membrane-associated and nuclear PCNA. Ex vivo, employing PDX-derived explants, it was found that combination with R11-NLS-pep8 stimulated antineoplastic effect of chemotherapies that were, although predicted based on the patient’s genetic mutation, inactive on their own. Furthermore, treatment in vivo of PDX-bearing mice showed an exactly similar trend in the result, corroborating the finding to be translated into clinical setup.
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