KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.

Introduction: Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. Methods: To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing. Results: KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015). Conclusions: Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.

Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.

Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations.

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.

Modulatory effect of a complex fraction derived from colostrum on fibroblast contractibility and consequences on repair tissue.

A complex compound (immune ('IM') fraction) from colostrum-derived whey was investigated for its potential wound healing properties. One of its most intriguing in vitro abilities was to significantly inhibit the contraction of collagen gel while fibroblast density remained as in control gels. This antagonist effect was dose dependent and fibroblasts in these gels did not exhibit any stress fibres. Subsequently, in vivo studies have been conducted in two wound models in guinea pigs. Daily application on full-thickness wounds of a liquid formulation of the IM fraction (first model) significantly delayed wound closure by contraction compared to what normally occurred in control wounds. In another wound model, a gel formulation of the IM fraction was applied on scar tissues, which resulted in a minimised residual scar on 5/8 wounds compared to corresponding wound areas seen prior to treatment. Conversely, most control wounds exhibited scar tissue from which 3/8 resembled hypertrophic scar tissue. Wound tissue treated with IM fraction covered a significantly larger area than in the control wounds, whereas the collagen deposition was unchanged as in the presence of α-smooth muscle actin. Thus, IM fraction may act by modulating the contraction rate and wound remodelling.

Expression of hck-tr, a truncated form of the src-related tyrosine kinase hck, in bovine spermatozoa and testis.

In bull testicular haploid germ cells, an mRNA encoding for hck was detected in addition to another one encoding for hck-tr, a truncated form of the tyrosine kinase hck. As the transcripts were expressed in spermatids, we tried to determine whether hck-tr is present in mature bovine spermatozoa. Two polyclonal antibodies were produced against peptides specific to the N- and C-terminal portions of the truncated protein. Western blot analyses confirmed the presence of hck-tr in total protein extracts of ejaculated bull spermatozoa, and sub-cellular fractionation experiments suggest its presence in both head and flagellum. The truncated protein appears tightly associated with cytoskeletal elements as it could be extracted only with SDS under reducing conditions. When assessed by indirect immunofluorescence, hck-tr was mostly localized at the acrosomal area of the sperm cell and a similar localization was observed on demembranated spermatozoa. Immunohistochemical studies on testis sections revealed protein expression in spermatocytes as well as in round and elongating spermatids. The results presented in this study clearly show the presence of mRNAs encoding for hck and hck-tr in testicular germ cells; hck-tr being translated during spermatogenesis and expressed on mature ejaculated bull spermatozoa.

Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

Developmental expression of SRC-related tyrosine kinases in the mouse testis.

An increase in protein tyrosine phosphorylation occurs during sperm capacitation in numerous species. The involvement of Src-related tyrosine kinases in this phenomenon has been demonstrated using different inhibitors specifically targeting this family of enzymes. In mammals, this group of nonreceptor tyrosine kinases is made up of 8 members with similar SRC homology domain 3 (SH3) and SH2 domains. Although some members of this group of enzymes can compensate for one another, showing some redundancy, each is unique and may perform specific functions during male germ cell development. To further characterize the importance of Src-related tyrosine kinases in the events leading to proper sperm formation, and because no inhibitor affecting a single gene product exists, expression of Src, Yes1, Fyn, Lyn, Lck, Hck, Blk, and Fgr was assessed by real-time polymerase chain reaction in developing mouse testes and in enriched populations of mouse spermatogenic cells, revealing distinct expression profiles for each kinase during testis development and in isolated male germ cells. Immunolocalization of SRC, LYN, and HCK in adult mouse testes as well as in mature spermatozoa further confirmed differential localization of these kinases during spermatogenesis. Although mRNA levels of these latter kinases were higher in spermatogonia and spermatocytes than in spermatids, protein levels were highest in spermatids, suggesting delayed transcript translation. Taken together, these results clearly show an uneven expression of each kinase in different spermatogenic cells, indicating that each member may play a different role during spermatogenesis, in addition to highlighting the complexity of Src-related kinase expression regulation in male germ cells. Furthermore, differential localization of these tyrosine kinases in mature spermatozoa also suggests a specific role for each member in sperm function and integrity.

The stimulation of angiogenesis and collagen deposition by copper.

Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO(4) with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO(4) in a cylindrical collagen-based scaffold. CuSO(4) enhanced significantly the invasion of microvessel compared to control implants and to 20ng FGF-2+/-CuSO(4). Vascular infiltration was also significantly improved by combination of CuSO(4) with FGF-2, compared to FGF-2 alone (0.2 and 1microg). Nevertheless, in comparison with CuSO(4) alone, there was a significant increase only with 1microg of FGF-2 combined with CuSO(4). Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO(4) only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.

Bull testicular haploid germ cells express a messenger encoding for a truncated form of the protein tyrosine kinase HCK.

Protein tyrosine phosphorylation is a process that has been studied worldwide during sperm capacitation and acrosomal exocytosis events. Although few capacitation-induced phosphotyrosine-containing proteins have been identified, little is known about the tyrosine kinases directly involved in this post-translational modification. Different studies from our and other groups using tyrosine kinase inhibitors suggest the involvement of members of the family of src-related tyrosine kinases in the sperm capacitation associated increase in protein tyrosine phosphorylation. Using a molecular biology approach, we report for the first time messengers encoding for members from the src-related tyrosine kinase family in bovine spermatogenic cells. Degenerated primers were designed within a highly homologous region specific to the family of src tyrosine kinases, and RNAs coding for c-src, c-yes, lyn, lck, and hck were identified in bull testis and haploid germ cells by RT-PCR. We also report the presence of a messenger in haploid bull germ cells that could encode for a truncated isoform of the hck tyrosine kinase. This messenger was detected by screening of a haploid germ cells cDNA library using the RT-PCR product homologous to hck as a probe. The presence of this transcript in haploid germ cell RNA preparations was validated by RT-PCR, 3'RACE, 5'RACE as well as Northern blot. Such a truncated protein could function as an adaptor protein or as a competitive inhibitor in spermiogenesis or mature sperm functions.

Cut-off phenomenon in the protective effect of alcohols against lysophosphatidylcholine-induced calcium overload.

We studied the effect of chain length on the protective effect of alcohols against lysophosphatidylcholine (LPC)-induced Ca2+ overload in neonatal rat cardiomyocytes. We previously found that ethanol retards Ca2+ elevation. Cells were loaded with the Ca2+-sensitive fluorophore fura-2, and changes in fluorescence were followed. The addition of 10 microM LPC increased Ca2+, which reached a plateau after an 8-10 min delay. The presence of 88 mM n-propanol, n-butanol, tert-butanol, or 2,2-dimethylpropanol significantly increased the delay by 94-213%. However, n-pentanol at 2 mM or 88 mM had no protective effect. Among n-alcohols, the increase in lag time was inversely proportional to the length of the carbon chain. Chain length, rather than molecular weight determines the effect, because 2,2-dimethylpropanol had a protective effect. The influence of alcohols on LPC micelle formation was estimated from the increase in octadecyl rhodamine B fluorescence; the increase by n-alcohols was directly proportional to chain length, indicating that micelle formation was not involved in the extension of lag time. The absence of the protective effect when the alcohol aliphatic chain exceeds four carbons suggests that the effect of ethanol may be mediated via a small lipophilic pocket on a protein, or to lateral pressure perturbation in the membrane.