Lipid-modified low molecular weight branched polyethyleneimines (PEIs) are promising non-viral gene delivery systems that have been successfully explored for treatment of various diseases. The present study aims to determine in vitro safety of these delivery systems based on assessment of cytotoxicity with peripheral blood mononuclear cells (PBMCs), hemolysis with human red blood cells (RBC) and cytokine secretion from several sources of PBMCs. The viability of cells treated with lipopolymer/pDNA complexes was dependent on the polymer:pDNA ratio used but remained low at therapeutically relevant concentrations for most lipopolymers, except for the propionic acid substituted PEIs. The extent of hemolysis was minimal and below the accepted safety levels with most of the lipopolymers; however, some linoleic acid substituted PEIs yielded significant hemolysis activity. Unlike strong cytokine secretion from PMA/IO stimulated cells, most lipopolymer/pDNA complexes remained non-responsive, showing minimal changes in cytokine secretion (TNF-alpha, IL-6 and IFN-gamma) irrespective of the lipopolymer/pDNA formulations. The 0.6 kDa PEI with lauric acid substituent displayed slight cytokine upregulation, however it remained low relative to the positive controls. This study demonstrated that the lipid modified LMW PEIs are expected to be safe in contact with blood components. However, close attention to lipopolymer concentration and ratio of polymer to pDNA in formulations might be required for individual lipopolymers for optimal safety response in nucleic acid therapies. Statement of significance This manuscript investigated the safety aspects of various lipid modified low molecular weight polyethylenimine (LMW-PEI) polymers employed for pDNA delivery through in vitro studies. Using peripheral blood mononuclear cells (PBMCs) from multiple sources, we show that the hemolysis ability was minimal for most polymers, although a particular lipid substituent (linoleic acid) at specific ratios exhibited hemolysis. The levels of pro-inflammatory cytokines (TNF-alpha, IL-6 and IFN-gamma) were slightly upregulated only with a lauric acid substituted 0.6PEI, but remained low relative to positive control treatments. We further report the beneficial effect of polyacrylic acid additives on hemolysis and cytokine secretion to a reasonable extent. This study confirms the feasibility of using LMW-PEI as safe delivery agents for various therapeutic purposes. (C) 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The generation of a potent humoral immune response by B cells relies on the integration of signals induced by the B cell receptor, toll-like receptors and both negative and positive co-receptors. Several reports also suggest that integrin signaling plays an important role in this process. How integrin signaling is regulated in B cells is however still partially understood. Integrin activity and function are controlled by several mechanisms including regulation by molecular adaptors of the paxillin family. In B cells, Leupaxin (Lpxn) is the most expressed member of the family and in vitro studies suggest that it could dampen BCR signaling. Here, we report that Lpxn expression is increased in germinal center B cells compared to naïve B cells. Moreover, Lpxn deficiency leads to decreased B cell differentiation into plasma cells in vitro. However, Lpxn seems dispensable for the generation of a potent B cell immune response in vivo. Altogether our results suggest that Lpxn is dispensable for T-dependent and T-independent B cell immune responses.

Abstract Background Leupaxin is a member of paxillin family proteins that function as adaptor proteins in integrin signaling. Compared to other members, leupaxin is mainly expressed in leukocytes. However, the role of leupaxin in T cells is unknown. Results Leupaxin has been shown to be tyrosine phosphorylated in cells including tumor cells, fibroblasts and B cells. However we detected no leupaxin tyrosine phosphorylation in T cells. Instead, leupaxin showed mobility shift in response to TCR signaling. We demonstrated that the mobility shift was caused by serine phosphorylation and was ERK dependent. Although leupaxin is downstream of integrin receptors, LFA-1 stimulation did not induce leupaxin phosphorylation. Several potentially phosphorylated residues were mutated into alanine individually. When Ser54 was mutated, leupaxin no longer showed mobility shift, suggesting that leupaxin is phosphorylated at Ser54. Leupaxin was recruited to the immunological synapse and colocalized with LFA-1. Leupaxin has N-terminal leucine-aspartic acid (LD) domains and C-terminal LIM domains that contain four double zinc finger motifs. The LIM domains were dispensable for leupaxin localization at the contact zone. Leupaxin failed to be recruited to the immunological synapse after deleting LD2–4 domains, suggesting the importance of LD2–4 domains for the recruitment. Conclusions Leupaxin is not tyrosine phosphorylated but serine phosphorylated at Ser54 in response to TCR engagement. Leupaxin is recruited to the immunological synapse through LD2–4 domains. Studies are underway to determine the contribution of leupaxin to CTL adhesion, polarization and degranulation.

Abstract Background Leupaxin is an adaptor protein that functions downstream of integrin receptor engagement. It contains N-terminal LD domains and C-terminal LIM domains. Although leupaxin is primarily expressed in leukocytes, it is very little known about the localization and function in leukocytes. Results Degranulation follows the formation of the immunological synapse (IS) and recruitment of MTOC to the IS. Leupaxin co-localizes with MTOC and microtubules, and is recruited to the IS during CTL degranulation. Through deletion of leupaxin domains, it shows that both LD2 and LD4 domains are sufficient for leupaxin recruitment to the IS. Leupaxin fails to be recruited to the IS after deleting LD2–4 domains. Leupaxin is a focal adhesion protein in adherent cells. Leukocytes do not form classical focal adhesions during migration. We have found that leupaxin is recruited to the contact zone during CTL migration on ICAM-1, and forms very dynamic focal adhesion (FA)-like structures. These structures are assembled in the leading edge, and disappear in the trailing edge. Leupaxin also co-localizes with vinculin and talin at the FA-like structures, which are both FA proteins. Different from the role of leupaxin at the IS, leupaxin C-terminal LIM domain contributes to the localization of leupaxin at the FA-like structures. Conclusions Leupaxin localization suggests that it contributes to LFA-1 related processes in CTL, including degranulation and migration, but with different domains. Leupaxin LD domains are necessary for the recruitment to the IS during degranulation, whereas LIM domains are necessary for the formation of FA-like structures during migration.

Protein tyrosine kinase 2 (Pyk2) is required for T cell adhesion to ICAM-1; however, the mechanism by which it regulates adhesion remains unexplored. Pyk2 function in murine CTL clones and activated ex vivo CD8(+) T cells was disrupted by pharmacological inhibition, knockdown of expression with small interfering RNA, or expression of the dominant-negative C-terminal domain. We found that Pyk2 is not absolutely required for adhesion of CTL to ICAM-1, but rather delays the initial adhesion. Disruption of Pyk2 function caused cells to display an unusual elongated appearance after 1 h on ICAM-1, consistent with abnormally strong adhesion. Furthermore, the random mobility of CTL on ICAM-1 was severely compromised using all three methods of disrupting Pyk2 function. Live-cell imaging studies revealed that the decreased migration is the result of a defect in the detachment from ICAM-1 at the trailing edge when Pyk2 function is inhibited. Examination of Pyk2 tyrosine phosphorylation in normal polarized cells demonstrated that Pyk2 phosphorylated at Y579 and Y580 preferentially localizes to the leading edge, whereas Y881-phosphorylated Pyk2 is enriched at the trailing edge, suggesting that the tyrosine phosphorylation of Pyk2 is spatially regulated in migrating CTL. Additionally, inhibition of Pyk2 caused cells to form multiple LFA-1-rich tails at the trailing edge, most likely resulting from a defect in LFA-1 release required for forward movement. Our results show that Pyk2 contributes to CTL migration by regulating detachment of CTL at the trailing edge, which could explain why Pyk2 is important for chemotactic and migratory responses.

In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan–containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow–derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan–containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.

Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2. These antibodies recognized overlapping but biochemically distinct molecular species of Pyk2 since the CT antiserum recovered Pyk2 after NT antibody immunodepletion. Furthermore, the CT antibody could not immunoblot NT antibody-captured Pyk2. Phosphorylation partially accounts for the differential binding of these antibodies as dephosphorylation of Pyk2 recovered with the NT antibodies allows for recognition by the CT antibody. Additionally, Pyk2 recovered with the NT antibody displays increased serine/threonine phosphorylation. We suggest that the NT epitope is inaccessible to the antibody because Pyk2 is in a closed confirmation in association with paxillin. Upon induction of serine and/or threonine phosphorylation of Pyk2, it opens to a confirmation that allows for antibody binding to the NT epitope but at the same time no longer binds paxillin or the CT antiserum. These antibodies also display differential staining of Pyk2 in both T cells and macrophages. Pyk2 recognized by the CT antibody, but not the NT antibody, colocalized with paxillin at the microtubule-organizing center (MTOC). The MTOC-bound Pyk2 was not tyrosine phosphorylated upon T cell activation. We hypothesize that a reservoir of primarily inactive Pyk2 associates with paxillin at the MTOC, which may allow for rapid delivery of Pyk2 to specific sites of adhesion.

The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and, upon target cell binding, is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Examination of the localization of paxillin at the SMAC revealed that paxillin localizes to the peripheral area of the SMAC along with LFA-1, suggesting that LFA-1 may contribute to its recruitment. LFA-1 or CD3 engagement alone was insufficient for paxillin recruitment because there was no paxillin accumulation at the site of CTL contact with anti-LFA-1- or anti-CD3-coated beads. In contrast, paxillin accumulation was detected when beads coated with both anti-CD3 and anti-LFA-1 were bound to CTL, suggesting that signals from both the TCR and LFA-1 are required for paxillin accumulation. Paxillin was shown to be phosphorylated downstream of ERK, but when we generated a mutation (S83A/S130A) that abolished the mobility shift as a result of phosphorylation, we found that paxillin still bound to the MTOC and was recruited to the SMAC. Furthermore, ERK was not absolutely required for MTOC reorientation in CTL that require ERK for killing. Finally, expression of the LD2-4 region of paxillin substantially reduced MTOC reorientation. These studies demonstrated that paxillin is recruited, through its LD domains, to sites of integrin engagement and may contribute to MTOC reorientation required for directional degranulation.

CTL lyse target cells through the release of cytolytic granule mediators and expression of the death receptor ligand Fas ligand (FasL). We previously demonstrated that FasL is stored in vesicles distinct from cytolytic granules and is translocated to the cell surface within 15 min of TCR stimulation, followed by a later wave of newly synthesized FasL cell surface expression at 2 h poststimulation. Initial studies suggested that the two FasL responses had different signaling thresholds. To test this possibility directly, we titrated Ag presented to murine CTL to measure FasL and degranulation response thresholds. Stored FasL translocation to the cell surface required substantially lower concentrations of peptide than was required for de novo expression of FasL and degranulation. Furthermore, a low-affinity agonist peptide stimulated strong stored FasL translocation but only limited de novo FasL expression and degranulation. These data imply that the two FasL populations may have distinct functions. We examined bystander killing and found that the rapidly expressed FasL triggered highly specific lysis of target cells, as did degranulation. In contrast, the newly synthesized later wave of FasL mediated extensive Fas-dependent bystander killing. Our data indicate that stored FasL is mobilized in response to low concentrations of Ag to mediate rapid, highly specific lysis of target cells, whereas the later, newly synthesized FasL requires higher concentrations of Ag and mediates indiscriminate lysis. These findings suggest that early and late FasL and degranulation represent nonredundant lytic mechanisms that have been selected for distinct situations, possibly for optimal pathogen clearance.

Pyk2 was identified as a Ca(2+)-dependent kinase, however, the regulation of Pyk2 by Ca(2+) in T cells remains controversial. We found that Ca(2+) mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca(2+) dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca(2+)-regulated association between Pyk2 and calmodulin. Ca(2+)-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca(2+)-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H(2)O(2) scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H(2)O(2) induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca(2+) mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca(2+)-dependent kinase in T cells but instead, increased intracellular Ca(2+) induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca(2+) flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.

The CD45 protein tyrosine phosphatase is essential for T cell development. Its external domain undergoes changes in glycosylation and isoform usage during thymocyte development, the consequences of which remain unknown. The contribution of this complex external domain to T cell development is unknown so we sought to examine the impact of CD45 engagement on T cell development. Treatment of wildtype fetal thymic organ cultures (FTOC) with certain CD45-specific monoclonal antibodies resulted in decreased thymocyte numbers specifically at the double positive and CD4 single positive stages of development. The decrease in thymocyte number correlated with increased annexinV staining, an early indicator of apoptotic death. In contrast, CD45-/- FTOC exhibited decreased cellularity and increased annexinV staining at all stages of development. Thymocyte selection, as assessed by CD5 expression on DP thymocytes, was not affected by anti-CD45 treatment, whereas it was impaired in FTOC from CD45-deficient mice. The decrease in cellularity was not due to impaired proliferation as neither anti-CD45 treatment nor CD45 deficiency resulted in decreased proliferation in FTOC. Thus, antibodies specific for the external domain of CD45 results in the selective impairment of thymocyte survival at specific stages of development, whereas CD45 expression appears to be required for survival at all stages of development. Our studies show that CD45 expressed on thymocytes is an important regulator of survival during T cell development.

CTL lyse target cells through the release of cytolytic granule contents and cell surface expression of Fas ligand (FasL). Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. We demonstrate that murine CTLs undergo two waves of FasL cell surface expression after stimulation. The first wave is from a pre-existing pool of FasL, and the second wave requires new protein synthesis. Signaling for FasL expression appears to be finely tuned as a weak signal preferentially induced surface translocation of the stored FasL, whereas a strong signal preferentially triggered the expression of de novo synthesized FasL. The early FasL is differentially regulated from degranulation, as there were multiple circumstances whereby rapid FasL cell surface expression and FasL-dependent killing occurred in the absence of detectable degranulation. Furthermore, we found through confocal microscopy that stored FasL resides in vesicles distinct from cytolytic granules. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation.

Cytotoxic T lymphocytes (CTL) lyse target cells through the release of cytolytic granules and cell surface expression of Fas ligand (FasL). We found that there is prestored FasL pool in CTL, which underwent rapid TCR-regulated cell surface expression upon target cell engagement. Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same control as degranulation. The aim of the current study is to investigate if degranulation of cytolytic granules and FasL expression pathways of CTL are controlled by differential mechanisms. FasL cell surface expression and degranulation, as measured by CD107a cell surface expression in CTLs were compared by flow cytometry under different conditions. FasL underwent an immediate and transient cell surface delivery following either TCR stimulation by anti-CD3 antibody crosslinking, or phorbol ester treatment, conditions that do not permit degranulation. Furthermore, treatment with pharmacological reagents indicated that the preformed FasL cell surface transport is PI3-kinase (PI3K)-, calcineurin- and microtubule-independent, which are all indispensable for degranulation. Finally, stored FasL resides in vesicles distinct from cytolytic granules as assessed by confocal microscopy and subcellular fractionation. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to regulation and localization. This work was funded by the Canadian Institutes of Health Research (CIHR).

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation. This signal reversal is not restricted to tyrosine phosphorylation, as disruption of the actin cytoskeleton also reverses the phosphorylation of the more downstream serine/threonine kinase extracellular signal regulated kinase (Erk). An intact cytoskeleton and cell spreading are not sufficient for maintaining signals, as stabilization of actin filaments, at a point when peak tyrosine phosphorylation is occurring, also leads to the rapid loss of protein tyrosine phosphorylation. Disruption of tyrosine kinase activity after TCR signals are maximally induced causes the immediate reversal of tyrosine phosphorylation as well as cytoskeletal disruption, as indicated by loss of cell spreading, adhesion and CTL degranulation. Taken together, our results indicate that actin remodelling occurs co-temporally with ongoing tyrosine kinase activity, leading to CTL degranulation. We hypothesize that continuous actin remodelling is important for sustaining productive signals, even after downstream signalling molecules such as Erk have been activated, and that the actin cytoskeleton is not solely required for initiating and maintaining the T cell in contact with its stimulus.

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3zeta. CD3zeta phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex. The protein complexes were not preformed, and a number of tyrosine-phosphorylated proteins were inducibly and specifically associated with the TCR/CD3 complex. These results suggest that complex formation only occurs at the site of TCR engagement. The recruitment and tyrosine phosphorylation of most proteins were abolished when T cells were stimulated in the presence of the SFK inhibitor PP2. Surprisingly, CD3zeta, but not CD3epsilon, was inducibly tyrosine phosphorylated in the presence of PP2. Furthermore, ZAP70 was recruited, but not phosphorylated, after TCR stimulation in the presence of PP2, thus confirming the phosphorylation status of CD3zeta. These data suggest that there is a differential requirement for SFK activity in phosphorylation of CD3zeta vs CD3epsilon. Consistent with this possibility, ZAP70 recruitment was also detected with anti-CD3-stimulated, Lck-deficient human Jurkat T cells. We conclude that TCR/CD3-induced CD3zeta phosphorylation and ZAP70 recruitment do not absolutely require Lck or other PP2-inhibitable SFK activity, but that SFK activity is absolutely required for CD3epsilon and ZAP70 phosphorylation. These data reveal the potential for regulation of signaling through the TCR complex by the differential recruitment or activation of SFK.

Pyk2 is a protein tyrosine kinase expressed primarily in brain and hematopoietic cells. It becomes activated in response to stimulation through numerous receptors, including integrins, chemokine receptors, and antigen receptors, and is found in association with src-family kinases. Although this enzyme associates with many proteins known to be important for activation and has many characteristics of a scaffolding protein, its function remains elusive. A number of studies in non-T-cells suggest that Pyk2 is important for cell spreading, cell migration, and integrin function; however, a defined role in T-cells has not been established. Here, we discuss evidence that implicates Pyk2 in directionality of signaling, which is essential to establishment of the directional killing mediated by cytotoxic lymphocytes.