Mesothelin Virus-Like Particle Immunization Controls Pancreatic Cancer Growth through CD8+ T Cell Induction and Reduction in the Frequency of CD4+foxp3+ICOS? Regulatory T Cells

Our previous study has shown that mesothelin (MSLN) is a potential immunotherapeutic target for pancreatic cancer. Here, we further studied the immunogenicity of chimeric murine MSLN-virus-like particles (mMSLN-VLPs), their ability to break tolerance to mMSLN, a self-antigen, and deciphered the mechanism of immune responses elicited by mMSLN-VLP immunization using a pancreatic cancer (PC) mouse model. In addition to what we have found with xenogeneic human MSLN-VLP (hMSLN-VLP), mMSLN-VLP immunization was able to break the tolerance to intrinsic MSLN and mount mMSLN-specific, cytotoxic CD8⁺ T cells which led to a significant reduction in tumor volume and prolonged survival in an orthotopic PC mouse model. Furthermore, CD4⁺foxp3⁺ regulatory T cells (Tregs) were progressively decreased in both spleen and tumor tissues following mMSLN-VLP immunization and this was at least partly due to elevated levels of IL-6 production from activated plasmocytoid dendritic cell (pDC)-like cells following mMSLN-VLP immunization. Moreover, mMSLN-VLP treatment mainly reduced the frequency of the CD4⁺foxp3⁺ICOS⁻ Treg subset. However, mMSLN-VLP induced IL-6 production also increased ICOSL expression on pDC-like cells which supported the proliferation of immunosuppressive CD4⁺foxp3⁺ICOS⁺ Treg cells. This study reveals that mMSLN-VLP immunization is capable of controlling PC progression by effectively mounting an immune response against mMSLN, a tumor self-antigen, and altering the immunosuppressive tumor microenvironment via activation of pDCs-like cells and reduction in the frequency of CD4⁺foxp3⁺ICOS⁻ Treg cells. However, combination therapies will likely need to be used in order to target residual CD4⁺foxp3⁺ICOS⁺ Treg cells.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

Age-associated parallel increase of Foxp3CD4 regulatory and CD44CD4 memory T cells in SJL/J mice

Effector/memory T cells (Tem) are required to maintain successful immunity, while regulatory T cells (Treg) are required to prevent excessive/uncontrolled inflammation and/or autoimmunity. Although both Tem and Treg cells are increased during aging, the relationship between the increased proportion of Foxp3⁺ Treg cells and CD44⁺ Tem cells with aging is not clearly understood. We found in this report that Foxp3⁺ Treg cells are increased in parallel with CD44⁺ Tem cells in SJL/J mice with aging, and that all Foxp3⁺ Treg cells are of CD44⁺ Tem phenotype, suggesting that the increased Foxp3⁺ Treg cells originated from the expanded pool of CD44⁺ Tem cells with aging. Our in vitro kinetic studies further suggested that Foxp3⁺ Treg cells are converted through the CD44⁺ stage. Furthermore, we observed that although the balance between Foxp3⁺ Treg and CD44⁺Foxp3⁻ Tem cells remained with aging, the aged mice have higher ratios of both Tem and Treg cells vs. naïve T cells resulting in the “shrunken” naïve T cell pools. Our results suggest that an age-associated imbalance of T cell repertoire is a mechanism that contributes to spontaneous occurrence of Hodgkin’s-like lymphoma in aged SJL/J mice.     

Numerical defects in CD8CD28 T-suppressor lymphocyte population in patients with type 1 diabetes mellitus and multiple sclerosis

Type 1 diabetes mellitus (T1D) and multiple sclerosis (MS) are organ-specific autoimmune diseases leading to an attack of auto-aggressive lymphocytes against the pancreatic β-cells and central nervous system, respectively. Using four-colour flow cytometry, T-lymphocyte populations having an important function in autoimmune processes were analyzed. T-regulatory cells (Treg) CD4⁺CD25⁺CD127^low, T-suppressor cells (Ts) CD8⁺CD28⁻, activated helper CD4⁺CD25⁺CD127⁺ and cytotoxic CD8⁺CD25⁺ T-cells and also naive CD4⁺CD45RA⁺ and memory T-cells CD4⁺CD45RO⁺ were compared in the group of patients with T1D (n = 30), MS (n = 31) and in the group of healthy controls (n = 29). Significant differences in Ts cells, activated helper and cytotoxic cells and also memory T-cells were recognized in the group of T1D patients compared to healthy controls. Ts population was significantly lowered in MS patients as well. However, no significant differences were noticed in Treg population. The observed data demonstrate significant differences among patients with T1D and MS in comparison to healthy individuals.     

Expansion of regulatory GITR+CD25low/-CD4+ T cells in systemic lupus erythematosus patients

Introduction

CD4⁺CD25^low/-GITR⁺ T lymphocytes expressing forkhead box protein P3 (FoxP3) and showing regulatory activity have been recently described in healthy donors. The objective of the study was to evaluate the proportion of CD4⁺CD25^low/-GITR⁺ T lymphocytes within CD4⁺ T cells and compare their phenotypic and functional profile with that of CD4⁺CD25^highGITR⁻ T lymphocytes in systemic lupus erythematosus (SLE) patients.

Methods

The percentage of CD4⁺CD25^low/-GITR⁺ cells circulating in the peripheral blood (PB) of 32 patients with SLE and 25 healthy controls was evaluated with flow cytometry. CD4⁺CD25^low/-GITR⁺ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4⁺CD25^highGITR⁻ cells. Regulatory activity of both cell subsets was tested in autologous and heterologous co-cultures after purification through a negative sorting strategy.

Results

Results indicated that CD4⁺CD25^low/-GITR⁺ cells are expanded in the PB of 50% of SLE patients. Expansion was observed only in patients with inactive disease. Phenotypic analysis demonstrated that CD4⁺CD25^low/-GITR⁺ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10. In contrast, CD4⁺CD25^highGITR⁻ cells appear to be activated and express low levels of Treg markers. Functional experiments demonstrated that CD4⁺CD25^low/-GITR⁺ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4⁺CD25^highGITR⁻ cells. Suppression is independent of cell contact and is mediated by IL-10 and TGF-β.

Conclusions

Phenotypic and functional data demonstrate that in SLE patients, CD4⁺CD25^low/-GITR⁺ cells are fully active Treg cells, possibly representing peripheral Treg (pTreg) that are expanded in patients with inactive disease. These data may suggest a key role of this T-cell subset in the modulation of the abnormal immune response in SLE. Strategies aimed at expanding this Treg subset for therapeutic purpose deserve to be investigated.     

ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8⁺ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8⁺ T cell–mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG^-/- mice, we found that CD4⁺ T cells and ICOS^+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4⁺Foxp3 ⁺ ICOS⁺ T cells were enriched in the lungs of animals surviving lung injury and ICOS^+/+ Tregs promoted survival in animals that received ICOS^-/- T cells. Direct comparison of ICOS^-/- Tregs to ICOS^+/+ Tregs found defects in vitro but no differences in the ability of ICOS^-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.     

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25⁻CD4⁺ effector (Teff) and CD25⁺CD4⁺ regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4⁺ T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4⁺ T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.     

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX⁻) or HPX-containing (HPX⁺) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX⁻ sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.     

Development of Virus-Specific CD4+ and CD8+ Regulatory T Cells Induced by Human Herpesvirus 6 Infection

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4⁺ and CD8⁺ HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4⁺ and CD8⁺ Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4⁺ effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.     

Identification of Cytolytic CD161?CD56+ Regulatory CD8 T Cells in Human Peripheral Blood

We previously developed methods for establishing CD8 regulatory T cell (Treg) clones from normal human peripheral blood and demonstrated that these clones were capable of killing T cell receptor (TCR)-activated autologous CD4 T cells. Based on phenotypic and functional characterization of the CD8 Treg clones, we have identified a corresponding population of endogenous CD8 Treg in normal human peripheral blood. These cells appear morphologically as large lymphocytes with abundant cytoplasm and have the following unique phenotype: CD3⁺CD8⁺CD161⁻CD56⁺. The majority of CD8 Treg express CD45RA and CD62L with low or negative expression of CD45RO, CD25, CD27, CD28 and CCR7. The expression of CD94 and NKG2a on CD8 Treg was elevated compared to conventional CD8 T cells. Following in vitro activation, this T cell subset is capable of killing TCR-activated CD4 T cells. These studies identify an endogenous CD8 Treg population in humans and it will now be possible to characterize these cells in a variety of clinical conditions.     

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4 T cells

CD4⁺ T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4⁺ T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4⁺ T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4⁺CD25⁻ T cells into Rag2^−/− mice did not cause colitis accompanied by increasing Foxp3⁺ inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4⁺CD25⁻ T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4⁺ T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.     

Human CD80/IL2 lentivirus transduced acute myeloid leukaemia cells enhance cytolytic activity in vitro in spite of an increase in regulatory CD4<Superscript>+</Superscript> T cells in a subset of cultures

Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4⁺ T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8⁺ T cells (P = 0.046) CD3⁻CD56⁺ NK cells (P = 0.028) and CD3⁺CD4⁺CD25^highFoxp3⁺ Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8⁺ T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8⁺ T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-γ and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.     

CD45RA−Foxp3 non-regulatory T cells in the CCR7−CD45RA−CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA−Foxp3^low non-Treg cells, and the frequency of the non-Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA−Foxp3^low non-Treg cells in synovial fluid.     

A tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by activation of antigen-specific CD4+CD25+ T regulatory cells that promotes skin allograft survival in mice

Semimature dendritic cells (smDCs) can induce autoimmune tolerance by activation of host antigen-specific CD4⁺CD25⁺ regulatory T (Treg) cells. We hypothesized that donor smDCs injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4⁺CD25⁺Treg cells, and promote skin allograft survival. Myeloid smDCs were derived from C57BL/6J mice (donors) in vitro. BALB/c mice (recipients) were injected with smDCs to generate antigen-specific CD4⁺CD25⁺Treg cells in vivo. Allograft survival was prolonged when BALB/c recipients received either C57BL/6J smDCs prior to grafting or C57BL/6J smDC-derived CD4⁺CD25⁺Treg cells post-grafting, and skin flaps from these grafts showed the highest IL-10 production regardless of rapamycin treatments. Our findings confirm that smDCs constitute an independent subgroup of DCs that play a key role for inducing CD4⁺CD25⁺Treg cells to express high IL-10 levels, which induce hyporesponsiveness of effector T cells. Pre-treating recipients with donor smDCs may have potential for transplant tolerance induction.     

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

The peripheral Foxp3⁺ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4⁺ T cells can be readily converted to Foxp3⁺ iTreg in vitro, and memory CD4⁺ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3⁺ T cells from various CD4⁺ T-cell subsets in human peripheral blood. Though naive CD4⁺ T cells were readily converted to Foxp3⁺ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3⁺ T cells did not express the memory marker CD45RO as do Foxp3⁺ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4⁺ T cells, defined as CD62L⁺ central memory T cells, could be induced by TGF-β to differentiate into Foxp3⁺ T cells. It is well known that Foxp3⁺ T cells derived from human CD4⁺CD25^- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4⁺ T cell-derived Foxp3⁺ T cells. But further research showed that mouse CD4⁺ central memory T cells also could be induced to differentiate into Foxp3⁺ T cells, such Foxp3⁺ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4⁺CD62L⁺ central memory T cells as a novel potential source of iTreg.     

CD61 enriches long-term repopulating hematopoietic stem cells

Among the subsets that define hematopoietic stem cells (HSCs), CD34⁻ c-kit⁺ Sca-1⁺ lineage marker⁻ (CD34⁻KSL) cells are regarded as one of the populations that have the highest enrichment of HSCs in adult mouse bone marrow. Here, we demonstrate that long-term repopulating hematopoietic stem cells (LTR-HSCs) have high expression of CD61 (integrin β₃) within the CD34⁻KSL population. Approximately 60% of CD34⁻KSL cells showed high expression of CD61. CD61^HighCD34⁻KSL populations also exhibited significantly greater properties of HSC, such as expression of HSC markers, the side population (SP) phenotype, and ability for long-term repopulation. In both SP cells and non-SP (NSP) cells, CD61^HighCD34⁻KSL cells also contained significantly more LTR-HSCs than CD61^Low/−CD34⁻KSL cells. Our results indicate that CD61 is exploitable for HSC enrichment as a supportive positive cell surface marker.     

Cord blood CD4CD25 regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4⁺CD25⁺ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.     

CD8CD25 T cells reduce atherosclerosis in apoE(−/−) mice

Background

It is increasingly evident that CD8⁺ T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8⁺CD25⁺ T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8⁺CD25⁺ T cells in experimental atherosclerosis were investigated in this study.

Methods and results

CD8⁺CD25⁺ T cells were observed in atherosclerotic plaques of apoE(−/−) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8⁺CD25⁺ T cells from apoE(−/−) mice. Depletion of CD8⁺CD25⁺ from total CD8⁺ T cells rendered higher cytolytic activity of the remaining CD8⁺CD25⁻ T cells. Adoptive transfer of CD8⁺CD25⁺ T cells into apoE(−/−) mice suppressed the proliferation of splenic CD4⁺ T cells and significantly reduced atherosclerosis in recipient mice.

Conclusions

Our study has identified an athero-protective role for CD8⁺CD25⁺ T cells in experimental atherosclerosis.     

Co-expression of<Emphasis Type="Italic"> α</Emphasis>(1,3)galactosyltransferase and <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> PIPLC enhances hyperacute rejection of tumor cells

The use of α(1,3)galactosyltransferase (αGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine αGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance αgal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the αGT and EGFssPIPLC genes expressed high levels of αgal epitope and exhibited a 5-fold increase in sensitivity to anti-αgal mediated complement lysis.     

Expression of CD55 and CD59 on peripheral blood cells from systemic lupus erythematosus (SLE) patients

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p = 0.005 and p = 0.019, respectively), and CD59-granulocytes (p = 0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.