Kyra A. Gelderman

Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species

Reduced capacity to produce ROS increases the severity of T cell-dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell-independent anti-collagen antibody-induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-gamma production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (−SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.

C4b-binding Protein and Factor H Compensate for the Loss of Membrane-bound Complement Inhibitors to Protect Apoptotic Cells against Excessive Complement Attack

Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins (m-C-Reg) and to activate complement. Nonetheless, most apoptotic cells do not undergo complement-mediated lysis. Therefore, we hypothesized that fluid phase complement inhibitors would bind to apoptotic cells and compensate functionally for the loss of m-C-Reg. We observed that m-C-Reg are down-regulated rapidly upon apoptosis but that complement activation follows only after a gap of several hours. Coinciding with, but independent from, complement activation, fluid phase complement inhibitors C4b-binding protein (C4BP) and factor H (fH) bind to the cells. C4BP and fH do not entirely prevent complement activation but strongly limit C3 and C9 deposition. Late apoptotic cells, present in blood of healthy controls and systemic lupus erythematosus patients, are also positive for C4BP and fH. Upon culture, the percentage of late apoptotic cells increases, paralleled by increased C4BP binding. C4BP binds to dead cells mainly via phosphatidylserine, whereas fH binds via multiple interactions with CRP playing no major role for binding of C4BP or fH. In conclusion, during late apoptosis, cells acquire fluid phase complement inhibitors that compensate for the down-regulation of m-C-Reg and protect against excessive complement activation and lysis.

The Inhibitory Effect of CD46, CD55, and CD59 on Complement Activation After Immunotherapeutic Treatment of Cervical Carcinoma Cells with Monoclonal Antibodies or Bispecific Monoclonal Antibodies

The role of membrane-bound complement regulatory proteins (mCRP) in the protection of tumor cells in vivo against elimination by the immune system is still unknown. In this study the effect of expression of these mCRP by cervical cancer cells was investigated. In situ expression of mCRP was observed on cervical carcinomas, normal cervical epithelial cells, and the surrounding stroma. Deposition of C3 and C5b-9 was sporadically found on the tumor cells and the surrounding stroma. A low expression of CD46 was statistically significantly associated with deposition of C3. Comparable expression patterns were shown on primary cervical tumor cell suspensions. A relatively high deposition of C4c was found on these tumor cells, indicating classical pathway activation. Furthermore, it was demonstrated that CD55 and CD59 were the most potent inhibitors of C3 deposition and classical pathway-mediated lysis, respectively, on cervical cancer cell lines. The feasibility of increasing complement activation at the tumor cell membrane surface was demonstrated with an anti-HLA Class I*anti-CD55 bispecific mAb. The potential immunotherapeutic applicability was investigated with both anti-G250*anti-CD55 and anti-Ep-CAM*anti-CD55 bispecific mAbs. An approximate 2-fold increase in C3 deposition, compared with the parental anti-Ep-CAM mAb, was attained with an anti-Ep-CAM*anti-CD55 bispecific mAb when the tumor-associated antigen was expressed in sufficient amounts. These results demonstrate that when tumor-associated antigens are expressed in adequate amounts, bispecific mAbs in vivo may be potent immunotherapeutic agents to enhance an inflammatory reaction at the tumor site.

Handbook of Experimental Pharmacology "Dendritic Cells" The Use of Dexamethasone in the Induction of Tolerogenic DCs

Dendritic cells (DCs) have a central role in immune regulation, ranging from tolerance induction to the induction of specific immune responses. DCs serve as an essential link between innate and adaptive immunity. This broad range of powerful immune stimulatory as well as regulatory functions has made DCs as targets for vaccine development strategies. One approach to promote the tolerogenicity of DCs is to suppress their maturation by pharmacological agents, including glucocorticoids (GCs). In the present chapter we will review GCs used in vitro with cultured DCs, applied in vivo, or used to generate tolerogenic DCs for cellular therapy.

BETA-GLUCAN ENHANCED KILLING OF RENAL CELL CARCINOMA MICROMETASTASES BY MONOCLONAL ANTIBODY G250 DIRECTED COMPLEMENT ACTIVATION

Metastases from renal cell carcinomas (RCC) are resistant to radiation and chemotherapy but are relatively immunogenic. We have investigated the possibility to eliminate human RCC micrometastases using MAb G250. G250 penetrates human micrometastases completely in a spheroid model and induces complement deposition rapidly on the outmost cell layers. However, complement dependent cytotoxicity (CDC) was barely detected using either 51chromium release assays or confocal microscopy, due to relatively low expression of the G250 antigen and the effect of membrane bound complement regulatory proteins. Addition of blocking anti‐CD59 MAbs enhanced formation of C5b‐9 and consequently complement mediated lysis (13%). Complement assisted cellular cytotoxicity (CACC) was not detectable, although the iC3b ligand and CR3 receptor were present on respectively target and effector cells. Addition of soluble β‐glucan induced the killing of MAb and iC3b opsonized spheroids by effector cells (6–21%). Despite a lower affinity for G250 antigen, a bispecific anti‐G250*anti‐CD55 MAb enhanced cell killing in spheroids comparable to the parental G250 MAb. Our results suggest that complement‐activating G250 in combination with anti‐mCRP MAbs is able to kill human RCC cells in micrometastasis in vitro. For CACC the presence of CR3‐priming β‐glucan seems to be obligatory. In vivo, bi‐MAb may be more effective as therapeutic agent due to its increased C5a generating properties.

Enhancement of the complement activating capacity of 17-1A mAb to overcome the effect of membrane-bound complement regulatory proteins on colorectal carcinoma.

Adjuvant immunotherapy with 17‐1A mAb directed against colorectal carcinoma is found to be effective in patients. However, 52 % of the patients treated with mAb 17‐1A showed recurrence within 7 years. This high recurrence rate might be due to inhibition of complement activation by membrane‐bound complement regulatory proteins (mCRP). The effect of these complement regulatory proteins might be reduced by blocking mCRP, or be overcome by activating more complement at the tumor cell membrane. In this study the complement‐activating capacity of the 17‐1A mAb was enlarged by conjugating it to cobra venom factor (CVF) or C3b. The most important C3 regulatory protein, CD55, was blocked using a bispecific mAb directed against the 17‐1A / Ep‐CAM antigen and CD55. Up to a 13‐fold increase in C3 deposition was observed due to 17‐1A‐CVF and 17‐1A‐C3b, as compared to 17‐1A. CD55 was shown to partly inhibit complement activation by these conjugates. The effect of the bispecific anti‐17‐1A / Ep‐CAM*anti‐CD55 mAb was compared with 17‐1A conjugates with CVF or C3, and bispecific mAb were shown to be equally or more efficient in complement activation than the 17‐1A‐CVF or 17‐1A‐C3b conjugates. Therefore, 17‐1A conjugates and anti‐17‐1A / EpCAM*anti‐CD55 bispecific mAb may be promising immunotherapeutic agents for patients with colorectal cancer.

Tumor-Specific Inhibition of Membrane-Bound Complement Regulatory Protein Crry with Bispecific Monoclonal Antibodies Prevents Tumor Outgrowth in a Rat Colorectal Cancer Lung Metastases Model

Membrane-bound complement regulatory proteins (mCRP) inhibit complement-mediated tumor cell eradication in vitro and in vivo. Immunotherapy of cancer with monoclonal antibodies (mAbs) that activate complement might be hampered by expression of mCRP on tumor cells. An important strategy to improve mAb immunotherapy can be blocking or overwhelming mCRP at the tumor cells surface in a tumor-specific manner. In our study, we investigated the feasibility of this approach in vivo using bispecific mAbs (bi-mAbs). This study, performed in a syngeneic lung metastases model of rat (WAG/Rij) colorectal cancer, showed that modulation of mCRP on tumor cells resulted in significantly decreased tumor outgrowth. Opsonization of tumor cells with a bi-mAb directed against a tumor-associated antigen and rat mCRP Crry (MG42a*5I2) almost completely prevented the outgrowth of lung tumors (0–7 tumors/rat; n = 17). Opsonization with mAb-cobra venom factor conjugates significantly reduced the number of lung tumors (23–59 tumors; n = 12) compared with the unconjugated MG42a (175–246 tumors; n = 17; P = 0.008 and 0.014, respectively). The effect of MG42a*5I2 was shown to be caused by increased complement activation due to inhibition of Crry. Moreover, prophylactic treatment with MG42a*5I2 or MG42a showed comparable results (3–24 and 215–472 tumors, P = 0.02; n = 6) as observed with pre-opsonized tumor cells without noticeable side effects, despite binding of MG42a*5I2 to endothelium and leukocytes. These results demonstrate that Crry inhibits complement-mediated tumor cell eradication by immunotherapeutic mAbs and show that tumor-specific inhibition of complement regulatory proteins using bi-mAbs can significantly improve mAb-mediated immunotherapy.

Complement production and regulation by dendritic cells: molecular switches between tolerance and immunity.

In recent years it has become clear that the innate and adaptive immune systems are highly integrated and interact at several levels. Dendritic cells (DCs) are on the one hand instrumental for directing and controlling adaptive immunity and on the other hand are specialized in detecting and integrating signals from the microenvironment. In view of the strong link between deficiencies in certain complement components and the development of autoimmunity, interaction between complement and DCs seems to be of fundamental importance. We will discuss the role of C1q, C3, as well as complement regulators in DC biology.

Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages.

Macrophages have been demonstrated to suppress T cell responses by producing reactive oxygen species (ROS) leading to the subsequent induction of T regulatory cells in a ROS-dependent manner. Macrophages may therefore be instrumental in downregulating T cell responses in situations of exacerbated immune responses. Here we investigated the effect of immunosuppressive drugs on ROS production by macrophage subsets and the subsequent effects on T cell activation. Macrophage types 1 and 2 were differentiated with GM-CSF or M-CSF, in presence or absence of dexamethasone, cyclosporine A, FK506, rapamycin, or mycophenolic acid. The ROS producing capacity of fully differentiated Mph was highest in anti-inflammatory Mph2 and not affected by exposure to immunosuppressive drugs. However, presence of rapamycin during Mph2 differentiation decreased the ROS production of these cells. In contrast, other immunosuppressive drugs, with dexamethasone being the most potent, increased the ROS producing capacity of Mph2. Intriguingly although the ROS producing ability of Mph1 was unaffected, dexamethasone strongly increased the ROS producing capabilities of dendritic cells. Both at the mRNA and protein level we found that dexamethasone enhanced the expression of NOX2 protein p47(phox). Functionally, dexamethasone further enhanced the capacity of Mph2 to suppress T cell mediated IFN-γ and IL-4 production. In vivo, only in rats with normal ROS production (congenic DA.Ncf1(E3/E3)) it was observed that dexamethasone injection resulted in long-lasting upregulation of ROS production by macrophages and induced higher levels of Treg in a ROS-dependent manner. In conclusion, we show that the anti-inflammatory drug dexamethasone increases the ROS producing capacity of macrophages.