Neutrophil-mediated IFN activation in the bone marrow alters B cell development in human and murine systemic lupus erythematosus.

Inappropriate activation of type I IFN plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). In this study, we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM-resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre-B cells, suggesting an inhibition in early B cell development and an expansion of B cells at the transitional stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN-high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice, similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type-I IFNR. BM neutrophils were abundant, responsive to, and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil-mediated IFN activation and alterations in B cell ontogeny and selection.

Clinical implications and longitudinal alteration of peripheral blood transcriptional signals indicative of future cardiac allograft rejection.

BACKGROUND: We have previously demonstrated that a peripheral blood transcriptional profile using 11 distinct genes predicts onset of cardiac allograft rejection weeks to months prior to the actual event. METHODS: In this analysis, we ascertained the performance of this transcriptional algorithm in a Bayesian representative population: 28 cardiac transplant recipients who progressed to moderate to severe rejection; 53 who progressed to mild rejection; and 46 who remained rejection-free. Furthermore, we characterized longitudinal alterations in the transcriptional gene expression profile before, during and after recovery from rejection. RESULTS: In this patient cohort, we found that a gene expression score (range 0 to 40) of or =3A) rejection; 16 of 53 (30%) from the intermediate group (those who progressed to ISHLT Grade 1B or 2) and 13 of 46 (28%) controls (who remained Grade 0 or 1A) had scores < or =20. A gene score of > or =30 was associated with progression to moderate to severe rejection in 58% of cases. These two extreme scores (< or =20 or > or =30) represented 44% of the cardiac transplant population within 6 months post-transplant. In addition, longitudinal gene expression analysis demonstrated that baseline scores were significantly higher for those who went on to reject, remained high during an episode of rejection, and dropped post-treatment for rejection (p < 0.01). CONCLUSIONS: The use of gene expression profiling early after transplantation allows for separation into low-, intermediate- or high-risk categories for future rejection, permitting development of discrete surveillance strategies.

Transcriptional signals of T-cell and corticosteroid-sensitive genes are associated with future acute cellular rejection in cardiac allografts.

BACKGROUND: Profiling mRNA levels of 11 informative genes expressed by circulating immune effector cells identifies cardiac allograft recipients at low risk for current moderate-severe acute cellular rejection (ACR). METHODS: We conducted a nested case-control study of 104 cardiac allograft recipients to investigate the association of transcriptional profiles of blood samples with either a future rejection episode within 12 weeks of a baseline clinical sample or persistent histologic quiescence for the same time period. RESULTS: The transcription profile yielded a score (0 to 40 scale) of 27.4 +/- 6.3 for future rejectors (n = 39) and 23.9 +/- 7.1 for controls (n = 65) (p = 0.01). In patients who were

G6b-B cell surface inhibitory receptor expression is highly restricted to CD4+ T-cells and induced by interleukin-4-activated STAT6 pathway.

The G6b-B gene encodes a novel cell surface receptor of the immunoglobulin superfamily that activates inhibitory signaling pathways by triggering SHP-1/SHP-2 via immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic domain. We previously identified decreased G6b-B expression in peripheral blood mononuclear cells (PBMC) during acute cellular cardiac allograft rejection. We studied the expression of G6b-B in different human mononuclear cell populations and its regulation. Real-time polymerase chain reaction (PCR) revealed that G6b-B mRNA is higher in CD4+ T cells or monocytes, but is not different between CD25+ CD4+ T cells and CD25- CD4+ T cells. G6b-B mRNA was increased in CD4+ T cells in presence of interleukin-4 in dose- and time-dependent manners. To understand the regulatory mechanism, we analyzed a 1.9-kb 5'-flanking region of the G6b-B translation start site and found a putative cis-acting element for Signal Transducer and Activator of Transcription (STAT)-6. Luciferase-reporter-gene-assay and electrophoretic mobility shift assays identified the STAT6 site as necessary for the induction of G6b-B by IL-4. Our study demonstrates that G6b-B expression is highly restricted to peripheral CD4+ T cells and up-regulated by the IL-4-induced STAT6 pathway, strongly suggesting that G6b-B is involved in regulation of the immune response by CD4+ T cell-mediated and IL-4 induced regulatory mechanisms.

Understanding gene expression patterns in immune-mediated disorders.

Gene expression profiling of peripheral blood cells can provide dynamic information regarding the host response to immune-mediated disorders. AlloMap molecular expression testing from XDx monitors the expression of 20 genes in peripheral blood mononuclear cells (PBMC) to discriminate cardiac allograft recipients of 15 years or greater who are at low risk for acute cellular rejection (ACR). The AlloMap test classifier is based on the expression level of 11 genes, encoding proteins with diverse functions, which are differentially expressed in stable patients with moderate to severe ACR compared to patients without ACR. The nine other test genes are used for normalizing gene expression levels and assuring sample quality. In this work we review the development processes leading to the selection of the 11 informative genes and the derivation of the AlloMap test classifier, and discuss the relationship of peripheral blood gene expression with diverse pathways associated with ACR, including T-cell priming, platelet activation, systemic responses to allograft inflammation, and the overall state of immunosuppression.

An overview of the pharmacokinetics and pharmacodynamics of efalizumab: a monoclonal antibody approved for use in psoriasis.

Efalizumab is a recombinant humanized monoclonal IgG(1) antibody shown to be efficacious for the treatment of moderate to severe chronic plaque psoriasis. Efalizumab, a targeted inhibitor of T cell interactions, binds to the CD11a subunit of lymphocyte function-associated antigen 1 (LFA-1), thereby preventing LFA-1 binding to intercellular adhesion molecule 1 (ICAM-1). The authors review the pharmacokinetic and pharmacodynamic data from the efalizumab clinical development program and discuss how these data led to selection of the optimal weekly subcutaneous (SC) dose of efalizumab (1.0 mg/kg) in adults. Efalizumab SC dosages of 1.0 mg/kg/wk or greater exerted maximal pharmacodynamic effects for CD11a expression and available CD11a binding sites on T lymphocytes. Dosages greater than 1.0 mg/kg/wk SC did not provide additional benefits; moreover, higher doses did not alter the safety profile. During long-term administration of efalizumab, serum levels were generally stable and pharmacodynamic markers remained maximally affected.

Pharmacokinetic-pharmacodynamic-efficacy analysis of efalizumab in patients with moderate to severe psoriasis.

PURPOSE: Efalizumab is a humanized anti-CD11a monoclonal antibody that demonstrated efficacy in the treatment of patients with psoriasis. The objective of this study was to perform a pharmacokinetic (PK)-pharmacodynamic (PD)-efficacy (E) modeling analysis with intersubject variability assessment to increase our understanding of the interaction of efalizumab with CD11a on T cells and consequent reduction in severity of disease in psoriasis patients. METHODS: A total of 6,329 samples from 240 patients in five Phase I and II clinical studies were used in the analysis. For the analysis, plasma efalizumab concentration was used as the PK measurement, the percent of predose CD11a was used as the PD measurement, and the psoriasis area and severity index was used as the measure of efficacy. A receptor-mediated PK/PD model was developed that describes the dynamic interaction of efalizumab binding with CD11a. In the efficacy model, the rate of psoriasis skin production is directly proportional to the amount of free surface CD11a on T cells, which is offset by the rate of skin healing. An additional CD11a-independent component to psoriasis skin production accounted for incomplete response to efalizumab therapy. A Monte Carlo parametric expectation maximization method implemented in the ADAPT II program was used to obtain the estimate of population parameters and inter- and intrasubject variability. RESULTS AND CONCLUSIONS: The final model described the PK/PD/E data in psoriasis patients reasonably well. In addition, simulations using the final model suggested that efalizumab administered less frequently could possibly be more convenient with similar efficacy.

rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21.

rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin. rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23. Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus. To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids. A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI). These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure). Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities. In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21. In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease. We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.

Adhesion molecules as therapeutic targets for autoimmune diseases and transplant rejection.

Inflammatory disorders such as autoimmune diseases and graft rejection are mediated by activated leukocytes, particularly T lymphocytes, which penetrate the inflamed tissue and perpetuate or amplify the immune reaction. In an unstimulated state, leukocytes do not readily adhere to the vascular endothelium. However, inflammatory signals induce the expression of proteins on the endothelial cell surface that promote the adhesion and extravasation of activated immune cells from the circulation into the underlying tissues. Key among these molecules are P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cells, and their respective counter receptors, P-selectin glycoprotein ligand-1 (PSGL-1), leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4), on the leukocytes. In vitro blockade of these molecules inhibits the adhesion of leukocytes. In many cases there is attenuation of leukocyte activation as well. Adhesion blockade in animal models prevents or ameliorates graft rejection and disease severity in autoimmune models. Clinical studies with humanised monoclonal antibodies which interfere with LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions have shown significant efficacy and good safety profiles in autoimmune disease, including psoriasis, multiple sclerosis and inflammatory bowel disease. Thus, adhesion blockade is emerging as a useful therapeutic strategy in several inflammatory settings.

In vitro and in vivo pharmacology and pharmacokinetics of a human engineered monoclonal antibody to epithelial cell adhesion molecule.

ING-1(heMAb), a Human Engineered monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 microg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

Anti-adhesion antibodies efalizumab, a humanized anti-CD11a monoclonal antibody.

The acquired immune response that leads to graft rejection depends on regulated adhesive interactions between T lymphocytes, endothelial cells, dendritic cells, graft tissue and the extracellular matrix to coordinate cellular trafficking and activation of antigen-reactive T lymphocytes. Inhibiting the function of molecules involved in the adhesion processes offers the potential for interfering with the allograft response. The leukocyte function associated antigen-1 molecule (LFA-1), a heterodimer of CD11a (alphaL) and CD18 (beta2) integrin subunits, is an attractive therapeutic target because it plays an important role in key steps of inflammation and tissue rejection. These include: (1) binding of leukocytes to endothelium; (2) trafficking through activated endothelium; and (3) costimulatory interactions between T lymphocytes and antigen presenting cells. Clinical experience with efalizumab, a humanized anti-CD11a monoclonal antibody (mAb), in patients with chronic plaque psoriasis has shown that anti-CD11a therapy is well tolerated and effective at reducing the severity of the disease without depleting lymphocytes. Initial results in renal transplant patients are also promising.

Psoriasis as a model for T-cell-mediated disease: immunobiologic and clinical effects of treatment with multiple doses of efalizumab, an anti-CD11a antibody.

BACKGROUND: Leukocyte function-associated antigen 1 (LFA-1), consisting of CD11a and CD18 subunits, plays an important role in T-cell activation and leukocyte extravasation. OBJECTIVE: To test whether blocking CD11a decreases immunobiologic and clinical activity in psoriatic plaques. DESIGN: Open-label, multicenter, dose escalation study. PATIENTS: Thirty-nine patients with moderate-to-severe psoriasis. INTERVENTION: Intravenous infusions of efalizumab, a humanized anti-CD11a monoclonal antibody, for 7 weeks at doses of 0.1 mg/kg every other week or 0.1 mg/kg weekly (category 1), 0.3 mg/kg weekly (category 2), and 0.3 increasing to 0.6 or 1.0 mg/kg weekly (category 3). Skin biopsies were performed on days 0, 28, and 56. MAIN OUTCOME MEASURES: Serum efalizumab levels, levels of total and unoccupied T-cell CD11a, T cell counts, epidermal thickness, cutaneous intercellular adhesion molecule 1 (ICAM-1) and keratin 16 (K16) expression, Psoriasis Area and Severity Index (PASI) scores. RESULTS: Dose-response relationships were observed for pharmacokinetics and pharmacodynamic measures. Category 1 failed to maintain detectable serum efalizumab or T cell CD11a down-modulation between doses. Category 2 achieved both. Category 3 achieved both and additionally maintained sustained T-cell CD11a saturation between doses. A dose-response relationship was also observed clinically and histologically. The mean decrease in the PASI score was 47% in category 3, 45% in category 2, and 10% in category 1 (P<.001). Epidermal and dermal T-cell counts, epidermal thickness, and ICAM-1 and K16 expression decreased in categories 2 and 3 but not in category 1. Circulating lymphocyte counts increased in categories 2 and 3. CONCLUSIONS: At doses of 0.3 mg/kg or more per week, intravenous efalizumab produced significant clinical and histologic improvement in psoriasis, which correlated with sustained serum efalizumab levels and T-cell CD11a saturation and down-modulation.