Fc gamma R expression on NK cells influences disease severity in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with autoantibodies, the best known of which is rheumatoid factor (RF). RF/IgG complexes interact with FcgammaR on the surface of neutrophils, NK cells and monocyte/macrophages. We have analyzed the expression pattern and allelic polymorphisms of three FcgammaR genes (FcgammaRIIA, FcgammaRIIC and FcgammaRIIIA) in a large sample of RA patients and normal donors. We have found that the level of FcgammaR (CD16 and CD32) expression on NK cells is lower in RA patients than in normal individuals. Genotypic analysis demonstrated that the CD32 isoform expressed by the majority of RA patients was not the activating FcgammaRIIc1 isoform, commonly seen in normal individuals, but rather the inhibitory FcgammaRIIb isoform. The combination of the FcgammaRIIIA-176F allele with a lack of CD32 expression in NK cells appeared to be characteristic of RA subjects with aggressive disease. Since FcgammaRII and FcgammaRIIIA are predominantly expressed by NK cells, these data further suggest that FcgammaR-mediated activation of NK cells could be a disease-determining factor in RA patients.

Qualitative and quantitative abnormalities in splenic dendritic cell populations in NOD mice.

The phenotype and function of splenic DC populations from diabetes-prone NOD mice were characterized and compared to DC from diabetes-resistant strains in the presence or absence of Flt3 ligand (FL) treatment. NOD mice were found to have significantly fewer CD8alpha+ DC than both B10.BR and C57BL/6 mice, and this defect was reversed by FL treatment. Freshly isolated CD8alpha+ and CD8alpha- DC from all three strains were found to express similar levels of costimulatory molecules and this was similar in both FL-treated and untreated animals. IL-12 p40 production was significantly lower in purified CD11c+ DC from NOD mice compared to DC from C57BL/6 or B10.BR mice. CD8alpha+ DC isolated from NOD mice produced lower levels of IL-12p40 than CD8alpha+ DC from C57CBL/6 and this was dependent on the nature of the stimulus given. In contrast both CD8alpha+ and CD8alpha- DC from FL-treated mice produced high levels of IL-12p40 following activation, but only the CD8alpha- DC produced IL-12p70. Functionally, freshly isolated CD8alpha- DC were more stimulatory than CD8alpha+ DC in a primary allogeneic mixed lymphocyte reaction. However, DC maturation resulted in increased T cell stimulatory capacity for both DC subsets, and this pattern was seen in all strains. These results demonstrate significant differences in phenotype and function of splenic NOD CD8alpha+ DC, and further suggest that FL treatment may reverse some of these abnormalities.

Dendritic cells, T cell tolerance and therapy of adverse immune reactions.

Dendritic cells (DC) are uniquely able to either induce immune responses or to maintain the state of self tolerance. Recent evidence has shown that the ability of DC to induce tolerance in the steady state is critical to the prevention of the autoimmune response. Likewise, DC have been shown to induce several type of regulatory T cells including Th2, Tr1, Ts and NKT cells, depending on the maturation state of the DC and the local microenvironment. DC have been shown to have therapeutic value in models of allograft rejection and autoimmunity, although no success has been reported in allergy. Several strategies, including the use of specific DC subsets, genetic modification of DC and the use of DC at various maturation stages for the treatment of allograft rejection and autoimmune disease are discussed. The challenge for the future use of DC therapy in human disease is to identify the appropriate DC for the proposed therapy; a task made more daunting by the extreme plasticity of DC that has recently been demonstrated. However, the progress achieved to date suggests that these are not insurmountable obstacles and that DC may become a useful therapeutic tool in transplantation and autoimmune disease.

Receptor-mediated endocytosis of iron-oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging.

Dendritic cells (DCs) function as antigen presenting cells in vivo and play a fundamental role in numerous diseases. New methods are described for high-efficiency intracellular labeling of DCs with superparamagnetic iron-oxide (SPIO) utilizing a receptor-mediated endocytosis (RME) mechanism. Bone marrow-derived DCs or a fetal skin-derived DC line were incubated with SPIO conjugated to anti-CD11c monoclonal antibody (mAb) under conditions favoring RME. These cells exhibited approximately a 50-fold increase in uptake relative to DCs incubated with SPIO without the mAb. Flow cytometry studies assaying cell surface markers showed a down-modulation of CD11c, but no other changes in phenotype. Immunological function of the DCs was unmodified by the labeling, as determined by cytokine secretion assays. The RME mechanism was confirmed using electron microscopy, endocytosis inhibition assays, and incubation experiments with SPIO conjugated to mAbs against accessory molecules that are not expressed on DCs. Labeled DCs were injected into murine quadriceps and monitored in vivo for several days using MR microimaging at 11.7 T. DCs were observed to remain within the muscle for >24 hr. The use of RME is an efficient way to label immune cells for in vivo MRI and can be applied to a wide variety of cell types.

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.

Phenotypic and functional characteristics of BM-derived DC from NOD and non-diabetes-prone strains.

We compared BM-derived DC from NOD with DC from non-diabetes-prone strains. NOD myeloid progenitors cultured in GM or GM + IL-4 yielded lower numbers of DC than similar cultures from other strains. NOD GM + IL-4 DC produced lower amounts of IL-12 p70 following CD40 ligation or LPS stimulation in the presence of IFN-gamma than DC from other strains, although similar levels of IL-6 and NO were produced by all strains. The low amounts of IL-12 p70 produced by GM + IL-4 DC are despite a more mature DC phenotype observed in NOD mice. The low DC number could diminish the ability of DC to stimulate immunoregulatory T cells. Furthermore, the differentiation/maturation of DC from myeloid progenitors is altered, reflecting disorders in the function of these cells in NOD BM.

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils.

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.

Human leukocyte antigen class I allelic and haplotype loss in squamous cell carcinoma of the head and neck: clinical and immunogenetic consequences.

The expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary for the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system. Down-regulation of HLA class I gene expression has been implicated in tumorigenesis, including squamous cell carcinoma of the head and neck (SCCHN). Loss of MHC class I antigens may be one mechanism by which tumor cells escape immune detection. We performed prospective immunostaining of 26 primary SCCHN tumors and samples of normal mucosa harvested several centimeters away from the primary tumor, using a large panel of antibodies directed against allele-specific as well as monomorphic determinants of HLA class I molecules. Loss of expression of HLA class I proteins in the tumor was found in 50% (13 of 26) of primary tumors and was highly correlated with HLA loss in the corresponding normal mucosa (P < 0.0001). Further analysis demonstrated that the loss of HLA class I expression in the tumor was significantly associated with regional lymph node metastases (nodal stage; P = 0.0388), and that the number of HLA class I alleles lost in the normal mucosa was associated with subsequent development of a new primary aerodigestive tract cancer (P = 0.042). A patient with two metachronous cancers available for analysis had no evidence of HLA loss in the first tumor, demonstrated allelic loss in the second cancer, and subsequently died of disease. These results suggest that the loss of expression of HLA class I alleles may have prognostic implications.

Studies of HLA-DR and DQ alleles in systemic sclerosis patients with autoantibodies to RNA polymerases and U3-RNP (fibrillarin).

OBJECTIVE: To determine the clinical and immunogenetic features of systemic sclerosis (SSc) patients with anti-RNA polymerase (RNAP) or anti-fibrillarin antibodies. METHODS: DNA typing for HLA-DR and DQ alleles was performed in 292 patients with SSc, including 81 with anti-RNAP and 24 with anti-fibrillarin antibodies. The remaining patients had anti-topoisomerase I (anti-topo I; 71), anti-centromere (ACA; 56), anti-Th/To (28), or other antinuclear (32) antibodies. RESULTS: Significant associations were observed in the patients with anti-topo I, ACA, and anti-Th/To antibodies, similar to those previously reported. No significant HLA associations were detected in the 81 patients with anti-RNAP. although weak associations were noted when this group was subdivided on the basis of immunofluorescence staining pattern; i.e., HLA-DR4 was increased in patients with strong nucleolar staining and HLA-DR3 was more frequent in patients with nucleoplasm staining only. No HLA-DR or DQ associations were observed in 24 patients with anti-fibrillarin antibodies. CONCLUSION: The identification of HLA associations in SSc patients with anti-RNAP antibodies may only be possible when the individual antibody specificities recognized by these sera are identified. It may then be possible to classify these patients into distinct clinical and immunogenetic subgroups.

Prevention of diabetes in the NOD mouse by a Th1 clone specific for a hsp60 peptide.

Peptide-based therapies have been shown to be effective in the prevention of diabetes in the NOD mouse. We have been interested in the T cell response elicited by such therapies and have been studying a T cell clone (C3.5) specific for hsp60 AA 437-460, generated following immunization with the hsp60 437-460 peptide. The C3.5 clone was CD4(+), Vbeta8.3 TCR(+), I-A(g7)restricted and of the Th1 type. The injection of this clone into prediabetic NOD mice prevented the adoptive transfer of the disease and suppressed the development of spontaneous diabetes. This effect was reflected in a reduction in the degree and severity of insulitis in mice injected with this clone. In addition, an antibody response was elicited to the C3.5 clone in mice given multiple injections of the clone. The epitope recognized by C3.5 is located in the N-terminus of the hsp60 AA 437-460 peptide, and this clone was unable to recognize the native hsp60 molecule. These data raise questions concerning the mechanism by which peptide-based therapies prevent autoimmune disease.

Immunotherapy of NOD mice with bone marrow-derived dendritic cells.

We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.

Ligand binding specificities and signal transduction pathways of Fc gamma receptor IIc isoforms: the CD32 isoforms expressed by human NK cells.

We recently reported that human NK cells express, in addition to CD16 [Fcgamma receptor (FcgammaR) IIIA], a second type of FcgammaR, namely CD32 (FcgammaRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcgammaRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcgammaRIIc-specific isoforms isolated from NK cells, namely FcgammaRIIc1 and FcgammaRIIc3, which differ in their cytoplasmic tails. The ligand binding specificity for both murine and human IgG isotypes was found to be similar to that observed for FcgammaRIIb isoforms. Immunoprecipitation studies of FcgammaRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcgammaRIIc1, and a protein of around 32 kDa for FcgammaRIIc3. Signal transduction studies performed on FcgammaRIIc1-expressing Jurkat cells indicated that this molecule is functional, i. e. capable of Ca2+ mobilization and activation of Lck, Zap-70 and Syk protein tyrosine kinases, although the CD3 zeta chain was not found to functionally associate with FcgammaRIIc1. In contrast, FcgammaRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcgammaRIIc3. These studies demonstrate the functional activity of FcgammaRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.

Functional CD32 molecules on human NK cells.

Human NK cells are large granular lymphocytes that kill neoplastic or virally infected targets using perforin-dependent mechanisms. CD16 or FcgammaRIII is one of the cell surface molecules that can trigger the killing machinery following binding of the Fc portion of IgG to the receptor: a mechanism known as antibody dependent cell-mediated cytotoxicity (ADCC). We have recently shown that some individuals express an additional FcgammaR on their NK cells, CD32 or FcgammaRII. This receptor has now been characterized at the molecular, biochemical and functional level. The present review outlines our findings to date on the features of this novel receptor. These findings suggest that the presence of a functional FcgammaRII on the surface of NK cells could have important clinical consequences in both tumor immunotherapy and autoimmune disease.

Immunobiology of DC in NOD mice.

NOD mice spontaneously develop diabetes between 15 and 20 weeks of age, which is preceded by insulitis characterized by the infiltration of lymphocytes. Dendritic cells (DC) are among the first cells to infiltrate the islet and they have been implicated in the pathogenesis of the disease. Our work has been concerned with the detailed characterization of four distinct DC populations in NOD mice: two derived from bone marrow (BM) cells cultured in either granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) or GM-CSF alone and two from the spleen of Flt3 ligand (Flt3L) -treated mice, isolated on the basis of CD8alpha expression. Phenotypic and functional differences between these DC subsets in NOD mice have been identified. In addition, we obtained a lower yield of NOD BM-derived DC and they expressed higher levels of cell-surface CD40 and IL-12 p40 mRNA than BM-derived DC from the diabetes-resistant strain, B10.BR. We have also investigated the ability of these DC populations to modulate the development and progression of diabetes in NOD mice.

Evidence of positive cross-regulation on Th1 by Th2 and antigen-presenting cells: effects on Th1 induced by IL-4 and IL-12.

The response of Th cells to cytokines is normally strictly regulated, such that following antigenic stimulation, Th cells respond for only a short period of time, after which they become refractory to cytokine-mediated effects. IL-12, a costimulator of Th1 having no proliferation-inducing capacity of its own, allows Th1 clones and lines to respond to IL-4 when they would otherwise be unable to respond to this cytokine. Cells that have proliferated in response to IL-4 plus IL-12 are fully able to be subsequently activated by specific Ag and APC. Additionally, the response to IL-4 of Th1 effector cells derived from normal murine spleen is enhanced significantly by IL-12. Furthermore, in the presence of IL-12, stimulated Th2 can induce proliferation of Th1 via IL-4 production, in a dual chamber culture system. We hypothesize that the effects of IL-4 and IL-12 represent a novel, positive cross-regulatory pathway that acts on Th1, and is mediated by Th2 (the IL-4 source) and APC (the IL-12 source). We propose this as a way for a Th2 immune response to positively influence an ongoing or waning Th1 response.

Crossregulation between Th1 and Th2 cells.

Th1 and Th2 subsets have been characterized on the basis of the cytokines they secrete and the immune functions they mediate. Th1 cells secrete IL-2, IFN-gamma, and lymphotoxin and are important in the cell-mediated response; Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are important in the control of macrophage function and in the stimulation of particular immunoglobulin isotypes. Cytokines secreted by Th1 and Th2 cells regulate the growth and differentiation of Th1 and Th2 cells in both positive and negative ways; this has been termed crossregulation. Much work has concentrated on the factors important in the differentiation of these Th subsets, and it has been established that Th cells become committed to a Th1 or Th2 phenotype within 48 hrs of antigenic stimulation. During the differentiation process irreversible changes in the expression and function of cytokine receptors occur that provide an explanation for the observed crossregulatory features of Th1 and Th2 cells. In this review we summarize the crossregulation between Th1 and Th2 cells in terms of the changes in cytokine receptor expression and function that occur during differentiation.

Expression of functional CD32 molecules on human NK cells is determined by an allelic polymorphism of the FcgammaRIIC gene.

Human natural killer (NK) cells were thought to express only FcgammaRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcgammaR, ie, FcgammaRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcgammaRII from NK cells derived from several normal individuals that may represent four different products of the FcgammaRIIC gene. One transcript (IIc1) is identical with the already described FcgammaRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcgammaRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcgammaRIIc isoforms on their NK cells.

Mathematical modeling of immunological reactions.

The immune system is a highly regulated, complex and integrated system which has evolved to provide the organism with substantial defenses against pathogenic organisms. Over the last several decades there has been an explosion of experimental data in this area, and new techniques in molecular and cellular biology have been crucial in deepening our understanding of immune processes. Most of these new techniques have allowed the isolation of the process or cell under study so that the results can be readily interpretable. At the present time, however, there is an emerging need to understand the system as it functions as a whole and the language of mathematics is the one best suited for this purpose. This review, written from the perspective of an experimental immunologist, describes some of the recent advances in the development of mathematical models of the immune system. Particular emphasis is placed on the rapidly growing field of modeling in HIV infection and T cell activation. Immunology as a whole will benefit from the introduction of the language of mathematics in much the same way as neuroscience has done in the last decade.