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IN THIS ISSUE

Regulating Lymphocyte Velocity

There is growing evidence that PI3Ks are involved in chemotaxis and motility of B and T cells. On p. 2261 , Matheu et al. saw that fluorescent-tagged B and T cells treated with wortmannin (WMN), the covalent PI3K catalytic domain inhibitor, had less migration from peripheral lymph nodes to blood and spleen than untreated control cells after injection into mice. Imaging of lymph nodes with a two-photon microscope system showed that WMN-treated B cells remained at high density at the edge of the B cell follicle and did not enter it. Untreated B cells exhibited strong directional movement toward and into the follicle. WMN-treated B cells expressed fewer receptors for CCL13 but more receptors for CXCL12/CCL19 than control cells and had increased migration away from and toward the chemokines, respectively. WMN-treated T cells had reduced motility within the T cell zone of the lymph node and reduced chemotactic response to CXCL12/CCL19. In mice lacking p85, one major PI3K regulatory subunit, T cells had only modest defects, whereas B cells had reduced velocity within lymph nodes and exhibited drastically altered morphology. In contrast, in mice lacking p85, another major PI3K regulatory subunit, T cells had reduced velocity within lymph nodes, whereas B cells had only slight reductions. T cells from mice lacking both p85 isoforms plus other class IA PI3K regulatory subunits were shown by two-photon imaging to have reduced velocity and polarization compared with wild-type cells. The experiments indicate that both PI3K catalytic activity and class IA regulatory subunits influence lymphocyte motility and cell morphology in lymph nodes.

Tolerogenic B Cells

Although B cells are reported to suppress the induction of T cell immunity by naive T cells, there is no information about the mechanism involved. Chen et al. (p. 2046 ) induced the proliferation of CFSE-labeled CD4⁺ T cells by coincubation in vitro with nonirradiated allogeneic B cells or splenic dendritic cells (DCs) as APCs. A higher percentage of FoxP3⁺ T cells was generated in MLRs with the B cells, although the total number of stimulated T cells was lower than in MLRs with DCs. Most of the FoxP3⁺ T cells were in the CD25⁺ T cell subpopulation, and a higher B to T cell ratio was required to induce their proliferation compared with CD25^– T cells. Irradiated B cells lost the ability to stimulate CD25⁺ T cells but were able to stimulate CD25^– T cells alone, CD25⁺ TZ cells in the presence of CD25^– T cells, or CD25⁺ T cells plus IL-2. At low B to T cell ratios, the lower numbers of proliferating T cells were predominantly FoxP3⁺ T cells. Whereas anti-CD28 mAb increased FoxP3^– T cell proliferation, an anti-MHC class II mAb decreased proliferation of that population but not that of FoxP3⁺ T cells. The B cell-expanded CD25⁺ T cells blocked the proliferation of allogeneic CD25^– T cells in MLRs with DCs syngeneic to the CD25⁺ T cells. The experiments demonstrate that B cells preferentially expand allogeneic FoxP3⁺ CD4 T⁺ cells in a reaction that is stimulated by blocking MHC class II-TCR interactions.

Facilitating Transplant Tolerance

Although CD8⁺TCR^– facilitating cells (FCs) are known to promote transplant tolerance of allogeneic stem cells (SCs), the function of FCs is not known. Taylor et al. (p. 2153 ) showed in a fully allogeneic mouse model that recipients of allogeneic SCs plus FCs developed more splenic CD4⁺CD25⁺ regulatory T (Treg) cells of donor origin than survivors of allogeneic SCs plus bone marrow-derived T cells. More than half of the generated cells were FoxP3⁺, anergic, and inhibited proliferation of CD4⁺CD25^– T cells ex vivo. GFP⁺ donor FCs were detected by RT-PCR in recipient spleens 2 wk posttransplantation with SCs and 2 wk before the induction of FoxP3 mRNA expression. In a syngeneic in vitro model, bone marrow-derived FCs were stimulated with a TLR9 ligand, CpG, before coculture with CD4⁺CD25^– T cells. Nearly five times as many FoxP3⁺ Treg cells expressing IL-4 mRNA and inflammatory cytokines were generated compared with cultures containing unstimulated FCs. Transwell experiments showed that cell-cell contact between FCs and CD4⁺CD25^– T cells was required for the development of Treg cells. Up-regulation of the B7 receptor, CD86, on CpG-stimulated FCs was detected by flow cytometry and RT-PCR. Anti-CD86 Ab prevented the expression of FoxP3 mRNA in Treg cells induced by FCs. The authors show that FCs directly induce FoxP3⁺ Tregs in spleens of mice to facilitate tolerance of allogeneic bone marrow transplants.

A Renewed Model of AP Activation

Over 50 years ago, Pillemer and colleagues showed that properdin:target complexes direct C activation by the alternative pathway (AP). But their model was not accepted, and properdin was set aside in favor of C3b. On p. 2600 , Spitzer et al. demonstrated that C3b bound only to the surface of Neisseria pretreated with properdin and that the addition of factors B and D generated surface-bound C3bBbP. Properdin binding promoted opsonization of the bacteria. Properdin also bound zymosan or rabbit, but not sheep, erythrocytes to initiate assembly of the AP convertases. C3b bound to the surface of mouse erythrocytes incubated with a bifunctional recombinant protein containing a human properdin domain fused to a single chain Ab domain that recognized a mouse erythrocyte surface Ag. Comparable results were seen with human erythrocytes incubated with a similar recombinant protein whose single-chain Ab domain recognized a human erythrocyte Ag. Properdin bound to LPS-mutant bacteria with little or no O-Ag found in wild-type bacteria. C3 deposition and AP activation on bacteria correlated with the degree of properdin binding. The authors present evidence for Pillemer’s properdin model of AP activation and show that it differs in significant aspects from the standard model of AP activation. They propose that the properdin mechanism is directed to specific target surfaces, whereas the standard mechanism is nonspecific.

Detecting FPR Tails

Activity of the chemotactic N-formyl peptide receptor (FPR) on neutrophils is regulated by cellular kinases that act on the FPR cytoplasmic tail, but specific sites of phosphorylation have not been determined. Riesselman et al. (p. 2520 ) produced recombinant human neutrophil FPR (rFPR) and developed two mAbs, NFPR1 and NFPR2, which recognize denatured forms of both rFPR and cellular FPRs on immunoblots. They showed by phage display epitope analysis that each mAb recognized a different region in the FPR cytoplasmic tail. Only NFPR1 recognized the closely related chemoattractant receptor FPRL1 expressed on membranes from transfected mouse cells. Binding of NFPR2 to immunoblots of whole cell extracts from hamster cells expressing rFPR was diminished by preexposure of the cells to fMLP. The binding was restored by incubation of membranes from the stimulated cells with a phosphatase. rFPR was immunoprecipitated from nonionic detergent-treated cell extracts by NFPR2 but not by NFPR1. Binding to plasma membranes from primed human neutrophils was stronger for NFPR2, as shown by immunoblots of subcellular fractions, flow cytometry on permeabilized primed cells, and immunohistochemistry. Only NFPR1 detected FPR in intracellular granule fractions. These two novel mAbs are sensitive reagents for the study of intracellular FPR in human neutrophils at various stages after stimulation by formylated peptides.

Ag Stability in Vaccine Design

Cross-presentation of MHC class I-restricted epitopes to CD8⁺ T cells is required to generate efficient immunization against DNA vaccine-encoded Ags. However, the effect of Ag stability on CD8⁺ T cell induction is not clear. Bins et al. (p. 2126 ) measured CD8⁺ T cell responses to two viral epitopes delivered by multineedle intradermal DNA vaccination. A DNA vector incorporating either an influenza A nucleoprotein or human papillomavirus epitope within the carboxyl terminus of GFP elicited higher Ag-specific T cell responses than a DNA vector with either epitope incorporated within the hydrophilic segment of a signal peptide. Epitope half-lives were measured using a vector with the influenza epitope at the carboxyl terminus preceded by a luciferase gene with either a stabilizing or destabilizing residue at its amino terminus and capped with an ubiquitin moiety. The destabilizing residue conferred shorter half-lives after transfection into mouse cells or after intradermal DNA vaccination in mice; fewer Ag-specific CD8⁺ T cells were measured by ex vivo MHC tetramer staining of blood cells from mice vaccinated with vectors containing the destabilizing residue. The vector incorporating the stabilizing residue induced a greater division of transferred CFSE-labeled Ag-specific transgenic CD8⁺ T cells in mice lacking CD4⁺ and CD8⁺ T cells and greater CD8⁺ T cell responses in MHCII^–/– mice. The importance of Ag stability was confirmed using vectors with one of three different amino acids at the amino terminus of the luciferase gene. Intramuscular and intradermal vaccinations gave similar results. A vector incorporating a shuffled version of the luciferase protein induced fewer Ag-specific CD8⁺ T cells than the control. The experiments demonstrate that the stability of Ags delivered by DNA vaccination directly correlates with immunogenicity.

Understanding the Mouse Igh Locus

The highly repetitive nature of the mouse H chain (Igh) locus enables V(D)J and other rearrangements. The result is great diversity of Ig H chains. Although Igh is known to be highly polymorphic among different inbred strains, it has not been examined at the nucleotide level. Retter et al. (p. 2419 ) sequenced recombinant mouse DNA clones to obtain a 1.6-Mb sequence of the entire C_H, J_H, and D_H region and half of the V_H region of the Igh locus in the 129S1/SvImJ (129S1) mouse. Previously described functional rearrangements, nonfunctional pseudogenes, and "possibly functional" V_H genes were detected using gene analysis programs. Two regions of expansion within the 129S1 V_H region sequence were noted by comparison with the C57BL/6 mouse Igh V_H region; alleles localized to regions with high overall sequence similarity between the two strains. Similar analysis of the D_H region in the 129S1 mouse found nonfunctional genes, two separate blocks of homology with the haplotype-different C57BL/6 mouse, and differences from the haplotype-identical BALB/c mouse. The four 129S1 and BALB/c J_H genes were identical with the exception of a single nucleotide base change, and the eight 129S1 and BALB/c C_H region coding sequences were highly similar but had isotype differences. The authors point out that duplication of V_H genes plus their extensive flanking sequences in only one orientation indicates a directed mechanism in the evolution of this locus. They suggest that the allelic variations occurred over the past 100,000 years and became fixed during the generation of the different inbred mouse lines.

Summaries written by Dorothy L. Buchhagen, Ph.D.

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