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Ig carbohydrate and secretion
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L chain that was either wild type or mutated in one of the three CDRs. All of the VH and VL genes were joined to human sequences. VH chains expressed with wild type and all three mutant murine-human hybrid VL chains were susceptible to endoglycosidase H cleavage, indicating that the carbohydrate was of high mannose type. This result was obtained in human, mouse, and hamster cells. No Ab was produced after transfection of a construct, in which the CDR2 of the Asn60 VH replaced the CDR2 of an anti-dansyl VH that had no glycan, into a cell line expressing an anti-dansyl VL chain unless tunicamycin, an inhibitor of N-glycosylation, was added to the cells. A wild-type anti-dansyl Ab was localized in the ER and the Golgi apparatus, whereas the hybrid Ab was found only in the ER apparatus by confocal microscopy. The data indicate that, in some cases, addition of carbohydrate to Ig variable regions can prevent Ab secretion. EBV-induced B-1 cell development
The viral-encoded latent membrane protein 2A (LMP2A) of EBV functionally mimics a constitutively active B cell receptor (BCR). LMP2A is thought to regulate viral latency and pathogenesis within infected B cells. Ikeda et al. (p. 5329 ) found that transgenic mice that expressed high levels of LMP2A (TgE) have large numbers of CD5+ cells with surface marker phenotypes of B-1a cells in the spleen, peritoneal cavity, bone marrow, lymph nodes, and blood compared with wild-type mice. A line of LMP2A transgenic mice that expressed a low level of the viral protein and a line of transgenic mice carrying a mutant LMP2A that could not bind the phosphotyrosine kinase Syk had B-1a cell profiles similar to those of wild-type animals. Flow cytometry using a variety of Abs to B cell surface markers showed that wild-type and TgE bone marrow cells had equal percentages of early and late pro-B cells. However, CD5 was expressed on pre-B and immature B TgE cells, and there was a dramatic increase in the numbers of pre-B TgE cells compared with wild-type cells. TgE bone marrow cells cultured in IL-7 contained large pre-B cells expressing CD5 (43% compared with 9% for wild type). Both wild-type and TgE splenic B-1 cells responded in vitro to LPS but not to PMA. TgE spleen cells had low levels of surface IgM and high levels of serum IgM compared with wild-type mice. The authors conclude that a strong BCR signal, such as delivered by LMP2A of EBV, favors B-1 lineage commitment and that the commitment can occur as early as the pre-B stage.
Limiting Th2 allergic responses
Basophil precursors exposed to IL-3 synthesize histamine. However, their response to the pro-Th1 cytokines, IL-12 and IL-18, has not been documented. Schneider et al. (p. 5262
) found that mouse splenocytes exposed to IL-12 plus IL-18 for 24 h produced substantially less IL-3-induced histamine compared with splenocytes treated with either IL-12 or IL-18 alone or untreated; histamine levels produced by IL-3-induced bone marrow cells were higher after IL-12 plus IL-18 treatment. Histamine release was only slightly inhibited in IL-3-induced spleen cells from Fas- or FasL-deficient mice, and a caspase inhibitor reduced the high apoptotic index of IL-3-induced immature basophils treated with IL-12 plus IL-18. Spleen cells treated with IL-12 plus IL-18 killed target cells more efficiently than spleen cells treated with either cytokine alone. Depleting splenocytes of NK1.1+ cells abrogated the cytotoxic effect and reduced the level of IFN-
in the culture medium. TNF-
plus IFN- could restore inhibition of histamine synthesis in response to FasL cross-linking with anti-Fas Ab in spleen cells from NK cell-deficient mice. Splenocytes from mice injected i.v. with IL-12 plus IL-18 had reduced histamine levels in response to IL-3; this in vivo response also involved the Fas pathway and was NK1.1+-cell dependent. These experiments show that IL-12 and IL-18, acting via NK cells, can limit Th2 allergic responses in vitro and in vivo by removal of splenic basophil precursors.
Sequential anti-malaria immunizations
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responses. Others had shown that immunization with RTS,S, a fusion protein containing the carboxyl terminus of the PfCSP protein fused to hepatitis B surface Ag, induced Ab and CD4+ T cell-dependent IFN- responses. In their study reported on p. 5561
, Wang et al. found that 5 of 10 primed volunteers treated with the DNA vaccine a year earlier, but 0 of 14 nonprimed volunteers, had Ag-specific CTLs after two doses of RTS,S. No CTLs were detected before RTS,S administration in either group. IFN- responses against four PfCSP-specific peptides were seen with PBMCs from 6 of 10 DNA-primed, but against only one peptide from 2 of 14 nonprimed, volunteers after a single RTS,S injection. PBMCs depleted of either CD4+ T or CD8+ T cells showed that the IFN--producing T cell profiles varied with the PfCSP peptide used for stimulation and that CD8+ T cells were involved only in the production phase of the response. The authors conclude that DNA priming directed T cell responses toward the priming Ag during the boost. Ag-specific unresponsiveness of T cells
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did not inhibit supraoptimal activation-induced apoptosis. DNA fragmentation was observed after 6 h of PHA or 12 h of anti-CD3 treatment and was prevented by inhibitors of cysteine proteases. Yet caspases 3, 7, 8, and 9 were not activated at high concentrations of PHA or anti-CD3. Immunoblot analyses on cytosolic extracts from treated cells detected the active cleaved forms of cathepsins B and L. The cathepsins were localized by confocal microscopy to cytoplasmic granules in untreated cells but became distributed diffusely in the cytoplasm after treatment with high concentrations of PHA or anti-CD3. The data suggest that peripheral T cell tolerance induced by high doses of Ag is a result of lysosomal release of cathepsins. The end result is DNA fragmentation and T cell death.
IFN- promotes DC/NK cross-talk
The use of dendritic cells (DCs) in vaccination protocols in cancer therapy is becoming widespread. However, there is still need to maximize the ability of DCs to prime CD8+ T cells to recognize tumor Ags. Tosi et al. (p. 5363
) looked at the differentiation state of DCs generated ex vivo from monocytes in the presence of IFN- (IFN-DCs). Adherent PBMCs expressed activation and maturation surface markers intermediate to those of immature and mature DCs following incubation with IFN- and GM-CSF for three days. IFN-DC phagocytic activity was more similar to that of mature DCs. IFN-DCs pulsed with epitopes from tumor Ags and cultured with autologous PBMCs induced Ab-specific T cell responses comparable to those induced by mature DCs. IFN-DCs derived from immunosorted monocytes were less able to induce specific CD8+ T cell responses. NK cells present in PBMCs also were activated by IFN-. Removal of NK cells from PBMCs treated with IFN- resulted in differentiation of IFN-DCs with reduced priming activity for tumor Ag-specific T cells and lower production of TNF-; priming activity and TNF- levels were restored by the addition of NK cells before IFN-DC differentiation. The authors conclude that cross-talk between DC and NK cells is required to optimize the T cell priming activity of IFN-DC.
An infectious tolerance loop
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increased the numbers of CD25+ cells among alloactivated human CD4+ T cells. Using cells from two different HLA-A2 donors, the authors showed that the increased CD25+ population was derived from CD25– cells from the second donor but was dependent upon the presence of CD25+ cells from the first donor. Treg cells purified after stimulation of naive CD4+ T cells with alloantigen and TGF- (Treg1) induced CFSE-labeled CD4+CD25– T cells to divide (Treg2); both Treg1 and Treg2 cells suppressed alloactivation of CD8+ T cells. The education of Treg2 by Treg1 required cell contact and was inhibited by Abs against either TGF- or IL-10. Expression of mRNA for the forkhead/winged helix transcription factor FoxP3 was increased in CD4+CD25+ T, Treg1, and Treg2 cells compared with CD4+CD25– T cell controls. The authors propose a loop model whereby CD25– T cells, activated by IL-2- and TGF--stimulated CD25+ T cells, become Treg cells, express TGF- and IL-10 and, in turn, induce other CD25– T cells to become Treg cells. Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
, and IL-10
-Induced Monocyte-Derived Dendritic Cells and NK Cells in Priming CD8+ T Cell Responses against Human Tumor Antigens
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