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Increased Apoptosis in Sjögren’s Syndrome
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Immunizing against SARS
A vaccine prepared against the spike (S) protein of the coronavirus that caused a global outbreak of severe acute respiratory syndrome (SARS) in 2002–2003 was not effective in neutralizing infections during a less severe outbreak 1 year later. To assess a subunit vaccine, He et al. (p. 6085 ) raised rabbit and mouse mAbs against the S protein receptor-binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2) on the cell surface. RBDs were PCR-amplified from coronaviruses isolated from one patient during each outbreak and from a civet, the intermediate amplification host. All three RBDs elicited high levels of cross-reactive anti-S RBD sera in mice and rabbits. Sera from mice or rabbits immunized with human or civet RBDs neutralized infection of human cells expressing human or civet ACE2 by SARS pseudoviruses comprised of human or civet S proteins. Mouse anti-RBD mAbs against three of six groups of highly conserved conformation-dependent neutralization epitopes (Conf IV-VI) within the RBD neutralized all three pseudoviruses and reacted with a panel of naturally occurring RBD mutants in ELISA. Epitopes within RBD required for neutralization mapped to its N terminus and C terminus. Engineered single-point mutations of conserved residues in the RBD that are critical for receptor-binding or viral entry disrupted reactivity of five of the Conf epitopes with the mAbs. The data suggest that certain Conf epitopes within the coronavirus S protein RBD induce cross-reactive neutralizing Abs that block S protein-ACE2 interactions of diverse SARS isolates.
Thymic Epithelial Progenitor Cells
Medullary thymic epithelial cells (mTECs) negatively select self-reactive thymocytes. However, there is little information regarding the development and differentiation of individual mTEC progenitors and their expression patterns for peripheral tissue-restricted Ags (TRAs). Gillard and Farr (p. 5815
) confirmed ongoing mitosis of 
1–2% of mTECs in sorted CD45-depleted adult mouse thymus cells on a population basis with BrdU and triple-staining experiments and on a single cell basis with an instrument that measured individual DNA-stained cells. RT-PCR analysis of cDNAs from the large population of sorted mTECs showed expression of genes known to regulate mTEC differentiation and of transcription factors characteristic of multipotential cells, such as embryonic stem cells and blastocysts, and of peripheral epithelial tissues, such as endoderm and neural tissues. Multiple TRAs characteristic of the tissues regulated by the transcription factors also were expressed. RT-PCR analysis on cDNAs from small pools (20 cells/pool) of sorted mTECs showed limited expression of individual TRAs ranging from 2 of 15 to 4 of 15 TRAs examined. The percentage of mTECS expressing a given TRA was calculated to be <0.4 to 1%. Examination at the single cell level by nested gene-specific PCR on cDNAs from small pools and individual mature mTECs confirmed limited expression of a given TRA in rare individual mTECs. Expression of aire in almost all pools did not correlate with coexpression of aire-dependent TRAs in the pools or in individual cells. The data support the authors’ model for mTEC diversity that posits steady-state differentiation of epithelial progenitor cells with broad developmental potential in the adult thymus.
LPS-Induced Lung Inflammation
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in bronchoalveolar lavage fluid. In contrast, when IL-1 itself was administered, wild-type and mutant mice had similar inflammatory responses. Caspase-11 mRNA and protein, needed to activate caspase-1 and IL-1, were induced by LPS and two other ER stress-inducing stimuli in vivo in lungs and in vitro in peritoneal macrophages from wild-type, but not Chop–/–, mice. LPS induced mRNA for a major ER chaperone in wild-type and mutant mice and macrophages. Immunohistochemical analysis of lungs from LPS-treated wild-type mice demonstrated that CHOP was expressed in the nuclei, and caspase-11 was expressed in the cytoplasm of the same cells; caspase-11 protein was not expressed in lung cells of mutant mice. A CHOP expression plasmid transfected into peritoneal macrophages from wild-type mice induced caspase-11 expression. The authors show that LPS induces caspase-11 in macrophages via the ER stress-CHOP pathway, resulting in caspase-1-dependent activation of IL-1 during lung inflammation. Th2 Cytokines and Intestinal Epithelium
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had the opposite effect, namely up-regulation of mRNAs for TLR3, TLR4, and MD-2 plus those for TLR2 and TLR5. Costimulation with IL-4 attenuated the IFN--induced up-regulation. Immunoblotting showed that intracellular TLR4 protein levels in cytokine-treated cells correlated with mRNA levels. Flow cytometry using PE-anti-human TLR Abs on live or permeabilized cells confirmed decreased TLR3 and TLR4 protein surface and cytoplasmic expression after incubation with the Th2 cytokines. Priming of IECs with IL-4 or IL-13 decreased internalization of fluorescent LPS compared with unstimulated controls. Cells treated with LPS in the presence of IL-4 did not have the increased I
B
phosphorylation seen in LPS-stimulated IFN--primed IECs. IECs primed with IL-4 or IL-13 produced less IL-8 after stimulation with a ligand for TLR3 or TLR4 compared with stimulated unprimed cells; stimulated cells pretreated with IFN- produced higher levels of IL-8 than controls. The authors demonstrate impaired TLR signaling in Th2 cytokine-primed IECs and suggest that Th2 cytokines might protect IECs from proinflammatory effects of Th1 cytokines. Combination Antitumor Therapy
Smyth and collaborators demonstrated that a mAb against the TRAIL receptor DR5 inhibits tumor growth and metastasis of TRAIL-sensitive tumors and induces tumor-specific CTLs against TRAIL-resistant variants. In a continuation of their research, Smyth et al. (p. 6347 ) suppressed tumor growth in mice injected with mammary tumor or renal carcinoma cells by administering IL-21 3 days after a third anti-DR5 mAb injection compared with controls not given IL-21. Suppression of tumor growth was abrogated by mAb depletion of CD11b+ cells and reduced by mAb depletion of NK or CD8+ T cells. The anti-DR5 mAb/IL-21 combination increased survival of mice with metastases. Mice treated with anti-DR5 mAb alone or in combination with IL-21 rejected a low dose of renal carcinoma or mammary tumor cells. Although both groups of mice were able to resist a second low-dose tumor challenge 12 wk after the primary inoculation, only mice receiving the combination treatment resisted a high-dose tumor challenge. SCID mice adoptively transferred with splenic T cells from anti-DR5- or anti-DR5/IL-21-treated wild-type mice were protected against injection with a low dose of mammary tumor cells; only cells from the mice treated with both reagents were protective against a high dose of tumor cells. Depletion of CD8+ T, but not NK or CD11b+, cells rendered anti-DR5/IL-21 survivors of a low-dose mammary tumor challenge susceptible to rechallenge. Splenocytes from mammary tumor-bearing mice treated by either protocol had in vitro cytotoxic activity against mammary tumor targets. Anti-DR5/IL-21 combination therapy reduced growth of established renal tumors beginning 12 days after tumor cell injection. The data demonstrate that the combination of a mAb-based therapy that induces tumor cell apoptosis followed by IL-12 promotion of memory CD8+ T cell development is an effective anticancer treatment in several mouse tumor models.
Immature DCs in Leishmaniasis
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-galactosylceramide; killing of promastigote-infected iDCs was blocked partially. IB4 also blocked the increased IFN- secretion by iNKT cells in response to infected iDCs. Incubation with lipophosphoglycan, the major cell surface component of the promastigote form of Leishmania donovani, resulted in up-regulation of CD1d on iDCs comparable to that seen on infected iDCs. iNKT cells killed lipophosphoglycan-treated iDCs and released IFN-; incubation of the target cells with IB4 abrogated both responses. The authors conclude that L. infantum-infected iDCs become resistant to NK cell killing by up-regulating HLA-E but sensitive to iNKT killing by up-regulating CD1d in the context of iGb3. Summaries written by Dorothy L. Buchhagen, Ph.D.
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