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Peripheral T Cell Tolerance
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Preventing Thymic Export
Thymocytes that survive positive and negative selection mature in the thymic medulla before migrating to the periphery. It is not known why they spend so much time there or how Ag-TCR interactions influence their export. In mice injected i.v. 16 h previously with Staphylococcus enterotoxin B (SEB) and intrathymically (i.t.) with FITC, Uldrich et al. (p. 4553
) found increased CD25 and CD69 expression on V
8+ peripheral T cells but a decrease in FITC+ Ag-reactive recent thymic emigrants (RTEs). No loss of peripheral FITC+ RTEs occurred in animals injected i.t. with FITC but whose thymii were removed before receiving SEB i.v. Intrathymic injection of a low dose of SEB plus FITC reduced the number of peripheral RTEs in wild-type mice but had only a modest effect in TNF–/– mice. V8+ single-positive thymocytes increased in cell size and up-regulated CD25 and CD69 after i.t. SEB challenge. Wild-type mice and mice defective in clonal deletion of immature and mature T cells had similar losses of peripheral RTEs following i.t. SEB injection. Mice transgenic for an anti-OVA peptide TCR had >95% reduction in peripheral CD8+ single-positive RTEs after i.t. OVA peptide challenge but had no changes in peripheral resident CD8+ T cells. The data demonstrate that intrathymic Ag-TCR ligation inhibits export of T cells to the periphery in mice.
Improving Allograft Tolerance
Although partial depletion of T cells is used clinically to prolong retention of solid organ transplants, the Turka laboratory showed in a murine cardiac allograft model that it also promotes homeostatic proliferation of residual T cells and induces tolerance resistance. In Neujahr et al. (p. 4632 ), the Turka laboratory determined that memory T cells emerged in normal or thymectomized mice treated with depleting doses of anti-CD4 plus anti-CD8 mAbs. Using BrdU labeling of cells in vivo or transferring CFSE-labeled memory or naive cells into thymectomized recipients, they showed that homeostatic expansion began 7 days after the start of depletion and was most prominent for memory cells. Proliferation of regulatory T (Treg) cells occurred but was always less than that of memory T cells. Mice depleted of T cells but injected 2 wk later with donor-specific T cell-depleted spleen cells plus human CTLA4Ig at the time of allografting retained their grafts indefinitely when given Treg cells from naive littermates 2 days after grafting; control mice rejected their allografts. SCID mice given unfractionated T cells from naive wild-type mice and treated with nondepleting anti-CD4 plus anti-CD8 mAbs and given donor-specific T cell-depleted spleen cells plus human CTLA4Ig at the time of allografting also retained their grafts long term. The non-depleting mAbs preferentially inhibited the rapid pattern of T cell proliferation during lymphopenia but had little impact on the slow pattern of proliferation. The authors show that rapid proliferation of memory cells during lymphopenia can be countered by non-depleting mAbs plus CTLA4Ig and that exogenous Treg cells can restore tolerance induction.
A Specific Treg Cell Marker
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75% of them were CD25+. Anti-GITR mAb treatment depleted the GITRhigh and Foxp3+ cells; anti-CD25 mAb treatment did not completely eliminate either population. Interestingly, half of BALB/c nude mice injected with the anti-GITR mAb-treated cells died of autoimmune myocarditis within 40 days and developed autoimmune disease in other organs, suggesting a role of GITRhigh cells in maintenance of self-tolerance. Adoptive transfer of spleen cells from animals with myocarditis induced the disease in secondary recipients. Mice injected with anti-CD25 mAb-treated cells, with or without anti-GITR mAb-treated cells, developed autoimmune disease in several organs but rarely in the heart. In a second mouse model, NOD-SCID mice had accelerated onset of diabetes after receiving anti-GITR mAb-treated NOD T cells, compared with mice that received CD25–CD4+ T NOD T cells; diabetes was accompanied by inflammatory destruction of several organs but not the heart. Transfer of anti-GITR mAb-treated BALB/c thymocytes also produced myocarditis and autoimmune diseases in several organs in BALB/c nude mice. The experiments suggest that GITR is a more specific marker of Foxp3-expressing CD4+ Treg cells independent of their expression of CD25 and that both CD25+ and CD25– populations are involved in maintenance of self-tolerance. Tracking Memory B Cells
The inability to definitively identify memory B cells is an obstacle to understanding development of B cell memory and secondary Ab responses. Chappell and Jacob (p. 4706
) used cre-lox recombination to generate transgenic mice that expressed -galactosidase (-gal) exclusively in germinal center (GC) B cells. In vitro -gal staining of spleen sections from transgenic mice immunized with (4-hydroxy-3-nitrophenyl)acetyl chicken 
globulin (NPCG) revealed scattered -gal-stained GCs that were not visible in spleen sections from control animals. Immune -gal+ splenic B cells isolated up to 35 days after immunization expressed low levels of GC B cell markers and decreased binding of PNA (peanut agglutinin) and were negative for expression of the plasma cell differentiation marker, syndecan-1 (Synd-1), as determined by flow cytometry. DNA sequence analyses of splenocytes from immune transgenic mice indicated a low number of mutations in rearranged 
1 V gene segments from -gal+PNA– B cells at 8 days p.i. compared with either -gal–PNA+ B cells or with -gal+PNA+ B cells, containing 58 or 100% mutations within the complementarity determining regions, respectively. Numbers of splenic Ab-forming cells (AFCs) secreting Ag-specific IgG were highest from NPCG-challenged RAG2–/– animals adoptively transferred with -gal+ memory B cells from immune transgenic mice plus CD4+ T cells from NPCG-immune parental mice and splenocytes from unrelated naive mice compared with controls. B220int-gal+Synd-1+ B cells and B220int-gal–Synd-1+ B cells from NPCG-challenged NPCG-immune transgenic mice had nearly identical frequencies of Ag-specific AFCs. In secondary AFCs, -gal+Synd-1+ B cell mutation frequency was significantly greater than that of -gal–Synd-1+ B cells. This novel transgenic mouse model indicates that a large proportion of the secondary Ab response involves B cells from outside GCs.
Transcriptional Silencing in CD8+ T Cells
Generation of memory CD8+ T cells following viral infection involves a series of differentiation stages. However, the role of DNA methylation in establishing effector and memory CD8+ T cells is not known. Chappell et al. (p. 4562 ) found that mice in which the DNA maintenance methyltransferase gene, Dnmt1, was deleted only in CD8+ T cells after peripheral activation had fewer splenic Ag-specific CD8+ T cells 8 days postinfection (p.i.) with lymphocytic choriomeningitis virus (LCMV) than wild-type controls. T cell activation phenotypes and cytokine production in response to LCMV peptide stimulation in vitro were the same in both groups; Dnmt1–/– mice were only slightly impaired in their ability to control viral infection. Bisulfite sequence analysis of genomic DNAs isolated from flow-sorted Ag-specific Dnmt1–/– CD8+ T cell populations demonstrated extensive demethylation of a methylation-sensitive gene compared with naive CD8+ T cells. Ag-specific CD8+ T cells were found in spleens of wild-type mice and at reduced numbers in the mutated mice up to 180 days p.i. Contraction of Ag-specific memory cells between days 8 to 60 p.i. and response to LCMV peptide in vitro were considerably reduced in Dnmt1–/– mice vs wild-type mice. Although proliferation of CFSE-labeled naive Dnmt1–/– CD8+ T cells in response to in vitro stimulation was comparable to that of wild-type cells, no division was detected following transfer into sublethally irradiated wild-type hosts. Rates of apoptosis of wild-type and mutated cells following LCMV infection were the same. The authors suggest that decreased accumulation of Dnmt1–/– vs wild-type mouse CD8+ T cells after LCMV infection is due to delayed cell recruitment resulting from loss of DNA methylation patterns.
Human Complete Stat-1 Deficiency
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, or TNF-
over background levels in response to BCG stimulation in vitro. DNA sequence analysis of the STAT1 gene detected a homozygous frameshift mutation, resulting in a truncated cDNA and absence of intracellular Stat-1 protein. No proteins from nuclear extracts of an IFN-- or IFN--stimulated EBV-immortalized patient’s B cells bound to radioactive gamma-activated or IFN-stimulated response element DNA sequence probes in EMSAs. IFN- was unable to up-regulate HLA-II cell surface expression, and IFN- failed to reduce HSV or vesicular stomatitis virus replication in the patient’s SV40-transformed fibroblasts compared with control cells. This report on the clinical and biological impact of human complete Stat-1 deficiency highlights the importance of Stat-1 in mediating signaling by IFNRs in innate immunity. Each mutation in the three patients was unique. Summaries written by Dorothy L. Buchhagen, Ph.D.
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