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IN THIS ISSUE

Using AKSAs to analyze kinases

While it is clear that kinases are important in a variety of biological processes, it is not known what impact varying levels of the enzymes have on those processes. It is possible to mutate the ATP binding site while retaining the ability of a kinase to bind ATP and to function normally. These ATP-analog-sensitive kinase alleles (ASKA) have the advantage of binding large ATP analogs. Denzel et al. (p. 519) used this methodology to regulate and to explore the biological effects of the Lck kinase on T cell development in reaggregated thymic organ cultures. The mutated Lck kinase gene (Lck^a-as) was transfected into the thymic cultures as a fusion molecule with the green fluorescent protein gene marker. The kinase activity of expressed Lck^a-as was inhibited by the ATP analog, whereas the activity of endogenous Lck was unaffected. The double-negative to double-positive transition of T cells was inhibited by expression of Lck^a-as or a transfected constitutively active Lck allele (Lck^a). Addition of the ATP analog reversed the inhibition of the development of double-positive T cells by Lck^a-as but not by Lck^a. T cells expressing different levels of Lck^a-as were separated by flow cytometry on the basis of marker expression levels, and Lck enzyme activity was evaluated over a range of cellular concentrations. Varying the levels of Lck^a-as expression and the inhibitor concentrations permitted greater double-positive T cell development in RAG1^-/- mice and in Lck^-/- mice. The findings indicate that the ASKA technology could be of use in analyzing the functions of other kinases in lymphoid cells.

Transplantation, infection, and innate immunity

Although immunosuppressive drugs allow patients to retain transplanted organs, the patients are rendered more susceptible to microbial infections. One goal in treating transplant recipients is to boost the innate immune response while continuing the suppression of the adaptive immune response. The potential of GM-CSF to accomplish this goal was the focus of studies by Xu et al. (p. 938). In an in vitro system, they found that GM-CSF restored TNF, but not IL-1, production in dexamethasone-suppressed blood cells from healthy donors and in LPS-stimulated blood cells from immunosuppressed liver transplant patients. In contrast, GM-CSF was unable to restore production of IFN- from Con A-stimulated T cells that had been immunosuppressed in vitro. Salmonella typhimurium-infected immunosuppressed mice died within 2 wk of infection. However, pretreatment of bacterial infected, immunosuppressed animals with GM-CSF resulted in nearly 100% survival, comparable to that seen in an infected nonimmunosuppressed control group that did not receive GM-CSF. All immunosuppressed animals that received GM-CSF at the time of bacterial infection retained prior skin allografts. The data suggest that GM-CSF is an effective agent for improving resistance to infection in transplant recipients without compromising the graft.

Ramp-ing up allergies

The hygiene hypothesis states that a decrease in infections in childhood results in an increase in allergic diseases later in life. This hypothesis is partially supported by an observed reduction of allergic symptoms and asthma in tuberculin-positive, but not in tuberculin-negative, children. Smit et al. (p. 754) tested the ability of heat-killed Mycobacterium vaccae to suppress allergic reactions in mice carrying sensitive and resistant alleles of the natural-resistance-associated macrophage protein 1 gene (Nramp1). Nramp alleles determine sensitivity or resistance of macrophages to growth of intracellular bacteria. OVA-sensitized Nramp1^s mice were treated with OVA plus heat-killed M. vaccae. These mice exhibited decreased airway hyperresponsiveness, fewer lung eosinophils, and reduced levels of IgE, IgG1, IL-4, IL-5, and TGF1- in lung lavage fluid than control groups. In contrast, Nramp1^s mice had more marked delayed-type hypersensitivity reactions to M. vaccae than Nramp1^r mice, a finding that the authors attributed to an impaired ability of the Nramp1^s macrophages to clear the bacteria. M. vaccae activation of macrophages from Nramp1^r mice in vitro was greater than activation of Nramp1^s macrophages. The data demonstrate that the Nramp1 gene may be an important mediator between mycobacteria and an allergic response.

Memory B cells and viral latency

Human -herpesviruses are able to avoid immune surveillance and establish long-term latency. The major reservoir for the latent virus is known to be B cells, although it is not clear which population of B cells harbors the virus. Kim et al. (p. 886) used murine -herpesvirus-68 (MHV-68) infection of CD40⁺/CD40^- mixed bone marrow chimeric mice to identify the cells infected by MHV-68. Chimeric and wild-type mice, both infected with MHV-68, had comparable patterns of virus clearance from the lungs and establishment of splenic latency. PCR analyses of CD40⁺ and CD40^- B cells separated by flow cytometry indicated that the cell populations had equivalent levels of latent virus at 14 days postinfection (p.i.). By 90 days p.i., the CD40^- pool of cells had a greater than 170-fold reduction in frequency of MHV-68, whereas the virus-positive cells appeared in the CD40⁺ pool. Immunofluorescent staining of spleen sections from chimeric mice 14 days p.i. showed that the B cells in the germinal centers were CD40⁺ whereas the CD40^- B cells were outside the germinal centers. When analyzed by flow cytometry, only the CD40⁺ cells stained for markers associated with differentiation to germinal center B cells. The results demonstrate that MHV-68 maintains latency by infecting a population of long-lived memory B cells.

Marking regulatory T cells in inflammatory bowel disease

An inflammatory response to foreign Ags in the gastrointestinal tract can result in inflammatory bowel disease (IBD). Regulatory CD4⁺ T cells are implicated in controlling inflammation and preventing IBD. Uraushihara et al. (p. 708) assessed the contribution of the glucocorticoid-induced TNFR family-related gene (GITR), expressed on regulatory T cells, to the control of IBD. Populations of CD4⁺ T cells expressing various combinations of CD45, CD25, and GITR markers were separated by flow cytometry. SCID mice reconstituted with CD4⁺CD45⁺Rb^high cells or CD4⁺GITR^- cells alone developed colitis. In contrast, SCID mice injected with a mixture of CD4⁺CD45⁺Rb^high plus CD4⁺GITR⁺ cells were protected, as were animals injected with a mixture of CD4⁺CD45⁺Rb^high plus CD4⁺CD45⁺Rb^low cells. However, depletion of GITR⁺ cells from the CD4⁺CD45⁺Rb^low fraction abrogated the protection provided to animals injected with CD4⁺CD45⁺Rb^high cells. Injection of an anti-GITR mAb into mice receiving the mixture containing CD4⁺GITR⁺ cells resulted in a loss of the protection and the onset of colitis. Injection of CD4⁺GITR⁺ cells, whether or not they expressed CD25, protected SCID mice against CD4⁺CD45⁺Rb^high-induced disease. The findings identify GITR as a marker of regulatory T cells in IBD.

Peripheral (in)tolerance

Vaccination against tumor-associated Ags in cancer therapy is often hindered by peripheral deletion of reactive CD8⁺ T cells by the host. One approach to preserving the tumor-reactive T cells is to use an anti-CD40 Ab to substitute for CD4⁺ T cell help in activating CD8⁺ T cells. O’Carroll et al. (p. 697) used an agonist Ab to the CD40 molecule in an in vivo system involving two transgenic mouse strains. SV40 T-Ag-transgenic mice (501), tolerant to T Ag epitope I, exhibited an initial short-term accumulation of the T Ag epitope I-specific (TCR-I) cells in their cervical lymph nodes when injected with naive CD8⁺ T cells from TCR-I transgenic mice. These lymph node cells were activated, but were unable to proliferate in response to immunization of the mice with an injected epitope I-containing viral vector or to produce IFN- in response to epitope I in vitro. Treatment of 501 mice with the anti-CD40 Ab before or 7 days after injection of TCR-I cells led to the accumulation of TCR-I T cells in lymph nodes, spleen, and blood for as long as 1 year. Epitope I-expressing cells injected into these mice were lysed in vivo. Furthermore, lymph node cells from anti-CD40-treated mice exposed to epitope I in vitro had an increased rate of proliferation and IFN- production. A control Ab had none of these effects. The results show that an agonist CD40-specific Ab alleviated peripheral tolerance in a dual-transgenic mouse system and resulted in long-term maintenance of tumor-reactive T cells in vivo.

Nitrotyrosine and inflammation

Inducible NO synthase A is up-regulated in a variety of inflammatory conditions. Generation of reactive nitrogen species results in the nitration of tyrosines in proteins at the site of inflammation. Yet the immunological reactivity of nitrotyrosine residues has not been adequately explored. Birnboim et al. (p. 528) used the well-established Ie^k-restricted peptide from the model Ag moth/pigeon cytochrome c (MCC_88–103) in an in vivo system to study the immunological impact of conversion of its sole tyrosine residue (Y97) to nitrotyrosine (nMCC_88–103). A MCC_88–103-specific T cell hybridoma secreted IL-2 in response to MCC_88–103 but was unresponsive to nMCC_88–103. T cells from mice immunized with MCC_88–103 responded strongly in vitro to MCC_88–103 and weakly to nMCC_88–103, whereas mice immunized with nMCC_88–103 responded only to the nMCC_88–103. Transgenic mice expressing pigeon cytochrome c failed to respond to immunization with MCC_88–103, but had a strong cellular immune response when immunized with nMCC_88–103. The D region of the -chain in the TCRs from nMCC_88–103 T cell hybridomas had a single substitution of an amino acid making contact with the nitrotyrosine residue in the nMCC_88–103. Thus, conversion of tyrosine to nitrotyrosine rendered self-tolerant peptides highly antigenic. This finding might explain how autoreactive T cells with TCRs that recognize an altered peptide can escape the processes of central tolerance.

The eyes have it

Herpetic stromal keratitis (HSK) is an ocular inflammatory disease that often results in blindness. The involvement of activated T cells in the corneal destruction of this herpes simplex virus-1 (HSV-1)-induced infection raises the possibility that a costimulatory receptor on T cells is involved in the disease process. Seo et al. (p. 576) examined the roles of 4-1BB, a TNF-family costimulatory receptor, and its ligand, 4-1BBL, in a murine system. Corneas of 4-1BB^+/+ and 4-1BB^-/- mice were infected with the RE strain of HSV-1. The incidence and severity of HSK in the 4–1BB^-/- mice was significantly reduced compared with their wild-type littermates. Similar results were obtained by injection of a 4-1BB mAb, but not by injection of control IgG, into HSV-1-infected normal BALB/c mice. Chemokine levels and expression of T cell surface markers, especially CD62L, were lower in T cells infiltrating the corneas of virus-infected 4-1BB^-/- mice at 8 days post infection compared with infiltrating T cells from the corneas of virus-infected wild-type mice. The results show that the 4-1BB costimulatory pathway is directly involved in the inflammatory response of the host to HSV-1 infection and suggest that treatments aimed at blocking the pathway could prevent or lessen the severity of HSK.

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

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