The intracellular pathogen responsible for typhoid fever, Salmonella enterica servovar Typhimurium, uses a cluster of virulence genes in Salmonella Pathogenicity Island 2 (SPI2) to survive and replicate in specific vacuoles in dendritic cells (DCs). Cheminay et al. (p. 2892 ) found that Salmonella infection of mouse splenic or bone marrow-derived DCs impaired the ability of the cells to present OVA to CD4+ T cells expressing an OVA-specific TCR. T cell proliferation was normal in the presence of UV-inactivated bacteria or after addition of chloramphenicol to bacteria-infected DCs. Partial restoration of T cell proliferation occurred in the presence of specific inhibitors of inducible NO synthase or with DCs infected with an SPI2 mutant Salmonella strain. Fewer MHC class II molecules were loaded with a model Ag on DCs infected with wild-type bacteria compared with DCs infected with SPI2 mutant Salmonella. Mice vaccinated with SPI2 mutant Salmonella had a higher survival rate after challenge with a lethal dose of wild-type Salmonella 3 wks later compared with mice vaccinated with a Salmonella strain mutated in an unrelated gene. Higher numbers of CD4+ and CD8+ T cells were detected in SPI2-vaccinated mice compared with either control infected or wild-type animals. The authors propose that the SPI2 system of Salmonella interferes with DC processing of bacterial Ags and provides a mechanism by which the intracellular pathogen escapes immune destruction.

Defective ribosomal products are improperly folded proteins thought to be the source of peptides presented by MHC class I molecules to CD8+ T cells. However, the contribution of misfolding vs improper subcellular location to peptide generation is unclear. Golovina et al. (p. 2763 ) studied the susceptibility to proteasomal degradation of two engineered proteins containing a retrieval tag. The model for a cytosolic protein was influenza virus nucleoprotein deleted for N-terminal amino acids that target it to the nucleus (NP13–498). The model endoplasmic reticulum protein was IL-2R α-chain (Tac Ag). An H-2 Kb-restricted OVA epitope and an H-2 Kd-restricted NP epitope were inserted into each protein. Altered folding states were induced in NP13–498 by an amino acid substitution, by deletion of the hydrophobic domain or by replacing the N-terminal methionine with arginine preceded by a ubiquitin moiety. Tac Ag was modified by a mutation of both glycosylation sites, by removal of the signal peptide to force delivery to the cytosol or by substitution of an amino acid within the transmembrane domain to create a degradation signal. Only the Tac Ag variant retained in the cytoplasm, the Tac Ag transmembrane mutant and the ubiquitinated NP13–498 protein were rapidly degraded in vitro. Surface expression of OVA or NP epitopes in cells infected with constructs in vaccinia virus vectors was highest for the same three variants. The data indicate that increased MHC class I molecule presentation of peptides is seen only for model proteins specifically targeted to the proteasome or mislocalized within the cell independent of misfolding due to altered amino acid sequences.

Remodeling and scarring of orbital connective tissue in thyroid associated ophthalmopathy (TAO) during late stage Graves’ disease involve degradation of extracellular matrix proteins by matrix metalloproteinases. Han and Smith (p. 3072 ) used IL-1β to induce high mRNA in cultured TAO orbital fibroblasts. Lower levels of TIMP-1 mRNA were induced by IL-1β in normal orbital fibroblasts or by IL-1β in the presence of a protein synthesis inhibitor or dexamethasone in TAO orbital fibroblasts. The TIMP-1 gene response region was located between −920 and −733 nt by TIMP-1 promoter-driven luciferase activity. TIMP-1 protein induction by IL-1β in TAO cells was reduced substantially by an inhibitor of MEK in the ERK signaling pathway, by transient transfection with an ERK1 double-negative mutant kinase or by addition of either IFN-γ or IL-4. Either cytokine blocked promoter activation and TIMP-1 mRNA synthesis, and the blockade was reversed by mAb against the respective cytokine. Conditioned medium from IL-1β-treated TAO orbital fibroblasts inhibited proteolytic activity of gelatinase compared with medium from untreated cultures. The authors conclude that TIMP-1, released by orbital fibroblasts, is responsible for tissue remodeling in TAO and that its release can be blocked at the transcription level by Th1 or Th2 cytokines.

Patients with glioblastoma multiforme receive treatment with surgery, chemotherapy, and radiation, yet 1-year survival rates are low. Rats with 3-day-old, but not those with 7-day-old, gliomas induced by intracranial injection of rat gliosarcoma cells expressing the extracellular membrane form of M-CSF (mM-CSF) have a high survival rate and long-lasting tumor-specific immunity after s.c. immunization with a vaccine consisting of mM-CSF glioma cells. In a continuation of their rat glioma studies, Jeffes et al. (p. 2533 ) found that two anti-angiogenic drugs inhibited in vitro growth of endothelial cells but not glioma or T cells. Combining the tumor vaccine with i.p. injection of either anti-angiogenic drug increased the 3-mo survival of rats with 7-day-old gliomas to 92%. Nontreated controls or rats receiving only tumor vaccine died within 40 days; 40% and 20% of rats receiving either drug alone survived. Intracranial gliomas had reduced phosphorylation of several growth factor receptors after 6 days of combination therapy. Either drug reduced intracranial growth of injected tumor cells and decreased the number of blood vessels detected by immunofluorescence using endothelial cell-specific probes; increased infiltration of IL-2-producing lymphocytes was seen over an additional 8 days after combined treatment compared with controls. Approximately 67% of rats survived a challenge with an intracranial injection of nontransfected glioma cells more than 3 mo after receiving mM-CSF glioma cells and combined treatment; no rats in a parallel group survived challenge with unrelated syngeneic breast cancer cells. The data demonstrate that combining tumor immunization with anti-angiogenic drugs results in higher rates of survival of rats with large gliomas.

Progressive HIV infection involves chronic immune activation by virus and loss of CD4+ T cells. In infection with other viruses, CD127 is expressed on a subset of effector CD8+ T cells that differentiate into memory cells. Paiardini et al. (p. 2900 ) looked at CD127 expression on T cells from HIV-infected patients, either untreated or treated with highly active antiretroviral therapy, and from healthy controls. Decreased CD127 expression was detected predominantly in sorted memory CD8+ T cells only from untreated HIV-infected patients; increased numbers of CD8+CD127 T cells correlated directly with plasma viremia and inversely with CD4+ T cell count. Some decreased CD127 expression was noted in naive CD8+ T cells from the same patients. Expansion of CD8+CD127 T cells specific for HIV and other viruses was detected using viral-specific tetramers. Multiparametric flow cytometric analyses revealed that the HIV-specific cells had markers characteristic of activated effector memory T cells. CD8+CD127 T cells from HIV-infected patients produced large amounts of IFN-γ but almost no IL-2 following ex vivo stimulation. The same cell population from both HIV-infected patients and controls had high proliferation levels in vivo and increased apoptosis in response to PHA ex vivo compared with CD8+CD127+ T cells from the same individuals; CD8+CD127 T cells from uninfected controls had low proliferative levels ex vivo. The authors suggest that expansion of effector CD8+CD127 T cells serves as a marker of immune dysfunction in HIV infection.

Although members of the large family of Staphylococcus aureus superantigen-like proteins (SSLs) are homologous with superantigens, they do not demonstrate superantigen activity. Langley et al. (p. 2926 ) molecularly cloned the ssl7 gene from clinical isolates of S. aureus. An SSL7-Sepharose complex bound H and L chains of serum and secretory IgA from human serum, breast milk, saliva, and tears, and C5α- and β-chains from serum. SSL7 also bound IgA-like polypeptides and C5 from several other mammalian species. Biosensor binding analyses indicated that SSL7 interacted with the Fc region of serum IgA at two sites and with C5α at a third site. FITC-IgA incubated with SSL7 had reduced binding to human leukocytes in solution or to recombinant soluble FcαRI immobilized on a biosensor chip. Incubation of FITC-IgA with the superantigen staphylococcal entertoxin A had no effect on binding. Recombinant SSL7, but not staphylococcal entertoxin A, prevented hemolysis of allogeneic human RBCs by sera from two different patients and reduced complement lysis of Escherichia coli by fresh human serum, allowing 14% of an inoculum to survive. The authors suggest that SSL7 interferes with host defense mechanisms by preventing IgA binding to FcαRI, thus impeding Fc receptor-mediated leukocyte activation.

Most cancer patients have only modest responses to treatment with peptides specific for their tumors or with dendritic cells (DCs) pulsed with those peptides. Wang and coworkers have reported the presence of tumor peptide-specific CD4+ regulatory T (Treg) cells in a tumor-infiltrating lymphocyte (TIL) line established from a patient’s tumor. On p. 2661 , the same group found that a CD4+ TIL line derived from a fresh melanoma sample inhibited proliferation of autologous naive T cells activated by anti-CD3 mAb. Ten clones derived from the TIL line had characteristics of Treg cells, namely they secreted GM-CSF, IFN-γ, and IL-10, were CD25+, and expressed Foxp3. Four CD4+ effector T cell clones derived from other melanoma TIL lines secreted GM-CSF, IFN-γ, and IL-2, and enhanced proliferation of activated naive T cells. Suppressive activity of the TIL Treg cells was not dependent on IL-10 or TGF-β and required contact with the responding cells. A unique protein produced by a cloned melanoma cDNA stimulated cytokine release by the TIL Treg cells. The protein was generated by a point mutation in a wild-type gene. DCs pulsed with a synthetic peptide based on the sequence of the mutated protein stimulated melanoma TIL Treg cells; only peptide-activated TIL Treg cells suppressed IL-2 production of anti-CD3 mAb-activated effector T cells. Intact tumor cells, but not EBV-B cells incubated with tumor cell lysates, activated the peptide-specific CD4+ Treg cells. The data indicate that Ag-specific Treg cells activated by contact with melanoma cells suppress tumor-specific effector T cells, thus inducing immune suppression within the tumor.

Although regulatory T (Treg) cells develop in response to Ags of enteric bacteria, the degree of dendritic cell (DC) involvement is unknown. Cong et al. (p. 2787 ) used a proteosome inhibitor (benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI)) to block NF-κB signaling, thereby preventing the maturation of mouse bone marrow-derived DCs. Naive OVA-specific TCR transgenic CD4+ T cells had reduced proliferation and production of IL-2, IFN-γ, and IL-10 when incubated with irradiated PSI-treated DCs pulsed with OVA peptide. Untreated OVA peptide-pulsed DCs were stimulatory. Transgenic CD4+ T cells incubated with PSI-treated DCs (PSI-APC Treg cells) inhibited proliferation of naive or memory CD4+ T cells. PSI-treated, OVA peptide-pulsed normal spleen cells from wild-type mice, but not those from IL-10−/− mice, also were inhibitory for naive transgenic T cells. PSI-APC Treg cells had elevated Foxp3 expression compared with memory CD4+ T cells, were Ag-specific and were not affected by high levels of IL-2, anti-IL10R1 mAb, or anti-TGF-β mAb. PSI-APC Treg cells generated from both IL-10+/+ and IL-10−/− mice were inhibitory. Transwell separation of, or addition of anti-CTLA-4 mAb to, naive T cells prevented inhibition by PSI-APC Treg cells. PSI-APC Treg cells adoptively transferred into mice inhibited OVA peptide-induced proliferation of cotransferred naive OVA-specific transgenic T cells. No OVA-specific TCR transgenic mice given Th1 OVA transgenic cells with PSI-APC Treg cells developed colitis when infected with OVA-expressing bacteria, whereas controls not receiving PSI-APC Treg cells did. The data show that immature PSI-treated DCs generate Ag-specific Tregs that are inhibitory for naive and memory cells in vitro and in vivo.

The T cell repertoire of patients infected with Mycobacterium leprae consists of MHC class II-restricted and MHC-like (CD1)-restricted T cells. Sieling and collaborators have shown that CD1-restricted T cells participate in defense of patients against leprosy infection. On p. 2637 , Sieling et al. compared CD1-restricted and MHC class II-restricted T cell repertoires of patients with the localized tuberculoid form of the disease vs patients with the disseminated lepromatous form. Peripheral T cells of tuberculoid leprosy patients incubated with whole extracts or lipid extracts of M. leprae and dendritic cells (DCs) produced more IFN-γ than did similarly treated T cells from lepromatous patients. T cells from both sets of patients responded equally to whole extracts of Mycobacterium tuberculosis. T cells from lepromatous patients had a diminished response to lipid extracts of M. tuberculosis and did not produce IL-4 or IL-10 after stimulation with autologous DCs in the presence of M. leprae lipid extract; depletion of CD8+ T cells did not alter the response. MHC class II-restricted and CD1-restricted T cell clones from tuberculoid patients cross-reacted with protein and lipid extracts, respectively, from both mycobacterial species. However, in lepromatous patients, MHC class II-restricted T cell clones did not react with peptide extracts of M. leprae, whereas the residual CD-1-restricted T cells reacted with lipid extracts of both mycobacterial species. The authors conclude that CD-1 lipid-reactive T cells are clonally deleted in lepromatous patients, resulting in greater susceptibility to infection by other mycobacterial species.

Increased serum levels of IFN-α are found in patients with systemic lupus erythematosus (SLE), and patients receiving IFN-α treatment for a variety of conditions can develop SLE. Yet any direct contribution of IFN-α to SLE pathogenesis has not been established. Mathian et al. (p. 2499 ) used two methods to deliver IFN-α to mice. A replication-deficient recombinant adenovirus expressing murine IFN-α was given to young, preautoimmune (NZB × NZW)F1 mice (NZB/W) and BALB/c controls. The treated NZB/W mice developed proteinuria, renal damage, and death much earlier than NZB/W mice given an empty adenovirus or left untreated; no proteinuria or deaths were seen among the BALB/c mice given the IFN-α adenovirus. Histological examination revealed identical kidney pathology among treated and old untreated NZB/W mice with lupus glomerulonephritis that was absent from kidneys of treated BALB/c mice or controls given empty virus. Autoantibodies eluted from affected kidneys of both strains of mice were anti-ssDNA IgG; anti-dsDNA IgG was found only in treated and old untreated NZB/W mice. Increased serum levels of B-lymphocyte stimulator protein were detected in NZB/W and BALB/c mice after receiving IFN-α adenovirus. The IFN-α effect was dose-dependent for time of onset, yet all treated NZB/W mice developed lupus and died. Infusion of purified IFN-α into NZB/W mice using microosmotic pumps also induced the disease. The authors conclude that prolonged exposure of genetically susceptible mice to IFN-α accelerates lupus glomerulonephritis.

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