In This Issue

doi:10.4049/jimmunol.1090038

Knocking Down Arthritis

The discovery of dendritic cells (DCs) with immunosuppressive properties has opened a new avenue for novel immunotherapeutic interventions in rheumatoid arthritis. Zheng et al. (p. 6457) describe a strategy to prevent collagen-induced arthritis (CIA) in mice by treatment with Ag-specific tolerogenic DCs, which were generated through small-interfering RNA-mediated knockdown of the costimulatory molecules CD40, CD80, and CD86 prior to pulsing with the arthritogenic Ag type II collagen (CII). The development of clinical and histological signs of CIA was reduced by treatment with CII-specific DCs that had knocked-down expression of CD40 or CD80/CD86. Attenuated CIA development correlated with reduced anti-CII Ab production and CII-specific T cell recall responses. Clinical and immunological responses were significantly suppressed in mice treated with DCs in which all three genes were silenced. Moreover, gene-silenced DCs reduced TNF-α, IFN-γ, IL-2, and IL-17 production in T cells from CIA mice but promoted the expansion of CD4⁺CD25⁺FoxP3⁺ regulatory T cells. These findings highlight a potential for gene silencing in the development of immunotherapies against autoimmune diseases.

High Avidity Reliability

Adaptive immune responses are shaped by a lifetime of exposures to various pathogens. An existing hypothesis, termed “original antigenic sin”, suggests that pre-existing memory T cells that have low avidity to cross-reactive epitopes from successive pathogen exposures compromise the development of high-avidity T cell responses. This hypothesis was tested by Zehn et al. (p. 6320) in mice exposed to single amino acid variants of the MHC class I-restricted OVA_257–264 (SIINFEKL) epitope expressed in bacterial or viral vectors. Heterologous primary and secondary challenges with SIINFEKL variants caused the expansion of high avidity cross-reactive CD8⁺ T cells upon secondary challenge. The majority of high-avidity CD8⁺ T cells generated during secondary challenge originated from naive precursors, but a small portion of memory CD8⁺ T cells cross-reacted with high avidity to a subset of epitopes tested. Moreover, high avidity responses to secondary challenge were not significantly affected by the presence of a large number of cross-reactive CD8⁺ T cells with low avidity induced by primary responses. These data indicate that original antigenic sin does not compromise recall responses to some heterologous challenges and suggests that high-avidity CD8⁺ T cells can effectively compete with low-avidity cells to expand in response to infection.

Breathing Life into Bone Marrow

The therapeutic benefits of bone marrow transplantation (BMT) are often compromised by post-transplantation complications, including bacterial pneumonia. PGE₂ levels are significantly elevated in the lung following BMT, and Hubbard et al. (p. 6299) show that PGE₂ induces expression of IL-1R–associated kinase (IRAK)-M, an inhibitor of MyD88-dependent TLR/IL-1R signaling, in alveolar macrophages (AMs). Both IRAK-M mRNA and protein levels were elevated in AMs from wild-type (WT) mice that had undergone BMT compared with control mice. Elevated IRAK-M levels were linked to excessive PGE₂ production. Intratracheal inoculation of WT BMT mice with Pseudomonas aeruginosa caused significant lung and systemic infection and mortality compared with WT control mice. In contrast, the mortality and bacterial burden measured in IRAK-M^−/− BMT mice were significantly lower than in WT BMT mice and were similar to those in control mice. AMs from IRAK-M^−/− BMT mice showed greater phagocytosis and killing of P. aeruginosa by ex vivo analysis compared with WT BMT AMs, as well as increased production of TNF-α and cysteinyl leukotriene. PGE₂ treatment of IRAK-M^−/− BMT AMs did not inhibit phagocytosis, indicating that PGE₂-mediated phagocytosis inhibition required IRAK-M. This newly defined role for IRAK-M in PGE₂-associated immunosuppression provides a novel therapeutic target for preventing post-BMT infection.

Following Fates by Recombination

The Cre recombinase (Cre)-lox site-specific recombination system has revolutionized the generation of tissue-specific conditional knockout mice. Klinger et al. (p. 6170) describe a new application of Cre-lox recombination that can be used for fate mapping through differential Cre expression. The tdTomato and eGFP fluorescent reporter alleles were cloned into a vector upstream of a disruption element flanked by native loxP sites, which prevented reporter transcription in the absence of Cre. The eGPF allele was flanked by mutant loxP sites such that loss of eGFP expression correlated with increased Cre activity. Murine embryonic stem (ES) cells transfected with the reporter vector responded to increasing doses of Cre with greater recombination at both native and mutant loxP sites and a higher ratio of tdTomato⁺eGFP⁻ (fully recombined alleles) to tdTomato⁺eGFP⁺ (partially recombined alleles) cells. Lymphocytes from transgenic mice carrying reporter alleles responded similarly to Cre treatment. Mice generated from a cross between the reporter allele strain and an Ox40-Cre strain had a higher ratio of tdTomato⁺eGFP⁻ to tdTomato⁺eGFP⁺ in CD4⁺ regulatory and memory T cells versus naive and CD8⁺ T cells that correlated directly with the level of Cre expression. This reporter system highlights a new approach to mapping cell fates that can be applied across multiple immunological fields.

Switching Sequences

Class switch recombination (CSR) is a mechanism that changes the C_H gene expressed by activated B cells and, thus, alters the Ig isotype. Complementary switch (S) region sequences are located upstream of nearly all C_H regions, and transcription and activation-induced deaminase attack of S regions facilitate CSR. Bhattacharya et al. (p. 6242) examined how the S region sequence influences CSR-directed isotype targeting with B cells from chimeric mice that had a similarly sized Sγ1 sequence in place of the endogenous Sγ3 sequence. B cells from these chimeric mice showed normal levels of γ3 germline transcript expression, from both the intact Sγ3 allele and the Sγ1 replacement allele, in response to LPS or LPS + IL-4 stimulation. As expected, μ→γ3 CSR was repressed in wild-type B cells treated with LPS + IL-4, and no γ3 postswitch transcripts (PST) were detected. In contrast, LPS + IL-4 treatment induced efficient CSR on the Sγ1 replacement allele and repressed CSR on the intact γ3 allele. In conclusion, these results establish a role for the Sγ1 sequence in facilitating CSR independent of its position within the C_H region. Further investigation is needed to determine the influences of other S regions on CSR.

Smoke and Migrating Macrophages

Chronic lung inflammation caused by cigarette smoking has been associated with a significant accumulation of alveolar macrophages in the lung. The study by Koth et al. (p. 6522) indicates that the DAP12 adapter protein contributes to the recruitment of alveolar macrophages to inflamed lungs. DAP12-associated receptors have both pro- and anti-inflammatory functions, and a previous study showed that CLEC5A and TREM2 mRNAs were significantly upregulated in the alveolar macrophages of smokers. Elevated CLEC5A and TREM2 mRNA levels were observed in smokers in this study and correlated directly with the number of packs smoked per day. These observations suggested involvement of the DAP12 signaling pathway in macrophage migration. Cigarette smoke exposure caused macrophage infiltration into the lungs of wild-type and DAP12-deficient mice, but significantly fewer macrophages were in the lungs of DAP12-deficient mice. DAP12-deficient macrophages showed no chemotaxis toward CCL2, compared with wild-type macrophages in vitro, and macrophage recruitment to the lungs of DAP12-deficient mice was reduced in response to CCL2 airway treatment, compared with wild-type mice. Macrophage migration in vitro required phosphorylation of the DAP12 ITAM and also involved interactions with TREM2. These results provide a new insight into mechanisms of macrophage recruitment, which may advance the development of therapeutics for chronic lung disease.

Anti-Tyrosinase TCR Tackles Tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) is an effective immunotherapy for the treatment of metastatic melanoma, but not all patients can be successfully treated with TILs. Tyrosinase is an enzyme required for melanin production and is highly expressed in metastatic melanomas. Frankel et al. (p. 5988) developed a new TCR clone that recognizes the tyrosinase:368–376 peptide and is reactive against melanoma target cells. The TCR α- and β-chains from an anti-tyrosinase TIL were cloned into a retroviral vector and transduced into PBLs. CD4⁺ and CD8⁺ T cells expressing the anti-tyrosinase TCR released IFN-γ upon coculture with melanoma target cells and were reactive against melanoma epitopes independent of CD4 or CD8 coreceptors. Anti-tyrosinase TCR surface expression was improved by the addition of a second disulfide bond between the α- and β-chain constant regions. The replacement of the human constant regions with murine homologues improved both surface expression and reactivity against melanoma target cells, with similar reactivity when compared with the anti–MART-1 or anti-gp100:154–162 TCRs, which are being tested in clinical trials for melanoma therapy. This chimeric anti-tyrosinase TCR also mediated tumor regression in a transgenic mouse model of melanoma. Thus, modified forms of the anti-tyrosinase TCR may be a promising new tool for cell-based melanoma therapies.

New Kid on the NF-κB Block

The NF-κB signaling pathway is conserved among eukaryotes and is an important component of the innate immune system of Drosophila melanogaster. Valanne et al. (p. 6188) identified Gprk2 as a novel regulator of D. melanogaster NF-κB signaling through a genome-wide RNA interference screen. A Drosomycin promoter-driven luciferase in vitro reporter assay was used to screen for novel regulators of the Toll and Imd pathways of NK-κB activation in the D. melanogaster S2 cell line. Of the >16,000 genes screened by RNA interference, 10 new genes were identified that affected these pathways. Three of these genes, CG15737, Gprk2, and u-shaped, were essential in vivo for Drosomycin expression via the Toll pathway. A conserved function of Gprk2 was confirmed by the observations of reduced NF-κB responses following small interfering RNA silencing of the human homolog GRK5 in HeLa cells or morpholino silencing of GRK5 in zebrafish larvae. Silencing of Gprk2 in D. melanogaster compromised survival against infection with the Gram-positive bacteria Enterococcus faecalis. Moreover, Gprk2 silencing rescued blood cell activation in D. melanogaster larvae caused by constitutive activation of the Toll pathway. These findings support a role for Gprk2/GRK5 as a regulator of NF-κB signaling, and further research will define its place in this elaborate signaling network.