T cell effector functions contribute to the pathogenesis of rheumatoid arthritis. PKC-θ transduces the signal from the TCR through activation of transcription factors NF-κB, AP-1, and NFAT. We examined the effects of PKC-θ deficiency on two Th1-dependent models of Ag-induced arthritis and found that PKC-θ-deficient mice develop disease, but at a significantly diminished severity compared with wild-type mice. In the methylated BSA model, cellular infiltrates and articular cartilage damage were mild in the PKC-θ-deficient mice as compared with wild-type mice. Quantitation of histopathology reveals 63 and 77% reduction in overall joint destruction in two independent experiments. In the type II collagen-induced arthritis model, we observed a significant reduction in clinical scores (p < 0.01) in three independent experiments and diminished joint pathology (p < 0.005) in PKC-θ-deficient compared with wild-type littermates. Microcomputerized tomographic imaging revealed that PKC-θ deficiency also protects from bone destruction. PKC-θ-deficient CD4+ T cells show an impaired proliferative response, decreased intracellular levels of the cytokines IFN-γ, IL-2, and IL-4, and significantly diminished cell surface expression of the activation markers CD25, CD69, and CD134/OX40 on memory T cells. We demonstrate decreased T-bet expression and significantly reduced IgG1 and IgG2a anti-collagen II Ab levels in PKC-θ-deficient mice. Collectively, our results demonstrate that PKC-θ deficiency results in an attenuated response to Ag-induced arthritis, which is likely mediated by the reduced T cell proliferation, Th1/Th2 cell differentiation and T cell activation before and during disease peak.

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4+ T cell target population, we examined gene expression in CD4+ T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-α-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-αR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-α. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4+ T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4+ T cells, which induces an anti-HIV state but not apoptosis.

IMGN779 is a CD33-targeted ADC utilizing DGN462, a novel DNA-alkylating agent consisting of an indolino-benzodiazepine dimer containing a mono-imine moiety. CD33 is expressed on the surface of about 90% of AML cases, with elevated levels of CD33 found in cases having molecular markers associated with poor prognosis, including mutations in FMS-like tyrosine kinase 3 (FLT3). The internal tandem duplication mutation (FLT3-ITD) is the most common FLT3 mutation, present in about 20-25% of AML cases. Patients with FLT3-ITD AML have a worse prognosis than those with wild-type (WT) FLT3, with an increased rate of relapse and a shorter duration of response to induction chemotherapy. IMGN779 was found to demonstrate targeted activity against AML cell lines in vitro, with IC50 values ranging from 2-3,000 pM. The MV4-11 cell line, which has a FLT3-ITD mutation, was the most sensitive to IMGN779 of the cell lines tested, with an IC50 of 2 pM. We evaluated the in vivo activity of IMGN779 against MV4-11 xenografts in SCID mice; IMGN779 was highly active (T/C = 1 %) at a single dose of 0.6 mg/kg (conjugate dose, 10 µg/kg DGN462 dose), resulting in complete tumor regressions (CR) in 3/6 animals and partial regressions (PR) in 6/6 animals. A DGN462-ADC to a non-relevant target was inactive (T/C = 95%) at the same dose, demonstrating that the activity of IMGN779 was due to its CD33 targeting. IMGN779 has previously been shown to be highly active against AML xenograft models without FLT3-ITD mutations, at minimally efficacious doses of 0.6 mg/kg (10 µg/kg DGN462), demonstrating that the presence of FLT3-ITD does not confer resistance to IMGN779 treatment. IMGN779 was also highly active in vitro against primary patient AML cells isolated from peripheral blood or bone marrow samples. Patient AML cells with FLT3-ITD were more sensitive to IMGN779 compared with FLT3 WT AML samples. IC50 values in FLT3-ITD samples ranged from 10 to 300 pM. CD33 expression was generally greater on FLT3-ITD leukemic blast cells than on FLT3 WT blasts, which likely contributed to their increased sensitivity to IMGN779. In long term cultures, IMGN779 showed a dose dependent decrease in leukemic stem cell (LSC) colony formation using an AML patient sample with both FLT3-ITD and NPM1 mutations, which are an even worse prognostic marker than FLT3-ITD alone. In contrast, colony formation increased in normal bone marrow, indicating that normal hematopoietic stem cells (HSCs) were spared. The differential expression of CD33 on LSC compared to HSCs makes CD33 an attractive target for treatment of AML, with the potential to eliminate LSCs and, thus, minimal residual disease in FLT3-ITD AML. The potent in vitro activity of IMGN779 against FLT3-ITD AML cell lines and primary patient FLT3-ITD AML progenitor cells and LSCs and its high level of CD33-targeted in vivo activity in a FLT3-ITD AML xenograft model support the advancement of IMGN779 as a potential treatment for AML, including FLT3-ITD AML. Disclosures Whiteman: ImmunoGen, Inc.: Employment. Noordhuis:ImmunoGen, Inc.: Research Funding. Walker:ImmunoGen, Inc.: Employment. Watkins:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Johnson:ImmunoGen, Inc.: Employment. Schuurhuis:ImmunoGen, Inc.: Research Funding. Ossenkoppele:ImmunoGen, Inc.: Research Funding. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1α, MIP-1β and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.

The functional activity of natural killer (NK) cells has been found to be modulated by several point mutations associated with coat color. The most commonly studied gene, beige (Bg), has been found to block a postrecognition event in the lytic cycle. Four other coat color mutations in the mouse (satin, leaden, fuzzy, pale ears) were studied for their effect on NK cell function, and only one, satin (Sa), was found to be suppressive. When both the Sa and Bg mutations were present in the same animal, their effects were synergistic in the suppression of NK levels. Normal numbers of NK cells were present in these double mutants, as determined by the frequency of IgG2b binding cells and by antiasialo GM1 staining. The ability of Sa/Bg NK cells to recognize and bind targets suggests that the defect is localized in the postbinding cytolytic pathway. These genes were not specific for NK cells and also suppressed alloimmune cytolytic T lymphocyte function. Since Sa/Bg mice are much more suppressed in NK function than Bg mice, we suggest that this double mutant may be a better model for NK deficiency in vivo.