Roman Krzysiek

Blockade of B7-H1 improves myeloid dendritic cell–mediated antitumor immunity

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-γ. T cells conditioned with the B7-H1–blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic–severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10–induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Fractalkine receptor expression by T lymphocyte subpopulations and in vivo production of fractalkine in human

Expression and function of the fractalkine receptor CX3CR1 by T lymphocyte subpopulations was evaluated in healthy individuals. In CD8+ T lymphocytes, CX3CR1 was expressed by and functional in both CD45RO– and CD45RO+ cells. In CD4+ T lymphocytes, CX3CR1 was expressed mainly by CD45RO+ cells, and almost exclusively by activated HLA‐DR+ T lymphocytes. This receptor was functional in CD45RO+ cells, but not in CD45RO– cells. Expression of fractalkine was detected by in situ hybridization and immunohistochemistry in endothelial cells of normal lung and thymus. In hyperplastic lymph nodes, fractalkine was expressed by endothelial cells of high endothelial venules and of subcapsular vessels, by follicular dendritic cells (FDC) and by some follicle lymphocytes. Fractalkine mRNA was constitutively present in the HK FDC‐like cell line, and it was induced in vitro in B lymphocytes stimulated by an anti‐μ or by a CD40 mAb. These findings indicate that fractalkine may contribute to the recruitment of effector T helper lymphocytes, either in peripheral tissues or in lymphoid organs. In these tissues, fractalkine and its receptor may favor contact within follicles between activated T helper lymphocytes, activated B lymphocytes and FDC, thus contributing to the maturation of the B lymphocyte response.

Early control of HIV replication in primary HIV-1 infection treated with antiretroviral drugs and pegylated IFNα : results from the Primoferon A (ANRS 086) study

IFN alpha has both antiviral and immunostimulating properties. The ANRS086 Primoferon A Study evaluated in 12 patients with primary HIV infection the tolerance and efficacy of an early and transient administration of pegylated IFN alpha, in addition to highly active antiretroviral therapy. Tolerance was good, and this regimen allowed the early control of HIV replication and rapid decay of the viral reservoir. These results support the initiation of comparative studies with pegylated INF alpha in primary HIV infection.

The chemokine SDF-1/CXCL12 contributes to T lymphocyte recruitment in human pre-ovulatory follicles and coordinates with lymphocytes to increase granulosa cell survival and embryo quality.

We investigated the production and the role of the chemokine stromal cell‐derived factor‐1 (SDF‐1/CXCL12) in pre‐ovulatory follicles of women undergoing in vitro fertilization. We detected CXCL12 and its receptor CXCR4 by flow cytometry, western blotting and RT‐PCR. We tested cell migration in Transwell experiments. We measured apoptosis using ΔΨm‐sensitive fluorescent probe DiOC6(3) and we screened apoptosis‐related gene expression with macro‐arrays. Granulosa cells from follicular aspirates produce CXCL12 that contributes to T lymphocytes recruitment. CXCL12 reduces early apoptosis of granulosa cells. This effect is accompanied by a shift of bcl2/bax ratio, and decreased expression of p53‐targeted genes (pig7, pig8, p21, gadd45). Removal of lymphocytes disables CXCL12‐mediated anti‐apoptotic effect on granulosa cells. Anti‐apoptotic activity of CXCL12 is positively correlated to high quality of embryos. In conclusion, CXCL12 is locally produced by luteinizing granulosa cells. It specifically contributes to T lymphocytes recruitment and coordinates with local lymphocytes to increase granulosa cell survival and embryo quality.

Stimulation of the primary anti-HIV antibody response by IFN-α in patients with acute HIV-1 infection

Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV‐1 and treated with IFN‐α2b. Patients with acute HIV‐1 infection were randomized to receive antiretroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated‐IFN‐α2b (Group B, n=30). Emergence of anti‐HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN‐α2b treatment stimulated the production of anti‐HIV antibodies. On Week 32, 19 weeks after the last IFN‐α2b administration, there were 8.5 (6.5–10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0–10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN‐α2b treatment also increased circulating concentrations of the B cell‐activating factor of the TNF family (P<0.001) and ex vivo production of IL‐12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti‐HIV immune response in patients with acute HIV‐1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies.

Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells

Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

Glucocorticoid-Induced Leucine Zipper Enhanced Expression in Dendritic Cells Is Sufficient To Drive Regulatory T Cells Expansion In Vivo

Tolerance induction by dendritic cells (DCs) is, in part, mediated by the activation of regulatory T cells (Tregs). We have previously shown in vitro that human DCs treated with glucocorticoids (GCs), IL-10, or TGF-β upregulate the GC-Induced Leucine Zipper protein (GILZ). GILZ overexpression promotes DC differentiation into regulatory cells that generate IL-10–producing Ag-specific Tregs. To investigate whether these observations extend in vivo, we have generated CD11c-GILZhi transgenic mice. DCs from these mice constitutively overexpress GILZ to levels observed in GC-treated wild-type DCs. In this article, we establish that GILZhi DCs display an accumulation of Foxp3+ Tregs in the spleens of young CD11c-GILZhi mice. In addition, we show that GILZhi DCs strongly increase the Treg pool in central and peripheral lymphoid organs of aged animals. Upon adoptive transfer to wild-type recipient mice, OVA-loaded GILZhi bone marrow–derived DCs induce a reduced activation and proliferation of OVA-specific T cells as compared with control bone marrow–derived DCs, associated with an expansion of thymus-derived CD25+Foxp3+ CD4 T cells. Transferred OVA-loaded GILZhi DCs produce significantly higher levels of IL-10 and express reduced levels of MHC class II molecules as compared with OVA-loaded control DCs, emphasizing the regulatory phenotype of GILZhi DCs in vivo. Thus, our work demonstrates in vivo that the GILZ overexpression alone is sufficient to promote a tolerogenic mode of function in DCs.

Mechanisms of opsonized HIV entry in normal B lymphocytes

Using our in vitro model of normal B cell infection that functions with low doses of HIV but requires virus opsonization by seropositive patient serum, and complement, we analyzed what receptors allowed virus entry. Here, we show that HIV infection of B cells occurs through 2 major receptors: the CD4 antigen and the CR1/CR2 complex. These 2 pathways work independently since a complete inhibition of virus entry requires both CD4 and CD21/CD35 blockade on CD4dim tonsillar B cells whereas only the latter is critical on CD4‐negative B cells.

Immunogenicity of tocilizumab in patients with rheumatoid arthritis

OBJECTIVE The immunogenicity of tocilizumab (TCZ) has been poorly studied. We assessed the immunogenicity of TCZ and serum TCZ trough levels in rheumatoid arthritis (RA) patients and the preexisting TCZ-specific CD4+ T cell repertoire in healthy controls. METHODS Anti-drug antibodies (ADAs) to TCZ and serum TCZ trough levels in RA patients were assessed at different times by ELISA. Frequencies of naive anti-TCZ CD4+ precursors were studied in healthy controls. RESULTS In total, 91 samples from 40 RA patients were analyzed: 21 patients within the first 6 months after treatment initiation and 19 during follow-up after a mean TCZ treatment duration of 21±13 months. None of the 91 samples showed persistent ADAs to TCZ. Only 3 RA patients showed transient and low titers of anti-TCZ ADAs. Serum TCZ trough levels were associated with neither patient characteristics (gender, body mass index) nor disease activity and were identical for patients with and without co-treatment with methotrexate. Three of 9 healthy donors showed preexisting TZC-specific CD4+ T cells at a low level. CONCLUSION Serum TCZ trough levels were not affected by patient characteristics. The occurrence of ADAs to TCZ was a rare event. Because healthy donors show the same frequency of naive TCZ-specific and infliximab-specific CD4+ T cell precursors, the low prevalence of ADAs to TCZ might result from interleukin-6 blockade.