Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.

Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis.

PURPOSE: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes. METHODS: We used an in vitro coculture system in which UM cell lines and T cells were cultured together, but separated by a membrane. Uveal melanoma gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a transwell plate was used. RESULTS: Gene-expression analysis of UM cell lines showed that coculture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, VEGF, intracellular adhesion molecule 1 (ICAM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant toward monocytes. CONCLUSIONS: Cytokines derived from activated T cells shifted the UM cell transcriptome toward a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor-promoting inflammatory microenvironment.

Inflammatory cytokines protect retinal pigment epithelial cells from oxidative stress-induced death.

PURPOSE: To investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19. METHODS: Confluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting. RESULTS: Viability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment. CONCLUSION: Oxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.

Chemokine expression in retinal pigment epithelial ARPE-19 cells in response to coculture with activated T cells.

PURPOSE: To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). METHODS: We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting. RESULTS: Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL10 (IP10); CXCL11 (ITAC); and CX3CL1 (fractalkine). CCL7, CXCL9, CXCL10, and CXCL11 were secreted significantly more in the apical direction. Using recombinant human cytokines and neutralizing antibodies, we identified IFNγ and TNFα as the two major T-cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, CXCL10, CXCL11, CXCL16, and CX3CL1, we observed a synergistic effect of IFNγ and TNFα in combination. CCL20, CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ. CONCLUSIONS: RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.

Astrocytoma cells upregulate expression of pro-inflammatory cytokines after co-culture with activated peripheral blood mononuclear cells.

In this study, we investigated the effect of CD3/CD28-activated peripheral blood mononuclear cells (PBMCs) on two human astrocytoma cell lines (1321N1 and CCF-STTG1), with respect to the expression of cytokines and chemokines. We used an in vitro co-culture system in which the astrocytoma cells and PBMCs were separated by a membrane in a transwell system. Proliferation of T cells was quantified by a [(3) H]-thymidine incorporation assay. Differential gene expression of astrocytoma cells cultured alone or with PBMCs was identified using gene arrays. Protein expression of CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1ß was determined by Luminex. Co-culture of activated T cells and astrocytoma cells resulted in inhibition of T cell proliferation. Moreover, astrocytoma cells upregulated a number of pro-inflammatory genes including CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1ß upon co-culture with activated PBMCs. These results were confirmed on the protein level. Astrocytoma cells inhibited the proliferation of T cells, indicating that astrocytes may play an important role in controlling T cell mediated immune responses in the brain. Contrary to this, soluble factors derived from CD3/CD28-activated PBMCs affected the transcriptome of astrocytoma cells, which then upregulated several pro-inflammatory genes and proteins. This could be part of neuroinflammatory events and may play a role in neurodegenerative diseases.

Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells.

In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration.