Biochemistry and Pharmacology - Research Publications
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ItemRapid Deletion and Inactivation of CTLs upon Recognition of a Number of Target Cells over a Critical Threshold(AMER ASSOC IMMUNOLOGISTS, 2013-10-01)Initiation of CTL responses against foreign pathogens also primes anti-self CTLs. Mechanisms of CTL inactivation inhibit anti-self CTLs to prevent tissue damage. These mechanisms are exploited by pathogens and tumors to evade the immune response, and present a major hurdle to adoptive CTL therapies. It is unclear whether CTL inactivation is Ag specific and, if so, which APCs are involved. Potential candidates include the target cells themselves, dendritic cells, myeloid-derived suppressor cells, and macrophages. In this study, we show that lymphoma-specific CTLs are rapidly deleted in an Ag-specific manner after adoptive transfer into lymphoma-bearing mice, and the surviving CTLs are functionally impaired. The only APCs responsible were the target cells directly presenting Ag, notwithstanding the presence of myeloid-derived suppressor cells, and CD8(+) dendritic cells cross-presenting tumor Ag. The capacity to inactivate CTLs critically depended on the number of tumor/target cells; small numbers of targets were readily killed, but a large number caused quick deletion and functional inactivation of the CTLs. Application of mild, noninflammatory, and nonlymphoablative chemotherapy to specifically reduce tumor burden before CTL injection prevented CTL deletion and inactivation and allowed eradication of tumor. Our results advocate the use of adoptive CTL therapy soon after mild chemotherapy. They also suggest a simple mechanism for Ag-specific impairment of anti-self CTLs in the face of an active anti-foreign CTL response.
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ItemInflammation Conditions Mature Dendritic Cells To Retain the Capacity To Present New Antigens but with Altered Cytokine Secretion Function(AMER ASSOC IMMUNOLOGISTS, 2014-10-15)Dendritic cells (DCs) are directly activated by pathogen-associated molecular patterns (PAMPs) and undergo maturation. Mature DCs express high levels of MHC class II molecules ("signal 1"), upregulate T cell costimulatory receptors ("signal 2"), and secrete "signal 3" cytokines (e.g., IL-12). Mature DCs efficiently present Ags linked to the activating PAMP and prime naive T cells. However, mature DCs downregulate MHC II synthesis, which prevents them from presenting newly encountered Ags. DCs can also be indirectly activated by inflammatory mediators released during infection (e.g., IFN). Indirectly activated DCs mature but do not present pathogen Ags (as they have not encountered the pathogen) and do not provide signal 3. Therefore, although they are probably generated in large numbers upon infection or vaccination, indirectly activated DCs are considered to play little or no role in T cell immunity. In this article, we show that indirectly activated DCs retain their capacity to present Ags encountered after maturation in vivo. They can also respond to PAMPs, but the previous encounter of inflammatory signals alters their cytokine (signal 3) secretion pattern. This implies that the immune response elicited by a PAMP is more complex than predicted by the examination of the immunogenic features of directly activated DCs, and that underlying inflammatory processes can skew the immune response against pathogens. Our observations have important implications for the design of vaccines and for the understanding of the interactions between simultaneous infections, or of infection in the context of ongoing sterile inflammation.
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ItemThe inflammatory cytokine, GM-CSF, alters the developmental outcome of murine dendritic cells(WILEY, 2012-11)Fms-like tyrosine kinase 3 ligand (Flt3L) is a major cytokine that drives development of dendritic cells (DCs) under steady state, whereas GM-CSF becomes a prominent influence on differentiation during inflammation. The influence GM-CSF exerts on Flt3L-induced DC development has not been thoroughly examined. Here, we report that GM-CSF alters Flt3L-induced DC development. When BM cells were cultured with both Flt3L and GM-CSF, few CD8⁺ equivalent DCs or plasmacytoid DCs developed compared to cultures supplemented with Flt3L alone. The disappearance of these two cell subsets in GM-CSF + Flt3L culture was not a result of simple inhibition of their development, but a diversion of the original differentiation trajectory to form a new cell population. As a consequence, both DC progeny and their functions were altered. The effect of GM-CSF on DC subset development was confirmed in vivo. First, the CD8⁺ DC numbers were increased under GM-CSF deficiency (when either GM-CSF or its receptor was ablated). Second, this population was decreased under GM-CSF hyperexpression (by transgenesis or by Listeria infection). Our finding that GM-CSF dominantly changes the regulation of DC development in vitro and in vivo has important implications for inflammatory diseases or GM-CSF therapy.
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ItemWithout peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation(NATL ACAD SCIENCES, 2003-02-04)Peripheral activation can cause bystander thymocyte death by eliciting a "cytokine storm." This event complicates in vivo studies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral CD4 cells, this DP deletion was modest in mice lacking peripheral CD4 cells. Using BrdUrd and annexin V staining, we found that negative selection primarily occurs in a cohort of DP cells and the absence of single-positive (SP) cells is largely caused by reduction in the cohort of DP precursors. Moreover, the fates of DP cells and SP cells after antigen exposure were vastly different. Whereas SP cells up-regulated uniformly their CD69 and CD44 levels, increased their cell size, and survived after antigen exposure, DP cells had less CD69 and CD44 up-regulation, no size change, and promptly died. Thus, negative selection represents an "abortive" activation different from activation-induced cell death of mature T cells.
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ItemGlucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice(WILEY-V C H VERLAG GMBH, 2002-12)The involvement of glucocorticoid receptor (GR) signaling in T cell development is highly controversial, with several studies for and against. We have previously demonstrated that GR(-/-) mice, which usually die at birth because of impaired lung development, exhibit normal T cell development, at least in embryonic mice and in fetal thymus organ cultures. To directly investigate the role of GR signaling in adult T cell development, we analyzed the few GR(-/-) mice that occasionally survive birth, and irradiated mice reconstituted with GR(-/-) fetal liver precursors. All thymic and peripheral T cells, as well as other leukocyte lineages, developed and were maintained at normal levels. Anti-CD3-induced cell death of thymocytes in vitro, T cell repertoire heterogeneity and T cell proliferation in response to anti-CD3 stimulation were normal in the absence of GR signaling. Finally, we show that metyrapone, an inhibitor of glucocorticoid synthesis (commonly used to demonstrate a role for glucocorticoids in T cell development), impaired thymocyte development regardless of GR genotype indicating that this reagent inhibits thymocyte development in a glucocorticoid-independent fashion. These data demonstrate that GR signaling is not required for either normal T cell development or peripheral maintenance in embryonic or adult mice.