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Maturation-dependent licensing of naive T cells for rapid TNF production.

Priyadharshini B, Welsh RM, Greiner DL, Gerstein RM, Brehm MA - PLoS ONE (2010)

Bottom Line: The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery.One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions.Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4(+) and CD8(+) single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

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Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.A, The percentages of non-transgenic CD8+ and CD4+ cells (both CD44lo and CD44hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8+ and CD4+ compartments is shown. B and C, The percentages of CD44lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.
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pone-0015038-g006: Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.A, The percentages of non-transgenic CD8+ and CD4+ cells (both CD44lo and CD44hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8+ and CD4+ compartments is shown. B and C, The percentages of CD44lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.

Mentions: Polyclonal naïve CD4+ and CD8+ T lymphocytes (CD44lo) from secondary lymphoid organs rapidly produce TNF after TCR engagement before gaining other effector functions [8]. However, it is not known if polyclonal SP thymocytes also lack the capability to produce TNF like their transgenic counterparts. To determine this, thymocytes and splenocytes from naïve non-transgenic B6 mice were stimulated using both monoclonal αCD3 and αCD28 antibodies for 4 hrs in vitro, respectively. Fig. 6A shows that, similarly to the transgenic T cells, a lower proportion of polyclonal CD4+ CD8− and CD4− CD8+ SP thymocytes produced TNF when compared to naïve (CD44lo) splenic T cells during TCR stimulation (Fig 6A). This inability to produce TNF was not overcome by increasing the concentrations of the peptide or αCD3 (data not shown). Given this difference and the ability of transgenic SP thymocytes to gradually gain the capability to produce TNF with time in the periphery (shown in Fig 5), we wanted to directly test the ability of polyclonal RTEs that are naturally seeding into the periphery for their ability to produce TNF upon stimulation. For this, we used mice expressing GFP under the control of the Rag2 promoter (NG-BAC transgenic mice). The level of GFP expression by T cells in the periphery of these mice can be used to identify T cells at different stages of post-thymic maturation. GFPhi T cells have resided in the periphery for 0–7 days, GFPlo T cells have resided in the periphery for 7–14 days and GFPneg T cells have joined the MN T cell pool (>14 days in the periphery) [3]. We compared three T cell subsets: SP thymocytes (GFPhi), RTEs (GFPhi+lo) in the spleen, and MN T cells (GFPneg) in the spleen (Fig.6B). A higher proportion of CD8+ and CD4+ RTEs produced TNF in response to αCD3 and αCD28 stimulation when compared to SP thymocytes (Fig.6B). However, the proportion of CD8+ RTEs producing TNF was lower than MN CD8+ T cell populations (Fig.6B). This hierarchical pattern of TNF production was also observed on a per cell basis in the three T cell subsets (Fig.6C). In contrast to the CD8+ T cell compartment, a similar frequency of CD4+ RTE and MN T cells produced TNF, but the MFI of the TNF signal was significantly higher in the CD4+ MN T cells relative to both CD4+ RTEs and SP CD4+ thymocytes (Fig. 6B and 6C). Together these results support our data from the adoptive transfer model indicating that post-thymic maturation confers the complete licensing of naïve T cells to rapidly produce TNF after TCR engagement.


Maturation-dependent licensing of naive T cells for rapid TNF production.

Priyadharshini B, Welsh RM, Greiner DL, Gerstein RM, Brehm MA - PLoS ONE (2010)

Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.A, The percentages of non-transgenic CD8+ and CD4+ cells (both CD44lo and CD44hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8+ and CD4+ compartments is shown. B and C, The percentages of CD44lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991336&req=5

pone-0015038-g006: Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.A, The percentages of non-transgenic CD8+ and CD4+ cells (both CD44lo and CD44hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8+ and CD4+ compartments is shown. B and C, The percentages of CD44lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.
Mentions: Polyclonal naïve CD4+ and CD8+ T lymphocytes (CD44lo) from secondary lymphoid organs rapidly produce TNF after TCR engagement before gaining other effector functions [8]. However, it is not known if polyclonal SP thymocytes also lack the capability to produce TNF like their transgenic counterparts. To determine this, thymocytes and splenocytes from naïve non-transgenic B6 mice were stimulated using both monoclonal αCD3 and αCD28 antibodies for 4 hrs in vitro, respectively. Fig. 6A shows that, similarly to the transgenic T cells, a lower proportion of polyclonal CD4+ CD8− and CD4− CD8+ SP thymocytes produced TNF when compared to naïve (CD44lo) splenic T cells during TCR stimulation (Fig 6A). This inability to produce TNF was not overcome by increasing the concentrations of the peptide or αCD3 (data not shown). Given this difference and the ability of transgenic SP thymocytes to gradually gain the capability to produce TNF with time in the periphery (shown in Fig 5), we wanted to directly test the ability of polyclonal RTEs that are naturally seeding into the periphery for their ability to produce TNF upon stimulation. For this, we used mice expressing GFP under the control of the Rag2 promoter (NG-BAC transgenic mice). The level of GFP expression by T cells in the periphery of these mice can be used to identify T cells at different stages of post-thymic maturation. GFPhi T cells have resided in the periphery for 0–7 days, GFPlo T cells have resided in the periphery for 7–14 days and GFPneg T cells have joined the MN T cell pool (>14 days in the periphery) [3]. We compared three T cell subsets: SP thymocytes (GFPhi), RTEs (GFPhi+lo) in the spleen, and MN T cells (GFPneg) in the spleen (Fig.6B). A higher proportion of CD8+ and CD4+ RTEs produced TNF in response to αCD3 and αCD28 stimulation when compared to SP thymocytes (Fig.6B). However, the proportion of CD8+ RTEs producing TNF was lower than MN CD8+ T cell populations (Fig.6B). This hierarchical pattern of TNF production was also observed on a per cell basis in the three T cell subsets (Fig.6C). In contrast to the CD8+ T cell compartment, a similar frequency of CD4+ RTE and MN T cells produced TNF, but the MFI of the TNF signal was significantly higher in the CD4+ MN T cells relative to both CD4+ RTEs and SP CD4+ thymocytes (Fig. 6B and 6C). Together these results support our data from the adoptive transfer model indicating that post-thymic maturation confers the complete licensing of naïve T cells to rapidly produce TNF after TCR engagement.

Bottom Line: The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery.One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions.Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4(+) and CD8(+) single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

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