Regulation of Early T Cell Activation by TNF Superfamily Members TNF and FASL: A Dissertation

The instructive signals received by T cells during the programming stages of activation will determine the fate of effector and memory populations generated during an immune response. Members of the tumor necrosis factor (TNF) superfamily play an essential role in influencing numerous aspects of T cell adaptive immune responses including cell activation, differentiation, proliferation, survival, and apoptosis. My thesis dissertation describes the involvement of two such members of the TNF superfamily, TNF and FasL, and their influence on the fate of T cells early during responses to viral infections and to the induction of transplantation tolerance. TNF is a pleiotropic pro-inflammatory cytokine that has an immunoregulatory role in limiting the magnitude of T cell responses during a viral infection. Our laboratory discovered that one hallmark of naïve T cells in secondary lymphoid organs is their unique ability to rapidly produce TNF after activation and prior to acquiring other effector functions. I hypothesized that T cell-derived TNF will limit the magnitude of T cell responses. The co-adoptive transfer of wild type (WT) P14 and TNF-deficient P14 TCR transgenic CD8+ T cells, that recognize the GP33 peptide of lymphocytic choriomeningitis virus (LCMV), into either WT or TNF-deficient hosts demonstrated that the donor TNF-deficient P14 TCR transgenic CD8+ T cells accumulate to higher frequencies after LCMV infection. Moreover, these co-adoptive transfer experiments suggested that the effect of T cell-derived TNF is localized in the microenvironment, since the TNF produced by WT P14 TCR transgenic CD8+ T cells did not prevent the accumulation of TNF-deficient P14 TCR transgenic CD8+ T cells. To determine if T cell-produced TNF is acting on professional APC to suppress the generation of virus-specific T cell responses, I performed co-adoptive transfer experiments with WT P14 TCR transgenic CD8+ and TNF-deficient P14 TCR transgenic CD8+ T cells into TNFR1/2 (1 and 2) deficient mice. These experiments demonstrated that the absence of TNFR1/2 signaling pathway in the host cells resulted in a greater accumulation of WT P14 TCR transgenic CD8+ T cells, thereby considerably diminishing the differences between donor WT P14 TCR transgenic CD8+ and donor TNF-deficient P14 TCR transgenic CD8+ T cells. The increased frequency and absolute numbers of WT P14 TCR transgenic CD8+ T cells in TNFR1/R2 deficient recipients suggests that one mechanism for the suppressive effect of T cell-derived TNF on antigen-specific T cells occurs as a result of TNFR signaling in the host cells. However, the donor TNF-deficient P14 TCR transgenic CD8+T cells still accumulated to higher frequency and numbers compared to their donor WT transgenic counterparts. Together, these findings indicate that T cell-produced TNF can function both in an autocrine and a paracrine fashion to limit the magnitude of anti-viral T cell responses. Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, I questioned at what stage of development do T cells become licensed to produce this cytokine. The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. I hypothesized that naïve T cells emigrating from the thymus will be competent to produce TNF only after undergoing a maturation process in the periphery. To test this hypothesis, I 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 with respect to the upregulation of CD25 and CD69 following stimulation. The reduced ability of SP thymocytes to produce TNF correlated with a decreased level of detectable TNF message following stimulation when compared to splenic counterparts. Stimulation of SP thymocytes in the context of a splenic environment did not fully enable TNF production, suggesting an intrinsic defect in their ability to produce TNF as opposed to a defect in antigen presentation. Using a thymocyte adoptive transfer model, I 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 identified by the expression of green fluorescent protein (GFP) (NG-BAC transgenic mice), showed a significantly enhanced ability to express TNF relative to SP thymocytes, but not to the extent of 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. This highlights the functional heterogeneity of the naïve T cell pool (with respect to varying degrees of TNF production) during early T cell activation that can contribute to the many subsequent events that shape the course of an immune response. The productive activation of naïve T cells requires at least initial two signals; the first being through the TCR and the second is the engagement of co-stimulatory molecules on the surface of the T cells. T cells activated in the absence of co-stimulation become anergic or undergo cell death. Agents that block co-stimulation of antigen-specific T cells are emerging as an alternative to immunosuppressive drugs to prolong allograft survival in transplant recipients. Targeted blockade of CD154-CD40 interactions using a αCD154 monoclonal antibody (MR1) with a simultaneous transfusion of allogeneic splenocytes (donor specific transfusion or DST) efficiently induces tolerance to allografts. This co-stimulation blockade-induced tolerance is characterized by the deletion of host alloreactive T cells within 24 hours of treatment. Toll-like receptor (TLR) agonists abrogate tolerance induced by co-stimulation blockade by impairing the deletion of host alloreactive T cells and resulting in allograft rejection. The goal of my study was to determine the underlying molecular mechanisms that protect host alloreactive T cells from early deletion after exposure to TLR agonists. I hypothesized that TLR ligands administered during co-stimulation blockade regimen differentially regulate the expression of pro- and anti-apoptotic molecules in alloreactive T cells, during the initial stages of activation thereby preventing deletion. To test this hypothesis, I used syngeneic bone marrow chimeric mice containing a trace population of alloreactive KB5 TCR transgenic CD8+ T cells (KB5 Tg CD8+ T cells) that recognize H-2Kb as an alloantigen. I show here that KB5-CD8+ T cells downregulate CD127 (IL-7R!) and become apoptotic as early as 12 hrs after co-stimulation blockade. In contrast, KB5 Tg CD8+ T cells from mice treated with bacterial lipopolysaccaride (LPS) during co-stimulation blockade failed to become apoptotic, although CD127 was downregulated. Examination of the mRNA expression profiles of several apoptotic genes in purified KB5 CD8+ T cells from mice treated with DST+anti-CD154 for 12 hrs revealed a significant upregulation of FasL mRNA expression compared to the untreated counterparts. However, in vitro FasL blockade or in vivo cytotoxicity experiments with mice deficient in Fas or FasL indicated that the Fas-FasL pathway might not be crucial for tolerance induction. Another pro-apoptotic molecule BIM was upregulated in alloreactive T cells during co-stimulation blockade. This suggests that both the Fas pathway and BIM may be playing complementary roles in inducing deletional tolerance. Although FasL expression was diminished in alloreactive T cells in the presence of LPS, BIM expression was not diminished, suggesting that alloreactive T cells may still be vulnerable to undergo apoptosis. Concomitantly, I also found that LPS treatment during co-stimulation blockade resulted in non-specific upregulation of Fas expression in alloreactive T cells and non-transgenic T cells (CD4+ and CD8+). I demonstrate here that treatment with Fas agonistic antibody in vitrofor 4 hours can selectively induce apoptosis of alloreactive T cells that were believed to be refractory to apoptosis during LPS treatment. I speculate that under these conditions, deletion may be occurring due to the involvement of both Fas and BIM. Further, the mRNA expression profile revealed interleukin-10 (IL-10) as a molecule induced in alloreactive T cells during LPS treatment. Analysis of serum confirmed the systemic expression of IL-10 protein in mice treated with LPS during co-stimulation blockade. I hypothesized that LPS-induced IL-10 can have an anti-apoptotic role in preventing the deletion of alloreactive T cells and mediating allograft rejection. Contrary to my hypothesis, I found that IL-10 KO mice rejected allogeneic target cells similar to their WT counterparts, suggesting that IL-10 may not be required for LPS-mediated abrogation of tolerance induction. In addition to the systemic induction of IL-10, LPS also induced cytokines such as interleukin-6 (IL-6), TNF and interferon-γ (IFN-γ). These findings suggest that both Fas-FasL and BIM mediated apoptotic pathways may play complementary roles in inducing the early deletion of activated alloreactive T cells during tolerance induction. On the other hand, the mechanism of LPS mediated abrogation of tolerance induction can not be attributed to IL-10 alone as it may be playing a synergistic role along with other proinflammatory cytokines that may in turn result in the prevention of alloreactive T cell death during this process. Most importantly, these findings indicate that despite emerging from a pro-inflammatory cytokine milieu, alloreactive T cells are still susceptible to undergo Fas-mediated apoptosis during the first 24 hours after co-stimulation blockade and LPS treatment. Therefore, targeting the Fas-FasL pathway to induce deletion of alloreactive T cells during the peri-transplant period may still be a potential strategy to improve the efficacy of co-stimulation blockade induced transplantation tolerance during an environmental perturbation such as inflammation or infection

The phosphatase PTEN-mediated control of PI-3 kinase in Tregs cells maintains homeostasis and lineage stability

Foxp3+ regulatory T cells (Tregs) are required for immune homeostasis. One notable distinction between conventional T cells (Tconv) and Tregs is differential phosphatidylinositol 3-kinase (PI3K) activity: only Tconv downregulate PTEN, the primary negative regulator of PI3K, upon activation. Here, we show that control of PI3K in Tregs is essential for lineage homeostasis and stability. Mice lacking Pten in Tregs developed an autoimmune-lymphoproliferative disease characterized by excessive TH1 responses and B cell activation. Diminished control of PI3K activity in Tregs led to reduced CD25 expression, accumulation of Foxp3+CD25− cells and ultimately, loss of Foxp3 expression in these cells. Collectively, these data demonstrate that control of PI3K signaling by PTEN in Tregs is critical to maintain their homeostasis, function and stability

Maturation-Dependent Licensing of Naive T Cells for Rapid TNF Production

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

Post-thymic maturation status of naïve P14 transgenic T cells determines their TNF producing capability.

<p>Female CD45.1⁺ P14-CD8⁺ thymocytes were transferred into female CD45.2⁺ B6 congenic mice. Host spleens were recovered after the indicated time periods and were stimulated in vitro for 4 hours with GP33+αCD28 and donor CD45.1⁺ T cells were stained for intracellular TNF as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Dead cells were excluded using Live Dead Aqua Dead cell stain for this experiment. For analysis, cells were gated on the live donor SP P14-CD8⁺ T cells and the maturation profile of donor cells that are TNF⁺ (indicated by arrows in the boxed quadrants in plots iii,iv,v,vi) were compared at all the time points shown (corresponding histograms). Additionally, some CD45.1⁺ P14-CD8⁺ thymocytes were stimulated before transfer in the context of CD45.2⁺ B6 splenocytes in vitro for 4 hours with GP33+αCD28 and their maturation profile was compared to CD45.1⁺ P14-CD8⁺ thymocytes and splenocytes stimulated alone in vitro (plots i, ii and vii).</p

Reduced upregulation of TNF message in SP thymocytes relative to naïve splenic T cells upon TCR stimulation.

<p>SP P14-CD8⁺ thymocytes and (CD44^lo) naïve splenic CD8⁺ T cells were purified by cell sorting and stimulated in the presence of GP33 and GP33+αCD28 for 4 hours followed by RNA isolation and cDNA synthesis from 50 ng of RNA and then amplified using TNF specific primers by quantitative real time PCR from the indicated populations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. A, The basal steady state level of TNF transcripts in 50 ng of total RNA (normalized to a β-actin control) isolated from unstimulated SP P14-CD8⁺ thymocytes and (CD44^lo) naïve splenic T cells are shown. B, The increase in TNF message in these subsets upon stimulation with GP33 and GP33+αCD28 in terms of fold induction with respect to unstimulated SP thymocytes been (normalized to a β-actin control) are shown. This profile is representative of 3 individual experiments.</p

SP thymocytes are poor TNF producers relative to naïve splenic T cells during TCR activation in vitro.

<p>Thymocytes and splenocytes from P14-CD8⁺, OT-1-CD8⁺, SMARTA-CD4⁺ and OT-2-CD4⁺ TCR transgenic mice were stimulated in vitro as indicated for 4 hours and then stained for intracellular TNF cytokine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. For analysis, the cells were gated on either SP CD8⁺ CD4⁻ or SP CD4⁺ CD8⁻ cells. A, The percentages of TCR transgenic thymocytes and splenic T cells (both CD44^lo and CD44^hi) staining positive for TNF are shown. B, Resting P14-CD8⁺ thymocytes and naïve splenocytes were stained with mAbs to Vα2 and Vβ8.1. The profile for the SP thymocytes is shown in gray solid histograms and for splenic T cells in black line histograms. C, P14-CD8⁺ thymocytes and naïve splenocytes were either unstimulated (gray solid histograms) or stimulated (black line histograms) for 4 hours with GP33+αCD28 in vitro and stained with mAbs to the indicated surface molecules. D, CD45.1⁺ SP P14-CD8⁺ thymocytes were enriched and stimulated with GP33+αCD28 for 4 hours either alone or in the presence of live or irradiated (3000cGy) H-2D^b WT or H-2D^b KO splenocytes respectively. Cells were then stained for intracellular TNF. The percentages of CD45.1⁺ CD8⁺ thymocytes (CD44^lo and CD44^hi) staining positive for TNF are shown.</p

Maturation state of SP thymocytes reflects their TNF producing capability^a.

a<p>The average MFI of maturation markers CD24 (n = 6), CD45RB (n = 6) and Qa2 (n = 3 for group1 and 2 and n = 6 for for group 3 and 4) in TNF producing donor SP P14-CD8⁺ are shown. The averages were analysed using One-way ANOVA with a Tukey post-test as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Error indicates SD. N/A, Not Applicable.</p>b<p>p<0.05 vs Table II, group 1.</p>c<p>p<0.05 vs Table II, group 1.</p>d<p>p<0.05 vs Table II, group 1.</p>e<p>p<0.05 vs Table II, group 1.</p>f<p>p<0.05 vs Table II, group 1.</p>g<p>p<0.05 vs Table II, group 1.</p>h<p>p<0.05 vs Table II, group 1 TNF+.</p>i<p>p<0.05 vs Table II, group 1 TNF+.</p>j<p>p<0.05 vs Table II, group 1 TNF+.</p

SP thymocytes acquire the ability to produce TNF as a function of time in the periphery^a.

a<p>The recovery of the donor (CD44^lo) SP P14-CD8⁺ thymocytes from recipient spleens and the MFI of TNF expression at the indicated time points post-transfer are shown. The average recovery and the MFI of TNF expression by donor thymocytes (n = 6 per time point) were analysed using One-way ANOVA with a Tukey post-test as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. Error indicates SD. N/A, Not Applicable.</p>b<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>c<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>d<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>e<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 1.</p>f<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 2.</p>g<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 2.</p>h<p>p<0.05 vs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#pone-0015038-t001" target="_blank">Table 1</a>, group 3.</p

TNF producing SP thymocytes exhibit a lower maturation profile relative to their splenic counterparts.

<p>A and B, CD45.1⁺ P14-CD8⁺ thymocytes were stimulated with GP33+αCD28 for 4 hours in vitro and then stained for maturation markers and intracellular TNF, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015038#s4" target="_blank"><i>Materials and Methods</i></a>. The TNF producers and non producers of the thymic and the splenic subsets were each classified into 4 subgroups based on their CD24 and Qa2 expression as shown namely Subgroup 1 (CD24^hi Qa2^lo) followed by Subgroup 2 (CD24^hi-int Qa2^lo), Subgroup 3 (CD24^lo Qa2^lo) and finally Subgroup 4 (CD24^lo Qa2^hi). C, shows the histogram comparison of the small population of TNF producing SP thymocytes (dotted line histograms), the majority of SP thymocytes that are TNF non-producers (gray histograms) and TNF producing splenic T cells (solid dark line histograms) and TNF non-producing splenic T cells (black histograms). The MFIs of each of the maturation markers in TNF producing and non-producing thymic and splenic T cells are indicated in the left hand side of the histograms respectively. D, shows the maturation profile of the total SP CD8⁺ thymocytes based on their CD24 and Qa2 expression. The proportion of cells capable of making TNF in the 4 subgroups is shown with respect to CD45RB expression.</p

Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability.

<p>A, The percentages of non-transgenic CD8⁺ and CD4⁺ cells (both CD44^lo and CD44^hi) 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 CD44^lo 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.</p