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. 2008 Dec 1;181(11):7800-9.
doi: 10.4049/jimmunol.181.11.7800.

TNF receptor-associated factor 5 is required for optimal T cell expansion and survival in response to infection

Zachary J Kraus  1 , Jodie S Haring, Gail A Bishop
Affiliations

TNF receptor-associated factor 5 is required for optimal T cell expansion and survival in response to infection

Zachary J Kraus et al. J Immunol. .

Abstract

Receptors belonging to the TNF-receptor (TNF-R) superfamily include important costimulatory molecules, many of which specifically affect T cell activation. TNF receptor-associated factors (TRAFs) are recruited to many TNF-R superfamily members and are important modulators of the proximal signaling events that occur at the time of receptor engagement and activation. TRAF5 has been shown to be a positive regulator of a number of these receptors that are involved in T cell costimulation. However, the potential importance of TRAF5 in cellular immune responses to infection or in T cell expansion and memory have not been studied. We report in this study that TRAF5 was required for optimal CD8(+) T cell responses following infection with Listeria monocytogenes expressing OVA (LM-OVA). TRAF5 was necessary for optimal T cell expansion following primary infection with LM-OVA, and its absence resulted in fewer memory CD8(+) T cells following LM-OVA infection, together with higher bacterial loads in the liver. The effect of TRAF5 on CD8(+) T cell expansion was T cell intrinsic and not due to effects of TRAF5 deficiency on APCs. Although their proliferative ability remained intact, CD8(+) T cells from TRAF5(-/-) mice were more sensitive to apoptosis and were unresponsive to the prosurvival effects of the TNF-R superfamily costimulator CD27. Collectively, these studies identify TRAF5 as an important positive signaling element that enhances T cell expansion and pathogen containment by providing a survival advantage to responding Ag-specific CD8(+) T cells during infection.

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Figures

Figure 1
Figure 1. The effects of TRAF5 deficiency on T cell expansion in response to LM-OVA
(A) TRAF5-/- mice (□) and littermate controls (TRAF5+/+) (■) were infected i.v. with a sub-lethal dose of virulent LM-OVA (0.1XLD50 or 1000 CFU). (C & D) CD8+ T cell expansion in response to the primary infection was measured using OVA-peptide-MHC class I tetramer staining at day 7. * represents P<0.001 in a paired students t test (95% confidence interval) on the composite data of 3 independent experiments (N= 8 mice per group) for ICS experiments and 2 independent experiments (N= 7 littermate control mice and N= 4 TRAF5-/- mice) for tetramer experiments).
Figure 2
Figure 2. The effects of TRAF5 deficiency on T cell memory and secondary T cell expansion
(A &B) Memory levels of CD8+ and CD4+ T cells were measured at day 40 p.i., by IFN-g ICS (Composite data from 3 independent experiments where N= 8 mice per group). T cell expansion in response to a secondary infection (i.v.) of LM-OVA (5XLD50 or 5×105 CFU) was observed using ICS for IFN-γ at day 5 post challenge (Composite data from 3 independent experiments where N= 7 mice per group). (C) These methods were also used to measure contraction of both CD8+ and CD4+ T cells. Expansion from memory to the peak secondary response was also measured for CD8+ and CD4+ T cells. * represents P<0.05 in a paired students t test (95% confidence interval).
Figure 3
Figure 3. Protection from secondary infection
TRAF5-/- mice and littermate controls (TRAF5+/+) mice were infected i.v. with a sub-lethal dose of virulent LM-OVA (0.1XLD50 or 1000 CFU). At day 43 p.i. TRAF5-/- and littermate controls were challenged with a lethal dose (5XLD50 or 5×105 CFU) of LM-OVA. Naïve mice were also infected as unimmunized controls. Spleens (A) and livers (B) were harvested on day 3 and 5 post challenge. Cumulative data are from 2 independent experiments.
Figure 4
Figure 4. Primary response in mice receiving dendritic cell (DC) vaccination
C57BL\6 mice were immunized with 5×105 bone marrow-derived DCs from TRAF5+/+ mice. DCs were matured with 100ng/mL CpG-B oligonucleotide (2084), 100ng/mL CpG-B (2084) oligonucleotide plus 5μg/mL anti-CD40 mAb (HM-40.1) or 100ng/mL LPS for 48 hours prior to immunization (A & B). TRAF5-/- mice and littermate controls (TRAF5+/+) were immunized with 5×105 bone marrow-derived DCs from TRAF5+/+ mice or (C) TRAF5-/- DCs were used to immunize TRAF5+/+ mice, while TRAF5+/+ DCs were used to immunize TRAF5+/+ mice as a control (D). Ag-specific CD8+ T cells were measured by ICS for IFN-γ at day 7 post immunization (E). * represents P<0.05 in a paired students t test (95% confidence interval). Data in (C) are cumulative from 3 independent experiments where N= 8 mice per group. Data in (D) are cumulative from 2 independent experiments where N= 4 mice per group.
Figure 5
Figure 5. Intrinsic nature of the expansion defect in TRAF5 deficient mice
CD45.2/Thy1.2/1.2 OT-1 TRAF5-/- T cells were mixed 1:1 (1000 total CD8+ T cells) with T cells from littermate controls (CD45.2/Thy1.1/1.2 OT-1 TRAF5+/+) and transferred i.v. into naïve CD45.1/Thy1.2/1.2 mice. Recipient mice were infected with 0.1XLD50 virulent LM-OVA 24 hrs later (A). At day 7 CD45.1 was used to exclude T cells from recipient mice (B) and OT-1 TRAF5-/- CD8+ T cells and littermate OT-1 CD8 + T cells were measured as Thy1.1 versus Thy1.2 (C & D). * represents P<0.001 in a paired students t test (95% confidence interval). This figure contains cumulative data from 2 independent experiments where N= 8 mice per group.
Figure 6
Figure 6. Proliferation and apoptosis in CD8+ T cells
Purified CD8+ T cells were labeled with CFSE prior to stimulation. 3×105 CD8+ T cells/well were stimulated with plate bound anti-CD3 mAb (0.5ug/mL) with or without soluble anti-CD28 (10ug/mL) or anti-CD27 (10ug/mL) mAbs, or insect cells infected with CD70-producing baculovirus for 72 hours. Filled histograms represent control wells where cells were treated with a sub-optimal concentration of plate bound anti-CD3 mAb and isotype controls for anti-CD28 and anti-CD27 mAbs or insect cells infected with wild type baculovirus (A). Apoptosis was also measured as caspase 3 activation using the same stimulation parameters (TRAF5-/- CD8+ T cells (□) and littermate control CD8+ T cells (■) (C). Open histograms represent TRAF5-/- CD8+ T cell and filled histograms represent littermate control CD8+ T cells (B). Splenocytes from OT-1 TRAF5-/- CD8+ T cells (□) and littermate OT-1 CD8+ T cells (■) were pulsed with SIINFEKL peptide with or without anti-Fas/CD95 mAb, Hi5 insect cells expressing CD70 or anti-Fas + Hi5 CD70. Apoptosis was measured as an increase in caspase 3 activity in CD8+ cells (D & E). Filled histograms represent cells treated with isotype control mAb for anti-Fas/CD95 (Jo2 mAb) and open histograms represent cells treated with anti-Fas/CD95 mAb (D). CD8+ T cells were fixed and stained with propidium iodide following 48 hour stimulations (TRAF5-/- CD8+ T cells (□) and littermate CD8+ T cells (■) (F & G). Open histograms represent TRAF5-/- CD8+ T cell and filled histograms represent littermate control CD8+ T cells (F) Data are representative of 3 independent experiments with similar results. *= P<0.05 and ** =P<0.001 in a paired students t test (95% confidence interval).
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