Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection

Abstract

CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with retroviruses. Similar to textbook knowledge the most important function of CD4+ T cells during an acute retrovirus infection seems to be their helper function for other immune cells. Whereas there was no direct anti-viral activity of CD4+ T cells during acute Friend Virus (FV) infection, they were absolutely required for the control of chronic infection. During chronic FV infection a population of activated FV-specific CD4+ T cells did not express cytotoxic molecules, but Fas Ligand that can induce Fas-induced apoptosis in target cells. Using an MHC II-restricted in vivo CTL assay we demonstrated that FV-specific CD4+ T cells indeed mediated cytotoxic effects against FV epitope peptide loaded targets. CD4 + CTL killing was also detected in FV-infected granzyme B knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies against FasL, which identified the Fas/FasL pathway as critical cytotoxic mechanism during chronic FV infection. Interestingly, targeting the co-stimulatory receptor CD137 with an agonistic antibody enhanced CD4+ T cell cytotoxicity. This immunotherapy may be an interesting new approach for the treatment of chronic viral infections.

Figure 1

Kinetics of FV infection. Viral loads of FV-infected C57BL/6 mice were analyzed in spleens and lymph nodes using an infectious center assay. At least six mice per group were analyzed. Data were pooled from two independent experiments.

Figure 2

CD4+ T cell response during chronic FV infection. C57BL/6 mice were chronically infected with FV (6 weeks post infection). (A) Percentages and absolute numbers of CD4+ Tetr II+ T cells reactive with I-Ab MHC class II tetramers specific for the FV H19-Env epitope. (B) Percentages and absolute numbers of CD4+ T cells showing the activation profile CD43+ and CD62L−. (C) Numbers per million cells of effector CD4+ T cells expressing proliferation marker Ki67. (D) Numbers per million cells of effector CD4+ T cells expressing the differentiation marker KLRG1. Each dot represents an individual mouse. Mean values are indicated by a line. Statistically significant differences between the groups were determined by the unpaired t test: *p < 0.05; **p < 0.005; ***p < 0.0001, ns, not significant. Data were pooled from two independent experiments.

Figure 3

Expression of cytotoxic molecules in effector CD4+ T cells from chronically FV infected mice. Flow cytometry was used to detect intracellular granzymes, or surface Fas ligand in activated effector CD4+ T cells (CD43+CD62L−). (A) Percentages of effector CD4+ T cells expressing granzyme B. (B) Percentages of effector CD4+ T cells expressing granzyme K. (C) Percentages of effector CD4+ T cells expressing TRAIL after restimulation with αCD3 and αCD28 antibodies in vitro. (D) Absolute numbers of effector CD4+ T cells expressing FasL after restimulation with αCD3 and αCD28 antibodies in vitro. (E) FasL expression is presented as in D), except that data are expressed as MFI. (F) mRNA levels for FasL in effector CD4+ T cells isolated from FV infected mice. β-actin was used as an internal standard. (G) Representative dot plots of FasL expression on activated CD4+ T cells from different phases of FV infection. Each dot represents an individual mouse. Mean values are indicated by a line. Statistically significant differences between the groups were determined by the unpaired t test: *p < 0.05; **p < 0.005. Data were pooled from at least two independent experiments.

Figure 4

In vivo cytotoxicity of CD4+ T cells during chronic FV infection. Lymph node cells and spleen cells from naïve mice were loaded with CD4+ T cell FV specific epitope peptides and labeled with CFSE dye. Unloaded cells were stained with cell trace violet. Peptide loaded and unloaded cells were injected intravenously in a 1:1 ratio into naïve and FV-infected mice and tracked by flow cytometry 20 hours after injection. The figure shows the percentage of the target cell killing in the lymph nodes. (A) Cells from naïve mice were loaded either with H2-A^b-restricted F-MulV H19 envelope epitope alone (H19-Env) or with nine additional epitope peptides spanning the coding regions gag, pol, and env (epitope mix), which were described by R.J. Messer et al. and injected intravenously into naïve, acutely FV-infected (n = 8), chronically FV-infected (n = 12), chronically FV-infected CD4⁺ T cell depleted mice (n = 4). (B) Cells from naïve mice were loaded with the peptide epitope mixture. Peptide loaded and unloaded cells were injected intravenously into naïve, acutely FV-infected (n = 8), chronically FV-infected (n = 12), chronically FV-infected GzmB KO mice (n = 8), chronically FV-infected mice, receiving a FasL blocking antibody (n = 4). Mice receiving an isotype control antibody did not show any difference to the non-treated control group. Mean values are indicated by a line. Statistically significant differences between the groups were determined by the unpaired t test: **p < 0.005; ***p < 0.0001, ns, not significant. Data were pooled from two independent experiments.

Figure 5

CD4+ T cell cytotoxicity against MHC II+ target cells. Target cells for the CD4+ T cell cytotoxicity assay were obtained from lymph nodes and spleens of naïve mice, loaded with epitope peptides and injected into FV-infected recipient mice. The target cell population was depleted for T cells to increase the frequency of possible MHC II+ targets. (A) Distribution of different target cell populations in the lymph nodes of naïve recipient mice. (B) Frequencies of killed cells indicated that CD19+ B cells were the main targets of cytotoxic CD4+ T cells. The data were pooled from two independent experiments with similar results.

Figure 6

Effector cell profile of CD4+ T cells following costimulatory therapy. To augment CD4+ T cell cytotoxicity FV-infected mice were treated with αCD134 or αCD137 antibodies as described in Materials and Methods. (A) Spider plot analysis showing the expression levels of CD4+ T cell activation, differentiation and cytotoxic markers after antibody therapy. αCD134 antibody treatment only enhanced activation of CD4+ T cells, but only αCD137 antibodies induce expression of molecules associated with a cytotoxic program. (B) The percentage of the target cell killing in the lymph nodes of FV-infected mice following αCD137 antibody treatment. Mice receiving an isotype control antibody did not show any difference to the non-treated control group. (C) Viral loads in FV-infected mice after CD137 treatment. (D) FV RNA levels after CD137 treatment.Statistically significant differences between the groups were determined by the unpaired t test: *p < 0.05; **p < 0.005; ***ns, not significant.