Enhanced response of T cells from murine gammaherpesvirus 68-infected mice lacking the suppressor of T cell receptor signaling molecules Sts-1 and Sts-2

Abstract

The human gammaherpesviruses establish life-long infections that are associated with the development of lymphomas and neoplasms, especially in immunocompromised individuals. T cells play a crucial role in the control of gammaherpesvirus infection through multiple functions, including the direct killing of infected cells, production of cytokines such as interferon-γ (IFN-γ), and costimulation of B cells. Impaired T cell function in mice infected with murine gammaherpesvirus 68 (MHV68) leads to increased reactivation and pathologies, including a higher incidence of lymphoid hyperplasia. Here we report that the absence of Suppressor of TCR signaling -1 and -2 (Sts-1(-/-)/2(-/-)) during MHV68 infection leads to the generation of T cells with significantly heightened responses. Transient differences in the T and B cell response of infected Sts-1(-/-)/2(-/-) (Sts dKO) mice were also observed when compared to WT mice. However, these alterations in the immune response and the overall absence of Sts-1 and Sts-2 did not impact viral pathogenesis or lead to pathology. Acute lytic replication in the lungs, establishment of latency in the spleen and reactivation from latency in the spleen in the Sts dKO mice were comparable to WT mice. Our studies indicate that Sts-1 and Sts-2 are not required for the immune control of MHV68 in a normal course of gammaherpesvirus infection, but suggest that interference with negative regulators of T cell responses might be further explored as a safe and efficacious strategy to improve adoptive T cell therapy.

Figure 1. Increased IFNγ response to infected cells in the absence of Sts1 and Sts2.

Sts dKO and C57BL/6 WT mice were infected 1000 PFU of MHV68 by the intranasal route and spleens were harvested 28 dpi. Splenocytes were left untreated or stimulated with 1 ug/ml αCD3 antibody or cocultured with gamma-irradiated MHV68-infected MEFs, both overnight in the presence of monensin. Cells were stained with the T cell marker CD90.2 and the intracellular cytokine IFNγ and analyzed by flow cytometry. (A) Representative flow plots of IFNγ expression in cells stained for CD90.2 are shown for each genotype and culture condition. (B) Scatter plot summary of the percentage of T cells producing IFNγ+ after overnight stimulation. Symbols represent data from individual mice. * = p<.05, ****  = p<.0001.

Figure 2. Sts dKO CD4⁺ and CD8⁺ T cells have increased effector responses to infected cells.

Sts dKO and C57BL/6 WT mice were infected 1000 PFU of MHV68 by the intranasal route and spleens were harvested 28 dpi. Stimulations were performed as described in Figure 1 with the addition of αLAMP1, followed by costaining for CD4, CD8, and p79 tetramers. (A) Representative flow plots of LAMP-1 and IFNγ expression on CD8⁺ T cells are shown for each genotype and culture condition. (B-D) Scatter plot of the percentage of the indicated T cell subsets positive for IFNγ or LAMP-1. Axes were adjusted to better illustrate differences within each experimental condition. Symbols represent data from individual mice. * = p<.05, ** = p<.01, *** = p<.001. ****  = p<.0001.

Figure 3. Sts dKO T cells do not alter the kinetics or levels of acute replication in the lungs.

Sts dKO and C57BL/6 WT mice were infected 1000 PFU of MHV68 by the intranasal route. Lungs were harvested and disrupted at the indicated timepoints to quantitate levels of pre-formed infectious virus. Bar indicates median of log₁₀ transformed data; dashed line indicates limit of detection. 3–8 animals are included per replicate experiment, n = 3 for 4 dpi, n = 2 for 9 dpi, n = 2 for 12 dpi, and n = 1 for 16 and 42 dpi; no significant differences were found based on Mann-Whitney non-parametric t-tests.

Figure 4. Viral latency and reactivation from latency are unchanged in Sts dKO animals.

Sts dKO and C57BL/6 WT mice were infected with 1000 PFU of MHV68 by the intranasal route and single cell suspensions of the spleens were prepared at 16 dpi. (A) Frequency of splenocytes harboring viral genomes determined by limiting dilution nested PCR assay. (B) Frequency of splenocytes reactivting virus determined by a limiting dilution ex vivo reactivation assay. (C) Frequency of splenocytes harboring viral genomes at 45-57. For all limiting-dilution assays, curve fit lines were derived from nonlinear regression analysis, and symbols represent the mean percentage of wells positive for virus (viral DNA or CPE) ± the standard error of the mean. The dotted line represents 63.2%, from which the frequency of viral genome-positive cells or the frequency of cells reactivating virus was calculated based on the Poisson distribution. Graphs represent 3 independent experiments of 3–4 mice.