CD6 Regulates CD4 T Follicular Helper Cell Differentiation and Humoral Immunity During Murine Coronavirus Infection

Abstract

During activation the T cell transmembrane receptor CD6 becomes incorporated into the T cell immunological synapse where it can exert both co-stimulatory and co-inhibitory functions. Given the ability of CD6 to carry out opposing functions, this study sought to determine how CD6 regulates early T cell activation in response to viral infection. Infection of CD6 deficient mice with a neurotropic murine coronavirus resulted in greater activation and expansion of CD4 T cells in the draining lymph nodes. Further analysis demonstrated that there was also preferential differentiation of CD4 T cells into T follicular helper cells, resulting in accelerated germinal center responses and emergence of high affinity virus specific antibodies. Given that CD6 conversely supports CD4 T cell activation in many autoimmune models, we probed potential mechanisms of CD6 mediated suppression of CD4 T cell activation during viral infection. Analysis of CD6 binding proteins revealed that infection induced upregulation of Ubash3a, a negative regulator of T cell receptor signaling, was hindered in CD6 deficient lymph nodes. Consistent with greater T cell activation and reduced UBASH3a activity, the T cell receptor signal strength was intensified in CD6 deficient CD4 T cells. These results reveal a novel immunoregulatory role for CD6 in limiting CD4 T cell activation and deterring CD4 T follicular helper cell differentiation, thereby attenuating antiviral humoral immunity.

Figure 1:. CD6 deficiency results in enhanced adaptive immune cell expansion and activation in the cervical lymph nodes after mCoV infection.

cLNs from WT and CD6 KO mice infected with 10,000 PFU of mCoV were taken at the indicated time points and analyzed by flow cytometry for the A) total number of CD4 T cells, B) percent of CD4 T cells that are CD44 high, C) total number of CD8 T cells, D) percent of CD8 T cells that are CD44 high, and E) total number of CD19 B cells. cLNs were also analyzed by F) qRT-PCR for transcripts of the mCoV nucleocapsid gene (N). Each data point represents a single mouse and a minimum of two individual experiments were pooled for each time point. Significance was determined using a Two-way ANOVA with a Bonferroni’s post-hoc test and denoted as * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.

Figure 2:. CD6 regulates CD4 T helper cell differentiation in cervical lymph nodes after mCoV infection.

cLNs from mCoV infected WT and CD6 KO mice were taken at the indicated time points and analyzed by flow cytometry for the proportion and number of CD4 T cells that are A) T_FH cells (CXCR5⁺, PD1⁺), B) BCL6⁺, and C) T-Bet⁺. cLNs s were also analyzed by qRT-PCR for D) Ifng and E) Foxp3 transcripts. Each data point represents a single mouse with experiments pooled from a minimum of two independent experiments for each time point. Flow cytometry plots and histograms are of a representative WT(black) and CD6 KO (red) mouse. FMO staining controls (gray) were included in the day 4PI histograms. Significance was determined using a Two-way ANOVA with a Bonferroni’s post-hoc test or an unpaired T-test and denoted as * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.

Figure 3:. Germinal centers are enhanced in the absence of CD6.

WT and CD6 KO mice were infected with mCoV. At day 4 and 7PI cLN CD19⁺ B cells were examined by flow cytometry for expression of A) IgD or B) GL7 (denoting germinal center B cells). Additionally, qRT-PCR was used to analyze expression of C) Aicda in cLNs. Day 14PI flash fozen cLNs were D) stained for B220 (red), GL7 (green), and CD3 (blue) to confirm GC formation within B cell zones. Serum collected at days 4, 7, and 14 PI was analyzed for mCoV-specific IgG E) avidity index₅₀ and F) titers by ELISA. “()” indicates the experimentally determined concentration of the serum dilution used for the analysis. Each data point represents a single mouse and a minimum of two experiments were pooled for each time point. Flow plots are of a representative mouse. Significance was determined by Two-way ANOVA using a Bonferroni’s post-hoc test and denoted as * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.

Figure 4:. CD6-mediated control of Ubahs3a expression corresponds with decreased TCR signal strength.

cLNs from mCoV infected WT and CD6 KO mice were isolated at the indicated time points. A) Ubash3a expression was quantified by qRT-PCR. The MFI of CD3 on B) CD44⁺ CD4 T cells and C) CD4 T_FH cells at day 4PI was used to monitor cell-surface CD3/TCR by flow cytometry. D) Irf4 transcription in the cLNs was quantified by qRT-PCR. The percent of E) CD44+ CD4 T cells and F) CD4 T_FH cells that are IRF4^high in the cLN as well as the G) MFI of IRF4 on CD44+ CD4 T_FH cells was analyzed by flow cytometry. Each data point represents a single mouse and representative histograms are of one WT mouse in grey and one CD6 KO mouse in red. B, C, and H are data representative of a minimum of 2 experiments. Significance was determined by Two-way ANOVA using a Bonferroni’s post-hoc test (A, D, E) or by unpaired T-Test (B-C and F-G) and denoted as * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.

Figure 5:. CD6 regulates peripheral, but not CNS, immune responses.

CNS tissues were isolated from WT and CD6 KO mice at the indicated time points. The total number of A) CD4 T cells and B) CD8 T cells were quantified by flow cytometry. mRNA transcripts of C) Ifng, D) Il17, and E) viral N gene were quantified in the indicated CNS tissues. The total number of F) CD19 B cells in the brain was quantified by flow cytometry. mCoV-specific IgG antibodies in the CNS tissues were G) semi-quantified and H) measured for affinity using an avidity index₅₀ assay. Each data point represents a single mouse. Significance was determined by A-B) Two-way ANOVA using a Bonferroni’s post-hoc test, C-D) unpaired T-Test and E-G) Two-way ANOVA using a Bonferroni’s post-hoc test and denoted as * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001.