Fig 1: Genetic ablation of HCK in myeloid cells promotes CD8+ T cell recruitment and activation.(A) Flow cytometry quantification of CD4+ T cells, CD8+ T cells, and NK cells from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. Each symbol represents an individual mouse. (B) Representative immunohistochemical staining for CD8+ T cells in subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. Scale bar, 100 µm. Quantification of staining per square micrometer tumor area is also shown. Each symbol represents an individual mouse. (C) qPCR analysis on FACS-purified tumor-associated CD45+TCRß+CD8+ T cells for markers associated with immune cell activation. Cells were isolated from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. n = 4 mice per group. (D and E) Subcutaneous MC38 tumor volume of WT and HckKO hosts following treatment with aPD1 and/or depleting antibodies against CD8 or NK1.1. Depletion antibodies were administered once every 3 days before subcutaneous MC38 tumor cell injection (total of three treatments) and continued until the experimental end point when tumors reached =600 mm3. n = 10 mice per group. Data represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.
Fig 2: Expansion of T cells in the deep cervical lymph nodes occurs primarily during the chronic stage of infection and tracks with parasite burden in the brain.(a-b) DQ-OVA was injected into the CSF of chronically infected mice by i.c.m. injection and fluorescent emission of proteolytically cleaved DQ-OVA was measured in CD11chiMHC IIhi antigen-presenting cells of the deep cervical lymph nodes (DCLNs) and inguinal lymph nodes (ILNs) by flow cytometry. (a) Representative contour plots of DQ-OVA+ antigen-presenting cells in the DCLNs or ILNs at 6 wpi. CD11chiMHC IIhi cells were pre-gated on singlets/live/TCRß-/NK1.1-/CD19-. (b) Quantification of frequency of DQ-OVA+ antigen-presenting cells in the DCLNs or ILNs at 6 wpi. Data are compiled from three experiments (n = 11 mice per group) and are represented as mean values ± s.e.m. Statistical significance was measured using randomized block ANOVA (two-way), with p<0.001 (***). (c–d) C57BL/6 mice were infected i.p. with 10 cysts of the ME49 strain of T. gondii and total number of activated CD4+ T cells (c) or activated CD8+ T cells (d) in the DCLNs or ILNs was quantified at multiple time points over the course of acute and chronic infection (red dots). The steady-state number of activated CD4+ and CD8+ T cells in the different lymph node compartments was measured in naïve mice at corresponding time points (black dots). Activated T cells displayed a CD44hiCD62Llo phenotype. Data are compiled from three experiments and are represented as mean values ± s.e.m. (n = 6-14 mice per group per timepoint). (e–f) C57BL/6 mice were infected i.p. with 1,000 tachyzoites of the Pru-OVA strain of T. gondii. (e) Total number of SIINFEKL-specific CD8+ T cells in the DCLNs or ILNs was quantified at multiple time points over the course of acute and chronic infection using tetramer reagent. Data are compiled from two experiments and are represented as mean values ± s.e.m. (n = 7 mice per timepoint). (f) Quantification of T. gondii gDNA in brain (red), dural meninges (orange), deep cervical lymph nodes (black), inguinal lymph nodes (blue), subcutaneous adipose tissue isolated from the flank (green), and quadriceps femoris skeletal muscle tissue (gray) by real-time PCR. Data are compiled from two experiments and are represented as mean values ± s.e.m. (n = 5-6 mice per tissue per timepoint). For (c–e), statistical significance of differences across time points in infected mice was measured using one-way ANOVA with post-hoc Tukey multiple comparison testing. p<0.05 (*) and p<0.01 (**). For (f) statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison test to assess differences across timepoints. Statistically significant differences are indicated, with p<0.05 (*) and p<0.001 (***). Figure 2—source data 1.CD4+ T cell activation in the DCLNs and ILNs over the course T. gondii infection. Figure 2—source data 2.CD8+ T cell activation in the DCLNs and ILNs over the course of T. gondii infection. Figure 2—source data 3.SIINFEKL-specific CD8+ T cell responses in the DCLNs and ILNs over the course of T. gondii infection. Figure 2—source data 4.Parasite burden in the brain and peripheral tissues over the course of T. gondii infection.
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