Fig 2: High-dose stereotactic radiation with TIGIT blockade improved primary and secondary antitumor efficacy. (A) Timeline and experimental design of the abscopal model. (B) Bilateral 344SQ-P tumors were established in 129Sv/Ev mice. Primary tumors were irradiated with H-XRT, while secondary tumors were left untreated. Systemic immunotherapy with a-TIGIT or a-TIGIT + a-PD1 was administered, as shown. Survival was monitored over 40 d observation period and graphed using the Kaplan–Meier method. (C) Primary and (D) secondary tumor growth curves are plotted over time, and different experimental groups were compared using two-way ANOVA. The experiment was repeated twice, and the data were pooled. p = 0.05 was considered significant. * p = 0.05, ** p = 0.01, *** p = 0.001, ns = not significant.

Fig 3: Ng(-)pIL-12 adjuvant triggered the terminally differentiated effector memory responses against HBV infection. C57 BL/6J mice were injected intramuscularly with three doses of PBS, 2 µg HBsAg alone, 2 µg HBsAg combined with 45 µg Ng(-)pIL-12 at two-week intervals, separately, then these immunised mice were challenged with 8 µg pAAV/HBV 1.2 on day 56 after the initiation of immunisation. (A) The percentage of SLECs among HBV-specific CD11ahi CD8alo cells was detected on day 35 after the initiation of immunisation. (B) The percentage of splenic SLECs among HBV-specific CD11ahi CD8alo cells on day 5 after HBV challenge. The expression of CD107a (C) and TIGIT (D) on splenic SLECs on day 5 after HBV challenge. HBsAg, HBsAg alone; Ng(-)pIL-12, 40 µg Ng(-) containing 5 µg pIL-12. All data are expressed as the mean ± SEM (n = 4) from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: cHBV-vaccine induced long-lasting CXCR5+ CD8+ T cell response during HBV re-challenge. HBV-carrier mice were immunized subcutaneously with PBS (Untr) and 2 µg rHBVvac combined with 10 µg CpG M362 (cHBV-vaccine) weekly for 3 weeks, separately. Then, these treated mice were re-challenged with hydrodynamic injection of 8 µg pAAV/HBV1.2 plasmid on day 59 after the first vaccination. (A) Abundance of CD11ahi CD8alo cells among CD8+ T cells from liver and spleen on day 7 after HBV re-challenge. (B) LAG-3, CTLA-4, TIGIT, PD-1 and Tim-3 expression on HBV-specific CD11ahi CD8alo cells in liver (left) and spleen (right) on day 7 after HBV re-challenge. (C) Representative flow-cytometry scatter plot of splenic CXCR5+ CD11ahi CD8alo T cells, showing the frequency of CXCR5+ CD11ahi CD8alo T cells on day 7 after HBV re-challenge. (D) Representative flow-cytometry scatter plot of PD-1, TIGIT, Tim-3, and LAG-3 expression on HBV-specific CXCR5+ CD11ahi CD8alo T cells on day 7 after HBV re-challenge. *p < 0.05, **p < 0.01, ***p < 0.001 versus untreated mice.

Fig 5: cHBV-vaccine alleviated immunosuppression and restored exhausted HBV-specific CXCR5+ CD8+ T cells. HBV-carrier mice were immunized subcutaneously with PBS (Untr) and 2 µg rHBVvac combined with 10 µg CpG M362 (cHBV-vaccine) weekly for 3 weeks, separately. (A) The expression of PD-L1 on hepatocytes on day 21 post-immunization. (B) The proportion of splenic CD4+ CD25+ Foxp3+ on day 21 post-immunization. (C) Relative expression of TGF-ß1 mRNA in liver tissue and serum TGF-ß1 levels on day 21 post-immunization. (D) LAG-3, CTLA-4, TIGIT, PD-1 and Tim-3 expression on HBV-specific CD11ahi CD8alo cells in liver (left) and spleen (right) on day 21 post-immunization. (E) Ki-67 expression on HBV-specific CD11ahi CD8alo cells in liver on day 21 post-immunization. (F-G) Representative flow-cytometry scatter plot of splenic CXCR5+ CD11ahi CD8alo T cells. The frequency (F) and abundance (G) of CXCR5+ CD11ahi CD8alo T cells on day 21 post-immunization. (H) Representative flow-cytometry scatter plot of PD-1, TIGIT, Tim-3, and LAG-3 expression on HBV-specific CXCR5+ CD11ahi CD8alo T cells on day 21 post-immunization. All data are expressed as mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 versus untreated mice.