A simple, clinically relevant therapeutic vaccine shows long-term protection in an aggressive, delayed-treatment B lymphoma model

Abstract

Despite initial remission after successful treatments, B lymphoma patients often encounter relapses and resistance causing high mortality. Thus, there is a need to develop therapies that prevent relapse by providing long-term protection and, ultimately, lead to functional cure. In this study, our goal was to develop a simple, clinically relevant, and easily translatable therapeutic vaccine that provides durable immune protection against aggressive B cell lymphoma and identify critical immune biomarkers that are predictive of long-term survival. In a delayed-treatment, aggressive, murine model of A20 B lymphoma that mimics human diffuse large B cell lymphoma, we show that therapeutic A20 lysate vaccine adjuvanted with an NKT cell agonist, α-galactosylceramide (α-GalCer), provides long-term immune protection against lethal tumor challenges and the antitumor immunity is primarily CD8 T cell dependent. Using experimental and computational methods, we demonstrate that the initial strength of germinal center reaction and the magnitude of class-switching into a Th1 type humoral response are the best predictors for the long-term immunity of B lymphoma lysate vaccine. Our results not only provide fundamentally insights for successful immunotherapy and long-term protection against B lymphomas, but also present a simple, therapeutic vaccine that can be translated easily due to the facile and inexpensive method of preparation.

Keywords: Cancer immunotherapy; Immunology; Lymphomas; Vaccines.

Figure 1. Therapeutic efficacy of various A20 lysate vaccine formulations.

(A) Time line for therapeutic immunization and tumor rechallenge studies in 1-day preestablished A20 B cell lymphoma model. (B) Kaplan-Meier curve (n = 6–10 per group) showing survival for A20 tumor lysate formulation–vaccinated mice (at 100 μg lysate protein/mouse, s.c.) after 2 lethal A20 tumor challenges (dose, 1 × 10⁵ cells/mouse, i.p., on day 1, rechallenged with 2 × 10⁵ cells/mouse, i.p., on day 80) in a therapeutic immunization setting. *P < 0.05, **P < 0.01, log-rank (Mantel-Cox) test. (C) Time line for therapeutic immunization in 7-day preestablished A20 B cell lymphoma model. (D) Kaplan-Meier survival curve for various A20 tumor lysate formulation–injected BALB/C mice (n = 10 per group) following lethal A20 tumor challenges with 2 × 10⁵ cells, i.p.

Figure 2. Therapeutic efficacy of soluble and microparticle-encapsulated A20 lysate vaccine formulations in 7-day preestablished A20 B-lymphoma model.

(A) Time line for therapeutic immunization and tumor rechallenge studies in 7-day preestablished A20 B cell lymphoma model. (B) Kaplan-Meier survival curve (n = 20 per group, pooled data from 2 independent experiments) for various A20 tumor lysate formulation–treated mice after 2 lethal A20 tumor challenges of 2 × 10⁵ cells/mouse. Naive BALB/C mice were first injected (i.p.) with a lethal dose of A20 cells (2 × 10⁵ cells/mouse), followed by 3 immunizations at days 8, 10, and 14 with various tumor lysate formulations (100 μg lysate protein/mouse, s.c.) and at day 17 with α-GalCer (2 μg/mouse, i.p.). A second challenge with 2 × 10⁵ A20 tumor cells/mouse, i.p., was done at day 56, and moue survival was followed up to 128 days. *P < 0.05, 1-way ANOVA with Tukey multiple comparison tests.

Figure 3. Cellular immune responses for various therapeutic A20 lysate vaccine formulations.

(A) Time line for mechanistic studies. BALB/C mice (n = 5–6) were first injected with 2 × 10⁵ A20 tumor cells on day 1 and then immunized with the formulations on day 8, 10, and 14. Thereafter, a single i.p. injection of 2 μg α-GalCer was done at day 17 only in α-GalCer treatment groups. On day 21, all the mice were sacrificed and spleens were collected and analyzed for CTL, NKT, and NK cell responses by flow cytometry. (B) CD4⁺ and CD8⁺ T cell–mediated CTL response by granzyme B assay. Effector CD4⁺ and CD8⁺ T cells were magnetically sorted from pooled splenocytes for each group. (C) Box plot showing NKT cell percentage in splenocytes (n = 6). (D) Box plot showing NK cell percentage in splenocytes (n = 6). (E and F) IFN-γ (Th1 cytokine) and IL4 (Th2 cytokine) secreted by splenocytes following ex vivo restimulation with A20 lysate antigens (n = 6). Splenocytes (10⁶/well of 96-well plate) were restimulated with A20 lysate (100 μg/well) for 72 hours in vitro, and the culture media were analyzed for various Th1/Th2 cytokines using a multiplex luminex kit. (G) Box plot showing percentage of granulocytic MDSCs in CD11b⁺ splenocytes (n = 6). (H) Treg percentage in spleen CD4⁺ T cells (n = 5–6). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey multiple comparison tests.

Figure 4. Humoral responses for various therapeutic A20 lysate vaccine formulations.

(A) Confocal images of lymph node showing germinal center formation. Scale bar: 200 μm. (B) Box plot showing B220⁺GL7⁺ germinal center–forming B cells (n = 6). (C) Total B220⁺ B cell percentage in lymph nodes (n = 6). (D) Anti-A20 tumor lysate IgG1 (n = 6) and (E) IgG2a (n = 4–6) antibody levels in serum at day 21 of the mechanistic studies (time line shown in Figure 3A). (F) Anti-A20 tumor lysate IgG1 (n = 3–6; MP TL plus GalCer, n = 2) and (G) IgG2a antibody levels at day 128 (n = 3–6; MP TL plus GalCer, n = 2). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA with Tukey multiple comparison tests.

Figure 5. Therapeutic soluble tumor lysate plus α-GalCer vaccine shows CD8 T cell–dependent antitumor immunity in 7-day preestablished A20 B lymphoma model.

(A) Time line showing therapeutic immunization and immune cell depletion/blocking antibody injection schedule. (B) Kaplan-Meier curve (n = 19–20 per group, pooled data from 2 independent experiments) showing survival of tumor-challenged mice for various antibody-treated (anti-mouse CD4, CD8, and CD1d antibodies) soluble tumor lysate plus α-GalCer vaccine groups. *P < 0.05, **P < 0.01, log-rank (Mantel-Cox) test.

Figure 6. Linear discriminant analysis of immune responses for various therapeutic lysate vaccine groups.

Canonical plots showing (A) NKT and NK cells responses, (B) Th1 and Th2 cytokine response, (C) humoral response, and (D) regulatory cell responses for various A20 tumor lysate vaccine groups.