"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Abstract

Background: Cooperation of CD4+ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4+ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal findings: Here we have developed a protocol to selectively reduce the CD4+ T cell response against viral CD4+ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4+ T cells without affecting CD4+ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4+ T cell help, tolerisation did not impair CD8+ T cell responses.

Conclusions: This result reveals a novel "negative vaccination" strategy where specific CD4+ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.

Figure 1. Peptide tolerisation leads to functional impairment of virus-specific CD4⁺ T cells.

A–G: 5×10⁴ splenocytes from mice transgenic for a T cell receptor recognizing the LCMV helper epitope GP61 (LCMV-glycoprotein_61-80/I-A^b-specific TCR, SMARTA mice) and expressing the T cell marker Thy1.1 were adoptively transferred into C57BL/6 mice on day -10. One group of mice was treated with 100 µg GP61 dissolved in IFA (squares), while control mice were treated with IFA alone (circles) at days -9, -6, -3. At day 0 mice were infected with 200pfu LCMV-WE or left untreated. Seven days after infection mice were analyzed for CD4⁺ T cell function. (A) Frequencies of GP61-specific CD4⁺ T cells (Thy1.1⁺ T cells) were analysed in spleen and blood. (B) For further phenotyping of thy1.1⁺ CD4⁺ cells, cells were gated as shown in the gating tree. (C) Cells were re-stimulated with GP61 in vitro and after six hours Thy1.1⁺ T cells were analysed for intracellular expression of IL-4, IL-10, IFN-γ and TNF-α by FACS analysis (one of four representative dot blots is shown. (D) Bar charts show data analyzed in C (n = 4,*p<0.001). (E) Cells were re-stimulated with PMA/Ionomycin in vitro and after six hours Thy1.1⁺ T cells were analyzed for intracellular expression of IL-4, IL-10, IFN-γ and TNF-α by FACS analysis (n = 4, *p<0.001). (F) GP61-specific Thy1.1⁺ CD4⁺ T cells and CD4⁺ T cells from untreated C57BL/6 mice were analysed for the activation markers CD69, IL-7Rα, CD62L, CD44 and CXCR3 (marker is set on the activated phenotype). (G) Statistically analysis from the data derived in F (n = 4, *p<0.05). H: 5×10⁶ splenocytes from mice transgenic for a T cell receptor recognizing the LCMV helper epitope GP61 (LCMV-glycoprotein_61-80/I-A^b-specific TCR, SMARTA mice) and expressing the T cell marker Thy1.1 were transferred into a total of six C57BL/6 mice on day -11. Three of those mice were treated with 100 µg GP61 dissolved in IFA (GP61-IFA-pretreated), while the other three mice were treated with IFA alone (IFA-pretreated) at days -10, -7, -4. In addition six C57BL/6 mice were treated with 100 µg GP61 dissolved in IFA (GP61-IFA-treated) and six control mice were treated with IFA alone (IFA-treated) at days -10, -7, -4. At day -1 splenocytes from each of the three GP61-IFA-pretreated mice were transferred into one GP61-IFA-treated, one IFA-treated and one untreated C57BL/6 mouse (one untreated C57BL/6 mouse died during transfer). In parallel splenocytes from each IFA-pretreated mouse was transferred into one GP61-IFA-treated, one IFA-treated and one untreated C57BL/6 mouse (transfer scheme Figure S2). Number of transferred splenocytes was adapted to the frequencies so that all mice received the same numbers of Thy1.1⁺ CD4⁺ T cells at day -1 (data not shown). On day 0 mice were infected with 200pfu LCMV-WE. Six days after infection mice were analyzed for Thy1.1⁺ CD4⁺ T cells by FACS analysis.

Figure 2. Tolerisation of CD4⁺ T cells improves LCMV-specific antibody response.

A: C57BL/6 mice were tolerised with either 100 µg GP61-80-IFA, 100 µg NP309-328-IFA, or both peptides in IFA on days -109, -106, -103. On day 0, mice were infected with 200pfu LCMV-WE and splenocytes were analysed for intracellular IFN-γ expression after re-stimulation with different T cell epitopes in vitro 12 days later. The original dot plots are shown (n = 5). B: C57BL/6 mice were treated with 100 µg GP61-80-IFA or IFA on days -9, -6 and -3. On day 0, mice were infected with 200pfu LCMV-WE and splenocytes were analysed for intracellular IFN-γ after re-stimulation with different T cell epitopes in vitro 12 days later (n = 3, p<0.001). C+D: C57BL/6 mice were tolerized with either 100 µg GP61-80-IFA, 100 µg NP309-328-IFA or both peptides in IFA on days -9, -6, -3. On day 0, mice were infected with 200pfu LCMV-WE and LCMV-nucleoprotein (NP) specific antibodies were measured by ELISA at the indicated time points (n = 5, * p<0.05, C). LCMV-glycoprotein (GP) specific antibodies were measured by ELISA at the indicated time points (n = 5–7, *p<0.001, D). E: BALB/c mice were peptide-tolerised with 100 µg GP61-80-IFA or IFA alone on day -9, -6 and -3. On day 0 mice were infected with 200pfu of LCMV-WE and LCMV-GP specific IgG antibodies were measured in the serum at the indicated time points (n = 8, ns).

Figure 3. Peptide tolerisation improves protective antibody responses independently and without impairment of CD8⁺ T cells.

A: C57BL/6 mice were treated with GP61-IFA and NP309-IFA, while control mice were treated with IFA alone at days -9, -6, -3. At day 0 mice were infected with 200pfu LCMV-WE, and GP-specific (tet-gp33⁺) CD8⁺ T cells were analyzed in the blood by FACS analysis (n = 4, ns). B+C: 5×10⁴ splenocytes from mice transgenic for a T cell receptor recognizing the LCMV helper epitope GP61 (LCMV-glycoprotein_61-80/I-A^b-specific TCR, SMARTA mice) and for the T cell marker Thy1.1 were transferred into C57BL/6 mice on day -10. One group of mice was treated with 100 µg GP61 dissolved in IFA, while control mice were treated with IFA alone at days -9, -6, -3. At day 0 mice were infected with 200pfu LCMV-WE or left untreated. GP33 specific CD8⁺ T cells were analyzed for frequencies (Figure S3) and for the expression of the activation markers CD25, CD69, CD44, CD62L, IL-7Rα, CXCR3 and PD-1 (1 of 4 histogram plots is shown, grey area represents staining of naïve CD8⁺ T cells, B). CD8⁺ T cells were analyzed for expression of the intracellular effector molecules IFN-γ and Granzyme B after restimulation (1 of 4 dot plots is shown, C). D–F: cd8^−/− mice were treated with GP61/NP309-IFA or with IFA alone on days -9, -6 and -3. On day 0 mice were infected with 200pfu LCMV-WE and LCMV-GP1 specific IgG antibodies were detected by ELISA (n = 3–4, *p = 0.03, D) and by neutralisation assay (n = 7–8, *p = 0.04, E) at the indicated time points. Virus blood titers were measured with plaque assay on day 35 after LCMV infection as indicated (n = 7–8, p = 0.0059, F).

Figure 4. LCMV-GP specific peptide tolerisation does not affect antibody production against third party pathogen.

C57BL/6 were tolerised with 100 µg GP61-IFA at day -9, -6 and -3. At day 0, mice were infected with 2×10⁶ PFU of vesicular stomatitis virus (VSV). Neutralizing antibodies against VSV-glycoprotein were measured by VSV neutralisation assay; total neutralizing Ig (A) and neutralizing IgG (B). VSV-glycoprotein binding IgG was measured by ELISA (C).