Immunological memory transferred with CD4 T cells specific for tuberculosis antigens Ag85B-TB10.4: persisting antigen enhances protection

Abstract

Background: High levels of death and morbidity worldwide caused by tuberculosis has stimulated efforts to develop a new vaccine to replace BCG. A number of Mycobacterium tuberculosis (Mtb)-specific antigens have been synthesised as recombinant subunit vaccines for clinical evaluation. Recently a fusion protein of TB antigen Ag85B combined with a second immunodominant TB antigen TB10.4 was emulsified with a novel non-phospholipid-based liposomal adjuvant to produce a new subunit vaccine, investigated here. Currently, there is no consensus as to whether or not long-term T cell memory depends on a source of persisting antigen. To explore this and questions regarding lifespan, phenotype and cytokine patterns of CD4 memory T cells, we developed an animal model in which vaccine-induced CD4 memory T cells could transfer immunity to irradiated recipients.

Methodology/principal findings: The transfer of protective immunity using Ag85B-TB10.4-specific, CD45RB(low) CD62L(low) CD4 T cells was assessed in sub-lethally irradiated recipients following challenge with live BCG, used here as a surrogate for virulent Mtb. Donor T cells also carried an allotype marker allowing us to monitor numbers of antigen-specific, cytokine-producing CD4 T cells in recipients. The results showed that both Ag85B-TB10.4 and BCG vaccination induced immunity that could be transferred with a single injection of 3x10(6) CD4 T cells. Ten times fewer numbers of CD4 T cells (0.3x10(6)) from donors immunised with Ag85B-TB10.4 vaccine alone, transferred equivalent protection. CD4 T cells from donors primed by BCG and boosted with the vaccine similarly transferred protective immunity. When BCG challenge was delayed for 1 or 2 months after transfer (a test of memory T cell survival) recipients remained protected. Importantly, recipients that contained persisting antigen, either live BCG or inert vaccine, showed significantly higher levels of protection (p<0.01). Overall the numbers of IFN-gamma-producing CD4 T cells were poorly correlated with levels of protection.

Conclusions/significance: The Ag85B-TB10.4 vaccine, with or without BCG-priming, generated TB-specific CD4 T cells that transferred protective immunity in mice challenged with BCG. The level of protection was enhanced in recipients containing a residual source of specific antigen that could be either viable or inert.

Figure 1. The development of an adoptive transfer model to evaluate Mtb-specific CD4 T cells.

(A) Donor mice (B6.SJL, CD45.1) injected with BCG 2 mo before were boosted with Ag85B-TB10.4 and killed 7 days later. Spleen and lymph nodes were removed, depleted of B cells, CD8 T cells and macrophages. A representative profile of the resulting CD4 T cell population stained for CD45RB is shown (B). CD4 T cells were also depleted of CD45RB^high cells and injected into recipients on day 0 (d 0). A representative profile of CD45RB^low CD4 T cells used for adoptive transfer is shown (C). One day after transfer, irradiated recipients (X-rad) were challenged i.v. with live BCG, killed 3 weeks later (d 22) and spleens tested for a reduction in BCG numbers (Protection assay, CFUs/spleen). An aliquot of spleen cells was cultured overnight in the presence of Ag85B-TB10.4 antigen and stained for ICC IFN-γ. Donor-derived CD4 T cells (CD4⁺ CD45.1⁺) were gated in R1 (D) and analysed for IFN-γ positive cells (E). The donor-derived CD45.1⁺ CD4⁻ cells in profile D (upper left quadrant) were predominantly contaminating B cells. Numbers in the representative profiles are the percentage of events in the quadrant.

Figure 2. Kinetics of the antigen-specific CD4 T cell response in BCG-immunised mice after a boost with Ag85B-TB10.4.

Mice were killed at selected days following antigen injection and spleen cells analysed for ICC IFN-γ. Results are the net percentage of IFN-γ⁺ CD4 T cells stimulated in the presence of Ag85B-TB10.4 after subtraction of the response without antigen. The results were pooled from 8 experiments. Each point represents the mean+SD of 3 or 4 mice, except day 3, n = 2.

Figure 3. CD4 T cells specific for BCG or for Ag85B-TB10.4 transfer protective immunity.

(A) CD45RB^low CD4 T cells were purified from donors primed and boosted with BCG (Pr/Boost) and injected (3×10⁶ or 0.3×10⁶) into irradiated recipients (B, C). Control recipients received 3×10⁶ unprimed (Naïve) CD4 T cells or no cells (None). (D) CD45RB^low CD4 T cells were purified from donors primed with Ag85B-TB10.4 and boosted with Ag85B-TB10.4 and 3×10⁶ or 0.3×10⁶ T cells injected into irradiated recipients. Control recipients received 3×10⁶ unprimed (Naïve) CD4 T cells or no cells (None). All recipients in experiments A and D were challenged i.v. with BCG the day after transfer, killed 3 weeks later and assayed for BCG CFU/spleen (B, E). Each point represents a single recipient. Donor-derived splenic CD4 T cells stimulated in vitro with Ag85B-TB10.4 were stained for intracellular cytokine (ICC) IFN-γ (C, F). The results were from single experiments. Horizontal bars or histograms + SD are means of 4 recipients/group. * p<0.05; ** p<0.01.

Figure 4. Protective immunity persists for at least 8 weeks following transfer of prime/boost CD4 T cells.

(A) CD45RB^low CD4 T cells (3×10⁶) from donors primed with BCG and boosted with Ag85B-TB10.4 (Pr/Boost) or 3×10⁶ CD4 T cells from unprimed donors (Naïve) or no cells (None) were transferred to irradiated recipients and challenged with BCG immediately (d 1) or 8 weeks later (d 56) (B, C). Three weeks after challenge spleen cells were assayed for BCG CFUs (B) and ICC IFN-γ⁺ donor CD4 T cells (C). The results are from one experiment. Each point represents a single recipient. Horizontal bars or histograms + SD are means of 4 recipients/group. ** p<0.01.

Figure 5. Persisting antigen enhances protection.

(A) To establish a potential source of residual antigen (Ag) mice were injected with BCG or Ag85B-TB10.4, 4 weeks (d -28) before irradiation and cell transfer. Control recipients received no source of residual Ag (None) and were irradiated 1 day before cell transfer. CD45RB^low CD4 T cells (3×10⁶) from BCG primed/Ag85B-TB10.4 boosted donors (Pr/Boost, solid symbols) or unprimed CD4 T cells (Naïve, open symbols) were transferred into recipients after irradiation on d 0. All recipients were challenged with BCG 4 weeks later (d 28). (B) Three weeks after challenge spleen cells were assayed for BCG CFUs. Each point represents a single recipient. Horizontal bars are means of 4 recipients/group. ** p<0.01.