Short-term antigen presentation and single clonal burst limit the magnitude of the CD8(+) T cell responses to malaria liver stages

Abstract

Malaria sporozoites induce swift activation of antigen-specific CD8(+) T cells that inhibit the intracellular development of liver-stage parasites. The length of time of functional in vivo antigen presentation, estimated by monitoring the activation of antigen-specific CD8(+) T cells, is of short duration, with maximum T cell activation occurring within the first 8 h after immunization and lasting approximately 48 h. Although the magnitude of the CD8(+) T cell response closely correlates with the number of parasites used for immunization, increasing the time of antigen presentation by daily immunizations does not enhance the magnitude of this response. Thus, once a primary clonal burst is established, the CD8(+) T cell response becomes refractory or unresponsive to further antigenic stimulation. These findings strongly suggest that the most efficient strategy for the induction of primary CD8(+) T cell responses is the delivery of a maximal amount of antigen in a single dose, thereby ensuring a clonal burst that involves the largest number of precursors to become memory cells.

Figure 1

Persistence of the CD8+ T cell response after a single immunization with sporozoites. Normal mice received transgenic (Tg) CD8+ T cells; 24 h later, they were immunized (⧫) i.v. with 5 × 10⁴ attenuated sporozoites or were not immunized (■). The frequencies of epitope-specific CD8+ T cells in the spleen (Upper) and the liver (Lower) were determined by ELISPOT. Results represent one of two similar experiments expressed as mean values + SD.

Figure 2

Short-term antigen presentation defines the magnitude of the CD8+ T cell response. (A) Normal mice were immunized i.v. with 3 × 10⁴ attenuated sporozoites, and at different times postimmunization (8, 24, 48, 96 h), they received naïve Tg CD8+ T cells. The number and activation status of the Tg CD8+ T cells in spleens (Left) and livers (Right) for each experimental group were assessed by ELISPOT 4 days after transfer of Tg cells. As controls for full activation, mice received Tg cells at the time of immunization (0 h) and were analyzed 4 days later. Mice that were immunized but did not receive Tg cells (no Tg) served as controls for the endogenous CD8+ T cell response. (B) Spleen cells from A were stained with anti-CD8 antibodies and SYVPSAEQI tetramers. Plots were gated on lymphocytes, and the number in the upper right corner represents the frequency of CD8+ tetramer+ cells in the total CD8+ population. (C) Similar to A except that mice received day 8 activated/memory Tg CD8+ T cells. Mice that received activated/memory Tg cells but were not immunized (no spz) served as controls. The activation of the Tg CD8+ T cells in the spleen was measured by ELISPOT 4 days after transfer. (D) Similar to A except that RAG2^−/− mice were used. Immunized mice received both naïve Tg CD8+ T cells and naïve spleen lymphocytes at indicated times. The activation of the Tg CD8+ T cells in the spleen was measured by ELISPOT 4 days after transfer. Results in A–D represent one of two to three similar experiments.

Figure 3

Parasite viability or route of immunization does not modify the parameters of antigen presentation and T cell activation. Normal mice were immunized with 3 × 10⁴ attenuated sporozoites (A), 3 × 10⁴ live sporozoites (B), or four bites of irradiated infected mosquitoes (C). Naïve Tg CD8+ T cells were transferred at indicated times after immunization, and the activation of the Tg CD8+ T cells in the spleen was measured by ELISPOT 4 days after transfer. Mice that did not receive Tg CD8+ T cells (no Tg) served as control for the endogenous CD8+ T cell response. Results in A–C represent one of two similar experiments and are expressed as the relative number of CD8+ T cells compared with 0 h (100%) to facilitate comparison.

Figure 4

Primaquine treatment and development of the CD8+ T cell response. Normal mice received Tg CD8+ T cells and were immunized i.v. with 3 × 10⁴ attenuated sporozoites. At indicated times before and after immunization, mice were s.c. treated with 60 mg/kg primaquine. The frequencies of epitope-specific CD8+ T cells in the spleens were measured by ELISPOT 16 days after immunization. Results represent one of two similar experiments expressed as mean values + SD. P value_{(untreated vs. treated, −2,8)} <0.05 (t test).

Figure 5

Dependence of the CD8+ T cell response on priming dose and not on the length of time of antigen presentation. (A) Normal mice received and were immunized i.v. with indicated amounts of attenuated sporozoites. The activation and expansion of the Tg CD8+ T cells in spleens (⧫) and livers (■) were measured by ELISPOT 4 days after immunization. (B) Normal mice received Tg CD8+ T cells and were immunized with the indicated doses of attenuated sporozoites. The activation of the Tg CD8+ T cells in the spleens and the livers were measured by ELISPOT 14 days after the primary immunization (Upper). Spleen cells were stained with anti-CD8 antibodies and SYVPSAEQI tetramers, and plots were gated on lymphocytes; the number in the upper right corner represents the frequency of CD8+ tetramer+ cells in the total CD8+ population (Lower). Results in A and B represent one of two to three similar experiments (n = 3 mice/group). P values_{(100K vs. 4 × 25K for both spleen and liver)} <0.05; P values_{(25K vs. 4 × 25K for both spleen and liver)} = not significant.

Figure 6

A state of refractoriness limits the magnitude of the CD8+ T cell response despite repeated exposure to bites of infected mosquitoes. Normal mice were immunized every 48 h with two bites of irradiated infected mosquitoes. The frequencies of epitope-specific CD8+ T cells in the spleens were measured by ELISPOT 16 days after the first immunization. Results represent one of three similar experiments expressed as mean + SD.