Phenotype and function of protective, CD4-independent CD8 T cell memory

Abstract

While the need for CD4 T cells in the generation of CD8 T cell memory has been well documented, the mechanism underlying their requirement remains unknown. Here, we detail an immunization method capable of generating CD8 memory T cells that are indifferent to CD4 T cell help. Using a subunit vaccination that combines polyIC and an agonistic CD40 antibody, we program protective CD4-independent CD8 T cell memory. When cells generated by combined polyIC/CD40 immunization are compared to cells produced following a CD4-dependent vaccination, Listeria monocytogenes, they display dramatic differences, both phenotypically and functionally. The memory cells generated in a CD4-deficient host by polyIC/CD40 immunization provide protection against secondary infectious challenge, whereas cells generated by LM immunization in the same environment do not. Interestingly, combined polyIC/CD40 immunization generates long-term memory cells with low Blimp-1 and elevated Eomes expression despite high expression of Blimp-1 during the primary response. The potency of combined polyIC/CD40 to elicit CD8+ T cell memory in the absence of CD4 T cells suggests that it could be considered as a vaccine adjuvant in clinical situations where CD4 responses/numbers are compromised.

Fig. 1

Combined polyIC/αCD40 immunization generates CD8 T cell memory in the absence of CD4 T cells. Wild type (WT) and class II KO (ko) mice were immunized with combined PolyIC/αCD40/ova as described in the “Materials and methods.” Sixty days later, the mice were challenged with LM-ova. Mice were bled at the indicated time points and the percent of CD8+ tetramer+ cells was determined. a Plots shown are gated on CD8+ cells. “Primary” indicates day 8 following immunization, “Memory” indicates day 47 post-immunization, and “Secondary” indicates 5 days following the day 60 LM challenge. b Percent of CD8+ tetramer+ cells over time in the peripheral blood. c Fold expansion of tetramer+ CD8 memory cells in the blood was calculated by dividing the percent tetramer+ cells in the peripheral blood post-LM-ova boost by the percent tetramer+ cells in the peripheral blood pre-boost. These data are representative of at least 3 experiments performed. Data points are from 3 to 4 mice per group. Error bars represent standard deviation

Fig. 2

In contrast to LM challenge, combined polyIC/αCD40 immunization generates CD8 T cell memory in the absence of CD4 T cell help. a WT and CII KO mice were immunized with either PolyIC/αCD40/ova or LM ova as in Fig. 1. Mice were bled at multiple time points and the percent of CD8+ tetramer+ T cells determined. Representative flow cytometry results are shown (b). Plots are gated on all live, B220−, CD8+ cells. “Primary” represents day 8, and “Memory” day 29, post-immunization. c The percent of CD8+ tetramer+ cells in the spleen 5 days after challenge with LM-ova and the % max of the WT response. These data are representative of at least 4 experiments performed. Data points are from 3 to 4 mice per group. Error bars represent standard deviation

Fig. 3

Combined polyIC/αCD40 immunization mediates CD4-independent protective memory against pathogen challenge. WT and class II KO mice were immunized with either PolyIC/αCD40/B8R or LM-B8R as described in the Materials and methods. Day 60 after immunization, mice were challenged IV with VV-WR. Five days after challenge mice, viral titers in the ovaries were determined by plaque assay as described in the “Materials and methods.” Data are expressed as plaque-forming units (PFU). These data are representative of 2 experiments performed. Data points are from 3 to 4 mice per group. Error bars represent standard deviation

Fig. 4

Combined polyIC/αCD40 immunization generates cells with central memory phenotype and function. WT (wt) and class II KO (ko) mice were immunized with PolyIC/αCD40/ova or LM-ova. Representative flow cytometry results are shown. a Plots are gated on CD8+ tetramer+ cells. “Primary” represents day 8, and “Memory” day 29, after immunization. b Graphical representation of the percent of CD8+ tetramer+ cells expressing either CD27 or KLRG1. c Cells were surface stained, fixed, and then permeabilized to allow for intracellular cytokine staining. Representative flow cytometry data are shown. Plots shown are gated on CD8+ cells. Primary is day 8 and memory is day 30 following immunization. d The percent of IFN-γ producing cells that are also producing IL-2 primary and the memory time points. These data are representative of at least 3 experiments performed. Data points are from 3 to 4 mice per group. Error bars represent standard deviation

Fig. 5

Memory CD8+ T cells elicited by combined polyIC/αCD40 immunization are superior in mediating protective immunity on a per cell basis as compared to memory cells elicited by LM; a 2.5 × 10⁵ negatively selected CD8+ Vβ5 cells were transferred into WT and class II KO recipients. The following day mice were immunized with either PolyIC/αCD40/ova or LM-ova. After 44 days, spleens were harvested, and following CD8 T cell negative selection, 7.5 × 10⁵ antigen-specific cells were transferred into WT or class II KO recipients. Twenty hours after transfer, mice were challenged with LM-ova, and four and a half days later, livers were harvested to determine CFU. *p value <0.05 for KO LM sample when compared to other experimental groups and naïve control, unpaired t test. b CD8 memory cells were harvested from WT and class II KO mice immunized with either PolyIC/αCD40/ova or LM-ova 48 days after immunization. Following CD8 negative selection, 3.5 × 10⁵ CD8+ tetramer+ cells were transferred into CD45.1+ recipients. The following day mice were challenged with LM-ova. Four and a half days after challenge, the expansion of transferred (CD45.2+) cells determined. Total number of transferred antigen-specific cells in the spleen following challenge. *p value <0.05 unpaired t test

Fig. 6

Blimp-1 expression is reduced, and Eomes expression increased, in memory cells derived from polyIC/αCD40 as compared to LM immunization. CD4 depleted or non-depleted Blimp-1-YFP mice were immunized with PolyIC/CD40/ova or LM-ova as described in Fig. 1. a Representative flow cytometry plots gated on CD8+ tetramer+ cells in the peripheral blood. Primary time point is 7 days, and memory 35 days, after immunization. b Blimp-1 YFP geometric mean fluorescence intensity (gMFI) for CD8+ tetramer+ cells over time in the peripheral blood of the cells from a. c WT and CII^−/− mice were immunized as in A. Thirty days later, peripheral blood was stained with tetramer, and gMFI of Eomes expression in CD8+ tetramer+ cells was determined. These data are representative of at least 2 experiments performed. Data points are from 3 to 4 mice per group. Error bars represent standard deviation