Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans

Abstract

Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.

Figure 1

Kinetics of direct ex vivo virus-specific CTL activity during primary LCMV infection and after rechallenge. (A) Splenocytes from LCMV-infected mice were analyzed for direct ex vivo CTL activity (D4 RC = day 4 after LCMV rechallenge). Data shown are for an E/T ratio of 200:1. Lysis of noninfected target cells at the same E/T ratio was <5% (data not shown). The kinetics of viral clearance during acute LCMV infection are indicated by the shaded graph. The virus levels in the spleen peak between days 1 and 3 (at 10⁷ pfu per spleen) and then drop precipitously between days 5 and 8 to <10² pfu per spleen. (B) The direct ex vivo CTL activity at different time points after infection after normalizing the number of antigen-specific CD8 T cells present in each sample. There were 10⁴ NP118-specific CD8 T cells present in each sample, giving an E/T ratio of 1:1 based on virus-specific T cells. The data shown are representative of three experiments.

Figure 2

Kinetic analysis of 1B11 binding to antigen-specific CD8 T cells. (A) Splenocytes from LCMV-infected mice were stained with the L^d(NP118) tetramer and anti-CD8 and the percentage of CD8 T cells that were tetramer positive is noted. (B) Expression of cell surface O-glycans was examined with the mAb 1B11. Within the histograms, the bold line represents the 1B11 staining gated on the L^d(NP118)⁺ CD8 T cells at each time point, and the thin line corresponds to the 1B11 staining on naive BALB/c CD8 T cells. The percentage of 1B11^hi cells within the gated population is noted. (C) This panel shows the direct ex vivo CTL activity as assessed on LCMV-infected targets.

Figure 3

The 1B11 O-glycan epitope is upregulated on antigen-specific effector T cells. (A) LCMV-specific CD8 T cells were obtained from naive P14-transgenic mice or at days 5, 8, and 100 after adoptive transfer and infection. Splenocytes from these various groups of mice were stained with anti-CD8 and the D^b(GP33) tetramer. The percent of CD8 T cells that are GP33 specific is noted in the upper right quadrant. (B) 1B11 staining of the GP33-specific CD8 T cells. The percent of cells which are 1B11^hi within the gated D^b(GP33)⁺CD8⁺ population is noted.

Figure 4

Using the 1B11 antibody to monitor recruitment of memory CD8 T cells into the secondary response (A) and to isolate antigen-specific effector CTLs (B). (A) LCMV immune mice were rechallenged with varying doses of LCMV-Armstrong intravenously, and recruitment of the antigen-specific CD8 T cells into the secondary response was monitored by 1B11^hi staining. The contour plots show the L^d(NP118) tetramer-positive CD8 T cells at day 2 after reinfection with different doses of LCMV. The histograms demonstrate the 1B11 staining on the gated populations from the panels on the left, with the percentage of 1B11^hi and 1B11^lo tetramer-positive CD8 T cells noted. (B) LCMV immune mice were reinfected with 3 × 10⁵ pfu LCMV-Armstrong intravenously, and 2 d later, L^d(NP118) tetramer-positive CD8 T cells were sorted into 1B11^hi and 1B11^lo populations. (C) The sorted 1B11^hi and 1B11^lo L^d(NP118) CD8 T cells were then analyzed for their LCMV-specific direct ex vivo CTL activity. The level of killing on uninfected target cells was <6%.