Long-term persistence of activated cytotoxic T lymphocytes after viral infection of the central nervous system

Abstract

Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is mediated by CD8(+) cytotoxic T lymphocytes. We have studied the kinetics of this secondary immune response and found that despite the elimination of replication-competent virus by day 10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence of activated lymphocytes may be important for efficient long-term responses to viruses prone to recrudesce in sites of relative immune privilege.

Figure 1

High frequencies of CD8⁺ T cells are recoverable from the brain for long after A/WSN infection. (a) Cells were purified from the brains of A/X-31–primed A/WSN-challenged mice at various time points as described in Materials and Methods and used directly in CTL assays without restimulation. Total cell counts (broken line, circles), CD4 (triangles), and CD8 (squares) frequencies are plotted. Each data point represents the means of two separate trials with two to eight mice at each time point, except for day 320, which represents a single assay on brain lymphocytes pooled from eight mice. Standard deviations <10% are not shown. (b) Lysis of virus-infected (circles) and peptide-pulsed (triangles) EL-4 target cells is titratable and antigen specific (squares, untreated target cells). Variation between duplicates was <15%. E/T ratios refer to the ratio of CD8⁺ cells to target cells.

Figure 2

Cryostat coronal brain sections from mice inoculated 5 (a–f  ) and 320 (g–l) d previously with intracerebral A/WSN. Day 5: (a) CD8⁺ cells infiltrating the choroid plexus of the third ventricle (IIIv) and the periaqueductal parenchyma. (b) CD4⁺ cells (c) markedly increased MHC class I expression in brain parenchyma, (d) increased MHC class II expression predominantly within the inflammation obliterating the lateral ventricle (Lv), (e) B220⁺ cells in the lateral recess of the third ventricle, and (f ) influenza ribonucleoprotein (RNP) expression surrounding the cerebral aqueduct (Aq). Arrow, virus-infected cell. Day 320: (g) groups of CD8⁺ cells adjacent to the corpus callosum, (h) parenchymal CD4⁺ cell, single CD4⁺ cells in the brain parenchyma were evident on occasional sections. Lack of MHC class I (i) and II (j) staining in the brain parenchyma. (k) Accumulation of B220⁺ cells within the choroid plexus of the lateral ventricle, (j) absence of antiinfluenza RNP staining (periaqueductal region). The ependymal cell loss seen in h, k, and l is consequent on viral cytopathology. Bar: 50 μm in g and h and 250 μm in the other panels.

Figure 3

(a) CD8⁺ cells purified from day 7 (early response) and day 180 (late response) have a similar activated/memory phenotype and CD69 is a better activation marker than CD25. (b) In vivo proliferation of brain CD8⁺ cells early in the response to A/WSN. (c) Brain CD8⁺ cells are not proliferating late in the response. A representative one of three experiments is shown. Comparison of BRDU uptake by the splenic and brain CD8⁺CD44^hi populations showed that there was a 2.9 ± 0.17–fold enrichment of BRDU⁺ cells at day 6 in the brain compared with a 0.48 ± 0.22–fold enrichment at day 180 (mean ± SEM; P < 0.005 using an unpaired t test). A significant reduction in CD8⁺BRDU⁺ cells in the late brains compared with their CD8⁺CD44^hi counterparts was also evident. Quadrant frequencies are shown (%).

Figure 3

(a) CD8⁺ cells purified from day 7 (early response) and day 180 (late response) have a similar activated/memory phenotype and CD69 is a better activation marker than CD25. (b) In vivo proliferation of brain CD8⁺ cells early in the response to A/WSN. (c) Brain CD8⁺ cells are not proliferating late in the response. A representative one of three experiments is shown. Comparison of BRDU uptake by the splenic and brain CD8⁺CD44^hi populations showed that there was a 2.9 ± 0.17–fold enrichment of BRDU⁺ cells at day 6 in the brain compared with a 0.48 ± 0.22–fold enrichment at day 180 (mean ± SEM; P < 0.005 using an unpaired t test). A significant reduction in CD8⁺BRDU⁺ cells in the late brains compared with their CD8⁺CD44^hi counterparts was also evident. Quadrant frequencies are shown (%).

Figure 4

PCR amplification of nucleoprotein cDNA from mouse brain. Numbers refer to day after intracerebral inoculation. M, HaeIII-digested φx 174 markers. R1 is a positive from the NP1/NP2 reaction run for size comparison. Representative gels are shown; each lane represents the result from an individual mouse. With seminested PCR, nucleoprotein cDNA was amplified from three of nine animals at day 115 and from none of six animals at day 196 (Table 3). Hypoxanthine guanosine phosphoribosyl transferase PCR was positive in all samples indicating that the cDNA synthesis had been satisfactory. PCR products were visualized on 2% agarose ethidium bromide gels under UV light and photographed on Polaroid film.