Differentiation of Naive CTL to Effector and Memory CTL: Correlation of Effector Function with Phenotype and Cell Division

In vivo detection of cell division after viral infection. 318-CFSE spleen cells were transferred into recipients 1 day before i.v. injection with the indicated doses of LCMV WE. Three days after infection, spleen cells and mesenteric lymph node cells were stained with anti-CD8-PE and analyzed by two-color flow cytometry. Lines represent CFSE fluorescence intensities of CTL that have divided zero to seven times. A representative of at least two experiments is shown.

Surface marker analysis of in vivo differentiating CTL. Recipients were transfused with 318-CFSE spleen cells, and mesenteric lymph nodes were analyzed 3 days after i.v. injection of 5 × 10³ PFU of LCMV WE. Cells were stained for CD8 and for the indicated surface markers. Expression levels in dependence of cell cycle number were determined as described in Fig. 1. Endogenous = endogenous CTL population. Naive = surface marker expression levels on naive 318 CTL. Surface marker expression levels on 318 CTL 3 days after transfer into naive recipients were indistinguishable from fresh naive 318 CTL. A representative result from at least three independent experiments is shown.

TCR modulation after viral infection. 318-CFSE spleen cells were transfused into recipients and activated in vivo by i.v. injection of 5000 PFU of LCMV WE (Effector). Uninfected recipients (Naive) served as a comparison. After 3 days, mesenteric lymph nodes were stained with anti-CD8-PE or anti-Vα2-PE, to directly compare transgenic TCR modulation on proliferating CD8⁺CTL (upper versus lower panels). Black and white arrows point at TCR^high and TCR^low CTL, respectively, which are absent in the noninfected controls (left plot), but are visible in the infected recipients (right plot). A representative result from at least four mice is shown.

A, CTL acquire cytotoxic effector function within one cell division. 318-CFSE spleen cells were activated in vitro with p33, as described in Fig. 1. After 16 h (0 div) or 30 h (0–1 div), standard cultures were harvested and an aliquot was analyzed for cell division by gating on CD8-PE⁺CTL and displaying CFSE intensities in a histogram (upper panels). Cultures were then tested in a 5-h ⁵¹Cr release assay (lower graph). Cultures that had not divided yet (boxes) or that had divided once (triangles) were analyzed on unpulsed (open symbols) or p33-pulsed (filled symbols) EL-4 target cells. Spontaneous release was <12% for all targets. B, Cytotoxic effector function of acutely activated and memory CTL. 318-CFSE spleen cell cultures were stimulated with p33 for 48 h (div 0–4) to obtain effector CTL. For long-term cultures, 318-CFSE spleen cells were stimulated by a single addition of p33 (final concentration: 10⁻⁸ M), washed after 3 days, split at a ratio of 1:4, and recultured for an additional 4 days (div 6–10). Cell cycle numbers were determined as described above (top two panels). Cytotoxic activity (bottom panel) was determined in a ⁵¹Cr release assay at the indicated E:T ratios on p33-pulsed (closed symbols) and unpulsed (open symbols) EL-4 target cells. 0–4 div cultures were incubated for 5 h (squares); 6–10 div cultures for 5 h (triangles) and 20 h (circles). After 5 h, spontaneous releases were <10%; after 20 h, <25%. C, Phenotype of short-term (0–4 div) and long-term (6–10 div) cultures. Cultures from B were stained for CD8 and CD44 or CD62L. After gating on the most prominent division peak (div2 and div8, respectively), surface marker expression levels were displayed in a histogram and compared with naive 318 CTL. A representative of three independent cultures is shown.

A, In vivo effector function of naive, effector, and memory CTL. Day 3 effector mice (Effector) were generated by transfusing 3 × 10⁷ 318 spleen cells into syngeneic hosts and infecting them with 1000 PFU of LCMV. Memory mice were obtained by adoptively transferring 1 × 10⁶ 318 spleen cells into naive recipients and infecting them i.v. with 200 PFU of LCMV WE 30 or 60 days (17) before determination of CTL effector function. Untreated 318 mice served as naive control mice. To analyze CTL effector function in vivo, target cells were prepared by pulsing syngeneic spleen cells with p33 and labeling them with a high CFSE intensity. To control for Ag specificity, unpulsed syngeneic spleen cells were labeled with a low CFSE intensity. A 1:1 mixture of 3 × 10⁷ cells of each of the target cell populations was injected i.v. into the naive 318 controls, the effector, or the memory mice. After 4, 11, and 20 h, blood was collected from the tail vein and analyzed in a single-parameter flow-cytometric analysis for the presence of the CFSE^high (p33-pulsed) and CFSE^low (unpulsed) target cell populations. To test for contact-dependent, specific cytotoxic effector function, the elimination of p33-pulsed CFSE^high spleen cells was monitored and the ratio (r) between percentage of nonpulsed and percentage of p33-pulsed indicator cells was calculated. Data show a representative of three mice. B, CD62L expression on naive, effector, and memory LCMV-specific CTL. Blood was collected from the mice described in A before transfer of the target cells and triple stained for transgenic Vα2, CD8, and CD62L or CD44 (not shown). CD62L expression is shown after gating on CD8- and Vα2-positive lymphocytes. A representative result from three independent day 30 memory mice is shown.