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Comparative Study
. 2007 May 14;204(5):1193-205.
doi: 10.1084/jem.20062349. Epub 2007 May 7.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

António Peixoto  1 , César Evaristo, Ivana Munitic, Marta Monteiro, Alain Charbit, Benedita Rocha, Henrique Veiga-Fernandes
Affiliations
Comparative Study

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

António Peixoto et al. J Exp Med. .

Abstract

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen.

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Figures

Figure 1.
Figure 1.
Coexpression of “effector” genes in male-specific CD8 single cells during the primary immune response. Anti-HY CD8 Tg single cells were sorted at different points of the response corresponding to the following: PE, early expansion phase; PP, peak of exponential growth; PL, plateau; and different time points after the end of the contraction phase (PM-CD8s). Each row shows the same individual cell that is numbered. Each column shows a different gene, representing the number of mRNA molecules/cell according to a color log scale. Empty symbols represent cells negative for that particular mRNA (<2 mRNA molecules). Gray symbols correspond to positive cells where mRNA levels were not quantified. For better visualization of coexpression patterns, individual cells were ordered by the degree of gene coexpression. The same expression patterns were obtained in two independent experiments.
Figure 2.
Figure 2.
Variation of each gene expression level at different time points of the primary immune response. Individual CD8 Tg spleen lymphocytes specific to the male antigen were recovered at PE (blue diamond), PP (orange square), PL (yellow triangle), and at the memory phase 1 mo (green circle) and 2 mo (purple triangle) after immunization; 30 individual cells were studied at each time point. Negative cells are not figured. Results compare the expression levels of each gene in individual cells throughout the response, showing the absolute number of mRNAs/cell plotted in a log scale. They correspond to one of the two independent kinetic experiments we performed.
Figure 3.
Figure 3.
The primary response to L. monocytogenes immunization. OT-1 CD8 Tg cells specific of the OVA antigen were transferred to B6 mice, which were immunized with L. monocytogenes OVA. (a) Kinetics of the response. Results show the number of OT-1 cells recovered/mouse at different points after immunization and are the mean ± the SEM of three mice/time point. (b and c) Coexpression of effector genes at different points of the response. OT-1 cells were single-cell sorted, and their gene expression was depicted as described in Fig. 1. (c) OT-1 cells were recovered from two different individual mice in each time point. CD69+ cells were activated cells that had not yet divided. Gene expression patterns are as described in Fig. 1.
Figure 4.
Figure 4.
CD8 effector functions. (a and b) Trapping. Naive or PE-CD8s anti-HY–specific cells were injected in the spleen of Cd3ɛ−/− female mice. These mice were simultaneously injected i.v. with female CFSE+ target cells (a mixture of CFSElow and CFSEhigh targets, the latter loaded with the HY-peptide). 1 d later, we quantified the target cell recovery in the spleen and lymph nodes. (a) Gated CFSE+ cells in half of the lymph node (control) or total lymph node cells from individual mice injected with PE-CD8s. (b) Absolute number of targets in the lymph nodes and spleen. (c and d) Antigen loads. (c) Thy1.1+ naive “sensor” cells were injected into mice undergoing the immune reaction at different points of the responses. Results show CD69 expression in “sensor” cells 1 d after injection. (d) Quantification of Zfy-1 DNA in the spleen at different days after immunization. Similar results were obtained in the bone marrow. (e) In vivo killing. Targets were as described in the graphs in (a). CD8 cells were naive (control) or recovered at points of the response. Targets and CD8s were coinjected into the spleen of Cd3ɛ−/− female mice. The percentage of specific killing was evaluated as compared with the control performed in the same day. CD8 T cells and nonpulsed targets remained in similar numbers in the spleen during the 6 h required for optimal killing. All results are from one of three experiments.
Figure 5.
Figure 5.
Correlation between Il7r and Ccr7 expression and the coexpression of effector molecules. Each horizontal line shows the same individual cell. Columns show different genes and, when colored, represent the number of mRNA molecules/cell according to a color log scale. Empty symbols represent cells negative for that particular mRNA (<2 mRNA molecules). Gray symbols represent positive cells where mRNA levels were not quantified. (a) Il7r-expressing cells were ordered by decreasing amounts of Il7r copies/cell. Il7r-negative cells were ordered by the degree of effector gene coexpression. (b) Ccr7 + and Ccr7 − cells were ordered according to the degree of effector gene coexpression. Please note that individual Ccr7 + cells did not show major differences in Ccr7 expression levels.
Figure 6.
Figure 6.
Secondary responses. PM-CD8s were boosted with male cells. (a) Individual cells were sorted at different time points after boosting. Results show gene expression frequencies in the PM-CD8 donor cells we used in this experiment (yellow bars) and in the secondary response at different time points after boosting (blue bars). Results correspond to >47 cells per time point. (b) Comparison of Ag loads in the primary (orange circle) and secondary (blue circle) responses. Primary and secondary hosts were studied simultaneously. Results show CD69 expression in “sensor” cells at different time points, as described in Fig. 4 c. The same results were obtained in three independent experiments. (c) Comparison of cytotoxic capacity of PP-effector cells and SM cells recovered 3 mo after antigen elimination. Killer tests were performed as described in Fig. 4 e. Both CD8 populations were studied in the same day, with results corresponding to one of two experiments. (d) Quantification of mRNA expression levels in secondary single cells recovered at days 4 (blue diamond), 7 (orange square), 15 (yellow triangle), and 33 (green circle) after boosting. Results show the number of mRNA copies/cell plotted in a log scale.
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References

    1. Croft, M., L. Carter, S.L. Swain, and R.W. Dutton. 1994. Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J. Exp. Med. 180:1715–1728. - PMC - PubMed
    1. Gett, A.V., and P.D. Hodgkin. 1998. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl. Acad. Sci. USA. 95:9488–9493. - PMC - PubMed
    1. Lee, G.R., S.T. Kim, C.G. Spilianakis, P.E. Fields, and R.A. Flavell. 2006. T helper cell differentiation: regulation by cis elements and epigenetics. Immunity. 24:369–379. - PubMed
    1. Surh, C.D., O. Boyman, J.F. Purton, and J. Sprent. 2006. Homeostasis of memory T cells. Immunol. Rev. 211:154–163. - PubMed
    1. Veiga-Fernandes, H., U. Walter, C. Bourgeois, A. McLean, and B. Rocha. 2000. Response of naive and memory CD8+ T cells to antigen stimulation in vivo. Nat. Immunol. 1:47–53. - PubMed

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