Cognate antigen stimulation generates potent CD8(+) inflammatory effector T cells

Abstract

Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88-deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8(+) inflammatory effector cells in a lymph node can mobilize 10(7) cells in 24 h, including lymphocytes, natural killer cells, and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

Keywords: CD8 T cells; effector functions; immune responses; lymph-node shut-down-phase; lymphocyte trapping.

Figure 1

Expression of inflammatory mediators by CD8 T cells. (A,B) Mo CD90.2 TCR-Tg cells were transferred to CD90.1 B6 mice, and immunized with LM-OVA expressing the OT1 and OT2 epitopes. At different time points after infection we determined: (A) The proportion of OT1 and OT2 Tg cells expressing these mRNAs evaluated in 72 individual cells by single-cell RT-PCRs. (B) The ex vivo intracellular expression of pro-inflammatory proteins in Brefeldin A injected mice. Barriers to identify positive cells were based on the labeling of the same cell suspension with isotype control antibodies. The lack of suitable Abs prevented us from evaluating XCL1 expression. (C) Mo CD90.2 P14 TCR-Tg cells were transferred to CD90.1 B6 mice. 24 h later, these transferred mice, as well as My88⁺ and MyD88⁻ mice were immunized simultaneously with LM-GP33. CD8⁺ CD69⁺ GP33-specific-cells were sorted from these three mice types at days 2 and 3 after priming. For that purpose, spleen cell suspensions were depleted of non-CD8 T cells. The P14 Tg cells were identified by their co-expression of CD90.2 and CD8β. Endogenous cells from MyD88⁺ and MyD88 B6⁻ mice were identified by triple co-expression of CD8β, and APC, and PE labeled GP33-Dext. Results show the levels of these mRNAs in CD69⁺ GP33-specific-cells 2 (upper graphs) and 3 days (lower graphs) after immunization, evaluated by qRT-PCR.

Figure 2

(A) Correlation between CD8 division and differentiation. (A) CFSE-labeled Mo CD90.2 OT-1 Tg cells were transferred to CD90.1 B6 mice and studied at different time points after infection with LM-OVA. Left: CFSE dilution and the gates used to sort individual cells at each division. Right: expression frequency, as evaluated in RT-PCRs in 96 individual cells. (B,C) TCR expression after immunization with LM-GP33. (B) TCR-Tg cells. 5,000 Mo CD90.2⁺ P14 TCR-Tg cells were injected into CD90.1⁺ B6 mice prior to infection. Results show APC-Dext-GP33 binding in CD90.2⁺ CD8⁺ Tg T cells in naïve (upper graph) and infected mice, at different days (D) after immunization. (C) Endogenous cells. CD90.1 B6 mice were immunized with LM-GP33. Results show Dext-GP33 PE and APC double-labeling in gated CD8 spleen cells in naïve mice (upper graph) and primed mice at different days (D) after infection.

Figure 3

TCR signaling transduction at different time points after immunization. Mo CD90.2⁺CD45.1⁺ OT-1 TCR-Tg cells were transferred into CD90.1⁺ CD45.2⁺ B6 hosts and infected with LM-OVA. The figure shows the expression of phosphorylated signal transduction molecules in CD90.2⁺ CD45.1⁺ CD8⁺ Tg cells at different time points after priming. The labeling with isotype controls is shown in gray.

Figure 4

Quantification of the naïve T cell pool. (A) Quantification of antigen-specific naïve cells in the BRLN. Reference populations (RPs: 10⁴ CD90.2 CD45.1 cells and 10³ CD90.2 CD45.2 P14 cells) were added to collagenase/DNase digests of the BRLN from CD90.1 B6 mice. Cell suspensions were depleted of non-CD8 T cells, and antigen-specific-cells purified by Dext pull-down. Left: the gates used to identify CD90.2⁺ RP and CD90.1 endogenous cells. Middle: CD45 allotypes distribution in RPs. Right: GP33-Dext labeling in CD90.1 endogenous CD8 T cells. (B) Estimation of the size of the naïve GP33-specific T cell pool. Results show the number of Dext-GP33-specific-cells recovered/mouse in three independent experiments.

Figure 5

Inflammatory effector CD8 T cells recruit non-resident cells. Sorted Mo CD90.2⁺ OT-1 naïve cells or inflammatory effectors (recovered 2.5 days after LM-OVA infection) were injected with matrigel subcutaneously in the ear of CD90.1⁺ mice. (A) Recovery of injected cells from the matrigel plug (left) and the auricular LN (ALN) (right) 24 h after injection. (B,C) Cell populations recovered in the ALN of injected mice, 24 h after injection. (B) Results show cell numbers and are mean±SEM of three independent experiments. (C) Results show the gates used to identify different cell types and are from one representative experiment out of the three we performed.

Figure 6

CD8⁺ inflammatory effector T cells generated in sterile immunizations also recruit non-resident cells. Sorted Mo CD90.2⁺ HY-specific CD8 naïve cells or inflammatory effectors (recovered at 2.5 days after immunization with male cells) were injected with matrigel subcutaneously in the ear of CD90.1⁺ mice. (A) Results show the number of different cell types recovered in the ALN of injected mice, 24 h after injection and are the mean±SEM of three independent experiments. Cell populations were identified as described in Figure 5. (B) Major accumulation of inflammatory monocytes in the ALN of mice injected with inflammatory effectors. Results are from one representative experiment, out of the three performed.

Figure 7

Effects of intra-nodal injection of physiologic numbers of CD8⁺ inflammatory effector T cells. (A,B) The indicated number of sorted CD90.2⁺ naïve or inflammatory effectors (recovered 2.5 days after priming) were injected into the BRLN of CD90.1⁺ B6 mice. The BRLNs were removed 24 h later and the different cell types identified as shown in Figure 5. Results compare cell recovery in mice injected with naïve CD8+ T cells or inflammatory effector CD8⁺ T cells (A) Mice were injected with OT-1 Tg cells. (B) Mice injected with HY Tg cells. Similar results were obtained after injection of P14 Tg cells. (C) The BRLNs were injected with 500 naïve cells, 80 OT-1 effectors or 56 HY effectors. Results show of S1P amounts in LN lysates 16 h later, compared with known amounts of a synthetic S1P. Gray bars represent the Flag-S1P1 expression of WEHI231 incubated with medium alone. Results are representative of the seven experiments we performed.