Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential

Abstract

Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.

Keywords: CD4 T cell; Cigarette smoke; Cytokines; Memory; T-bet; Th1 effector.

Figure 1. Exposure to CSE during priming reduces CD4 T cell division and increases apoptosis

Triplicate wells of equal numbers of naïve HNT cells and irradiated APC were cultured with cognate peptide under Th1-polarizing conditions for 4 days in the presence of stated concentrations of CSE. The number of resulting effector cells was determined in replicate experiments with a broad (A) and narrower (B) titration of CSE (2 of 5 similar experiments). (C) In some experiments naïve CD4 T cells were labeled with CFSE prior to culture. Shown is representative CFSE staining from effectors at 4 days of culture in the presence of 0% (black) or 7.5% (red) CSE versus CFSE staining of undivided naïve cells not exposed to antigen. Day 4 effector cells were analyzed for apoptotic phenotype by staining for Annexin V and 7-AAD. Shown are (D) representative staining of effectors cultured with or without CSE and (E) summary of 4 wells per condition (one of 2 experiments).

Figure 2. Exposure to CSE during priming enhances Th1 polarization

Naïve CD4 T cells were stimulated with APC and peptide under Th1 conditions in the presence or absence of CSE. Effector cells were harvested on day 4 of culture and analyzed for forward scatter (FSC), CD25, and CD69 with (A) representative staining and (B) mean fluorescence intensity (MFI) analysis from triplicate cultures shown. Values from naïve CD4 T cells are shown as grey histograms in (A) and as dotted line in (B). Effector cells were assessed for their capacity to produce of IFNγ and TNF by intracellular cytokine staining. Shown is (C) representative staining in the absence or presence of stimulation by PMA and Ionomycin by effectors generated in the presence or absence of CSE, as well as (D) the frequency of cells from each condition capable of co-producing TNF and IFNγ and (E) the MFI of IFNγ⁺ cells from triplicate wells (one of 4 experiments). Expression of T-bet in effectors was determined by intracellular staining with MFI analysis and representative staining shown (F and G). Representative staining of re-stimulated cells for production of IL-4 (H) and IL-17 (I) (one of 2 experiments).

Figure 3. Delayed exposure to CSE reduces effector expansion

CSE was introduced to Th1 cultures at either day 0 or day 2 and (A) the total number of effector cells recovered at day 4 is shown from quadruplicate wells (one of 3 experiments). (B) Representative analysis of CFSE dilution from effectors either not exposed to CSE (black) or exposed to CSE for only from days 2–4 of culture. Effector cells were analyzed for apoptotic cells with (C) representative staining and (D) analysis from triplicate wells shown (one of 2 experiments).

Figure 4. Delayed exposure to CSE enhances Th1-polarization

Effectors generated without exposure of CSE or CSE added only from days 2–4 of culture only were analyzed for Th1 cytokine production by intracellular staining with (A) representative staining and (B) analysis of dual cytokine producers from triplicate conditions shown. T-bet levels in effectors was also determined with (C) representative staining and (D) MFI analysis from triplicate wells shown (one of 2 experiments).

Figure 5. CSE acts directly on CD4 T cells to reduce expansion and increase Th1-polarization

Naïve CD4 T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of CSE. The recovery of effectors at day 4 (A), CFSE dilution (B), FSC and CD69 MFI values (C), analysis of apoptosis (D and E), Th1 cytokine production (F and G), and T-bet expression (H and I) is shown from one of 3 experiments.

Figure 6. Exposure to CSE reduces memory potential in Th1-polarized effector cells

Effectors were generated by stimulation with either APC and peptide or with anti-CD3 and anti-CD28 antibodies. On day 4, live effector cells were isolated from cultures and analyzed for expression of the α chain of the IL-7 receptor (CD127). Representative staining and MFI analysis from triplicate wells is shown for cells exposed or not to CSE from cultures stimulated with APC and peptide (A and B) or by antibody-mediated activation (C and D). Equal numbers of live effectors exposed to CSE were thoroughly washed and re-plated in media alone for 3 additional days to form in-vitro generated memory cells. Shown is the ratio of memory cells recovered from effectors not exposed to CSE versus from effectors exposed to CSE during priming using both methods of CD4 T cell activation (E and F). Results representative from experiments repeated twice for both APC and peptide and antibody-mediated effector generation.