Intrinsic CD4+ T cell sensitivity and response to a pathogen are set and sustained by avidity for thymic and peripheral complexes of self peptide and MHC

Abstract

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells.

Figure 1. LLO56 and LLO118 T cells diverge in their IL-2 responses to specific or nonspecific stimuli

(a, b) Upregulation of CD69 and CD25 (a) and ELISA of IL-2 expression (b) in LLO56 and LLO118 T cells treated with indicated amounts of LLO(190-205) peptide,100 μM of MCC(83-101) peptide or 10 μg/mL αCD3 + α CD28 mAbs. (c) Surface plasmon resonance binding analysis of LLO56 and LLO118 scTCRs to LLO(190-205)/I-A^b. Results show a concentration series of scTCR injections beginning at 40 μM (topmost curves), with two fold serial dilutions going from top to bottom. (d) IL-2 capture assay of LLO56 and LLO118 CD4⁺ T cells stimulated with 10 μg/mL αCD3 + αCD28. (e) Intracellular IL-2 (top), IFN-γ (middle) and TNF (bottom) assays of LLO56 and LLO118 CD4⁺ T cells stimulated with PMA + ionomycin. For (d) and (e), primary data (left, numbers are the % cytokine⁺ CD4⁺ cells), graphed % cytokine⁺ CD4⁺ cells (middle), and graphed MFI of cytokine⁺ CD4⁺ cells (right) are presented. All data are representative of at least three experiments. Bar graphs depict means ± SEM, with statistical analyses done using unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Stronger LLO56 IL-2 responses are linked to greater activation-induced phospho-ERK and basal phospho-TCRζ than LLO118

(a) ERK phosphorylation kinetics of PMA-stimulated LLO56 and LLO118 T cells. (b) IκBα degradation kinetics of PMA-stimulated LLO56 and LLO118 T cells. IκBα band densities are normalized to β-actin for quantitation. (c) Flow cytometric calcium flux analysis of ionomycin-treated LLO56 and LLO118 T cells, with one measurement taken every second. (d) Basal p21-TCRζ phosphorylation in unstimulated LLO56 and LLO118 whole cell lysates. Densities of p21 bands are normalized to p16-TCRζ, and are reported relative to LLO56. All data are representative of at least three independent experiments. Bar graphs depict means ± SEM, with statistical analysis done using unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3. Strength of intrinsic IL-2 responses and signaling in polyclonal B6 CD4⁺ and CD8⁺ T cells correlates with CD5 expression

(a and b) CD4⁺ and CD8⁺ T cells were gated into four equal fractions (Q1 through Q4, from lowest to highest CD5 expression) and analyzed for IL-2 production in response to PMA + ionomycin (a), or ERK phosphorylation in response to PMA stimulation for 3 minutes (b). Primary (upper panels) and graphed (lower panels) data are presented. (c) Basal TCRζ phosphorylation in whole cell lysates of unstimulated B6 CD4⁺ and CD8⁺ T cells FACS-sorted from the Q1 and Q4 CD5 fractions. Densities of p21 bands are normalized to p16-TCRζ, and are reported relative to Q4. (d-f) Analysis of IL-2 production (d), ERK phosphorylation (e) and basal TCRζ phosphorylation (f) in sorted naive conventional (CD44^lo-int, CD25⁻, NK1.1⁻) CD4⁺ and CD8⁺ T cells. All data are representative of at least three independent experiments. Bar graphs depict means ± SEM, with statistical analyses done using unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Functional attributes of LLO56 and LLO118 T cells emerge during positive selection, during which LLO56 receives a stronger signal from selecting self-pMHC

(a) Analysis and quantitation of LLO56 and LLO118 thymocyte subsets and total thymic cellularity, compiled from 12 (LLO118) or 13 (LLO56) thymi. (b) Expression of markers reflecting avidity for self-pMHC in DP (pre-selection) and CD4SP (post-selection) thymocytes, representative of at least three LLO56 and LLO118 thymi each. (c and d) Intracellular IL-2 analysis of PMA + ionomycin stimulated LLO56 and LLO118 thymocytes (c), and ERK phosphorylation of LLO56 and LLO118 thymocytes stimulated with PMA for 3 minutes (d), gated and analyzed by subset. Data are representative of at least three experiments. (e) Basal p21-TCRζ phosphorylation in FACS-sorted, unstimulated DP and CD4SP thymocytes (1.7 × 10⁶ cells per lane) from LLO56 and LLO118 mice. Densities of LLO56 and LLO118 p21 bands are normalized to p16-TCRζ, and are reported relative to LLO56 and LLO118 DP thymocytes, respectively. Data are representative of five experiments. (f) Annexin V and 7AAD staining of LLO56 and LLO118 thymocyte subsets and peripheral CD4⁺ T cells stimulated 24h with 1 μg/mL αCD3 + αCD28, representative of four experiments. Bar graphs depict means ± SEM, with statistical analyses done using unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Deprivation of self-pMHC compromises intrinsic IL-2 and ERK responses, and in vivo response to Listeria

(a) IL-2 responses of LLO56 and LLO118 T cells transferred to B6 (LLO56 n = 6, LLO118 n = 7) or MHC II-deficient (LLO56 n = 8, LLO118 n = 6) mice for 4 days, then stimulated ex vivo with PMA + ionomycin. The % IL-2⁺ cells from each recipient over three experiments was compiled. (b) Phospho-ERK responses of LLO56 and LLO118 T cells transferred to B6 (LLO56 n = 7, LLO118 n = 6) or MHC II-deficient (LLO56 and LLO118 n = 5) mice for 4 days, then stimulated ex vivo with PMA for 3 minutes. The pERK MFI of cells transferred to MHC II-deficient recipients was normalized to the pERK MFI of cells transferred to B6 recipients in the same experiment, and compiled from three or four experiments. (c) Expansion of LLO56 and LLO118 T cells either deprived (LLO56 n = 12, LLO118 n = 6) or not deprived (LLO56 n = 12, LLO118 n = 7) of self-pMHC at 7 days post-transfer to Listeria-infected B6 mice, compiled from 6 (LLO56) or 4 experiments (LLO118). Bar graphs depict means ± SEM. For (a) and (b), statistical analyses were done using unpaired two-tailed Student’s t tests; for (d), paired two-tailed Student’s t tests were done. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6. CD5 antagonizes signal from self-pMHC and intrinsic IL-2 and pERK responses

(a and b) Expression of functional markers in wild-type and CD5-deficient LLO56 (a) and LLO118 (b) thymocyte subsets and peripheral cells. (c) Intrinsic IL-2 response in wild-type and CD5-deficient LLO56 and LLO118 T cells stimulated with PMA + ionomycin. (d) Phospho-ERK response in wild-type and CD5-deficient LLO56 and LLO118 T cells stimulated with PMA for 3 minutes. Bar graphs depict means ± SEM, with statistical analyses done using unpaired two-tailed Student’s t tests. Displayed data are representative of two (LLO118) or three (LLO56) experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.