CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells

Abstract

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms "transfer factor" and "DLE" were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity.

Keywords: Tc17; cellular immunity; transfer factor.

© Society for Leukocyte Biology.

Figures

Figure 1.. Cellular lysates transfer antigen-specific immunity.

Mice were injected with 2e6–5e6 ceq of DLE from antigen-naïve mice (Naïve:DLE), mice injected with CFA (CFA:DLE), or mice immunized with an emulsion in CFA of either BSA or OVA (BSA:DLE or OVA:DLE). (A and B) Twenty-four to 36 h after DLE (5e6 ceq) treatment, mice underwent footpad testing with OVA or BSA. The change in footpad thickness (Δ) compared with the opposing, noninjected footpad was measured in a blinded fashion, 18–24 h later. Individual symbols represent 1 mouse. (C–E) Twenty-four to 36 h after OVA:DLE (2e6 ceq) treatment, splenocytes were harvested and challenged ex vivo with DCOVA or DCBSA. Supernatants were harvested 3 d later, and concentrations of IL-17A (C), IL-22 (D), and IL-6 (E) were measured. (F) Twenty-four to 36 h after DLE (2e6 ceq) treatment, splenocytes were harvested from individual mice and processed into single-cell suspensions. The splenocytes underwent ex vivo challenge by coculturing 2e6 whole splenocytes with 4e5 bone marrow-derived DCOVA in a total of 1 ml media. Supernatants were harvested 3 d later, and IL-17A concentrations were measured. (G) Footpad testing with OVA, as in A, for wild-type (C57BL/6) versus IFN-γ knockout (IFN-γ−/−), a Stat3 transgenic mouse model of HIES, and IL-17A/F dko mice. Data shown are representative of 3 or more independent experiments, each using at least 5 mice/group (A–F) or a combination of 2 independent experiments using 4–7 mice/group (G) and displayed as means (A, B, and G) or means ± sem (C–F). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA (A, F, and G) or Student’s t test (B–E).

Figure 2.. DLE stimulates antigen-specific IL-17 production from antigen-naïve splenocytes.

Whole splenocytes (2e6) were harvested from OVA-naïve mice and cocultured with DCOVA (4e5) in 1 ml total media. (A) 1e5 ceq of DLE from mice immunized with CFA and OVA (OVA:DLE) or CFA alone (CFA:DLE) was added to the culture media. Supernatants were harvested after 72 h and analyzed for IL-17A. (B) Dose-response curve for OVA:DLE (0, 1e4, 1e5, and 1e6 ceq) added to cocultures (black bars) or splenocytes (Sp) or DC alone (gray bars; 1e6 ceq). IL-17A measured after 72 h of incubation. (C) 1e6 ceq of OVA:DLE added to cocultures. IL-17A measured at 24 h increments and depicted as change (Δ) versus time-matched media control. (D) Titrated doses (0, 1e4, 1e5, and 1e6 ceq) of DLE derived from mice immunized with CFA and BSA (BSA:DLE) were added to cocultures of BSA-naïve splenocytes and DCBSA. Supernatant IL-17A was measured after 72 h. (E) IL-22 induced from 1e4–1e6 ceq OVA:DLE added to cocultures as in B. (F) 1e6 antigen-naïve splenocytes were cocultured with 4e5 DCBSA or DCOVA in 1 ml total media. 1e6 ceq of either OVA:DLE or BSA:DLE was added; IL-17A concentrations at 72 h measured and depicted as the change from media control. Antigen-naïve splenocytes were assayed independently from at least 3 mice/group. Data shown are representative of 3 or more independent experiments and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA.

Figure 3.. DLE effects are produced by antigen-specific CD8+ T cells.

(A–D) C57BL/6 mice were immunized with an emulsion of OVA and CFA; 3 wk later, cells were sorted by beads (A and B) or flow cytometry (C–E), and then DLE was extracted from sorted populations. Change (Δ) in induction of IL-17A from DCOVA:splenocyte cocultures by depleted (A) or enriched (B) DLE. (C) Mice were injected with diluent (Dil) or 1e6 ceq of DLE derived from unsorted (Unsrt) or sorted (CD3+/CD4+ or CD3+/CD8+) splenocytes. Footpad testing to OVA was performed 24 h after DLE treatment. (D and E) Splenocytes from mice immunized to OVA and CFA were sorted for CD3+/CD8+ and then subdivided by staining of the MHC-I OVA tetramer. 1e5 whole splenocytes were cocultured with 2e4 DCOVA in 150 μl total media, along with 5e4 ceq of tetramer-positive CD8+ DLE (Tet+) or 1e5 ceq of CD8+/tetramer negative (Tet−) DLE. Change (Δ) from diluent control for IL-6 at 24 h (D) and IL-17 at 72 h (E) is shown. (F) Mice were immunized with OVA in CFA on d 0 and 21. DLE was extracted on d 42 as before (Boosted). 1e6 ceq of boosted OVA:DLE or OVA:DLE from mice immunized only once (Single) was added to the media of 2e5 splenocytes cocultured with 4e4 DCOVA; change in IL-17A concentration versus diluent control at 72 h is shown. Antigen-naïve splenocytes were assayed independently from at least 3 mice/group. Data shown are representative of 3–4 independent experiments and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA (A–C) or Student’s t test (D–F). NS, Not significant.

Figure 4.. DLE activates DCs to stimulate memory Tc17 cells.

Splenocytes were harvested from OVA-naïve mice and then sorted by flow cytometry before being placed into coculture with DCOVA in 1 ml total media. 2e6 ceq of OVA:DLE was added to the media. (A) 1e6 CD3+ splenocytes sorted by CD4 and CD8 positivity cocultured for 72 h with 4e5 DCOVA. Differences (Δ) in IL-17A from media control without DLE are shown. (B) IL-17A levels at 72 h from 2e5 CD3/8+ cells subsorted by CD62L and CD44 expression before coculture with 4e4 DCOVA in the presence of 2e6 ceq of OVA:DLE. (C) 2e6 CD3+/CD4+ cells were cultured under Th17 conditions, with and without 2e6 ceq of OVA:DLE. IL-17A at 72 h is shown. (D–F) 2e6 whole splenocytes were cultured with 4e5 DCOVA separated by a semipermeable Transwell membrane. 1e6–5e6 ceq of OVA:DLE was added to either the DC (above the transwell membrane) or splenocyte compartment (below the membrane). Controls included cocultures without the Transwell separator and DCOVA cultured in isolation (DC, no spleens). Change vs. diluent control for IL-17A after 72 h (D) and IL-6 at 24 h (E) are shown (dotted line in E represents no change from diluent control). (F) Antibodies against IL-6 and/or IL-1β were added to the DC compartment before coculture with whole splenocytes and OVA:DLE; change in 72 h values of IL-17A versus media control without DLE are shown. (G and H) IL-17 (G) and IL-6 (H) induced by OVA:DLE (0, 1e4, 1e5, and 1e6 ceq) from cocultures of whole splenocytes and DCe-OVA. (I) IL-6 production from 5e5 DCOVA derived from C57BL/6 wild-type (WT) mice or TLR4−/− mice was incubated for 18 h with 1e6 ceq of OVA:DLE or diluent control. Antigen-naïve splenocytes were assayed independently from at least 3 mice/group. Data shown are representative of 3–4 independent experiments and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, as determined by ANOVA.

Figure 5.. DLE activates inflammatory genetic pathways in DCs.

RNA-seq heat map for 1e6 DCOVA incubated for 18 h with 1e6 ceq of indicated DLE compared with diluent control. Selected for display were genes that had statistically significant differential expression [difference of estimation (Diff. of Est.); log2 ratio > 2 in the hypothesized direction, and FDR < 0.05] for all 3 tests: OVA:DLE versus HKCA:DLE, OVA:DLE versus diluent, and CD8-enriched (CD8enr) OVA:DLE versus OVA:DLE. The table of log2 ratio values was sorted, according to the value for CD8enr OVA:DLE versus OVA:DLE, and colorized as indicated in the legend. Data shown are combined from 3 independent experiments; significance determined by ANOVA.

Figure 6.. DLE impacts mucosal and cutaneous immunity against pathogens.

(A and B) Mice were injected with 2e6 ceq of OVA:DLE. Twenty-four hours later, mice were challenged via injection of 1 mg OVA in IFA either subcutaneously (A) or intranasally (B). Seventy-two hours later, skin or lungs were harvested, and mRNA was extracted. Relative inductions of various cytokines compared with untreated (dotted, horizontal lines) normalized to GAPDH for skin (A) and lung (B). (C and D) Mice were injected with 2e6 ceq of DLE from mice serially infected with MRSA (MRSA:DLE). Twenty-four hours later, mice were challenged subcutaneously with 1e7 CFUs of MRSA. (C) d 6, relative inductions in lesional mRNA for cytokines compared with infected but DLE-untreated mice (dotted, horizontal line) normalized to GAPDH. (D) Lesional area measured daily for mice infected with 1e7 CFU of MRSA, 24 h after treatment with diluent, MRSA:DLE, or OVA:DLE. Data shown are representative of 3 or more independent experiments, each using at least 5 mice/group, and displayed as means (A and B) or means ± sem (C and D). **P < 0.01, *P < 0.05, as determined by ANOVA.

Figure 7.. DLE immune activity is present in humans.

(A and B) Triplicate cultures of monocytes 1:1 with autologous CFSE-loaded CD3+ T cells in the presence of HKCA for 6 d from a Candida exposed donor (ED) who cohabitates with 2 patients with CMC or from random healthy volunteers (HV). On d 6, cells were stained and analyzed for CD3 and CD8 versus CFSE low (CFSElo) and CFSE high cells (A), and concentrations for indicated cytokines in the supernatants were measured (B). (C–H) hDLE was extracted from cellular lysates of PBMCs from ED or different random healthy volunteers. (C) d 3 IL-6 from 1e6 hDCHKCA from unrelated healthy donors (n = 3) and cultured with 1e6 ceq of hDLE from ED or healthy volunteers. (D and E) 4e5 DC from a patient with STAT1 GOF mutation [proband (PB)], her affected daughter (AD), or healthy volunteers were pulsed with HKCA and cultured with 2e6 autologous PBMC and 1e5 ceq of hDLE to assess d 3 IL-1β, IL-6, and IL-10 (D) and IL-17A, IL-17F, and IL-22 (E). (F and G) d 3 IL-17A (F) and IL-10 (G) after 4e5 hDCHKCA were cocultured with 2e6 autologous PBMCs with hDLE or murine HKCA:DLE before (Unsorted) or after bead enrichment for CD8 (CD8 Enr) or depletion (CD8 Dep). (H) IL-6 concentration for 1e6 hDCHKCA was cultured 24 h with 1e5 ceq of hDLE or murine HKCA:DLE. Data shown are representative of 2 (A and B) or 3 or more (C–H) independent experiments using a different healthy volunteer per experiment with triplicate cultures and displayed means ± sem. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA.

Figure 8.. DLE contains S100 peptides and binds target antigen.

(A and B) OVA:DLE or naïve DLE (0 and 1e2–1e8 ceq) was incubated in 96-well immunoplates overnight to replace the capture antibody in a standard sandwich ELISA. OVA or BSA was then added, and signal for bound OVA (A) and BSA (B) is shown versus expected value for the added concentration (dotted, horizontal lines). (C) 1e5 DCOVA were subsequently cultured with diluent; 1e6 OVA:DLE; or 1, 10, or 100 μg e-OVA (no DLE), and IL-6 concentrations were measured after 18–24 h. (D) 1e5 DCOVA cultured with 1e6 ceq of OVA:DLE extracted via ultrafiltration, dialysis, or both. (E) 1e6 DCSIINFEKL, DCOVA, or DCHKCA from C57BL/6 mice were incubated for 24 h with 1e6 ceq of DLE from OVA-immunized mice or unimmunized OT-I or OT-II mice. Change (Δ) in IL-6 compared with diluent condition is shown. (F) Ninety-six-well plates were coated with 1e5 ceq of OT-I, single OVA immunized (OVA:DLE), repeat OVA immunized (Boosted), or naïve:DLE. An ELISA was performed using anti-TCR-α, TCR-β, S100a9, or isotype control as primary antibodies. Absorbance at 405 nm for TCR chains and S100a9 minus the absorbance for isotype control are shown with significance versus naïve:DLE or as indicated. (G) 1e5 DCOVA were incubated with 1e6 ceq of OTI:DLE that had undergone antibody precipitation with antibodies to the TCR α chain (α)TCR-α, TCR-β, and/or S100a9. IL-6 production at 20 h is shown with significance versus the isotype condition (dotted, horizontal line). (H) IL-6 production from 1e6 DCOVA from C57BL/6 wild-type (WT) or TLR4 knockout (TLR4−/−) mice cultured with 5e4 ceq of DLE from CD8+/OVA tetramer-positive (Tet+) or tetramer-negative (Tet−) in the presence of anti-MHC-I or isotype control (Iso). Data shown are representative of 3 or more independent experiments, each using at least 3 mice/group, and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA.

Figure 9.. Proposed model of DLE activity.

Antigen-specific CD8+ T cells produce an antigen-specific activator of DCs. An antigen-binding region, likely derived from the TCR-β of the source CD8+ T cell, binds to the cognate antigen presented by DCs. Once bound by the DC, associated DLE molecules, such as S100a9-derived peptides or inosine/hypoxanthine, activate the DC to produce inflammatory cytokines, including IL-6. This cytokine response activates bystander memory CD8+ T cells in a contact- and TCR-independent manner, stimulating the production of IL-17 family cytokines.