Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life

Abstract

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

Keywords: T cell biology; basic (laboratory) research/science; cellular biology; cellular transplantation (non-islet); immunobiology; lymphocyte biology; rejection: chronic; translational research/science.

Figure 1. Col V but not Col I responses can be uncovered through depletion of CD39⁺ or CD25⁺ T cells in humans and mice

(A) Whole PBMCs (black), MACS separated monocytes plus T cell subsets (grey) or CD39⁻ depleted T cells and autologous monocytes (red) were tested for footpad swelling responses to Col V and Col I using the tvDTH assay. To test for cytokine-dependency, some footpads were injected with cells plus neutralizing antibody. In some experiments, CD39⁺ T cells were added back to the footpad-antigen mixture (red and black symbols). P values are shown for comparison with the Col I response or Col V response observed in PBMC group. Data from 2–14 individuals are shown. (B) Whole PBMC (black) or CD25 depleted T cells plus autologous monocytes (red) were tested for footpad swelling responses to Col V and Col I using the tvDTH assay. Data are from 4 individuals. (C) Separations of monocytes, whole T cells and CD25⁻ T cell populations was performed from CBA mouse spleens similar to that described in Methods. Following separations, groups of isolated total T cells plus autologous monocytes and CD25⁻ T cells plus autologous monocytes along with whole splenocyte samples were stimulated with Col I or Col V and used in the tvDTH assay. Results are from 8 pooled mice. Significant differences between groups were determined by Mann Whitney U tests (A, B) or ANOVA (C).

Figure 2

Col V specific IL-17 production from normal, healthy individuals is revealed when CD39⁺ T cells are removed. (A) Representative flow plots of IL-17 and IFNγ production from overnight cultures of whole PBMCs (top) or CD39⁻ T cells plus autologous monocytes (bottom) stimulated with Col I or Col V from a normal, healthy individual. Inset numbers represent percentage in each quadrant. (B and C) Quantified flow cytometry data from panel A indicating frequencies of IL-17 or IFNγ producing CD4 T cells from 7–10 normal, healthy individuals. P value represents significance vs PBMC-Col V stimulated control. (D) Representative histograms of TNFα, IL-22, PLZF-1, RORγt, CD45RO, CD161, CD39 and CD25 (blue) expression gated from total IL-17⁺ populations of Col V stimulated cells from the CD39⁻ Tcells plus monocyte groups. Red line represents isotype control (TNFα, IL-22, PLZF-1, RORγt, CD161), black line represents staining from CD3⁺/CD4⁺ T cells that are both IL-17 and IFNγ negative (CD45RO, CD39, CD25). Significant differences between groups were determined by ANOVA or Mann Whitney U tests.

Figure 3. Uncovering Col V responses via TGFβ, IL-35 and CD39 inhibition

In whole PBMCs from normal individuals, neutralizing antibodies to TGFβ (p<0.004), LAP (p<0.004), IL-35 (p<0.008) or chemical inhibition of CD39 activity (ARL67156, p<0.03) uncover Col V, responses. Neither CTLA-4 nor IL-10 caused a significant increase in the Col V response over that observed in whole PBMCs alone (P values not shown) or Col V plus IgG isotype (P values shown). Data are from 4–7 individuals. Significant differences between groups were determined by ANOVA or Mann Whitney U tests.

Figure 4

CD39⁺ T cell depletion uncovers reactivity to select transplant associated proteins in normal healthy individuals. (A) Multiple proteins were examined to determine if CD39⁺ T cell depletion uncovered cellular immune responses to these antigens. Self-antigens commonly associated with autoimmune disease (Col IV, myelin basic protein, cardiac myosin), DAMP activity (HMGB1), or prostate tumor immunity (PAP) yielded no response over PBMC ‘background’. However, three self-antigens that have been associated with transplant-induced autoimmunity (Col V, kα-1-tubulin, and vimentin) all elicited a strong response. * Represents significance (p<0.05) vs PBMC- stimulated value. Data are from 5–11 individuals. (B) Removal of CD39⁺ T cells uncovers a population of ColV, kα-1-tubulin or vimentin specific IL-17 producing CD4 T cells. Representative flow plots from two individuals of IL-17 and IFNγ production from whole PBMCs or CD39⁻ T cells plus monocytes treated overnight in the presence or absence of the indicated protein. Inset numbers represent observed percentage in each quadrant. (C) Quantified flow data from 5–7 normal, healthy individuals illustrating the corrected frequency of IL-17 (C) or IFNγ (D) positive CD4 T cells. Significant differences between groups (A-D) were determined by Mann Whitney U tests.

Figure 5

Cellular immune responses to Col V, vimentin and kα-1-tubulin are conserved in non-human primates. PBMCs from blood of 5–9 Rhesus Macaques were attained and responses to Col I, Col V, kα-1-tubulin and vimentin were investigated using TGFβ (25ug) neutralization-uncovering techniques. A significant increase in swelling was observed with Col V (p<0.02), kα-1-tubulin (p<0.01) and Vimentin (p<0.05) when TGFβ was neutralized vs IgG treated controls. Data are from 5–11 animals/group. Significant differences between groups were determined by Mann Whitney U tests.

Figure 6

Cellular immune responses to Col V, vimentin and kα-1-tubulin are present in human cord blood and fetal thymus. (A) Commercially available, cryopreserved, CD34 depleted cord blood samples were attained from 3 different donors and responses to Col I, Col V, kα-1-tubulin and vimentin were investigated using TGFβ neutralization strategies or pharmacological inhibition of CD39 activity with ARL67156. A dramatic increase in swelling was observed with Col V, kα-1-tubulin and Vimentin when either TGFβ was neutralized or CD39 enzymatic activity was inhibited (ARL67156) vs IgG treated controls. (B) Fetal thymus samples (16 and 17 weeks) from two individuals were attained along with autologous splenic wedge. Thymocytes were isolated and mixed with T cell depleted splenocytes (for antigen presentation) and Col I, Col V, or kα-1-tubulin with or without TGFβ neutralization.

Figure 7

Th17 cells respond in HLA-DR-restricted fashion to selected regions of the Col V chain, but not to synthetic 15mer peptides . (A) tvDTH responses to intact Col I or Col V by PBMCs from HLA-DR-15⁺ Col V reactive patients (grey), and by normal, healthy DR15⁺ individuals, in the absence (black) or presence of a TGFβ neutralizing antibody (white). (B) Responses of PBMC from DR-15⁺, Col V reactive patients (n=2) to DR-specific Col V-α-1 peptides (orange) or recombinant fragments (blue) of the collagenous domain of the α-1 V chain of Col V, with the indicated peptide contained therein. (C) Responses of PBMCs from normal, healthy DR-15⁺ individuals (n=3) in the presence of TGFβ neutralizing antibodies to the indicated Col V α1-peptide (checkered bar) or fragment (open bar). (D) Responses to intact Col I or Col V in whole PBMCs from a DR-1⁺ Col V reactive patient (grey), and by a normal, healthy DR1⁺ individual in the absence (black) or in the presence of a TGFβ neutralizing antibody (white). (E) Responses of PBMC from the DR-1⁺, Col V reactive patient, to DR-specific Col V-α-1 peptides (orange). Please note, fragments were not tested on this individual. (F) Responses of PBMC of the normal, healthy DR-1⁺ individual in the presence of TGFβ neutralizing antibody to the indicated Col V peptide (checkered bar) or fragment (open bar). All tvDTH experiments were repeated at least twice.