Kathleen M. Heidler

Evidence for Immune Responses to a Self-Antigen in Lung Transplantation: Role of Type V Collagen-Specific T Cells in the Pathogenesis of Lung Allograft Rejection

We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col(V)), and that col(V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col(V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col(V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col(V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col(V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+CD25− and produced IFN-γ in response to col(V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col(V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo- and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col(V).

Anti-Type V Collagen Lymphocytes that Express IL-17 and IL-23 Induce Rejection Pathology in Fresh and Well-Healed Lung Transplants

Immunity to collagen V [col(V)] contributes to lung ‘rejection.’ We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well‐healed lung grafts have differential susceptibility to anti‐col(V)‐mediated injury; and expression of the autoimmune cytokines, IL‐17 and IL‐23, are associated with this process. Adoptive transfer of col(V)‐reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post‐transplantation. Immunhistochemistry detected col(V) in fresh and well‐healed isografts but not native lungs. Hen egg lysozyme‐reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL‐17 and IL‐23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL‐17 were upregulated in fresh, but not well‐healed isografts after transfer of col(V)‐reactive cells. These data show that IRI predisposes to anti‐col(V)‐mediated pathology; col(V)‐reactive lymphocytes express IL‐17 and IL‐23; and anti‐col(V)‐mediated lung disease is associated with local expression of IL‐17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long‐term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H‐2b mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3‐A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL‐17A, but not IL‐17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL‐10 transcripts and protein were increased only in non‐OB mice. Neutralizing IL‐17 prevented OB, down regulated acute rejection, and upregulated systemic IL‐10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL‐17A or augmenting IL‐10 could be therapeutic interventions to prevent OB.

Prevention of bronchiolitis obliterans in rat lung allografts by type V collagen-induced oral tolerance.

Background. We have reported that feeding type V collagen (col(V)) to lung allograft recipients induces immune tolerance that prevents acute lung allograft rejection. Repeated acute rejection is a risk factor for or associated with chronic rejection, known as bronchiolitis obliterans (BO), the leading cause of death in lung allograft recipients. The current study examines if col(V)-induced oral tolerance prevents BO. Methods. WKY rats (RT1l) were fed either col(V) or diluent before orthotopic transplantation of F344 (RT1lvl) lung allografts. No rats received any immunosuppression. At 10 weeks posttransplantation the time to onset of BO, delayed type hypersensitivity (DTH) responses to donor antigens, and col(V) were examined. In addition, proliferative responses of recipient T lymphocytes to donor antigens, and ability of recipient antigen presenting cells to present alloantigens in lung allografts were evaluated. Results. The data show that recipient rats have sustained DTH responses to donor antigens and col(V). T lymphocytes from col(V)-fed lung allograft recipients were unable to proliferate in response to donor antigens, but feeding col(V) had no effect on the presentation of donor alloantigens by recipient antigen presenting cells. All diluent fed rats developed BO, but only mild acute rejection (grade 2) was present in all rats fed col(V). Transforming growth factor (TGF)-&bgr; production was up-regulated systemically in col(V)-fed, but not diluent fed, lung allograft recipients, and neutralizing TGF-36 recovered the DTH response to donor antigens in col(V)-fed rats. Conclusions. Collectively these data show that col(V)-induces oral tolerance that prevents BO, and that tolerance may be mediated by systemic production of TGF-36.

Th-17, Monokines, Collagen Type V, and Primary Graft Dysfunction in Lung Transplantation

RATIONALE The pathogenesis of primary graft dysfunction (PGD), a serious complication of lung transplantation, is poorly understood. Human studies and rodent models have shown that collagen type V (col[V]), stimulates IL-17-dependent cellular immunity after lung transplantation. OBJECTIVES To determine whether patients with end-stage lung disease develop pretransplant col(V)-specific cellular immunity, and if so, the impact of this response on PGD. METHODS Trans-vivo delayed-type hypersensitivity (TV-DTH) assays were used to evaluate memory T-cell responses to col(V) in 55 patients awaiting lung transplantation. Pa(O(2))/Fi(O(2)) index data were used to assess PGD. Univariate risk factor analysis was performed to identify variables associated with PGD. Rats immunized with col(V) or irrelevant antigen underwent lung isografting to determine if prior anti-col(V) immunity triggers PGD in the absence of alloreactivity. MEASUREMENTS AND MAIN RESULTS We found that 58.8% (10/17) of patients with idiopathic pulmonary fibrosis, and 15.8% (6/38) of patients without idiopathic pulmonary fibrosis tested while on the wait list for a lung transplant were col(V) DTH positive. Col(V) reactivity was CD4(+) T-cell and monocyte mediated, and dependent on IL-17, IL-1beta, and tumor necrosis factor (TNF)-alpha. Pa(O(2))/Fi(O(2)) indices were impaired significantly 6-72 hours after transplantation in col(V)-reactive versus nonreactive patients. Univariate risk factor analysis identified only preoperative TV-DTH to col(V) and ischemic time as predictors of PGD. Finally, in a rat lung isograft model, col(V) sensitization resulted in significantly lower Pa(O(2))/Fi(O(2)), increased local TNF-alpha and IL-1beta production, and a moderate-to-severe bronchiolitis/vasculitis when compared with control isografts. CONCLUSIONS The data suggest that activation of innate immunity by col(V)-specific Th-17 memory cells represents a novel pathway to PGD after lung transplantation.

Differential Expression of Smad7 Transcripts Identifies the CD4+CD45RChigh Regulatory T Cells That Mediate Type V Collagen-Induced Tolerance to Lung Allografts

Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF-β that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4+CD45RChigh-RChigh) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1l) induces oral tolerance to lung allografts (F344-RT1lvl) by T cells that produce TGF-β. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RChigh cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4+CD45RClow (RClow) cells from tolerant rats and RChigh or RClow cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF-β enhanced proliferation in response to col(V) presented to tolerant RChigh, but not other cells. In contrast to other cells, only RChigh cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF-β-mediated signaling.

Flt3-Ligand, IL-4, GM-CSF, and Adherence-Mediated Isolation of Murine Lung Dendritic Cells: Assessment of Isolation Technique on Phenotype and Function

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8α expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both “lymphoid” (CD8α+) and “myeloid” (CD8α−). DCs from mice genetically deficient in CD8α expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8α− DCs from CD8α-sufficient mice are better APCs compared with CD8α+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.

Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs.

Lung allograft rejection is believed to be initiated by donor lung accessory cells, namely macrophages and dendritic cells, interacting with recipient lymphocytes leading to up‐regulated Th1 type (IFN‐γ) cellular immunity culminating in graft destruction. The purpose of this study was to determine the individual role of donor lung macrophages and dendritic cells in the rejection response. Utilizing a murine model that reproduces the immunology and histology of acute rejection, C57BL/6 mouse (I‐ab, H‐2b) lung dendritic cells (DC‐enriched lung cells), purified alveolar macrophages (I‐a‐negative macrophages), or various ratios of I‐a‐negative macrophages/DC were instilled into BALB/c mouse (I‐ad, H‐2d) lungs followed by an assessment of local IFN‐γ production and grading of rejection pathology. The data show that DC, and not I‐a‐negative macrophages, induced IFN‐γ production in recipient lungs. However, the local production of IFN‐γ was not always associated with histological changes characteristic of rejection pathology. In contrast to either cell type alone, instillation of C57BL/6 I‐a‐negative macrophages and DC, together, were required to induce rejection pathology in BALB/c lungs. In addition, the rejection response was dependent on interactions between donor I‐a‐negative macrophages and DC. J. Leukoc. Biol. 64: 578–586; 1998.

Metalloproteinase inhibition has differential effects on alloimmunity, autoimmunity, and histopathology in the transplanted lung.

Background. Upregulation of matrix metalloproteinases (MMPs) has been associated with chronic lung allograft rejection known as bronchiolitis obliterans syndrome. It has been suggested that MMP inhibition could prevent the rejection response. However, the effect of MMP inhibition on lung allograft rejection has not been reported. Methods. Utilizing a rat model of lung transplantation, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were overexpressed by gene therapy in F344 rat lung allografts prior to transplantation into WKY recipient rats. Separately, WKY rats that received F344 lung allografts were treated systemically with COL-3, a global MMP inhibitor. Results. TIMP-1 and TIMP-2 had differential effects on delayed type hypersensitivity (DTH) responses to donor antigens and type V collagen, an autoantigen involved in the rejection response. Neither TIMP-1 or TIMP-2 affected the onset of rejection pathology. COL-3 suppressed DTH responses to donor antigens and type V collagen, abrogated local production of tumor necrosis factor-&agr;, and interleukin-1&bgr;. Although it did not prevent rejection pathology, COL-3 (30 mg/kg) induced intragraft B cell hyperplasia suggestive of posttransplant proliferative disorder (PTLD). Conclusions. These data identify a complex role for MMPs and TIMPs in the immunopathogenesis of lung allograft rejection, and indicate their effects are not limited to matrix remodeling.

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

BackgroundDirect allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.MethodsUsing transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d) prior to transplanting into C57BL/6 mice (H-2b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).ResultsDepleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.ConclusionCollectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.