Kathryn Brown

Pivotal role of CD4+ T cells in renal fibrosis following ureteric obstruction

Tubulointerstitial fibrosis is a common consequence of a diverse range of kidney diseases that lead to end-stage renal failure. The degree of fibrosis is related to leukocyte infiltration. Here, we determined the role of different T cell populations on renal fibrosis in the well-characterized mouse model of unilateral ureteric obstruction. Depletion of CD4(+) T cells in wild-type mice with a monoclonal antibody significantly reduced the amount of interstitial expansion and collagen deposition after 2 weeks of obstruction. Reconstitution of lymphopenic RAG knockout mice with purified CD4(+) but not CD8(+) T cells, prior to ureteric obstruction, resulted in a significant increase in interstitial expansion and collagen deposition. Wild-type mice had significantly greater interstitial expansion and collagen deposition compared with lymphopenic RAG(-/-) mice, following ureteric obstruction; however, macrophage infiltration was equivalent in all groups. Thus, our results suggest that renal injury with subsequent fibrosis is likely to be a multifactorial process, with different arms of the immune system involved at different stages. In this ureteric obstruction model, we found a critical role for CD4(+) T cells in kidney fibrosis. These cells could be a potential target of therapeutic intervention to prevent excessive fibrosis and loss of function due to renal injury.

Homeostatic Proliferation of Lymphocytes Results in Augmented Memory-Like Function and Accelerated Allograft Rejection

Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3+ regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.

Tertiary lymphoid organs in renal allografts can be associated with donor-specific tolerance rather than rejection

Tertiary lymphoid organs can form at sites of chronic inflammation. Their presence has been mainly associated with tissue destruction. In transplantation, there is a dynamic immune response as in chronic inflammation. Indeed, the presence of tertiary lymphoid organs has been associated with chronic rejection. In addition to a destructive alloimmune response, secondary lymphoid organs are also important in transplant tolerance. We hypothesised that tertiary lymphoid organs may also form during transplantation tolerance as this process also requires an active local immune response. If so, their presence may enhance tolerance, resulting in better graft function rather than chronic rejection. Using a mouse kidney allograft model of tolerance, we demonstrate the formation of tertiary lymphoid organs within tolerated allografts. Tertiary lymphoid organs are supplied by high endothelial venules, and contain T and B cells, macrophages, DC, Foxp3+ T cells, donor MHC class II+ cells and recipient cells presenting donor‐derived allopeptides. Formation of tertiary lymphoid organs and the presence of immune cells within them are associated with superior graft function, suggesting that tertiary lymphoid organs act to amplify the prevailing immune response, be it a tolerant and beneficial immune response or the previously described destructive alloimmunity.

Extensive and bidirectional transfer of major histocompatibility complex class II molecules between donor and recipient cells in vivo following solid organ transplantation

Intercellular transfer of surface molecules has been demonstrated in vitro, or in vivo under artificial situations. Transplantation is a unique clinical situation in which foreign major histocompatibility complex (MHC) molecules are deliberately introduced. This provides a model to study intercellular MHC transfer because donor MHC molecules can easily be tracked. Here we describe the bidirectional transfer of MHC class II molecules between donor and recipient cells after transplantation of vascularized kidney and cardiac allografts in mice. Cells that are positive for both donor and recipient MHC class II accounted for up to 30% of the donor MHC class II+ population, suggesting that they play a significant role in the antigen presentation process. The majority of these cells were dendritic cells, but macrophages and B cells were also able to acquire foreign MHC molecules. Most doublepositive cells were also positive for costimulatory molecules, indicating a capability to elicit a T‐cell response. This transfer of MHC molecules between donor and recipient cells provides a link between the direct and indirect pathways of alloantigen presentation and suggests that MHC transfer is also likely to occur under normal physiological conditions, which has implications in the fields of infection, vaccination, and tumor immunology.— Brown, K., Sacks, S. H., Wong, W. Extensive and bidirectional transfer of major histocompatibility complex class II molecules between donor and recipient cells in vivo following solid organ transplantation. FASEB J. 22, 3776–3784 (2008)

Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation

Experimental unilateral ureteral obstruction (UUO) is widely used to study renal fibrosis; however, renal injury can only be scored semiobjectively by histology. We sought to improve the UUO model by reimplanting the obstructed ureter followed by removal of the contralateral kidney, thus allowing longitudinal measurements of renal function. Mice underwent UUO for different lengths of time before ureteral reimplantation and contralateral nephrectomy. Measurement of blood urea nitrogen (BUN) allows objective evaluation of residual renal function. Seven weeks after reimplantation and contralateral nephrectomy, mean BUN levels were increased with longer duration of UUO. Interstitial expansion, fibrosis, and T-cell and macrophage infiltration were similar in kidneys harvested after 10 days of UUO or following 10 weeks of ureter reimplantation, suggesting that the inflammatory process persisted despite relief of obstruction. Urinary protein excretion after reimplantation was significantly increased compared to control animals. Our study shows that functional assessment of the formerly obstructed kidney can be made after reimplantation and may provide a useful model to test therapeutic strategies for reversing renal fibrosis and preserving or restoring renal function.

Coexpression of Donor Peptide/Recipient MHC Complex and Intact Donor MHC: Evidence for a Link between the Direct and Indirect Pathways

T lymphocytes recognize foreign antigens presented by donor or recipient cells through the direct and indirect pathways respectively. This raises the question of how directly and indirectly activated T cells interact. A 4‐cell model involving the interaction of CD4+ and CD8+ T cells recognizing major histocompatibility complex (MHC) class II on recipient antigen presenting cell (APC), and MHC class I on donor APC, has been proposed. However, this would require complex co‐ordination between all the participating cell types. The semidirect pathway of alloantigen presentation suggests a simpler mechanism. Although exchange of MHC class II molecules between donor and recipient cells has been described, coexpression of recipient MHC molecules presenting donor derived allopeptides (indirect presentation) and donor MHC (direct presentation) on the same cell, a key requirement for the semidirect alloantigen presentation pathway, has not been demonstrated. We have used a mouse transplantation model to demonstrate the presence of cells expressing both donor MHC class I/II molecules, and a donor MHC class II peptide in the context of a recipient MHC class II molecule. This would allow indirectly activated CD4+ T cells to regulate directly activated CD4+ T cells, or to help directly activated CD8+ T cells, thus providing physical evidence for the semidirect pathway.

Diagnostic value of regulatory T cells: a new facet of a much studied cell population.

FOXP3 has emerged as the most suitable marker for Treg and has been studied in both experimental and clinical transplantation. Biopsy samples have been stained for FOXP3+ cells whereas FOXP3 messenger RNA levels have been measured in blood and urine samples after transplantation. Although not unanimous, some studies have suggested that FOXP3 is associated with better outcome and can also serve as a marker of rejection. Routine staining of FOXP3+ cells in biopsy samples and FOXP3 messenger RNA levels in urine of kidney transplant patients may determine each patient’s immunological profile, enabling less immunosuppression to be given to those at lower risk.

Donor specific transplant tolerance is dependent on complement receptors

The complement system has recently been described as a crucial component for transplant tolerance induction, but the underlying mechanisms are poorly understood. Using a rodent model of donor lymphocyte infusion‐induced male histocompatibility antigen‐specific transplant tolerance, we demonstrate that tolerance induction is dependent on the complement receptors decay accelerating factor, complement receptor 3, and complement component 3a receptor (C3aR). Furthermore, we have provided evidence that complement dependent tolerance is mediated through C3aR on infused donor splenocytes and on recipient cells. Ex vivo studies showed that C3aR deficiency leads to an imbalance between T regulatory and T effector cells. Increased numbers of antigen‐specific CD8+ cells in the blood and less T regulatory cells, with reduced suppressive function, in the spleen and in the skin grafts were detected in C3aR deficient compared to wild type mice. This imbalance might be explained by the requirement of complement for dendritic cells to generate T regulatory cells effectively. Our experiments suggest that multiple complement receptors play an important role in transplant tolerance induction providing new insights into the mechanisms of complement dependent tolerance.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients’ during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

What Have We Learnt from Experimental Renal Transplantation

The first series of kidney transplantation performed in the experimental setting over a century ago and its subsequent translation into humans has initiated a whole new facet of medical practice that has benefited a large number of patients with end-stage kidney and other organ failure. It has proven to be an indispensable tool in our quest to advance our skills and knowledge and continues to play a role in the development of better treatment protocols. Here, we discuss the advantages and drawbacks of this technique and use key examples to illustrate how it has been exploited to achieve our goals.