John G. Gartner

Surveillance biopsies are superior to functional studies for the diagnosis of acute and chronic renal allograft pathology in children

Abstract:  In this report of our 3‐yr protocol biopsy program, we describe the evolution of acute rejection (AR) and chronic renal allograft nephropathy (CAN) in a cohort of 21 children treated with antibody induction, tacrolimus, mycophenolate mofetil, and prednisone. The aims of this study were to compare the pathogenicity of clinical acute rejection (CAR) and subclinical acute rejection (SAR), and to determine whether functional studies accurately represent acute and chronic renal allograft pathology in pediatric recipients with disproportionately large grafts. Using concurrent biopsies, we evaluated: (i) the utility of changes in the baseline sCr (ΔsCr) to predict both the onset of AR and the response to immunosuppressive therapy; and (ii) the relationship of the calculated creatinine clearance and the presence of pathologic proteinuria to the severity of CAN. We performed 112 biopsies: 11 donor, 73 protocol, 16 acute graft dysfunction and 12 1‐month follow‐up AR therapy. CAR and SAR were similar in incidence, timing and histologic severity. Progression of CAN was associated with the first episode of CAR (p < 0.02) and the cumulative number of episodes of CAR (p < 0.01), SAR (p < 0.05), CAR plus SAR (p < 0.002) and borderline SAR (B‐SAR) (p < 0.006). One‐month post‐treatment ΔsCrs could not distinguish 1‐month follow‐up biopsies with histologically confirmed worsened or unchanged AR from those with improved histology (35.2 ± 74.8% vs. 23.8 ± 24.9%, p = NS). These findings led to the addition of anti‐lymphocyte antibody therapy in five of 10 (50%) cases. Despite 100% 3‐yr actuarial graft survival and excellent function (GFR = 111 ± 36 mL/min/1.73 m2), 18 of 21 (86%) patients had grade I CAN or greater chronic histology at a mean ± sd follow‐up period of 18.2 ± 13.1 months. Thirteen of 21 (62%) patients progressed to grade I CAN at 5.2 ± 3.6 months and five (38%) of these patients progressed to grade II CAN at 17.8 ± 11.3 months. Schwartz GFR did not differ between patients with or without CAN (108 ± 38 mL/min/1.73 m2 vs. 127 ± 8 mL/min/1.73 m2, p = NS). In biopsies with CAN and no associated AR, neither the Banff chronic tubulointerstitial (Banff ci) score nor the Banff chronic grade correlated with the GFR. Proteinuria was not associated with CAN. Clinical AR and SAR are similar histologic lesions with a capacity for CAN progression. In pediatric renal transplant recipients, longitudinal protocol biopsies are superior to functional studies for the diagnosis and post‐therapeutic monitoring of AR and for the surveillance of CAN.

Effect of recombinant human keratinocyte growth factor (rHuKGF) on the immunopathogenesis of intestinal graft-vs.-host disease induced without a preconditioning regimen.

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6→(C57BL/6 X DBA/2)F1-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNFα, did not develop endotoxemia, and did not die. LPS augmented serum TNFα release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-α, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-γ mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-γ gene knockout grafts, suggesting that IFN-γ may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.

The role of interferon‐γ, nitric oxide and lipopolysaccharide in intestinal graft‐versus‐host disease developing in F1‐hybrid mice

(C57BL/6 × DBA/2)F1‐hybrid mice injected with lymphoid cells from wild‐type, C57BL/6 donors develop acute, lethal graft‐versus‐host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 μg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor‐α (TNF‐α) into the serum. To explore the role of interferon‐γ (IFN‐γ) in the pathogenesis of intestinal GVHD we used IFN‐γ gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild‐type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 μg LPS into recipients of wild‐type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of Nωnitro l‐arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN‐γ gko grafts. These findings indicate that donor‐derived IFN‐γ is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN‐γ gko grafts develop high levels of LPS‐induced TNF‐α release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent→F1‐hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF‐α, and that this depends on donor‐derived IFN‐γ.

Depletion of natural killer cells from the graft reduces interferon-gamma levels and lipopolysaccharide-induced tumor necrosis factor-alpha release in F1 hybrid mice with acute graft-versus-host disease.

BACKGROUND We wished to determine whether removal of NK1.1+ cells from the graft provides protection against acute graft-versus-host disease (GVHD) by obviating the Th1 immune response that underlies the development of this disease. METHODS Graft-versus-host (GVH) reactions were induced in two groups of (C57BL/6 x DBA/2)F1 hybrid mice. The first received grafts harvested from polyinosinic:polycytidylic acid-stimulated, C57BL/6 donors and depleted in vitro of NK1.1+ cells. This treatment provides protection against GVHD-associated mortality and cachexia. The second received unmodified grafts. We compared interferon-gamma and interleukin-10 production as well as the levels of engraftment in these two groups. Lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) release was also compared since TNF-alpha levels in GVH mice following injection of a sublethal dose of endotoxin provide an index of macrophage priming by Th1 cytokines. RESULTS Interferon-gamma production was absent in recipients of NK1.1-depleted grafts at the time when high levels were seen in recipients of unmodified grafts. Following lipopolysaccharide injection, high levels of TNF-alpha were observed in recipients of unmodified grafts, whereas negligible amounts were present in recipients of NK1.1-depleted grafts. The use of NK1.1-depleted grafts did not result in a reduced level of engraftment of CD4+ or CD8+ cells. CONCLUSIONS These results suggest that NK1.1 depletion of the graft confers protection against mortality by interfering with an immunoregulatory mechanism that results in the development of a Th1 response in GVH mice, and does not result in abortion of the graft. Because macrophage priming is prevented, recipients are also protected from the exaggerated sensitivity to endotoxin seen in mice with acute GVHD.

Murine graft-versus-host disease induced using interferon-γ-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia

Acute, lethal graft‐versus‐host disease (GvHD) develops in B6D2F1 hybrid recipients of wild‐type, C57BL/6, parental strain grafts; however, when interferon‐γ (IFN‐γ) gene knockout (gko) donors are used, the disease is prolonged and associated with a higher level of engraftment, particularly of T cells. Lesions containing large, mixed cellular infiltrates develop in the skin, liver, pancreas, salivary gland, lung and kidney. In our current study, we wished to determine whether GvHD features a preponderance of T helper 2 (Th2) cytokines in the absence of donor‐derived IFN‐γ, and whether autoantibody production, commonly associated with chronic GvHD, also occurs. Because mitogen responsiveness is consistently suppressed in mice with acute GvHD, we wished to measure this response in recipients of IFN‐γ gko grafts. Our findings indicate that spleen cells from the latter produce interleukin (IL)‐4, IL‐5 and IL‐13 in culture, but respond poorly to concanavalin A (Con A) and lipopolysaccharide (LPS). Their sera contain anti‐nuclear antibodies (ANA), some of which are specific for double‐stranded (ds)DNA and are predominantly immunoglobulin (Ig)M and IgG1. We also noted the presence of numerous eosinophils in the infiltrates developing within the target organs. In some respects, this syndrome bears resemblance to both systemic lupus erythematosus (SLE) and chronic GvHD. However, histological evidence of glomerulonephritis is lacking and proteinuria fails to develop in recipients of IFN‐γ gko grafts, suggesting that IFN‐γ may be necessary for the development of lupus nephritis. On a broader scope, our findings underscore the importance of IFN‐γ in the pathogenetic mechanism of GvHD, and demonstrate that the absence of this cytokine promotes the development of chronic GvHD and autoimmunity.

NATURAL KILLER (NK) CELL ACTIVITY IN MICE WITH ACUTE GRAFT-VERSUS-HOST REACTIONS : CHARACTERIZATION OF A THY-1+ NK-LIKE CELL WITH A BROADENED SPECTRUM OF LYTIC ACTIVITY IN THE SPLEEN AND LYMPH NODES

We have shown that acute (GVH) reactions produced in the parental Kt hybrid combination. A/J→(C57BL/6 × A/J) F1 result in the activation of two cytotoxic cell populations: a host‐derived Thy‐1+/‐ natural killer (NK) cell with a lytic spectrum confined to YAC‐1 targets, and a donor‐derived Thy‐1+ NK‐like cell that has the ability to lyse target cells that are normally insensitive to lysis by NK cells. Cold‐target inhibition (CTI) experiments have shown that the greater range of target cell killing seen in the NK‐like population is mediated by a single effector cell with a broadened lytic repertoire. Percoll density fractionation studies have revealed that NK and NK‐like cell co‐fractionate, suggesting that btith are large granular lymphocytes. We have also shown that NK‐like cells do not express either Lyt‐2 or L3T4 markers. We have also observed that there is a close temporal relationship between elevated levels of mterleukin‐2 (IL‐2) secretion by spleen cell cultures from mice with GVH disease and the subsequent emergence of splenic NK activity in both acute [A/J→(C57BL/6×A/J)F1] and chronic (A/J→ CBA×A/J)F1 GVH reactions. We have also noted that, despite high levels of IL‐2 secretion, mice with chronic GVH reactions do not generate NK‐like activity. Interferon (IFN) measurements have shown that, although increased IFN activity can be detected in both acute and chronic models, a preponderance of IFN‐α/β and some IFN‐γ is produced in the acute reaction, whereas only IFN‐γ can be detected in the chronic model. These results suggest that, although IL‐2 may participate in augmenting conventional NK activity. IL‐2 by itself does not generate NK‐like activity. We suggest that IFN‐α/β may be the cytokinc that, either alone or in concert with IL‐2, triggers the NK‐like cell response. The NK‐like cell described in our study resembles a phenotypically identical, donor‐derived large granular lymphocyte, identified by others, in close proximity to dead or dying epithelial cells in mice with GVH disease [14]. It has been suggested that these cells may mediate tissue injury. If in fact these graft‐derived NK‐like cells are involved in the pathogenesis of acute GVH disease, our present findings suggest that they must first be activated by an appropriate complement of cytokines that includes IFN‐α/β. We postulate that whether or not a GVH reaction pursues an acute, rapidly lethal, course depends not only on the cohort of effector cells in the graft, but also on the stimulatory cytokines released during the early, lymphoproliferative phase of the reaction.

Palifermin Mediates Immunoregulatory Effects in Addition to its Cytoprotective Effects in Mice with Acute Graft-Versus-Host Disease

IntroductionTreating recipient mice with palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal, graft-versus-host disease (GVHD). This is due, at least in part, to the ability of palifermin to protect epithelial cells from injury. Using the C57BL/6→(C57BL/6 × DBA/2)F1-hybrid model, we previously showed that the protective effect of palifermin was also associated with redirection of the cytokine profile from Th1 to Th2.DiscussionTo study this immunoregulatory effect more directly, we induced acute GVH reactions in which we treated the donors rather than the recipients with palifermin. The recipient mice were protected from GVHD-associated morbidity, and their cytokine profile was predominantly Th2. The palifermin-treated donor mice alone showed a similar Th2 cytokine profile, and we observed elevated levels of thymic stromal lymphopoietin mRNA in the thymus. We further demonstrated that treating the donor mice with palifermin protects against GVHD-associated morbidity, even if the donors are deficient in Vα14i natural killer T cells. Our findings clearly show that palifermin mediates immunoregulatory effects in addition to its cytoprotective effects and that both are likely to be involved in the mechanism through which palifermin provides protection from acute murine GVHD.

In vivo Direction of CD4 T Cells to TH1 and TH2-Like Patterns of Cytokine Synthesis

Factors that influence the initial development, and continued maintenance, of Th1 or Th2-like responses in vivo play a pivotal role in determining immune effector mechanisms and clinical outcome. Here, we review recent developments in this area with particular emphasis on (i) the ability of chemically modified exogenous antigens to preferentially activate Th1-dominated responses in vivo and (ii) the role played by NK cells in initial commitment of naive exogenous antigen-specific T cells to Th1 or Th2-like cytokine synthesis. We find that NK cell depletion of naive mice prior to immunization with OVA (which induces balanced Th0 like responses), or a high Mr polymer (that preferentially elicits OVA-specific Th1-dominated responses), fails to influence the development of cytokine or specific antibody responses. The results argue that NK cells do not play an essential role in shaping induction of immune responses to exogenous antigens, the most common class of inhalant allergen.

Lack of functional TSLP receptors mitigates Th2 polarization and the establishment and growth of 4T1 primary breast tumours but has different effects on tumour quantities in the lung and brain.

The 4T1 mammary carcinoma cell line produces TSLP. We had hypothesized that TSLP promotes the development of a permissive environment for the growth and metastasis of primary tumour and that this is associated with a Th2‐polarized antitumour immune response. We found that, in Tslpr−/− mice, the mean tumour diameters were smaller from days 27 to 40, and relatively fewer tumour cells were present in the lung, compared with wild‐type mice. Polarization of the Th2 cytokine profile was also diminished in Tslpr−/− mice. These findings confirmed those reported previously by others. Here, we further show that primary tumours are established less often in Tslpr−/− mice and that, unexpectedly, the relative number of tumour cells in the brain is greater in Tslpr−/− mice compared with wild‐type mice. Findings from our cytotoxicity assays show that 4T1‐directed lysis is undetectable in both WT and Tslpr−/− mice, ruling out the possibility that altered cytotoxic responses in Tslpr−/− mice are responsible for the differences we observed. In a human tissue microarray, positive staining for TSLP was seen in tumour cells from breast cancer tissue, but it was also seen in normal glandular epithelial cells from normal breast tissue, which has not been shown before. Thus, our findings provide new insight into the effects of TSLP in metastatic breast cancer.

Positive selection of NK1.1+ cells on a magnetic cell separator (MACS)

Natural killer (NK) cells are involved not only in resistance to tumors and infection, but also in some transplantation reactions. A specific, inexpensive method for purifying large numbers of NK cells is often required. All NK cells in H-2b mice express the surface marker NK1.1. We report a method for positively selecting NK1.1+ spleen cells from normal and Poly I:C-stimulated C56Bl/6 mice using a magnetic cell separation technique known as MACS. Our results show that cytotoxic activity directed at YAC-1 target cells by normal and Poly I:C-stimulated spleen cells could be increased five-fold using this method. We also found that spleen cells from mice given Poly I:C could lyse NK-resistant, BW1100 target cells, and that this activity could be increased two-fold. Flow cytometry analysis of Poly I:C-stimulated, MACS enriched, NK1.1+ spleen cells revealed the presence of two subpopulations: one consisting of LGL and the other consisting of smaller, agranular lymphocytes (SAL). After enrichment, the percentage of NK1.1+ spleen cells increased from 69% to 91% in the LGL subpopulation and from 33% to 73% in the SAL subpopulation. These results clearly demonstrate the effectiveness of the MACS technique for purifying large numbers of NK1.1+ cells for both flow cytometric and functional analyses.