Carol Bentlejewski

Immune cell function testing: An adjunct to therapeutic drug monitoring in transplant patient management

Abstract:  Each year, 55 000 organ transplants are performed worldwide. Cumulatively, the number of living organ recipients is now estimated to be over 300 000. Most of these transplant recipients will remain on immunosuppressive drugs for the remainder of their lives to prevent rejection episodes. Controlled doses of these drugs are required to prevent over‐medication, which may leave the patient susceptible to opportunistic infection and drug toxicity effects, or under‐dosing, which may lead to shortened graft survival because of rejection episodes.

Immunogenicity and immunomodulatory effects of amnion-derived multipotent progenitor cells

This is the first study on the immunologic properties of a clinically relevant population of cells derived from the amnion of human placenta. Unlike other cells from the amnion, these amnion-derived multipotent progenitor cells (AMP cells), from human amnion, grow in serum-free conditions and have never been cultured in the presence of medium containing animal-derived components. This study reports the immunologic characteristics of AMP cells and their roles as immunomodulators. Characterization of AMP cells revealed the presence of major histocompatibility complex (MHC) class I but the lack of class II antigens and absence of co-stimulatory molecules B7-1 and B7-2. The nonclassical human leukocyte antigen (HLA)-G was expressed at low levels on cultured AMP cells. Expression was significantly increased after interferon-gamma (IFN-gamma) treatment. Cultured peripheral blood mononuclear cells did not respond to irradiated AMP cells, indicated by lack of proliferation as measured by standard mixed lymphocyte reaction. Culturing AMP cells with IFN-gamma did not reverse this result and did not upregulate class II expression. The AMP cells were shown to have immunomodulatory capabilities by inhibiting peripheral blood mononuclear cell proliferative responses to mitogen, alloantigen, and recall antigen, but the AMP cells were unable to inhibit preactivated T-cell blast response to growth factor media. This immunomodulatory effect of AMP cells was found to be dependent on cell-to-cell contact.

IgG Donor-Specific Anti-Human HLA Antibody Subclasses and Kidney Allograft Antibody-Mediated Injury

Antibodies may have different pathogenicities according to IgG subclass. We investigated the association between IgG subclasses of circulating anti-human HLA antibodies and antibody-mediated kidney allograft injury. Among 635 consecutive kidney transplantations performed between 2008 and 2010, we enrolled 125 patients with donor-specific anti-human HLA antibodies (DSA) detected in the first year post-transplant. We assessed DSA characteristics, including specificity, HLA class specificity, mean fluorescence intensity (MFI), C1q-binding, and IgG subclass, and graft injury phenotype at the time of sera evaluation. Overall, 51 (40.8%) patients had acute antibody-mediated rejection (aABMR), 36 (28.8%) patients had subclinical ABMR (sABMR), and 38 (30.4%) patients were ABMR-free. The MFI of the immunodominant DSA (iDSA, the DSA with the highest MFI level) was 6724±464, and 41.6% of patients had iDSA showing C1q positivity. The distribution of iDSA IgG1-4 subclasses among the population was 75.2%, 44.0%, 28.0%, and 26.4%, respectively. An unsupervised principal component analysis integrating iDSA IgG subclasses revealed aABMR was mainly driven by IgG3 iDSA, whereas sABMR was driven by IgG4 iDSA. IgG3 iDSA was associated with a shorter time to rejection (P<0.001), increased microcirculation injury (P=0.002), and C4d capillary deposition (P<0.001). IgG4 iDSA was associated with later allograft injury with increased allograft glomerulopathy and interstitial fibrosis/tubular atrophy lesions (P<0.001 for all comparisons). Integrating iDSA HLA class specificity, MFI level, C1q-binding status, and IgG subclasses in a Cox survival model revealed IgG3 iDSA and C1q-binding iDSA were strongly and independently associated with allograft failure. These results suggest IgG iDSA subclasses identify distinct phenotypes of kidney allograft antibody-mediated injury.

Re-examination of the Lymphocytotoxic Crossmatch in Liver Transplantation: Can C4d Stains Help in Monitoring?

C4d‐assisted recognition of antibody‐mediated rejection (AMR) in formalin‐fixed paraffin‐embedded tissues (FFPE) from donor‐specific antibody‐positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO– incompatibility status, histopathology and clinical–serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO‐incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection‐related causes of allograft dysfunction in DSA– and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA– recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

Pancreas transplantation under alemtuzumab (Campath-1H) and tacrolimus: Correlation between low T-cell responses and infection

Background. Alemtuzumab induction and tacrolimus-based immunosuppression has been effective in pancreas transplantation. Despite the encouraging results of this minimalistic approach to immunosuppression, infection still remains a significant cause of morbidity. The Cylex ImmunoKnow assay was used in this study to compare pancreas recipient clinical states (stable, rejection, infection) with T cell responses. Methods. Blood samples were taken from pancreas recipients pretransplant and at approximately three-month intervals posttransplant for analysis of T cell responses. When possible, T cell responses were also quantified during changes in clinical status (infection or rejection). Results. A range between 100–300 ng/ml adenosine triphosphate (ATP) was found in stable patients (mean 194±123, n=51) with good graft function and no infection or rejection. A low T cell response was highly correlated with infectious states. The fourteen patients with infections/posttransplant lymphoproliferative disease had a mean ATP of 48 ng/ml. Risk hazard analysis showed that patients with ATP levels <100 ng/ml were four to seven times more susceptible to infection compared to stable patients. Four patients with rejection showed a T cell response of 550 ng/ml ATP, which was statistically significant compared to stable patients, although the sampling numbers (9) were too small to be conclusive. Conclusion. The Cylex ImmunoKnow assay is a valuable tool to more precisely modulate immunosuppression in pancreas transplant patients. In particular, the assay is extremely useful in detecting overly immunosuppressed patients vulnerable to infections.

Value of Donor–Specific Anti–HLA Antibody Monitoring and Characterization for Risk Stratification of Kidney Allograft Loss

The diagnosis system for allograft loss lacks accurate individual risk stratification on the basis of donor-specific anti-HLA antibody (anti-HLA DSA) characterization. We investigated whether systematic monitoring of DSA with extensive characterization increases performance in predicting kidney allograft loss. This prospective study included 851 kidney recipients transplanted between 2008 and 2010 who were systematically screened for DSA at transplant, 1 and 2 years post-transplant, and the time of post-transplant clinical events. We assessed DSA characteristics and performed systematic allograft biopsies at the time of post-transplant serum evaluation. At transplant, 110 (12.9%) patients had DSAs; post-transplant screening identified 186 (21.9%) DSA-positive patients. Post-transplant DSA monitoring improved the prediction of allograft loss when added to a model that included traditional determinants of allograft loss (increase in c statistic from 0.67; 95% confidence interval [95% CI], 0.62 to 0.73 to 0.72; 95% CI, 0.67 to 0.77). Addition of DSA IgG3 positivity or C1q binding capacity increased discrimination performance of the traditional model at transplant and post-transplant. Compared with DSA mean fluorescence intensity, DSA IgG3 positivity and C1q binding capacity adequately reclassified patients at lower or higher risk for allograft loss at transplant (category-free net reclassification index, 1.30; 95% CI, 0.94 to 1.67; P<0.001 and 0.93; 95% CI, 0.49 to 1.36; P<0.001, respectively) and post-transplant (category-free net reclassification index, 1.33; 95% CI, 1.03 to 1.62; P<0.001 and 0.95; 95% CI, 0.62 to 1.28; P<0.001, respectively). Thus, pre- and post-transplant DSA monitoring and characterization may improve individual risk stratification for kidney allograft loss.

Liver transplant recipients weaned off immunosuppression lack circulating donor-specific antibodies

Human leukocyte antigen (HLA)-specific antibodies (Abs) were examined in 73 clinically stable liver transplant recipients divided into group A (n = 19; clinically tolerant), group B (n = 34; undergoing weaning, on minimal immunosuppression), and group C (n = 20; had failed drug withdrawal or weaning never attempted). Of 19 patients in group A, six (32%) had anti-HLA Abs; none were donor-specific. In contrast, 23 of 34 patients (67%) in group B and nine of 20 patients (45%) in group C exhibited anti-HLA Abs (p = 0.02). Furthermore, 15 of 19 patients in groups B and C (9/12, p = 0.01 and 6/7, p = 0.01, respectively) exhibited donor-specific anti-HLA Abs. The prevalence of donor-specific HLA Abs was significantly higher in nontolerant patients. Five years after initial evaluation, >90% (18/19) group A patients remained off immunosuppression. One of seven of these patients available for retesting exhibited donor-specific Abs. In group B, two-fourths of 34 patients (12%) weaned successfully were HLA-Ab negative; four patients who experienced rejection while undergoing weaning exhibited anti-HLA Ab initially and at 5 years. Thus, most of the liver recipients off immunosuppression lacked donor-specific alloAbs. The occurrence of these alloAbs should now be examined prospectively in a drug weaning trial.

Three years of follow-up of bone marrow-augmented organ transplant recipients: The impact on donor-specific immune modulation ☆

The discovery of the presence of previously unsuspected microchimerism in successful longterm liver and kidney transplant recipients prompted us to postulate that these cells are essential for graft acceptance and the induction of donor-specific hyporeactivity.1–3 This observation provided the basis for the initiation of a new therapeutic strategy which involved infusion of donor bone marrow (BM) cells under conventional immunosuppressive treatment with tacrolimus and prednisolone.4 The initial outcome of sequential in vitro immunologic evaluations performed to determine the development of donor-specific hyporeactivity in the first 15 BM-augmented and 16 contemporaneous controls has been described previously.5,6 We report here the immune profile of 102 BM-augmented and 57 control patients who were at least 6 months posttransplantation.

Enhanced Donor‐Specific Alloreactivity Occurs Independently of Immunosuppression in Children with Early Liver Rejection

To determine whether early acute cellular rejection (ACR) is associated with sub‐optimal immunosuppression in children with liver transplants (LTx). Methods: Twenty‐five children with primary LTx after pre‐transplant rabbit anti‐thymocyte globulin (rATG), and steroid‐free tacrolimus (TAC) were evaluated. Mitogen‐stimulated T‐ and B‐cell responses and mixed lymphocyte response to donor and third‐party antigens were performed at several time points between two consecutive TAC doses. TAC concentrations (C) associated with half‐maximal effect (EC50) on lymphocytes was determined by pharmacodynamic equations. Results: Mean age was 7.2 ± 6.2 years, mean time to lymphocyte function studies was 25 ± 19 days. Acute rejection occurred at a mean interval of 31 ± 19 days after LTx. Rejectors (n = 16) demonstrated significantly higher EC50 of TAC for the intra‐cellular IFN‐γ in T cells (p = 0.005) and its CD8+ sub‐population (p = 0.027) as well as the co‐stimulatory/activation receptor CD54 on B cells (p = 0.0001). The response of recipient lymphocytes to donor antigen was significantly higher in rejectors, compared with non‐rejectors (p = 0.015). The patient groups demonstrated no differences in third‐party MLR, or in C of TAC. Conclusions: Independent of the amount of immunosuppressant, ACR of liver allografts in children is associated with enhanced donor‐specific alloreactivity. This is accompanied by a cytotoxic T‐cell sub‐population with increased requirement for TAC.

Allospecific CD154+ B cells associate with intestine allograft rejection in children.

Background. As a significant determinant of T- and B-cell cooperation, CD154 has been used to identify allospecific T-cytotoxic memory cells (TcM) for rejection risk assessment with high sensitivity or specificity but not for alloreactive B-cells, especially among recipients predisposed to acute cellular and humoral rejection, that is, children with intestinal transplantation (ITx). Methods. Single blood samples from 32 pediatric ITx after lymphocyte depleting induction therapy were obtained within 30 days of protocol biopsies. Samples were assayed for allospecific CD154+CD19+ B cells and allospecific CD154+ TcM in 16-hr live-cell mixed leukocyte reaction using multiparametric flow cytometry. Results were expressed as the immunoreactivity index (IR) or the ratio of donor- to third-party-induced CD154+ B cells or TcM. The rejection threshold IR of B cells was determined by logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses. Results. Biopsy-proven acute cellular rejection was present in 15 subjects (rejectors) and absent in 17 (nonrejectors). In archived serum samples from 16 of 32 subjects donor-specific anti-HLA antibodies (DSA) were assayed by Luminex bead array. DSA were absent in all 7 nonrejectors but present in 7 of 9 rejectors. The IR of allospecific CD154+CD19+ B cells more than or equal to 1.351 was associated with rejector status and was present in 13 of 15 rejectors (sensitivity 87%) and absent in 15 of 17 nonrejectors (specificity 88%). Excellent correlations were seen between CD154+CD19+ B cells and CD154+ TcM (Spearman &rgr;=0.647, P=0.0001) but could not be tested independently for DSA, which was highly correlated with rejector status and with CD154+ TcM. Conclusions. Allospecific CD154+CD19+ B cells identify rejection-prone children with ITx and can likely substitute for T-cell alloreactivity in estimating rejection risk in this rare subject population.