A. Karasyov

Regulation of anti-HLA antibody-dependent natural killer cell activation by immunosuppressive agents.

Background It was demonstrated that human natural killer (NK) cells, via antibody-dependent cellular cytotoxicity (ADCC)-like mechanism, increase IFN&ggr; production after exposure to alloantigens. This finding was associated with an increased risk for antibody-mediated rejection (ABMR). Although the effects of various immunosuppressive drugs on T cells and B cells have been extensively studied, their effects on NK cells are less clear. This study reports the effect of immunosuppressive agents on antibody-mediated NK cell activation in vitro. Methods Whole blood from normal individuals was incubated with irradiated peripheral blood mononuclear cells (PBMCs) pretreated with anti-HLA antibody+ sera (in vitro ADCC), with or without immunosuppressive agents. The %IFN&ggr;+ and CD107a+ (degranulation marker) in CD56+ NK cells were enumerated by flow cytometry. Results Cyclosporine A and tacrolimus significantly reduced IFN&ggr; production in a dose-dependent manner (53%–83%), but showed minimal effect on degranulation (20%). Prednisone significantly reduced both IFN&ggr; production and degranulation (50%–66% reduction at maximum therapeutic levels). Calcineurin inhibitors (CNIs) in combination with prednisone additively suppressed IFN&ggr; production and degranulation. The effect of sirolimus or mycophenolate mofetil on NK cells was minimal. Conclusions These results suggest that potent suppressive effects of CNIs and prednisone on antibody-mediated NK cell activation may contribute to the reduction of ADCC in sensitized patients and possibly reduce the risk for ADCC-mediated ABMR. These further underscore the importance of medication compliance in prevention of ABMR and possibly chronic rejection, and suggest that ADCC-mediated injury may increase in strategies aimed at CNI or steroid minimization or avoidance.

IFNγ production by NK cells from HLA-sensitized patients after in vitro exposure to allo-antigens

Using a novel cytokine flow cytometry test (allo-CFC), we have previously shown that incubation of allogeneic cells with peripheral blood from highly-HLA sensitized (HS) patients results in reproducible gamma-interferon (IFNγ production in CD3(-) cells, and high (+) allo-CFC levels correlated with risk for antibody-mediated rejection (AMR). Here we report on identification of the cells and mechanisms responsible. The allo-CFC with/without modification was performed using blood from HS or normal individuals. IFNγ producing cells were CD3(-)/CD19(-), but CD3(-)/CD56(+). In vitro and in vivo B cell-depletion did not affect IFNγ production, demonstrating NK cells as the cells responsible for IFNγ production. NK cells from allo-CFC(+) or (-) individuals released significant amounts of IFNγ against target cells treated with serum from allo-CFC(+) individuals, but not allo-CFC(-) individuals. IFNγ release was abrogated by protein A/G treatment of the pretreated target cells, suggesting mediation by antibodies via FcγRIIIa (CD16). In conclusion, NK cell IFNγ release after allo-antigen exposure is mediated primarily through antibody-dependent cellular cytotoxicity (ADCC)-like mechanisms, suggesting that NK cells may be partially responsible for graft injury during AMR including C4d(-) AMR via ADCC, and could be a potential target for modification of this process.

Immunologic parameters and viral infections in patients desensitized with intravenous immunoglobulin and rituximab

Desensitization with IVIG and rituximab followed by transplantation with alemtuzumab or daclizumab induction is an effective clinical protocol. Here, we examined the effects of this protocol on immune cell number, T cell function by Cylex ImmuKnow®, CMV-specific CD8+ T cell (CMV-Tc) activity, total and viral-specific immunoglobulin levels and viral infections. In 17 highly HLA-sensitized (HS) patients who received desensitization, CD19+ cells were undetectable immediately after desensitization, while other immune cells were unchanged. No alteration in Cylex or CMV-Tc levels was seen. In separate 14 HS patients who were desensitized followed by transplantation, T cell numbers were near zero after alemtuzumab, while NK cell reduction was minimal. Early B cell recovery was not a risk for antibody-mediated rejection. Total IgG, IgM, and IgA remained in the normal range up to 12.6 months post-transplant, and CMV IgG level did not change. CMV-Tc activity was eliminated post-transplant in some patients, but recovered by 4 months post-transplant. None of them developed CMV infection. In conclusion, IVIG-rituximab-desensitization does not significantly alter T cell function pre-transplant, or reduce Ig levels below the normal range post-transplant. Although post-transplant induction therapy is associated with a transient depletion of viral-specific CD8+ memory cells, it does not increase risks for viral infections.

Genes associated with antibody-dependent cell activation are overexpressed in renal biopsies from patients with antibody-mediated rejection

INTRODUCTION Antibody-mediated rejection (ABMR) is dependent on complement activating donor-specific anti-HLA antibodies (DSA). This is commonly detected by C4d deposition in allografts. However, recent data define a C4d negative ABMR phenotype suggesting a role for complement-independent DSA injury, antibody-dependent cellular cytotoxicity (ADCC). METHODS Here, we established an in vitro ADCC model that identified human ADCC-activated genes using microarray analysis. We subsequently interrogated renal allograft biopsies from patients with ABMR and controls for mRNA expression of the ADCC-activated gene set. RESULTS We identified 13 ADCC-activated genes. Six gene expression assays including 8 of the 13 genes (CCL3, CCL4/CCL4L1/CCL4L2, CD160, IFNG, NR4A3 and XCL1/XCL2) were analyzed in 127 kidney biopsies obtained from HLA-sensitized (HS), non-HS patients and control individuals. Most ADCC-activated genes showed significantly higher expression in the transplant samples compared to the controls (p<0.0005). The gene expression levels were significantly higher in HS and non-HS transplant patients who developed ABMR compared to those who did not (p=0.04-0.002). There was no difference in the gene expression levels between C4d positive and negative ABMR (p=0.26-0.99). Samples from high PRA (>80%) or positive DSA patients showed higher gene expression levels for the ADCC-activated genes compared to low PRA (<80%) and negative DSA patients (p=0.04-0.001). CONCLUSION ADCC pathways are active in transplant patients with ABMR, and likely mediate allograft injury, providing a potential mechanism for C4d negative ABMR.

Significant Reduction of ATP Production in PHA-Activated CD4+ Cells in 1-Day-Old Blood from Transplant Patients

Background Global immunosuppression can be measured by assessing adenosine triphospate (ATP) levels in mitogen-stimulated CD4+ T cells. Methods We investigated the effect of storage time on ATP levels in 234 blood samples from 18 healthy individuals and 152 transplant patients. The difference between day 0 (<13 hours post-blood draw) and day 1 (24–37 hours) measurements was analyzed and compared with various factors; a subset of samples was also analyzed in 6-hour intervals. Results The ATP levels were significantly lower on day 1 compared with that on day 0 in healthy individuals (279±159 vs 414±159 ng/mL, P<0.001) and patients (356±209 vs 455±221 ng/mL, P<0.0001). Of the 18 healthy individuals, 17 showed ATP reduction, whereas 192 (89%) of 216 patients did so on day 1 (24.8±24.1%). In the time course analysis, ATP levels decreased with the blood storage time in healthy and patient samples, and the reduction began as early as 7 hours post-blood draw. The reduction rate was significantly higher in patient samples with low day 0 ATP levels compared with samples with moderate or high levels (44.7±31.3% vs 23.2±23.6% or 18.7±15.7%; P<0.001). The reduction rate in patients treated with alemtuzumab induction was slightly higher than that in daclizumab-treated patients (28.8±24.6% vs 21.3±21.3%, P=0.09). CD4+ cell number did not change within 24 hours post-blood draw, but CD4 expression decreased 2.0±2.8% (P<0.05). Conclusions The ATP levels are significantly lower in 1-day-old blood compared with fresh blood, suggesting that fresh blood should be used for assessing the T cell immune function to obtain the most accurate results.

Cellular allo reactivity against paternal HLA antigens in normal multiparous females as detected by intracellular cytokine flow cytometry remains elevated over years despite diminution of anti-HLA antibody levels

BACKGROUND Sensitization to allo-antigens (Ags) resulting from previous transplants, blood transfusion or pregnancy (PG), is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). We have previously shown that allo-Ag-specific CD3 negative (-) cells as analyzed by cytokine flow cytometry (CFC) are elevated in many highly HLA-sensitized (HS) patients, but not most normal individuals. HS patients with high(+) CFC, especially to donor Ags, may be at high risk for AMR and may need additional pre-transplant desensitization. Here, we investigate allo-sensitization that results from paternal HLA (pHLA) Ag exposure in normal multiparous females and assess the utility of the CFC assay in detecting allo-Ag-specific immune cell activity. METHODS Whole blood from 8 normal females with previous PG (pPG), 8 normal females without pPG and 5 normal males were incubated with irradiated normal peripheral blood mononuclear cells (PBMC). IFNgamma+/CD3- cells were enumerated and results expressed as a ratio against un-stimulated cells. RESULTS 4/8 females with pPG showed elevated CFC reactivity against at least 1 PBMC tested (ratio 4.7+/-1.4), while the remaining 4 (0.76+/-0.42), 8 females without pPG (0.65+/-0.24) and 5 males (0.99+/-0.59) showed minimal reactivity. The reactivity against the same PBMC was fairly consistent over months and years. Anti-HLA antibody was detected in females with elevated CFC, but the levels as analyzed by ELISA did not correlate with CFC reactivity. Individuals with minimal reactivity consistently show negative anti-HLA antibody levels. Two females with pPG with elevated CFC (#1 and #2) showed high reactivity to PBMC from their husbands, one offspring and 3rd-party normals carrying antigenic pHLA-Ags (#1: 13.2, 9.4 and 22.9; #2: 8.2, 8.0 and 8.8-12.6, respectively). Antibody specificity for antigenic pHLA-Ags was found by Luminex in anti-HLA antibody(+) samples in these 2 females. CONCLUSIONS 1) The CFC is a novel way to measure allo-specific CD3- cell responses and can detect allo-sensitization resulting from PG, 2) the CFC detects allo-specific memory cell activity as reactivity is consistent over years regardless of anti-HLA antibody levels, 3) high(+) CFC in normal multiparous females may explain increased risk for AMR in female HS patients, and 4) analysis of donor- or allo-specific CFC in multiparous females awaiting kidney transplant may identify those at risk for AMR.

Cytomegalovirus Immunity After Alemtuzumab Induction in Desensitized Kidney Transplant Patients

Background Desensitization with IVIG + rituximab combined with alemtuzumab induction gives HLA-sensitized patients an opportunity for successful kidney transplantation. However, it may be associated with a high risk for viral infections due to combined T cell and B cell depletion. Methods Anti-cytomegalovirus (CMV) activity was assessed in 280 pretransplant and posttransplant blood samples from 33 desensitized patients who received alemtuzumab induction. CMV-specific CD8+ (CMV-Tc), CD4+ (CMV-Th) T cell activity, and natural killer (NK) cell number were measured by flow cytometry. Anti-CMV IgG was measured by enzyme-linked immunosorbent assay, and CMV DNA by polymerase chain reaction. Results All 30 CMV sero (+) patients were (+) for CMV-Tc and/or Th predesensitization, while 3 sero (−) patients showed no CMV-T cell activity. CMV-Tc and/or Th became (−) in 50% to 70% of these sero (+) patients at 1 month post-alemtuzumab. However, 75% showed CMV-T cell (+) by 2 months and 95% did so by 3 months post-alemtuzumab. More than 50% of pretranslpant NK cell levels were detected post-alemtuzumab. Anti-CMV IgG levels did not decrease posttransplant in sero (+) patients. Four patients developed CMV viremia with clearance by 1.2 months, which correlated with an increase or appearance of CMV-T cells, even in the sero (−) patient. Conclusions CMV-T cell activity, anti-CMV IgG, and NK cell-mediated antibody-dependent cell cytotoxicity were present in aleumtuzumab-treated CMV sero (+) patients. One sero (−) patient developed CMV-T cell responses post-CMV viremia. These results suggest that the IVIG + rituximab desensitization combined with alemtuzmab induction with triple immunosuppression maintenance does not result in prolonged suppression of anti-CMV immunity or increased risk for CMV infection.