Jean Kwun

Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection

De novo donor‐specific antibody (DSA) after organ transplantation promotes antibody‐mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti‐CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti‐CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM+ B cells, bela‐ and 2C10R4‐added groups maintained a predominant population of IgM+ B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4+CD28+CD95+) as well as PD‐1hiCD4+ T cells were decreased in both bela‐added and 2C10R4‐added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC‐follicular helper T (Tfh) cells, and IL‐21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.

BAFF is increased in renal transplant patients following treatment with alemtuzumab

Alemtuzumab is a monoclonal antibody that depletes T and B cells and is used as induction therapy for renal transplant recipients. Without long‐term calcineurin inhibitor (CNI) therapy, alemtuzumab‐treated patients have a propensity to develop alloantibody and may undergo antibody‐mediated rejection (AMR). In pursuit of a mechanistic explanation, we analyzed peripheral B cells and serum of these patients for BAFF (Blys) and BAFF‐R, factors known to be integral for B‐cell activation, survival, and homeostasis. Serum BAFF levels of 22/24 alemtuzumab‐treated patients were above normal range, with average levels of 1967 pg/mL compared to 775 pg/mL in healthy controls (p = 0.006). BAFF remained elevated 2 years posttransplant in 78% of these patients. BAFF‐R on CD19+ B cells was significantly downregulated, suggesting ligand/receptor engagement. BAFF mRNA expression was increased 2–7‐fold in CD14+ cells of depleted patients, possibly linking monocytes to the BAFF dysregulation. Addition of recombinant BAFF to mixed lymphocyte cultures increased B‐cell activation to alloantigen, as measured by CD25 and CD69 coexpression on CD19+ cells. Of note, addition of sirolimus (SRL) augmented BAFF‐enhanced B‐cell activation whereas CNIs blocked it. These data suggest associations between BAFF/BAFF‐R and AMR in alemtuzumab‐treated patients.

Noninvasive detection of acute and chronic injuries in human renal transplant by elevation of multiple cytokines/chemokines in urine.

Background. Injuries in kidney transplant is currently diagnosed by needle biopsy. A noninvasive test that sensitively detects these injuries would benefit the patients. Methods. Urine samples were collected from healthy controls and kidney transplant recipients. Urine samples were screened first with an antibody array consisting of 120 chemokines and cytokines and then with a multiplex beads assay. Representative parameters, including macrophage inflammatory protein-1Δ, osteoprotegerin, monokine induced by interferon-γ (IFN), and IFN-γ–induced protein of 10 kDa, were simultaneously determined by a quadruplex assay in urine samples from 84 patients with renal allograft injury, 29 patients with stable graft function, and 19 healthy individuals. Results. Twenty-three cytokines/chemokines were found to be elevated in urine samples of patients with acute rejection by the antibody array. The second round of screening confirmed that 11 of the 23 parameters were elevated in the patients but not in the healthy controls. Induced protein of 10 kDa and monokine induced by IFN-γ were significantly elevated in urine samples of patients with acute renal injury, and macrophage inflammatory protein-1Δ and osteoprotegerin were significantly elevated in patients with both acute and chronic renal injuries. The combination of the four parameters had a high positive detection rate (97.6%) for renal transplant injury and could differentiate between acute and chronic injury. Conclusion. These results might indicate that the present multiplex assay provides a basis to establish a noninvasive method for the diagnosis and monitoring of renal transplant injury.

Early and Limited Use of Tacrolimus to Avoid Rejection in an Alemtuzumab and Sirolimus Regimen for Kidney Transplantation: Clinical Results and Immune Monitoring

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27–39 months of follow‐up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed‐type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3–4 ng/mL. One patient showed clinical signs of rejection at month 9 post‐transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor‐specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid‐free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1‐year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Developmental Exposure to Noninherited Maternal Antigens Induces CD4+ T Regulatory Cells: Relevance to Mechanism of Heart Allograft Tolerance

We hypothesize that developmental exposure to noninherited maternal Ags (NIMA) results in alloantigen-specific natural and adaptive T regulatory (TR) cells. We compared offspring exposed to maternal H-2d (NIMAd) with nonexposed controls. In vitro assays did not reveal any differences in T cell responses pretransplant. Adoptive transfer assays revealed lower lymphoproliferation and greater cell surface TGF-β expression on CD4+ T cells of NIMAd-exposed vs control splenocytes. NIMAd-exposed splenocytes exhibited bystander suppression of tetanus-specific delayed-type hypersensitivity responses, which was reversed with Abs to TGF-β and IL-10. Allospecific T effector cells were induced in all mice upon i.v. challenge with B6D2F1 splenocytes or a DBA/2 heart transplant, but were controlled in NIMAd-exposed mice by TR cells to varying degrees. Some (40%) NIMAd-exposed mice accepted a DBA/2 allograft while others (60%) rejected in delayed fashion. Rejector and acceptor NIMAd-exposed mice had reduced T effector responses and increased Foxp3+ TR cells (CD4+CD25+Foxp3+ TR) in spleen and lymph nodes compared with controls. The key features distinguishing NIMAd-exposed acceptors from all other mice were: 1) higher frequency of IL-10- and TGF-β-producing cells primarily in the CD4+CD25+ T cell subset within lymph nodes and allografts, 2) a suppressed delayed-type hypersensitivity response to B6D2F1 Ags, and 3) allografts enriched in LAP+, Foxp3+, and CD4+ T cells, with few CD8+ T cells. We conclude that the beneficial NIMA effect is due to induction of NIMA-specific TR cells during ontogeny. Their persistence in the adult, and the ability of the host to mobilize them to the graft, may determine whether NIMA-specific tolerance is achieved.

Unique aspects of rejection and tolerance in liver transplantation.

Spontaneous acceptance of liver allografts occurs in several species. However, tolerance is rare in human transplant patients even though rejection is relatively easily reversed. Histological features of acute rejection in liver transplantation are similar to those in other organs. Nevertheless, mechanisms of rejection of liver transplants may differ in degrees and cellular involvement. Liver-specific cell populations, such as Kupffer cells (KCs), liver sinusoidal epithelial cells (LSECs), and hepatic stellate cells (HSCs), may contribute to liver tolerogenicity. Other mechanisms, such as microchimerism, soluble major histocompatibility complex (MHC molecules), donor human leukocyte antigen (HLA)-C genotype, and regulatory T cells, may participate in inducing tolerance. The low incidence of hyperacute or antibody-mediated rejection in liver might be linked to the infrequency of chronic rejection of liver transplants. Understanding the mechanisms of liver transplant rejection/tolerance and the availability of better immune monitoring could help develop strategies to recognize tolerance and reduce rejection.

Overcoming Chronic Rejection—Can it B?

Despite the success of immunosuppressive drug therapy to reduce the incidence of acute rejection in organ transplantation, chronic rejection is still an impediment to long-term graft survival and tolerance. There is a growing body of evidence that B-cell production of alloantibody is an important element in the genesis of chronic rejection. Effector function of B cells in transplantation is not specifically targeted by current T-cell-directed therapeutic approaches. We briefly discuss the origin, animal models, diagnostic methods, and currently available B cell reagents that might be used in combination with existing immunosuppressive regimens to address B-cell-mediated allograft injury.

Unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of Cxcr3-/- mouse recipients.

Previous studies showed that absence of chemokine receptor Cxcr3 or its blockade prolong mouse cardiac allograft survival. We evaluated the effect of the CXCR3 receptor antagonist MRL‐957 on cardiac allograft survival, and also examined the impact of anti‐CXCR3 mAb in human CXCR3 knock‐in mice. We found only a moderate increase in graft survival (10.5 and 16.6 days, p < 0.05) using either the antagonist or the antibody, respectively, compared to control (8.7 days). We re‐evaluated cardiac allograft survival with two different lines of Cxcr3−/‐ mice. Interestingly, in our hands, neither of the independently derived Cxcr3−/‐ lines showed remarkable prolongation, with mean graft survival of 9.5 and 10.8 days, respectively. There was no difference in the number of infiltrating mononuclear cells, expansion of splenic T cells or IFN‐γ production of alloreactive T cells. Mechanistically, an increased other chemokine receptor fraction in the graft infiltrating CD8 T cells in Cxcr3−/‐ recipients compared to wild‐type recipients suggested compensatory T‐cell trafficking in the absence of Cxcr3. We conclude Cxcr3 may contribute to, but does not govern, leukocyte trafficking in this transplant model.

Surveillance of acute rejection in baboon renal transplantation by elevation of interferon-γ inducible protein-10 and monokine induced by interferon-γ in urine

Background. CXCR3 binding chemokines play a key role in recruitment of inflammatory cells into an organ transplant. This study addresses the question of whether urinary excretion of these chemokines correlates with acute rejection in a baboon kidney transplantation model. Methods. Seven outbred baboons underwent renal allotransplantation from major histocompatibility complex (MHC)-mismatched donors. The treatment of baboons consisted of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, rapamycin, and mycophenolate mofetil (MMF). Urinary levels of interferon-&ggr; inducible protein-10 (IP-10) and monokine induced by interferon-&ggr; (Mig) were determined by ELISA. Renal biopsies were examined by immunohistochemical staining for CXCR3 and Mig. Results. Urinary levels of IP-10 and Mig increased significantly in all of the five baboons at the time of acute rejection of renal transplant. The IP-10 and Mig levels did not rise in two nonrejecting baboons. In two baboons, urinary levels of IP-10 and Mig rose before the elevation of the serum creatinine. In renal biopsies, expression of Mig was detected in glomeruli, tubules, and infiltrating cells, and the expression was significantly elevated in biopsies with acute rejection (P<0.01). CXCR3 was constitutively expressed in tubular cells in biopsies derived from both normal grafts and grafts with acute rejection. Whereas the infiltrating cells were increased in the biopsies with acute rejection, the expression of CXCR3 was also significantly higher (P<0.01) in these infiltrating cells compared with those in the normal controls. Conclusions. This study shows an important correlation between urinary excretion of IP-10 and Mig and acute rejection in baboon kidney transplantation.

Neutralizing BAFF/APRIL With Atacicept Prevents Early DSA Formation and AMR Development in T Cell Depletion Induced Nonhuman Primate AMR Model

Depletional strategies directed toward achieving tolerance induction in organ transplantation have been associated with an increased incidence and risk of antibody‐mediated rejection (AMR) and graft injury. Our clinical data suggest correlation of increased serum B cell activating factor/survival factor (BAFF) with increased risk of antibody‐mediated rejection in alemtuzumab treated patients. In the present study, we tested the ability of BAFF blockade (TACI‐Ig) in a nonhuman primate AMR model to prevent alloantibody production and prolong allograft survival. Three animals received the AMR inducing regimen (CD3‐IT/alefacept/tacrolimus) with TACI‐Ig (atacicept), compared to five control animals treated with the AMR inducing regimen only. TACI‐Ig treatment lead to decreased levels of DSA in treated animals at 2 and 4 weeks posttransplantation (p < 0.05). In addition, peripheral B cell numbers were significantly lower at 6 weeks posttransplantation. However, it provided only a marginal increase in graft survival (59 ± 22 vs. 102 ± 47 days; p = 0.11). Histological analysis revealed a substantial reduction in findings typically associated with humoral rejection with atacicept treatment. More T cell rejection findings were observed with increased graft T cell infiltration in atacicept treatment, likely secondary to the graft prolongation. We show that BAFF/APRIL blockade using concomitant TACI‐Ig treatment reduced the humoral portion of rejection in our depletion‐induced preclinical AMR model.