**8.1. Intravenous immunoglobulins (IVIG)**

The immunomodulatory effects of IVIG are multiple, and the exact mechanisms are not elucidated. However, effective alloantibody inhibition by IVIG was shown in the context of desensitization protocols only relying on high dose IVIG treatment (Jordan et al., 2003). IVIG inhibits mixed lymphocyte reactions and induces apoptosis mainly in B cells (Toyoda et al., 2004). There are numerous proposed mechanisms how IVIG exerts its immunomodulatory action. They include modification of circulating alloantibody concentration through induction of antiidiotypic circuits, antigen binding through the Fab part of the immunoglobulin mole‐ cule, Fc receptor-mediated interaction with antigen-presenting cells to block T- and B-cell activation, and inhibition of complement activity (Jordan et al., 2006).

In vivo, IVIG reduces the number of B cells and monocytes, and it reduces CD19, CD20 and CD40 expression by B cells, thereby modulating B-cell signaling (Jordan et al., 2003). IVIG inhibits binding of donor-reactive antibodies to target cells in ∼80% of patients, indicating that the presence of blocking antibodies might explain the efficacy of IVIG, although the mechanism is not known (Jordan et al., 2003). Billing and colleagues (Billing et al., 2008) studied Six pediatric renal transplant recipients with CAMR and gave them four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion. Median glomerular filtration rate during 6 months before intervention dropped by 25 (range, 11–26) mL/ min/1.73m2 (P<0.05) and increased in response to antihumoral therapy by 21 (-14 to+30) 6 months (P<0.05) and by 19 (-14 to+\_23) mL/min/1.73 m2 12 months (P=0.063) after start of treatment. Glomerular filtration rate improved or stabilized in 4 patients; the two non-responders had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in peritubular capillaries and pronounced interstitial inflammation. The treatment regimen was well tolerated. Another study conducted by Fehr and colleagues (Fehr et al., 2009) who reported four kidney allograft recipients suffering from chronic AMR 1 to 27 years post-transplant, who were treated with a combination of rituximab and intravenous immunoglobulin (IVIG) with improved kidney allograft function in all four patients, whereas donor-specific antibodies were reduced in 2 of 4 patients.

#### **8.2. Rituximab**

Rituximab, a chimeric monoclonal anti-CD20 antibody directed against B cells, prevents new antibody production by depletion of B cells as precursors of mature plasma cells in the circulation and the lymphoid tissue {although, some recent reports demonstrated that depletion in secondary and tertiary lymphoid structures is far less efficient and may not affect an ongoing localized humoral immune response (Genberg et al., 2006; Thaunat et al., 2008)}, prevention of B-cell proliferation, and induction of apoptosis and lysis of B cells through complement-dependent and -independent mechanisms (Salama & Pusey, 2006). Rituximab binds CD20 at the surface of precursor and mature B cells and leads to transient B-cell depletion, with typical B-cell recovery after 6–12 months in more than 80% of patients, although the degree of depletion is highly variable and is observed for up to 24 months in some individuals (Sureshkumar et al., 2007).An additional potential mechanism of action of rituximab is the direct targeting of CD20-positive cells that infiltrate the graft (Steinmetz et al., 2007). Preliminary studies indicate that rituximab decreases the concentration of pre-existing and post-transplantation antibodies (Gloor et al., 2003; Vieira et al., 2004). Conclusions and extrapolations from these studies are limited, because rituximab is usually combined with other therapies in these small and uncontrolled trials. The risk of bacterial infection as a result of immunoglobulin deficiency is also an important consideration. Based on the pathophysio‐ logic condition of this rejection process and efficacy of rituximab in B cells and antibodymediated autoimmune diseases (Eisenberg & Albert, 2006; Levesque & St Clair, 2008), a combination treatment with rituximab/IVIG represents a logical approach.

the number of immunoglobulin producing cells. By contrast, tacrolimus and cyclosporin marginally inhibited B-cell proliferation and immunoglobulin production, and the extent of

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While the B cell-depleting anti-CD20 antibody rituximab is increasingly incorporated in treatment protocols of humoral rejection (Faguer et al., 2007), this reagent is neither effective in eliminating antibody-producing plasma cells (PC) – either newly created from memory or naıïve B cells or from those that existed prior to transplant- nor does it decrease circulating antibody titers (Singh et al., 2009). For an effective blockade of alloantibody formation, a specific PC-depleting reagent would be desirable. Bortezomib (BZ), a selective inhibitor of the 26S proteasome, has been approved by FDA for the treatment of relapsed multiple myeloma. Mechanisms of BZ action include inhibition of NF-κ B and cytokine expression as well as induction of apoptosis as a result of activation of the terminal unfolded protein response (Meister et al., 2007). Susceptibility to BZ-induced apoptosis is related to the high immuno‐ globulin synthesis rate of PCs associated with accumulation of unfolded proteins/DRiPs inducing endoplasmatic reticulum stress (Meister et al., 2007). Moreover, BZ not only acted on the humoral response but also effectively inhibited the influx of MHC class II+ cells, mono‐ cytes/macrophages, CD8+ as well as CD4+ T cells. In animal models, Vogelbacher and colleagues (Vogelbacher et al., 2010) found that combination of Bortezomib and sirolimus inhibit the chronic active antibody-mediated rejection in experimental renal transplantation in the rat. In humans, data are lacking. In one case report, Bortezomib failed to treat CAMR

Perry and colleagues (Perry et al., 2009) described two sensitized patients with AMR treated in February 2007 using a combination of bortezomib and multiple plasmapheresis. Both patients had resolution of AMR and decreased serum DSA levels months after treatment. Neither developed transplant glomerulopathy. In a slightly different clinical setting, Everly and colleagues (Everly et al., 2008) used bortezomib to treat six patients who had combined AMR and cellular rejection occurring from 3 months to 7.5 years after transplant. All six patients showed resolution of AMR with a decrease in DSA levels after treatment. Unfortu‐ nately, three of the six patients developed transplant glomerulopathy. Flechner and coworkers (Flechner et al., 2010) treated 20 cases (16 kidney-only and 4 kidney-combined organ recipients) with AMR 19.8 months (range 1-71 months) posttransplant using a combined regimen of intravenous corticosteroids followed by a 2-week cycle on days 1-4-8-11 of plasmapheresis and 1.3 mg/m2 bortezomib; then 0.5 mg/kg intravenous immunoglobulin four times. They found that the bortezomib-containing regimen demonstrated activity in AMR but seems to be most effective before the onset of significant renal dysfunction (serum creatinine <3 mg/dL) or

Compared to rituximab, Waiser and colleagues (Waiser et al., 2012) found that patients with AMR treated with bortezomib had better graft survival At 18 months after treatment (P = 0.071) and renal function at 9 months was superior in patients treated with bortezomib as compared to rituximab-treated patients (P= 0.008). Whereas these early clinical experiences with protea‐

inhibition depended on the degree of the B-cell stimulation.

even after treatment with rituximab and IVIG.

proteinuria (<1 g/day).

**8.4. Bortezomib**

#### **8.3. Mycophenolic acid and sirolimus**

In a multicenter study, MMF in combination with cyclosporine resulted in significantly lower frequencies of HLA antibodies when compared with azathioprine and cyclosporine treatment (Terasaki & Ozawa, 2004). Moreover, MMF was described to be effective in inhibiting primary antigen-specific antibody responses in renal transplant patients (Rentenaar et al., 2002). Heidt et al (Heidt et al., 2008) stimulated purified human B cells devoid of T cells with CD40L expressing L cells, or by anti-CD40mAb with or without Toll-like receptor triggering, all in the presence of B-cell activating cytokines. These three protocols resulted in various degrees of Bcell stimulation. Then, they added four commonly used immunosuppressive drugs (tacroli‐ mus, cyclosporin, mycophenolic acid [MPA], and rapamycin) to these cultures and tested a variety of parameters of B-cell activity including proliferation, apoptosis induction, and both IgM and IgG production. They found that MPA was extremely potent in inhibiting both proliferation and immunoglobulin production. Moreover, these effects persisted when MPA was added to already activated B cells, implying that an ongoing B-cell response may be dampened by MPA, whereas calcineurin inhibitors are ineffective. MPA levels used are lower than levels that are usually achieved physiologically.

In the same in vitro experiments, rapamycin, like MMF, was described to be extremely potent in inhibiting humoral responses. Rapamycin was the most effective drug tested, as it inhibited not only B-cell proliferation and immunoglobulin production, but also inhibited the number of immunoglobulin producing cells. None of the other drugs tested were capable of decreasing the number of immunoglobulin producing cells. By contrast, tacrolimus and cyclosporin marginally inhibited B-cell proliferation and immunoglobulin production, and the extent of inhibition depended on the degree of the B-cell stimulation.
