An Update on Alloantibody and Post-Transplant Failure

There have been a number of studies reported in the past month which broaden our understanding of the complexity of alloantibodies post kidney transplant. It is well recognized that the presence of donor specific anti-HLA antibodies (DSA) infers a worse allograft prognosis, both in terms of early antibody mediated rejection (AMR) and graft survival. It is also becoming clear, from groups such as the DeKAF study investigators, that most causes of late graft failure are due to chronic AMR. Treatment options for this entity are unfortunately dismal at present. Our current understanding of immunological risk is somewhat simple and DSA alone do not fully explain a patients’ risk of AMR events. AMR can occur without DSA, presumably due to non-HLA antibodies (see NEJM MICA antibodies paper). Moreover, the presence of DSA alone does not necessarily associate with immunological injury. Recent studies help with our understanding of these important points.

Non-HLA antibodies

Two reports in the current edition of American Journal of Transplantation identify antibodies against Angiotensin II type-1 receptor (Anti-AT1R), with AMR and allograft failure. Anti-AT1R have previously been associated with AMR and polymorphisms in AT1R have been associated with steroid resistant acute rejection. In the first AJT study, 351 consecutive kidney recipients had sera tested for anti-AT1R. In patients with abnormal biopsies/acute rejection, the rate of anti-AT1R was significantly higher than the control group (18% vs. 6%, p < 0.001). What is novel however is the survival data; with 79% of abnormal biopsy patients with anti-AT1R losing their grafts (vs. 0% in control group). Patients with both anti-AT1R and DSA had lower graft survival than those with DSA alone. In a multi-factorial model, de novo anti-AT1R was an independent predictor of graft failure.

A second report included 599 kidney recipients and determined a cut-off of anti-AT1R >10 U, with individuals above this level having 2.6-fold higher risk of graft failure from 3 years onwards (p = 0.0005) and a 1.9-fold higher risk of experiencing an acute rejection episode in the first 4 months (p = 0.0393). Notably, AMR only accounted for one third of all acute rejection cases.

       Complement-binding DSA

What makes some DSA pathogenic and others not? Does the complement-binding capacity of anti-HLA antibodies play a role in their capacity to cause immunological injury? This question was partially addressed in this French NEJM study of 1016 kidney transplant recipients. Complement-fixing DSA (tested for the presence of C1q-binding by single-antigen bead assays) were associated with more C4 deposition, more AMR and worse allograft survival (54% V 93% 5 year survival). Interestingly, patients with no DSA and those with non-complement binding DSA had similar 5 year survivals.

Overall, these studies certainly add to our understanding of post-transplant allo-antibody but additional work needs to be done before they become clinically useful and exact indications for the tests remain unclear. Certainly in the case of Anti-AT1R, the increased risk of allograft failure needs to be clarified. Is it truly independent from Anti-HLA antibodies? Regarding Anti-HLA antibodies, it is likely that the pathogenicity of DSA is more nuanced than just their ability to fix complement. However, these studies are beginning to explain some of our unanswered questions regarding sensitization, alloantibody and post-transplant risk.

Vascular rejection – Reassessing its etiology

Vascular rejection has been traditionally considered a severe form of acute rejection characterized by infiltration of mononuclear cells beneath the endothelium or by the presence of arteritis. Though initially reported as an aggressive form of T-cell mediated rejection with poor response to T-cell targeted therapy, newer findings suggest a strong association with alloantibodies. 

 Study from France analyzed 302 patients with biopsy-proven rejection and identified 4 subtypes of acute rejection with different outcomes (Figure): T-cell-mediated vascular rejection (9%), antibody-mediated vascular rejection (21%), T-cell-mediated rejection without vasculitis (46%), and antibody-mediated rejection without vasculitis (24%). Antibody-mediated vascular rejection manifested a median of 1.1 months (0.4–4.4) post-transplant and had the worst prognosis of the four subtypes. Moreover, 71% of cases of vascular rejection, which were mostly graded as v1 and v2 arteritis by the Banff schema, were associated with donor-specific antibodies (DSA). 

Therefore, it seems that the majority of cases of vascular rejection are associated with DSA and therapies to remove and decrease alloantibody production may be warranted. Indeed, this study suggested that antibody-directed treatment involving plasmapheresis, IVIG and rituximab led to better outcomes in this subpopulation. As of today, v1 and v2 vascular lesions are not accounted by the Banff classification in the antibody-mediated rejection category. 

Alloantibodies may bind to endothelium antigens and activate complement, attracting mononuclear cells which express Fc and adherence receptors, initiating the process of vascular infiltration. 

How will this affect our practice? Whenever a biopsy shows a component of vascular rejection, one must send the serum for alloantibody testing, even if biopsy is not classic for antibody-mediated rejection. Furthermore, antibody-directed treatment strategies should be considered, in particular if no response to initial therapy and evidence of DSA. The ideal treatment of the different severities of vascular rejection still remain to be determined.

Acute rejection: what do the circulating cells have to say about it?

One of the frequent situations that we face in the renal transplant clinic is the patient in otherwise good condition who presents with a slight rise in serum creatinine. Although this is sometimes due to reversible causes, such as high CNI levels or dehydration, acute rejection is of course in the differential. 

The fact that we still rely on an invasive procedure – the graft biopsy – for formal diagnosis of rejection clearly limits our ability for repetitive monitoring and potentially delays treatment. There is no doubt that a simple, non-invasive assay to monitor the immune status would be of great help in the day-to-day practice. Indeed this is currently a field of intense research in transplantation. We recently provided new insights into this issue. We optimized a simple assay to determine the level of activation of circulating blood mononuclear cells in renal transplant recipients. The method is relatively straightforward: peripheral blood is collected, cells are isolated and incubated overnight; cytokine production by the cultured cells is measured in the cell supernatant. The main objective was to determine if this assay, when used in patients for whom a biopsy was performed for an acute rise in serum creatinine, could identify those that would show histological signs of rejection. We found that the measurement of a single cytokine, IL-6, can predict rejection with a sensitivity of 92% and specificity of 63%. This tool could thus potentially be used to exclude rejection, which would be particularly helpful for low-risk or remote patients. 

Where do we go now? 

This work is a first step towards the development of a clinically useful tool. Ideally, a non-invasive test would be able to identify acute rejection well before the serum creatinine starts to rise. To achieve this, we now need to collect blood samples and study cell activation serially post transplant. What we need to determine more precisely is when the cells become activated before the usual signs of graft dysfunction occur. This will allow us to identify rejection early and by doing so, to prevent further graft damage. Although this sounds simple, from a research perspective this next step implies an enormous investment of human and lab resources. 

Sacha De Serres
Leonardo Riella 

Antibody-Mediated Rejection: Choose your weapons

Acute humoral or antibody-mediated rejection (AMR) is attributed to the presence of alloantibodies against the graft, which could be either antibodies against human leukocyte antigens (HLAs) Class I and/or II , non-HLA antigens or endothelial antigens. Diagnosis of AMR is made through tissue biopsy and presence of alloantibodies. Early treatment is of paramount for the preservation of graft function. Treatment strategies include removal of alloantibodies, decreasing or stopping production of alloantibodies, or attenuating the immune systems response to alloantibodies. 

Plasmapheresis/Plasma Exchange 

Removal of alloantibodies is done through the use of plasmapheresis/plasma exchange or immunoadsorption. Plasmapheresis, or removal and replacement of one plasma volume, is effective at removing approximately 60% of the intravascular IgG which accounts for about 75% of the intravascular immune response. Extravascular IgG equilibrates in about 48 hours thus reducing total body IgG concentrations and reducing the effective immune response. Immunoadsorption works similarly to plasmapheresis except that plasma immune complexes and IgG are removed via protein A bound silica matrices. In the latter, the remaining plasma components are returned to the patient without the need for plasma exchange. FFP is needed even on the first run of plasmapheresis if recent biopsy was performed (prevention of bleeding). The cost of 5 treatments is about $4,600. 

Intravenous Immune Globulin 

Infusion of intravenous immunoglobulins (IVIG) has been studied at doses of 10 grams to 2 gm/kg as monotherapy or in conjunction with plasmapheresis or B-cell depleting agents. The mechanism of action is not entirely known but it is thought that neutralization of alloantibodies occurs when bound by the anti-idiotypic antibodies in IVIG as well as diminished plasma cell production by increasing total body concentrations of immunoglobulins and direct T-cell and complement cascade effects. Cost for IVIG is $77.06/gm resulting in $770.60 or $10,788.40 (dose intensity), based on a 70kg patient for each dose. 

Decreasing or stopping the production of alloantibodies requires therapies directed against mature plasma cells, memory B-cells or plasmablasts. Targeting memory B-cells or plasmablasts has a delayed onset of action as this therapy prevents new plasma cells from being formed but does not affect currently active ones. 

Rituximab 

A chimeric anti-CD20 monoclonal antibody, is dosed 375 mg/m2 or 1000 mg IV and given for one to two doses. The CD20 receptor is found on the surface of B-lymphocytes, including memory B-cells and immature plasmablasts, but not plasma cells. Rituximab has cytotoxic activity directly reducing B-lymphocyte and antibody levels. Cost for therapy ranges from $4,923.78 for dosing based on normal body surface area to $7,589.64 for a 1000mg dose. 

Bortezomib 

A proteasome inhibitor, is dosed 1.3 mg/m2 IV and given for four doses on days 1, 4, 8, and 11. Proteasome inhibition prevents protein biosynthesis resulting in apoptosis of the plasma cell and cessation of alloantibody production. Cost per dose based on a normal body surface area is $1,134.27. 

Eculizumab 

A humanized monoclonal antibody directed against C5, is dosed 600mg to 1200mg IV and administered weekly depending on alloantibody concentrations. Prevention of AMR is mediated by inhibition of membrane attack complex formation and halting activation of the complement cascade. Eculizumab is supplied as a 300mg vial for $6,638.40 or $13,276.80 to $26,553.60 per dose. 

Increasing the dose of maintenance immunosuppressive agents, including calcineurin inhibitors, antimetabolites, and steroids are also used to attenuate the immune system and help alleviate AMR. With all of the available treatment options, a multimodal approach is usually recommended to maximize chances of preventing graft injury. However, as you might see from the numbers above, careful clinical decision must be based on both efficacy and cost in order to responsibly avoid collapsing our already broken health care system. Instead of each center using its on protocol, our society should get together and perform a randomized trial with those interventions. Though I doubt this will happen any time soon, in particular with all the NIH budget cuts...

David Reardon, PharmD, PGY2 Critical Care Resident

Steve Gabardi, PharmD

Leonardo V Riella MD PhD (editing role)