Background

Kidney transplantation is the treatment of choice for patients with end-stage kidney disease. Modern immunosuppression strategies considerably increased the one-year graft survival by decreasing the frequency and the potency of acute cellular-mediated rejection episodes, though long-term graft survival remains problematic mainly due to chronic antibody-mediated rejection, a case without effective treatment (Abramowicz et al., 2018). Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing humoral alloimmunity during various drug interventions, as well as for preclinical evaluation of new medications.

We developed such a protocol in order to test the effect of certain compounds on humoral alloimmunity (Eleftheriadis et al., 2017a, 2017b and 2017c). Although this protocol has been applied in human peripheral blood mononuclear cells (PMBCs), it could also be easily modified for using cells from other species, for instance from different mouse strains.

Our protocol was based on previous studies, supporting that one week is enough for the development of IgM and IgG alloantibodies in one-way mixed lymphocyte reaction (MLR) with a stimulator to responder cell ratio equal to 1:1 (Rumke et al., 1982), along with the well-established antibody-mediated complement-dependent cytotoxicity (CDC) assay (Konishi et al., 2008). Mitomycin C was used to treat the stimulator cell population. Mitomycin C is an anti-tumor antibiotic agent, which acts via the inhibition of DNA and RNA synthesis, rendering the lymphocytes unable to proliferate or differentiate in effector cells. Prolonged treatment with mitomycin-C may also induce apoptosis. The responder PBMCs produced alloantibodies against the mitomycin-C treated stimulator cells.

To determine humoral alloimmunity, de novo alloantibodies production is induced in one-way MLR. Amount and capacity of generated alloantibodies in the supernatant of each one-way MLR are assessed using an antibody-mediated cell dependent cytotoxicity (CDC) assay. The principle of the assay relies on the assessment of cellular survival of resting PBMCs isolated from the same donors, supplemented with the alloantibodies from the one-way MLR supernatant (Figure 1). Cell survival was measured by performing a well-known cell proliferation assay [Tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, XTT cell proliferation assay kit].

We avoided direct cytotoxicity assessment through the non-radioactive lactate dehydrogenase release assay (LDH) mainly because cellular cytotoxicity inevitably occurs in the context of MLRs (Sato et al., 1999) and lactate dehydrogenase is released in the supernatant by dead cells only. Thus, different drug treatments may exert different effects on the MLR mixed cell populations resulting in different amounts of LDH released in the supernatant, a fact that would provide erroneous data in the subsequent antibody-mediated CDC assay.


Figure 1. Assessment of humoral alloimmunity in mixed-lymphocyte reaction. The flow-chart depicts the protocol to assess humoral alloimmunity using MLR. In one-way MLR, responder PBMCs produce alloantibodies against the mitomycin-C treated stimulator cells. Blood A and B correspond to blood donors A and B, respectively. Blood B sample will be used to generate the resting cells of that donor that will constitute the responder cells against the mitomycin-C treated stimulator cells of Blood A. Resting cells, or responder cells of Blood A will be used against the mitomycin-C treated stimulator cells of another blood donor’s sample (e.g., Blood B). Quantitative measurement of alloantibodies in the supernatant of the MLR is assessed by adding this supernatant to resting PBMCs (from the same individual who provided the stimulator PMBCs used for the MLR) using an antibody-mediated CDC assay and measuring cell viability with XTT assay.