ELISpot assay and prediction of organ transplant rejection

Differences in human leukocyte antigens (HLA) and other polymorphic antigen systems between recipients and organ donors lead to the development of allograft rejection after organ transplantation. Predicting the immune response of recipients to transplanted organs may reduce the incidence of rejection episodes using individualised immunosuppressive protocols. Patients on the waiting list for organ transplantation can become sensitised to HLA antigens due to pregnancies, blood transfusions and previous transplantations. Sensitisation is one of several factors that promote development of HLA-specific memory T cells, the major effectors of organ transplant rejection (Lin et al., 2015). Even in patients without previous exposure to alloantigens, alloreactive memory cells can be detected as a result of heterologous immunity (Karahan, Claas et al., 2017; Stranavova et al., 2019). In the pre-transplant period, B-cell responses are regularly assessed by detection of donor-specific antibodies (DSA) using solid-phase methods (ELISA, Luminex) and complement-dependent cytotoxic/or flow cytometry crossmatch tests. In recent years, the enzyme-linked immunosorbent spot (ELISpot) assay has been extensively investigated in connection with its ability to predict rejection via assessment of T-lymphocyte reactivity. The ELISpot assay is used to determine both cytokine-producing [interferon (IFN)-gamma, interleukin-2 (IL-2), etc.] memory/effector T cells and, more recently, HLA-specific IgG-producing B cells. In this article, we briefly review the current literature and assess the value of the ELISpot assay in predicting incidence and risk of allograft rejection and survival.

1.1 Predictive value of IFN-gamma ELISpot for graft rejection and survival 1.1.1 Principle of the IFN-gamma ELISpot assay

The IFN-gamma ELISpot (IFN-g ELISpot) assay is used to evaluate frequencies of memory IFN-gamma-producing T cells, either donor-specific, or in case donor cells are unavailable, third-party stimulators may be used to provide a panel reactive T cell (PRT) assessment (Poggio et al., 2007) (Figure 1). There are two variations of the technique: direct and indirect. The direct method is used when the expected number of IFN-gamma-producing cells is low. This involves stimulation of recipient lymphocytes in cell-culture plates pre-coated with an IFN-gamma antibody (Augustine et al., 2008; Crespo et al., 2015). When the estimated frequencies of IFN-gamma-producing lymphocytes are relatively high, the indirect test is performed. In this instance, recipient cells are first stimulated with kidney-donor lymphocytes, PRT cells or polyclonal stimulators and subsequently transferred to plates coated with an anti-IFN-gamma antibody. Donor stimulator cells can be inactivated by either irradiation (or mitomycin-C treatment) or CD2 depletion. Polyclonal stimulators such as pokeweed mitogen, staphylococcal enterotoxin B (SEB) and phytohemagglutinin may be used as positive control stimuli. After cell lysing and washing, a secondary enzyme-labelled detection antibody is added followed by a chromogen substrate. The resulting spots are then counted using a microscope (problematic when the number of spots is high) or, preferably, an ELISpot reader. The ELISpot assay is extremely sensitive and specific, with the ability to detect IFN-gamma spot-forming cells (SFC) in the range of 10–1000 SFC/106 peripheral blood mononuclear cells (PBMC) (Smith et al., 2001). A threshold value is essentially to be defined, that is, the number of spots above which the test will be evaluated as positive or negative (Augustine et al., 2008; Crespo et al., 2015).

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Technique of the donor-specific IFN-gamma T-cell enzyme-linked immunosorbent spot (ELISpot) assay

1.2 Clinical value of IFN-gamma ELISpot assay in predicting rejection

A number of pilot studies have tried to establish a correlation between the presence of donor-specific, IFN-gamma-producing lymphocytes before kidney transplantation and impaired graft function/rejection post-transplant. Most of these reports conclude that a positive pre-transplant IFN-gamma ELISpot is associated with acute rejection (AR) and worse graft function 6 and 12 months post-transplant (Heeger et al., 1999; Hricik et al., 2003). The first few studies reported that seven out of nine patients with positive pre-transplant IFN-gamma ELISpot developed AR, while none of the negative patients did (Heeger et al., 1999). In 37 patients, the mean frequency of pre-transplant IFN-gamma ELISpot was significantly higher in patients with subsequent AR than in those without rejection (Hricik et al., 2003). Another study found that the highest risk of severe AR was among patients with a high number of alloreactive cells (> 200/3 × 105 PBMC, n = 5/42) (Nickel et al., 2004). Augustine et al. (2005) found that 38% of patients with a positive pre-transplant IFN-gamma ELISpot developed AR versus only 14% of patients with a negative pre-transplant ELISpot (p = .008). A drawback of the above-mentioned pilot reports is the relatively low number of recipients studied. Furthermore, patient cohorts included both deceased and living-donor transplants, so the effect of additional variables, such as ischemia time, immunosuppressive protocols, cannot be excluded. Of the more recent studies, a positive pre-transplant IFN-gamma ELISpot predicted AR in 65 patients within 2 months after transplantation (AUC = 0.701, p = .065), but not rejection within 1 year (Crespo et al., 2015). In contrast, subsequent studies have failed to clearly demonstrate the predictive nature of the pre-transplant IFN-gamma ELISpot (Augustine et al., 2008; Hricik et al., 2015; Slavcev et al., 2015). The multicentre CTOT-01 study revealed that IFN-gamma ELISpot positivity in the pre-transplant period (176 patients) did not correlate with incidence of AR or renal function 6 and 12 months post-transplant. Interestingly, impaired graft function correlated with IFN-gamma ELISpot positivity in patients without Antithymocyte globulin (ATG) induction therapy (Hricik et al., 2015). Various authors have highlighted the effect of induction therapy on the frequencies of donor-specific effector/memory T cells. In the study by Augustine et al. (2008) as mentioned above, almost half of the IFN-gamma ELISpot-positive patients without induction therapy experienced AR, comparing with cases among ELISpot-positive patients that had received ATG pre-transplant. Cherkassky et al. (2011) demonstrated that ATG administration pre-transplant may be directly linked to decreased frequencies of donor-reactive T cells after transplantation (but not third-party memory T cells) which may explain the lack of correlation with rejection in ATG-treated recipients.

When interpreting the correlation of ELISpot to graft function and rejection, the effect of the number of HLA mismatches among donors and recipients should also be considered. Hricik et al. (2003) were the first to note a trend for a positive correlation between positive IFN-gamma ELISpots and the number of HLA mismatches. A previous study by our group found no statistically significant association between pre-transplant IFN-gamma-producing lymphocytes and incidence of AR in living-donor transplant recipients. However, a clear relationship between the frequencies of IFN-gamma-secreting cells and the number of HLA mismatches was observed (p <.01) (Slavcev et al., 2015). This finding may be explained by the fact that recipient lymphocytes were stimulated in mixed lymphocyte cultures, which is a technique reliant on the direct pathway of allorecognition and thus on the level of HLA mismatching between responder (recipient) and stimulator (donor) cells (DeWolf et al., 2016).

These contradictory results, as discussed above, may be attributable to one or more of the following factors: variability in performance of the IFN-gamma ELISpot assays, differences in study cohorts including various immunosuppressive regimens, diverse statistical approaches used to calculate prediction models and/or other unknown reasons. To overcome discrepancies in evaluating such data, Montero et al. (2019) performed a meta-analysis of pre-transplant IFN-gamma ELISpot studies. Out of 1181 patients, 512 (43%) had a positive pre-transplant IFN-gamma ELISpot and 209 developed AR (18%). IFN-gamma ELISpot positivity pre-transplant was significantly associated with increased risk of AR [OR = 3.29, 95% confidence interval (CI) = 2.34–4.60]. It should be noted, however, that the estimated sensitivity and specificity of the test in this study was only 64.9% and 65.8%, respectively. The association was stronger in patients without induction therapy, but the negative predictive value of the test was greater than 90% in low-risk patients (AR risk less than 16%). The authors concluded that even though the power of the pre-transplant IFN-gamma ELISpot for predicting AR development is sub-optimal for clinical use, its prognostic value may improve when used in combination with other biomarkers, especially in the stratification of low-risk patients (Montero et al., 2019). The outcome of the multicentre CELLIMIN trial with immunosuppression (IS) based on the results of pre-transplant ELISpot was published recently. The occurrence of biopsy-proven AR was evaluated in three patient cohorts: (1) IFN-gamma ELISpot-negative patients on Tacrolimus monotherapy (n = 48); (2) IFN-gamma ELISpot-negative patients on standard triple IS (n = 53) and (3) IFN-gamma ELISpot-positive patients on standard triple IS (n = 66). The incidence of biopsy-proven rejection in patients on tacrolimus monotherapy was slightly higher, but not statistically significant (p = .15 at 6 M; p = .073 at 12 M), compared to patients on standard IS. Further analysis revealed that poor HLA class-II eplet matching (p = .026), especially in HLA-DQ (p = .015), discriminates pre-transplant IFN-gamma ELISpot-negative patients developing biopsy-proven AR from those without biopsy-proven AR (Bestard et al., 2021). In relation to this finding, the influence of epitope matching on de novo antibody production and kidney allograft rejection is currently analysed in the frame of the upcoming International HLA & Immunogenetics Workshop, so multicentre data on this relevant topic will be available in the near future.

1.2.1 Relevance of follow-up after transplantation

In a non-randomised clinical trial by Bestard et al., 60 kidney recipients were placed on an immunosuppression regimen based on the results of pre-transplant IFN-gamma ELISpot, which was subsequently amended according to IFN-gamma ELISpot 6 months after transplantation. The authors found a strong correlation between T-cell-mediated rejection and anti-donor T-cell alloreactivity (Bestard et al., 2013). An analogous study by the same group examined the power of IFN-gamma ELISpot to predict subclinical rejection in 6-month protocol biopsies. IFN-gamma ELISpot positivity predicted subclinical T-cell mediated rejection (TCMR) (AUC = 0.800), with sensitivity and specificity at 82% and 88%, respectively. The cut-off of 19 spots per 3 × 105 PBMC in the initial cohort of 60 patients was validated in 101 additional patients followed prospectively (AUC = 0.725 m, sensitivity and specificity 80% and 64% respectively; p = .006) (Crespo et al., 2017). The STAR 2019 workgroup report was published recently, where a number of limitations of the T-cell ELISpot assay were discussed. It was indicated that the technique has only a positive predictive value, that is, if positive, it may predict a potential alloimmune response; however, if found negative, the presence of T alloreactive memory cells cannot be excluded. Most notably, the test has not reached clinical applicability so far due to technical difficulties in standardising the assay and the time needed to generate results (Tambur et al., 2020). Perspectives for upcoming research were also outlined, as to determine whether anti-HLA memory T cells recognise donor antigens via the direct or the indirect pathway, developing assays to enumerate follicular helper T cells and others (Tambur et al., 2020).

1.3 Predictive value of IgG ELISpot for graft rejection and survival 1.3.1 Principle of HLA-specific IgG B-cell ELISpot method

The IgG ELISpot assay can be used to estimate the frequencies of B cells secreting total IgG and the number of B lymphocytes producing IgG specific to a particular antigen. The ELISpot IgG assay can be performed in several ways. In the study by Perry et al. (2008), HLA-specific antibody-producing B cells were enumerated in bone marrow plasma CD138+ cells in transplanted patients. Plasma cells were pre-cultured with IL-2, IL-4 and PHA for 3 days and then transferred to ELISpot plates coated with purified HLA molecules. After the addition of anti-human IgG biotin, spots (IgG-producing cells) were visualised using horseradish peroxidise-streptavidin conjugate (Perry et al., 2008). Heidt et al. (2012) documented another method for detecting HLA class I-specific memory B lymphocytes in peripheral blood, which is easier to perform than the bone marrow cell assay (Figure 2). After enrichment for B cells using anti-CD2 monoclonal antibody (T-cell depletion), B cells were cultured with a fibroblast cell line expressing CD40L and then stimulated with a cocktail of cytokines and TLR-9 ligand. The antibody secreting cell (ASC) population obtained was transferred to ELISpot plates pre-coated with HLA class I monomers. Antibodies specifically bound to HLA class I molecules were detected by goat anti-IgG conjugated to horseradish peroxidase (HRP) and visualised using an immuno-enzyme technique (Heidt et al., 2012). In a subsequent study, the same group presented a detection method for enumerating B cells secreting HLA class II specific antibodies (Figure 2) (Karahan et al., 2015). ELISpot plates were coated with anti-human IgG monoclonal antibody. After incubation with antibody-producing B cells, HLA-specific antibodies produced by activated B cells were detected by adding biotinylated HLA class II molecules. Using streptavidin-alkaline phosphatase and substrate, HLA class II antibody-producing cells were visualised (Karahan et al., 2015). Luque (2018) documented a B-cell FluoroSpot assay enabling enumeration of multiple HLA-specific antibody-secreting cells originating from circulating donor-reactive memory B cells. Karahan, de Vaal et al. (2017) developed an HLA- and donor-specific ELISpot assay by using donor-lysates as detection matrix. In this setting, polyclonally activated recipient B cells were put into ELISpot plates coated with anti-human IgG, then incubated with HLA class I or II from donor lysates. These were detected by anti-beta 2-microglobulin antibody (for HLA class I) conjugated to HRP. For HLA class II, pan anti-human HLA class II antibody (IC2) was detected by anti-mouse IgG conjugated to HRP (Figure 3). By sorting naïve and memory B cell before culture, this group showed that IgG spots are produced only by memory B cells upon 6-day in vitro polyclonal activation (Karahan, de Vaal et al., 2017). In the above-mentioned protocols, a total IgG ELISpot assay was performed in parallel with each HLA-specific memory B-cell ELISpot to confirm successful polyclonal activation (Karahan et al., 2015; Luque, 2018). Given that not all antibody-producing cells produce IgG, the number of HLA-specific memory B cells was expressed as a ratio of HLA-specific memory B cells to the number of IgG-producing cells (Luque, 2018).

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Technique of the B-cell enzyme-linked immunosorbent spot (ELISpot) assay

image Human leukocyte antigens (HLA) and donor-specific enzyme-linked immunosorbent spot (ELISpot) techniques using donor lysates of HLA class I and II molecules (after Karahan et al., 2017). (a) HLA class I lysate-based ELISpot. (b) HLA class II lysate-based ELISpot. Anti-beta-2 M-HRP: antibody to beta-2-microoglobulin conjugated to horseradish peroxidase. IC2: mouse anti-human pan HLA class II antibody. Abbreviation: Anti-MS-HRP, anti-mouse antibody conjugated to horseradish peroxidase 1.4 Clinical value of the donor-specific IgG ELISpot assay 1.4.1 Antibodies, rejection and allograft outcomes

DSA detection before and after transplantation is essential when characterising the B-cell immune response of recipients to their respective organ donors (Lefaucheur et al., 2010; Wehmeier et al., 2017). DSA are produced by two different sources of B cells. Quiescent memory B cells reactivate rapidly upon antigen re-encounter (as in the case of retransplantation), while long-lived plasma cells residing in the bone marrow constitutively secrete antibodies but do not mobilise upon re-encounter (Chong & Sciammas, 2015).

The first HLA-specific IgG B-cell ELISpot was performed in cells constitutively producing alloantibodies to determine the impact of various desensitisation protocols in immunologically high-risk patients. Alloantibody production by ASC proved resistant to multiple plasmaphereses, low-dose intravenous immunoglobulin alone and in combination with rabbit ATG (Perry et al., 2008). Lynch et al. (2013) tested nine transplant recipients with undetectable DSA for frequency of donor-specific ASC 8 weeks after transplantation. HLA-specific B cells were detected in all patients, with increased frequencies of antibody-producing cells observed after transplantation. A recent large study tested the use of the memory B-cell ELISpot assay in a group of 175 consecutive kidney transplant patients (Luque et al., 2019). All 16 patients having DSA-positive acute antibody-mediated rejection (ABMR) and 24 out of 30 patients with DSA-positive chronic ABMR (80%) had detectable HLA-specific memory B cells. Interestingly, 21 out of 29 patients (72%) with DSA-negative chronic ABMR also exhibited donor-specific memory B cells. The B-cell ELISpot assay thus displayed high accuracy in discriminating the presence of ongoing ABMR (AUC > 0.85) (Luque et al., 2019).

In summary, the IgG B-cell ELISpot assays described above require specific reagents and are not easy to perform, so even though they certainly provide valuable information, we consider their widespread application as unlikely in the near future.

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