Murder in the Cathedral: Antibodies and the Limits of Transplantation

Abstract

Although transplantation is the optimal therapy for end-stage renal disease, long-term survival of kidney allografts remains elusive. Antibody-mediated rejection has been implicated as a major factor in late period graft loss. The refractory nature of late period antibody-mediated rejection is the evidence of an important immunological process with implications for other solid organ transplants and for other diseases. This review details advances in the understanding of late period antibody-mediated rejection in the context of local experience.

Main article text

INTRODUCTION

For… every act

Consequence of good and evil can be shown.

And as in time results of many deeds are blended

So good and evil in the end become confounded (1)

Thus, the Chorus in TS Eliot's 1935 historical play ‘Murder In The Cathedral’ confront the knights who, believing themselves to be acting in the best interests of England, martyr Archbishop Thomas à Becket on the floor of Canterbury Cathedral. This philosophy is particularly relevant to the field of transplantation, where the immune system must be balanced between the immunocompetence required to prevent infection and malignancy, and the immunosuppression required to prevent rejection of the transplanted organ.

Although renal transplant confers improved morbidity and mortality,(2) this benefit only endures while the allograft survives. Declining rates of renal transplantation in the local setting in general and in the state sector in particular (3) in the context of a globally increasing prevalence of end-stage kidney disease (2) mandate the development of protocols which optimize the survival of both the transplanted kidney and the transplant recipient.

Whereas advances in surgical engraftment and immunosuppression induction protocols have improved outcomes in the immediate post-transplant period, long-term allograft survival has remained static at 10–12 years.(4) Although substantial evidence has implicated the humoral arm of the recipient immune response as a significant barrier to extended survival,(5–9) an effective treatment strategy has yet to be elucidated.(10,11)

This review contextualizes the local experience of the treatment refractoriness of late period antibody-mediated rejection (LpAMR) to suggest an immunological model for the pathogenesis thereof. Evidence for the applicability of this model in other clinical scenarios is also presented.

THE IMMUNOBIOLOGY OF REJECTION

Rejection of the solid organ allograft results from the redirection of immune responses which evolved to provide host defence against invading pathogens. These responses are targeted through the recognition of foreign tissue as ‘non-self’, achieved by engagement of the immune system with antigens expressed on cellular surfaces; among the most important of which are the widely expressed human leukocyte (HLA) antigens.

Engagement of recipient immune cells with donor HLA may occur through a direct or indirect pathway.(12,13) In the direct pathway, donor antigen-presenting cells (APCs), transplanted into the recipient as ‘passenger leukocytes’ within the engrafted organ, present donor HLA to recipient naïve CD4⁺ T-helper cells (Th₀) via complexing of their major histocompatibility complex (MHC) with the T-cell receptor (TCR); in the indirect pathway, recipient APCs endocytose shed donor HLA, which, following intracellular processing, is presented to the recipient immune system via Th₀ in a similar manner.(12,13)

Binding of APC MHC to the Th₀ TCR through either pathway with co-stimulatory signals results in Th₀ activation, augmented by Th₀ release of interleukin (IL)-2 which acts in an autocrine fashion.(13)

In the classical theory of transplant immunobiology, activated Th₀ subsequently differentiates into daughter Th₁ or Th₂ lineages as directed by the relative concentration of cytokines in the local microenvironment, facilitating either a cell-mediated cytotoxic or humoral response, respectively. IL-12 and IFNγ promote Th₁ differentiation; secretion of IFNγ, IL-2 and lymphotoxin α by matured Th₁ facilitate activates phagocytes and CD8⁺ cytotoxic cells leading to cellular rejection.(13) In contrast, IL-4 and IL-6 favour the differentiation of Th₂ cells;(13) matured Th₂ cells produce IL-4 and IL-6 which are potent stimulators of CD20⁺ B-cell activation following cognate interactions.(13,14) Subsequent B-cell maturation is most efficiently achieved within the germinal centres of secondary lymphoid tissue (lymph nodes and spleen) under the stimulus of CXCR-5⁺ follicular helper T cells (T_FH).(13) The origins of T_FH are unclear, but a Th₂ descent has been suggested by some studies showing similar cytokine profiles across the two lineages.(13) T_FH differentiation may be encouraged by T-cell interaction with specialized dendritic cells of the nascent germinal centre, and CD25 produced by these dendritic cells reduces IL-2 availability which favours T_FH development.(14) B-cell maturation into plasma cells within the germinal centre is facilitated by Th₂-type cytokines IL-4 (15) and IL-6 (16,17) and T_FH-derived IL-21.(18) Matured plasma cells produce antibody directed against the inciting donor antigen, and this donor-specific antibody (DSA) results in humoral rejection.

Cellular and humoral reactivity may be developed prior to transplantation through exposure to donor-type HLA, for example during transfusion of non-leukodepleted blood, pregnancy or viral infections (due to molecular mimicry). In these settings, low-level transient TCR activation by APC in association with IL-15 and IL-7 and decreased IL-2 result in the generation of memory T cells.(19–21) In a similar fashion, weaker signalling via the B-cell receptor during cognate B/T-cell interactions in combination with decreased availability of IL-21 favours the generation of memory B cells (B_mem).(22,23) Plasma cells matured during such reactions migrate to the bone marrow following CXCL-12 chemokine gradients,(24) where they take up residence as long-lived plasma cells (LLPC) supported by local survival factors such as BAFF,(25) APRIL (26) and IL-6.(27)

Both B_mem and LLPC can produce DSA;(28) re-exposure to donor HLA results in a rapid anamnestic response which can lead to antibody-mediated rejection (AMR) within hours of engraftment. Advances in pre-transplant DSA detection techniques have, however, largely abrogated the incidence of such events through the identification of high-risk crossmatches.

Modern immunosuppression protocols therefore preferentially target the T cell as the master regulator of post- engraftment non-self-recognition. In prescribing these protocols, clinicians must consider the risks to the recipient of long-term immunosuppression against the risk of graft rejection. Balance is achieved through gradual minimization of the high-dose immunosuppression prescribed in the early post-engraftment period (usually the first 3 months after transplant), an effort to engender tolerance through incremental exposure of the recovering immune system to donor HLA, a process analogous to allergen desensitization. The failure of this approach to improve long-term allograft survival has led to a re-evaluation of the T-cell centric model of transplant immunobiology.

THE CONTRIBUTION OF AMR TO GRAFT OUTCOMES

Alloantibody has been known to contribute to graft survival since the 1970s;(29) however, the significance of antibody-mediated injury was poorly appreciated until the discovery of a reliable and reproducible marker by Feucht in the form of C4d (30). The split product of antibody-dependent complement factor 4 activation, C4d is a robust marker for intragraft deposition of antibody; its discovery led to the inclusion of C4d⁺ AMR into the 2009 update of the Banff system for the interpretation of allograft histopathology (31).

The availability of this objective test has resulted in an increase in AMR diagnosis. C4d staining was introduced to Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) in 2009; analysis of the incidence of rejection for the period 2004–2013 demonstrated a constant decline in the number of cases of cell-mediated rejection (CMR) but an increase in those of AMR and mixed rejection (a combination of AMR and CMR) after this date (Figure 1).(32)

Fig 1:

Changing patterns of late-period rejection at CMJAH: An increase in the diagnosis of AMR and MR has been noted after the introduction of C4d staining

Subsequent advances in the histological definition of AMR were recognized by the addition of C4d-AMR in the Banff 2013 update.(33) Using these criteria, re-analysis of injury patterns in a pre-C4d historical cohort of late period for-cause biopsies at CMJAH found the incidence of undiagnosed antibody-mediated rejection to be 35%;(32) other researchers have reported AMR to contribute to 30–60% of cases of late period graft dysfunction and loss.(7–9)

The diagnosis of AMR in the late period carries a poor prognosis,(34) the average graft survival at CMJAH being 14 months.(35)

The substantial and increasing prevalence of LpAMR and the poor prognosis thereof mandate the development of an effective therapeutic strategy; consideration of the immunobiology of transplantation reveals possible interventions.

THERAPEUTIC OPTIONS IN LpAMR

Treatment of LpAMR rests upon the suppression of DSA production. Plasma exchange removes DSA, but alloantibody levels rapidly rise upon its cessation;(11) AMR remission requires suppression of the plasma-cell clone. This may be achieved directly (e.g. using proteasome inhibitors such as Bortezomib), or, as suggested by the classical model, indirectly, by depleting CD20⁺ B-cell precursors (using Rituximab) or CD4⁺ Th cells [using antithymocyte globulin (ATG)].(11)

Despite initial promising results in small cases series of early period AMR,(36,37) recent randomized control trials do not support Bortezomib efficacy in LpAMR.(38) While it is possible that this disparate responsiveness is due to cumulative irreversible damage in late rejection, differences in proteasome inhibitor responsiveness between early and late period AMR have also been reported.(39)

Bortezomib is not widely available in South Africa; but attempts at inhibition of earlier B-lineage cells using Rituximab have not shown additional efficacy beyond plasma exchange in the CMJAH LpAMR cohort (40) (Figure 2), a finding consistent with a prospective randomized control trial.(41)

Fig 2:

Lack of efficacy of Rituximab in LpAMR at CMJAH: Prescription of RTX has no additional effect on graft survival (Cox F 1.35, p = 0.390)

Inhibition of T-cell help through CD4⁺ lymphocyte depletion using ATG has been proposed as an alternative to B lineage strategies, particularly in cases with significant interstitial inflammation at biopsy.(11) In addition to effects on Th cells, ATG may suppress natural killer (NK) cells (mediators of antibody-dependent cytotoxicity) and may also be capable of direct depletion of B lymphocytes and plasma cells.(42) In addition, ATG may increase regulatory T cell (T_reg), offering the opportunity of endogenous control of the immunological response.(43) Retrospective analysis of the CMJAH LpAMR cohort has, however, failed to demonstrate a beneficial effect for ATG.(40) Indeed, prescription of ATG in this cohort was associated with increased risk of graft loss compared to the use of either Rituximab (40) (Figure 3).

Fig 3:

Prescription of ATG in LpAMR is associated with increased risk of graft loss: ATG accelerates graft loss in LpAMR; this effect is more pronounced at increased i-scores (HR for graft loss with ATG = 3.298, 95% CI 1.09–9.96, p = 0.016)

The refractoriness of LpAMR to these depletion therapies suggests that the classical immunobiology model of rejection requires re-evaluation. Progress in this regard has been made through reappraisal of the contribution of lymphocyte lineages to AMR, beginning with the discovery of the role of interstitial inflammation in the prognosis thereof.

THERAPEUTIC FAILURES PROVIDE NEW INSIGHTS INTO THE IMMUNOBIOLOGY OF LpAMR

Plasma exchange and depletion therapies carry significant financial cost; in addition, this combination therapy carries the potential for significant morbidity associated with profound immunosuppression. The CMJAH LpAMR cohort has been subject to regular analyses in an attempt to optimize the cost/benefit ratio of treatment.

Analysis of baseline characteristics of this and other series has indicated that alloantibody in this series is frequently elucidated against human leukocyte antigen DQ (HLA-DQ) specificities.(35) Alloantibody specificity and titre do not, however, correlate with graft survival.(35) In contrast, the severity of interstitial inflammation as determined by the i-score (area of the biopsy core occupied by interstitial inflammation) has been found to predict LpAMR outcomes (35,40,42) (Figure 4), a finding confirmed by other studies.(8) Analysis of the cellular composition of the interstitial infiltrate in an effort to explain this association has provided new insights into the pathogenesis of LpAMR.

Fig 4:

i-Score prognosticates graft survival in LpAMR: The presence of interstitial inflammation comprising more than 25% of the biopsy core is associated with reduced graft survival

Two patterns of interstitial infiltrate are discernible in LpAMR (Figure 5). First, an organized form in which cells are arranged into nodules has been described.(45–47) T-, B- and follicular dendritic cells have been observed within these nodules which have structural homology with the germinal centres of secondary lymphoid tissue.(46,47) Elegant experiments have demonstrated the rise of a plasma- cell population within these nodules following CD20⁺ B-cell activation; these plasma cells have been shown to produce significant amounts of DSA within the allograft against a greater number of HLA specificities than that detectable in the peripheral blood.(47) Thus, the nodular phenotype provides evidence for the ability of the renal allograft to act as a ‘tertiary lymphoid tissue’ (TLT) and participate in its own demise through the elucidation of a highly efficient local humoral response.

Fig 5:

Interstitial inflammation phenotypes in LpAMR: (A) Nodular phenotype: the nodular phenotype consists of organized T-, B- and dendritic cells with structural and functional homology to germinal centres (H&E, 10×). (B) Diffuse phenotype: the diffuse phenotype consists of an unorganized infiltrate distributed throughout the allograft (H&E, 40×). (C) Diffuse phenotype, Th or NKT cells: the diffuse phenotype consists of CD3⁺ NKT or T cells (CD3, 40×). (D) Diffuse phenotype, plasma cells: mature plasma cells are appreciable throughout the diffuse phenotype (CD38, 40×). (E) Diffuse phenotype, B cells: few immature B cells are detected in the diffuse phenotype interstitial infiltrate (CD20, 40×)

The nodular phenotype is, however, comparatively rare, with only 10 cases documented in the CMJAH cohort (11.6%). A diffuse interstitial infiltrate is more commonly observed, the composition of which is controversial. The infiltrate has been reported to be enriched for NK-cell transcripts; with NK transcript burden showing correlation with microcirculation injury scores,(48) consistent with NK-mediated antibody-dependent cellular cytotoxicity. Interestingly, NK transcript burden does not show correlation with i-score, raising the possibility of the presence of additional cell types in the infiltrate.(48) Furthermore, NK transcripts have also been detected in some cases of T-CMR, raising the possibility of Natural Killer T (NKT) cells being miscategorized as NK.(48)

NK and NKT cells are capable of both a cytotoxic and a cytokine producer effector function. Cytotoxic effect is inhibited by prednisone, calcineurin inhibitors and mycophenolate mofetil, commonly prescribed maintenance immunosuppressants in renal transplantation.(49) In comparison, cytokine production is suppressed by prednisone and calcineurin inhibitors, although not by mycophenolate mofetil.(49) Modern immunosuppression protocols favour a gradual reduction in prednisone and calcineurin inhibitor dose in order to minimize the metabolic derangements of long-term high-dose corticosteroids and the nephrotoxic effect of calcineurin inhibitors;(50) this may favour the induction of a cytokine-producing phenotype.

NKT cells can be distinguished from NK cells by their expression of the TCR (CD3); limited analysis of the CMJAH LpAMR cohort has found the diffuse interstitial infiltrate phenotype to be CD3 enriched. NKT cells are subcategorized as NKT₁, NKT₂, NKT₁₇ and NKT_FH dependent on their ability to match the cytokine profile of the representative Th line.(51) NKT cells are also a source of BAFF and APRIL, which are important survival factors for LLPC;(25,26) the presence of CD38⁺ mature plasma cells in biopsy cores from the CMJAH cohort may be further evidence for NKT in the interstitial infiltrate (Figure 5).

Other studies have reported that the level of expression of the transcription factors RORγt and FoxP3 in the LpAMR interstitial infiltrate may prognosticate graft survival.(52) RORγt is a gene marker for Th₁₇ lineage cells, while FoxP3 expression identifies T_regs; increased intragraft Th₁₇ with reduced T_reg in LpAMR parallels peripheral blood flow cytometry changes seen in these cases.(53) The Th₁₇ lineage cytokines IL-17 and IL-21 are potent stimulators of B-cell maturation; Th₁₇ has been observed at the periphery of germinal centres and may be able to differentiate into T_FH,(54) which may explain the ability of the latter to produce IL-21.(18,55) Th17 provides survival signals to LLPC indirectly via IL-17, which increases IL-6 release by parenchymal cells,(56) and possibly directly via IL-21;(18) in addition, Th₁₇ facilitates the homing of LLPC to survival niches through IL-17-mediated upregulation of CXCL12.(57,58) Th₁₇ and T_reg are known to exhibit developmental plasticity dependent on parent Th₀ TCR signal strength and microenvironment cytokine concentration;(59,60) in contrast to the B-cell support provided by Th₁₇, T_reg inhibits plasma-cell survival through direct cell–cell contact.(61) Thus, Th₁₇ expansion at the cost of T_reg depletion favours the generation and maintenance of LLPC.

It is noteworthy that the expression of the transcription factor RORγt is required for the differentiation of both NKT₁₇ and Th₁₇ lineages;(62) since both express CD3, current data is inadequate to resolve which cell line is the dominant cellular constituent of the LpAMR infiltrate.

Synthesis of the available literature and the CMJAH experience may thus suggest a model for the diffuse infiltrate phenotype in LpAMR. Immunosuppression protocol modulation (calcineurin inhibitor and prednisone minimization) increases recipient exposure to donor HLA while polarizing the immune system towards a cytokine-producing NKT₁₇ or Th₁₇ phenotype, with concomitant tolerizing T_reg suppression. The lack of CD20⁺ B cells observed in the interstitial infiltrate suggests that plasma-cell maturation occurs in the germinal centres of secondary lymph tissue, possibly from a B_mem population, where NKT₁₇ or Th₁₇ cells migrate after activation by donor HLA in the allograft. Within the secondary lymph tissue, NKT₁₇ or Th₁₇ cells may differentiate into NKT_FH or T_FH in order to facilitate plasma-cell generation. Once matured, plasma cells migrate to the allograft following CXCL12 chemokine gradients upregulated by the NKT₁₇ or Th₁₇ cells which have remained in situ in the allograft. Survival signals produced by these cells allow the immigrating plasma cells to adopt a long-lived phenotype, resulting in significant levels of intragraft production of DSA. NKT cells within the graft which have developed a cytotoxic phenotype in response to immunosuppression minimization respond to DSA deposition by engaging in antibody-dependent cytotoxicity.

Once established, this highly efficient and self-promoting system is refractory to standard depletion therapies. Whereas Bortezomib may temporarily deplete plasma cells, these are rapidly and efficiently replaced from the ancestor B_mem pool; depletion of immature CD20⁺ B cells by Rituximab has no effect on the LLPC population. ATG is relatively ineffective against NKT;(42) prescription of a high cumulative dose abrogates the T_reg recruitment effect of this therapy and induces T_reg depletion instead (63), further reducing inhibition of the LLPC population.

BEYOND THE RENAL ALLOGRAFT

The development of an intragraft immune response has been most extensively documented in renal transplants, reflecting the availability of a cheap and reliable indicator of evolving graft injury in the form of serum creatinine to prompt investigation, and the amenability of the transplanted organ to percutaneous biopsy. The renal transplant is not, however, unique in its ability to host such an immune response; TLT has been observed in cardiac, lung, trachea and composite tissue grafts,(64) and an imbalance between Th₁₇ and T_reg has been reported in lung and cardiac recipients.(53)

Other evidence suggests that immune system activation in non-canonical tissue is not unique to transplantation. TLT has been observed in the pancreas of porcine models of diabetes mellitus,(65) in the synovium of patients with rheumatoid arthritis (66) and in the lung parenchyma of patients afflicted with pulmonary hypertension (67) and idiopathic pulmonary fibrosis.(68) The presence of TLT has also been reported in native kidneys manifesting a variety of glomerular pathologies, including IgA nephropathy,(69,70) minimal change and membranous nephropathies and focal segmental glomerulonephritis.(70) Local immune system activation is particularly well evidenced in lupus nephritis; TLT development in association with BAFF expression has been observed in the parenchyma of lupus nephritis,(71) and an increase in Th₁₇ in the peripheral blood of patients with active lupus has been shown to be associated with proteinuria and SLEDAI score.(72)

CONCLUSIONS

Late period antibody-mediated rejection is an important factor in the limited longevity of renal transplants. Accumulating evidence suggests a complex process of immune system activation driven by Th₁₇-type cells which culminates in plasma- cell clones responsible for graft loss taking up residence within the rejecting transplant. This process may occur in other solid organ transplants and may also occur in native organ autoimmune diseases. Further delineation of the underlying mechanisms may therefore identify interventions of use beyond renal transplantation.

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