Elsevier

Transplant Immunology

Volume 64, February 2021, 101360
Transplant Immunology

Research Article
Impact of rituximab on the T-cell flow cytometric crossmatch

https://doi.org/10.1016/j.trim.2020.101360Get rights and content

Highlights

  • Rituximab interferes not only with B-cell but also with T-cell flow cytometric crossmatch.

  • ‘T-cell tailing’ phenomenon was observed in >50% patients, which cleared with pronase treatment.

  • Absence of donor-specific antibodies in all patients proves that this is a false positive finding.

  • Structural homology of Fc receptors to CD20 on activated T-cells might explain this phenomenon

Abstract

Rituximab is frequently used in the setting of ABO-incompatible renal transplants, and highly sensitized patients. Its interference with B-cell flow cytometric crossmatch (B-FCXM) is well known. However, its effect on the T-cell flow cytometric crossmatch (T-FCXM) has not been described. We aimed to evaluate the effect of rituximab on the T-FCXM using non-pronase and pronase treated donor lymphocytes and compare results with the single antigen bead (SAB) assay. In this retrospective study, 28 patients on rituximab therapy were evaluated against 30 donors. Using non-pronase treated donor lymphocytes, all 30 FCXMs showed strong B-cell positivity {median (IQR) B-cell ratio: 184.65 (253.17)} which significantly reduced {1.0 (1.18); p < 0.00001} with pronase treatment. ‘T-cell tailing’ phenomenon was observed in 17/30 FCXMs in the non-pronase group as a ‘tail of T-cells’, indicating a rare sub-population. However, it disappeared in the pronase-treated group. SAB assay did not show donor-specific antibodies (DSA) in all 17 patients with ‘T-cell tailing’ phenomenon. Although, rituximab is described to impact only B-FCXM, we have consistently found ‘T-cell tailing’ in 57% of T-FCXMs, which clears with pronase treatment. The ‘T-cell tailing’ led to weak positive T-FCMX ratios due to increased MFI in the FL1 channel. However, the absence of DSA in all recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I DSA. Structural homology of Fc receptors on activated T-cells to CD20 could be a possible explanation of the same and provide insight into a novel mechanism of action of rituximab.

Introduction

The flow cytometric crossmatch (FCXM) assay is utilized to detect donor-specific anti-HLA antibodies in both solid organ and stem cell transplant recipients [1]. A positive FCXM in the presence of negative complement-dependent cytotoxicity crossmatch (CDCXM) is associated with higher rates of graft rejection and early graft failure [1,2].

Rituximab (RTX) is a genetically engineered recombinant chimeric mouse-human monoclonal antibody (IgG1-k) against CD20 [3,4]. Apart from its use in the treatment of B-cell malignancies [5,6] and autoimmune disorders [7], it is extensively utilized to control and prevent humoral rejection in solid organ as well as haploidentical haematopoietic stem cell transplant [[8], [9], [10], [11], [12], [13]].

Inclusion of B-cell depleting agents like RTX in immunosuppressive protocols along with plasmapheresis/immunoadsorption has allowed renal transplantation across the ABO barrier and expanded the donor pool [[14], [15], [16]]. The long term graft outcome in ABO-incompatible renal transplantation (ABOi-RT) reported by most centres are comparable with ABO-compatible (ABOc-RT) [[16], [17], [18]].

However, RTX when present in the sera of transplant recipients causes false-positive CDCXM (by exerting a cytolytic effect on B-cells) as well as B-cell FCXM (B-FCXM; secondary to recognition by anti-human antibody) [[19], [20], [21], [22]]. Its impact on T-cell FCXM (T-FCXM) has not been reported to date.

Pronase treatment of donor lymphocytes cleaves Fc receptors and thus CD20 from B-cell surface, thereby removing the RTX effect and facilitating the correct interpretation of the B-FCXM [22,23]. The Luminex based tests are not affected by RTX, and to rule out the presence of donor-specific antibodies (DSA) more definitively, a virtual crossmatch using the single antigen bead (SAB) assay is done alongside. Testing of historic serum collected before RTX therapy is another feasible option to rule out DSA [19].

The aim of this study was to evaluate the effect of RTX on T- and B-cell FCXM using Pronase (P-) and Non-pronase (NP-) treated donor lymphocytes and compare it to the SAB assay.

Section snippets

Patients

This is a retrospective analysis of 26 ABOi-RT and 2 haploidentical stem cell transplant recipients on RTX therapy whose samples were received for pre-transplant workup in the HLA and Immunogenetics laboratory in the Department of Transfusion medicine & Immunohaematology between December 2015 to February 2020.

As part of Pre-transplant workup, FCXM using P- and NP- treated donor lymphocytes and SAB assay was performed for all recipients treated with RTX.

Cell isolation

Donor lymphocyte isolation was done by

Patients

A total of 28 patients (26 renal and 2 stem cell transplant recipients) on RTX therapy were analyzed during the study period. The mean age of recipients was 33.5 ± 10.5 years with a male to female ratio (M: F) of 3:1. The pre-transplant evaluation was done against 30 donors (2 donors each for 2 stem-cell transplant recipients). The mean time of testing post-RTX therapy was 21 days. Further demographics are given in Table 1.

For each recipient, B- and T-FCXM using NP- and P- treated donor

Discussion

RTX has replaced splenectomy in most of the immunosuppression protocols for ABOi-RT due to its B-cell depleting action [15,16,24,25]. It is also indicated in induction chemotherapy of highly sensitized transplant recipients to reduce panel reactive antibody (PRA) [[26], [27], [28]] as well as to treat humoral allograft rejection [10,[28], [29], [30]]. Its half-life is between 1.6 and 20 days but is relative to dose and frequency of administration. It can be detected in the serum of transplant

Conclusion

Though traditionally RTX is described to impact B-FCXM only, we describe a consistent finding of ‘T-cell tailing’ phenomenon in 57% of total FCXMs, which clears with pronase treatment. The absence of DSA in all these recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I donor-specific antibody. To our knowledge, this is the first study to report the effect of RTX on T-FCXM.

In the light of the growing use of RTX in various

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

All authors declare that they do not have any potential conflict of interest that could inappropriately influence the present study.

Acknowledgements

None.

References (45)

  • E. Lazar-Molnar et al.

    The effect of pronase on lymphocyte surface markers and implications for flow cytometric crossmatch

    Hum. Immunol.

    (2015 Oct)
  • S.J. Hetrick et al.

    Impact of pronase on flow cytometric crossmatch outcome

    Hum. Immunol.

    (2011 Apr)
  • H. Park et al.

    Frequent false-positive reactions in Pronase-treated T-cell flow cytometric cross-match tests

    Transplant. Proc.

    (2012 Jan 1)
  • A.T. Vaughan et al.

    Activatory and inhibitory Fcγ receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain

    J. Biol. Chem.

    (2015 Feb 27)
  • R.A. Bray et al.

    The flow cytometric crossmatch. Dual-color analysis of T cell and B cell reactivities

    Transplantation.

    (1989 Nov)
  • J. Rebibou et al.

    T-cell flow-cytometry crossmatch and long-term renal graft survival

    Clin. Transpl.

    (2004 Oct)
  • J.P. Deans et al.

    CD20-mediated apoptosis: signalling through lipid rafts

    Immunology.

    (2002 Oct)
  • G.A. Leget et al.

    Use of rituximab, the new FDA-approved antibody

    Curr. Opin. Oncol.

    (1998 Nov)
  • B.D. Cheson et al.

    Monoclonal Antibody therapy for B-cell non-Hodgkin’s lymphoma

    N. Engl. J. Med.

    (2008 Aug 7)
  • M.G. Warnock et al.

    Cryptic T-cell epitopes and their role in the pathogenesis of autoimmune diseases

    Rheumatology.

    (1997 Nov 1)
  • A.A. Zachary et al.

    Desensitization for solid organ and hematopoietic stem cell transplantation

    Immunol. Rev.

    (2014 Mar)
  • J.M. Aranda et al.

    Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral rejection: a case report

    Transplantation.

    (2002 Mar 27)
  • Cited by (4)

    • Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis

      2021, Transplant Immunology
      Citation Excerpt :

      Complement-dependent cytotoxicity and FCXM for such patients could be considerably influenced. These methods are difficult because of nonspecific antibody binding to Fc receptors on B cells, which is associated with false-positive results. [19] After transplantation, FCXM has not been used for annual monitoring due to a problem with availability of intact donor PBMC.

    1

    First co-authors; Contributed equally.

    View full text