Applications of immunosequencing in transplant research

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    Despite advances in transplant research, transplant recipients are still at risk of mortality and morbidity due to complications such as severe infection, graft-versus-host disease (GVHD), and graft rejection.

    Reconstitution of the T-cell repertoire after hematopoietic stem cell transplant (HSCT) is associated with many of these complications. In fact, high T-cell receptor (TCR) diversity has been linked to longer survival among transplant recipients (Figure 1). (1,2)

    immunosequencing in transplant research

    Figure 1. Correlation between TCR repertoire diversity and survival. Data show peripheral TCR diversity among cord blood transplant recipients before transplant and at five timepoints after transplant. Patients who served more than one year post transplant showed a rapid recovery of TCR diversity. Those who died within a year of transplant showed a decrease in TCR diversity through 100 days. TCR: T-cell receptor. Adapted from Milano et al. (3)

    Additional research is needed to better understand how donor and recipient characteristics impact T-cell repertoire reconstitution. These insights could ultimately be used to refine donor selection criteria and optimize the clinical care of transplant recipients. (2)

    Researchers are using the immunoSEQ® Assay to track T-cell reconstitution in transplant recipients to identify T-cell repertoire characteristics in both donors and recipients that correlate with outcomes. (1)

    To learn more about this technology, view our webinar on the Applications of immunoSEQ in Transplant Research.

    Monitoring T-Cell Reconstitution Over Time and Space

    One key advantage of the immunoSEQ Assay is its ability to work with a variety of sample types. In a recent study by Harris et al., researchers used the immunoSEQ Assay to track changes in CD4+ and CD8+ T-cell repertoires in both the blood and cerebrospinal fluid (CSF) in HSCT recipients with relapsing-remitting multiple sclerosis (MS). (4)

    The immunoSEQ Data revealed extensive, durable remodeling of the CD4+ T-cell repertoire, which was more robust in the CSF than in the periphery. In contrast, remodeling of the CD8+ T-cell repertoire was less extensive and more variable (Figure 2). (4)

    Figure 2. Autologous HSCT induces the ablation of pre-existing peripheral T-cell repertoires at 12, 24, and 48 months. Proportion of undetectable (teal) versus detected (orange) clones by ultra-deep sequencing of TCR repertoires in CD4+ T cells and CD8+ T cells. Adapted from Harris et al. (4)

    A key feature of this study was that the authors were able to both track T cells over time—up to 4 years post transplant—and compare T-cell repertoire overlap between T-cell compartments. Being able to track the T-cell repertoire over time is key in transplant research. Recipients often experience prolonged T-cell deficiencies that can last for years. (2)

    In addition, since the CSF is more likely to contain pathological T-cell clones that are directly involved in MS progression, the immunoSEQ Data were able to shed light on whether analyzing difficult-to-obtain CSF samples is necessary. This study revealed that, although there are differences between the two T-cell compartments, the peripheral repertoire can act as a surrogate for the CSF repertoire, providing hope for a peripheral biomarker.

    What is the Link Between T-cell Reconstitution and GVHD?

    The incredible complexity of the T-cell repertoire makes it difficult to analyze. While T-cell reconstitution following bone marrow transplant is important for outcomes, there are conflicting data on the association between T-cell repertoire diversity and GVHD. (1)

    High-throughput sequencing technologies like the immunoSEQ Assay have made it possible to quantitatively track individual T-cell clones over time. The immunoSEQ Assay enabled researchers to track T-cell reconstitution among bone marrow transplant patients to see how repertoire complexity is associated with GVHD.

    This uncovered an initial decrease in repertoire complexity followed by a time-dependent increase for most transplant recipients. However, there were no apparent changes in T-cell repertoire complexity among those who developed GVHD. The study describes new methods to quantify and characterize the post-transplant T-cell repertoire that have been made possible by high-throughput sequencing technologies. (1)

    The sequencing results for this study, as well as many others, are publicly available on our immuneACCESS® platform. Researchers can mine these data using our suite of analysis tools to uncover additional insights.

    Can the T-cell Repertoire Shed Light on Response to Human Leukocyte Antigen Mismatches in HSCT?

    One key limitation in HSCT is the need for a human leukocyte antigen (HLA) match between donor and recipient. A strong HLA mismatch can activate and expand alloreactive T-cell clones, resulting in graft rejection or GVHD. (5,6)

    In theory, a detailed profile of the recipient’s T-cell repertoire should be able to provide clues as to whether the recipient is likely to mount an immune response against a graft. However, a recent study by Bettens et al. shows that this is not as straightforward as one would hope.

    Using the immunoSEQ Assay to investigate the alloreactive CD8+ T-cell response against cells with various HLA mismatches, Bettens et al. highlighted the significant challenges to using the T-cell repertoire to predict alloreactive T-cell responses before and after transplant. (6)

    The work supports the hypothesis that T cells with high TCR flexibility can bind to foreign peptide–HLA complexes in alloimmune processes and that inflammation may influence the allogenic immune response by activating new alloreactive T cells. (6)

    How Can T Cells Help Differentiate Between GVHD and Drug Hypersensitivity Reactions?

    GVHD and drug hypersensitivity reactions often present with similar symptoms. Distinguishing between these two conditions is vital because they require different therapeutic interventions. (7)

    From the perspective of the T-cell repertoire, these two conditions are mechanistically very different—GVHD is likely mediated by clonal amplification of T cells (1,7), while in drug hypersensitivity reactions, T-cell responses are polyclonal and non-specific. (7)

    Immunosequencing has the power to identify T-cell-related biomarkers of disease. Indeed, the immunoSEQ Assay has been instrumental in defining T-cell signatures of cytomegalovirus (CMV) status and SARS-CoV-2 infection.

    With regard to transplant, the immunoSEQ Assay has shown that T-cell clonality is significantly higher among those with GVHD than those with drug hypersensitivity reactions (Figure 3). (7) This research not only shows the importance of T cells in these conditions but also may pave the way for T-cell-based biomarkers to aid in clinical decision making.

    immunosequencing in transplant research

    Figure 3. The TCR repertoire was sequenced from skin biopsy samples of 7 subjects with DHR and 10 subjects with GVHD. T-cell clonality is significantly higher among individuals with GVHD than among those with drug hypersensitivity reactions, despite the two conditions having similar clinical presentations. DHR: drug hypersensitivity reaction; GVHD: graft-versus-host disease. Adapted from Chang et al. (7)

    Can T-cell Therapy Improve Outcomes in HSCT Recipients?

    Tracking transgenic T-cells designed to reduce relapse

    While HSCT has proven invaluable for the treatment of hematologic malignancies, many patients will relapse. Post-HSCT, prophylactic treatment with T cells engineered to express TCRs that target selected acute myeloid leukemia (AML) antigens can reduce relapse among AML patients. (8)

    The immunoSEQ Assay provided a means of monitoring these engineered T cells to see which clones expand and persist. Among eight AML patients who maintained long-term responses after HSCT, an average of 5.5 transgenic T-cell clonotypes, which constituted >80% of cells present in the initial infusion product, were detected at the furthest analyzed time point (Figure 4), suggesting that the most persistent responses were mediated by clonal T cells.

    Figure 4. T-cell clonal evolution among T-cell infusion product and product recipients. The far-left column for each patient shows the frequencies of individual clonotypes within the transgenic T-cell infusion product administered to HSCT recipients. Other columns show the peripheral T-cell clonotype frequencies at various time points after infusion for eight select individuals. HSCT: hematopoietic stem cell transplant. Adapted from Chapuis et al. (8)

    Using Adoptive T-cell Therapies to Reduce Viral Reactivation

    Another serious complication in transplant recipients is reactivation of latent viruses, specifically CMV. Compelling evidence suggests that CMV reactivation in solid organ transplant recipients is linked to an inability to establish stable immunological memory against CMV.

    Despite this, current prophylactic and therapeutic strategies for CMV depend on antiviral therapy instead of immune reconstitution. CMV-specific immune reconstitution could provide an alternative approach to prevent viral complications without requiring long-term antiviral therapy. (9)

    A CMV-specific adoptive T-cell therapy (ACT) approach designed to promote immune reconstitution in solid organ transplant recipients with CMV complications has demonstrated positive clinical outcomes with reasonable safety. (10)

    A study by Smith et al. used the immunoSEQ Assay to monitor the T-cell repertoire among these individuals and to determine if T-cell repertoire properties correlate with improved clinical outcomes (Figure 5). (9)

    The immunoSEQ Data showed that all individuals who responded to ACT displayed significant clonotypic expansion post ACT. However, clonotype expansion was less robust among those who did not respond to ACT therapy (Figure 5C). (9)

    In other words, immune control after ACT requires significant repertoire remodeling, which may be hampered in non-responders. Read our recent article to discover more about CMV infection and its impact on the adaptive immune system.

    Figure 5. T-cell clonality in recipients of solid organ transplants after ACT. (A) The proportion of productive rearrangements among small, medium, large, or hyperexpanded clones. (B) Relationship between fold change in productive clonality long-term post ACT and T-cell clonality pre ACT. (C) Data showing the number of clonotypes in each patient that exhibited significant expansion post ACT. The orange circle highlights non-responders. ACT: adoptive T-cell therapy. Adapted from Smith et al. (9)

    T-cell therapies are revolutionizing cancer therapy in other ways as well. To see how the immunoSEQ Assay has increased the understanding of chimeric antigen receptor (CAR) T-cell treatment and engineered T-cell therapies, read our article Gaining insights into T-cell therapies with immunosequencing.

    Immunosequencing in Transplant Research Summarized

    For researchers studying T-cell biology and its role in clinical outcomes post transplant, it is important to understand how immune reconstitution is shaped by different factors in transplant donors and recipients. A more detailed understanding of these factors may improve therapeutic strategies and clinical outcomes for transplant recipients.

    Our immunoSEQ Assay provides a scalable, reproducible, and quantitative view of the T-cell repertoire. The immunoSEQ Assay works with a range of sample types, including but not limited to gDNA and cDNA isolated from whole blood, peripheral blood mononuclear cells, or formalin-fixed, paraffin-embedded tissue.

    We can provide you with the tools to propel your research. To learn more about the immunoSEQ Services and Kits, visit our products page. If you would like help getting started, get in touch with our product team.

    For Research Use Only. Not for use in diagnostic procedures.

    References

    1. Meier JA, Haque M, Fawaz M, Abdeen H, Coffey D, Towlerton A, et al. T Cell Repertoire Evolution after Allogeneic Bone Marrow Transplantation: An Organizational Perspective. Biol Blood Marrow Transplant. 2019;25(5):868–82.

    2. Velardi E, Tsai JJ, van den Brink MRM. T cell regeneration after immunological injury. Nat Rev Immunol. 2021;21(5):277–91.

    3. Milano F, Emerson RO, Salit R, Guthrie KA, Thur LA, Dahlberg A, et al. Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation. Frontiers in Oncology 2020;10:2219.

    4. Harris KM, Lim N, Lindau P, Robins H, Griffith LM, Nash RA, et al. Extensive intrathecal T cell renewal following hematopoietic transplantation for multiple sclerosis. JCI Insight 2020;5(2):e127655.

    5. Arrieta-Bolaños E, Crivello P, Metzing M, Meurer T, Ahci M, Rytlewski J, et al. Alloreactive T Cell Receptor Diversity against Structurally Similar or Dissimilar HLA-DP Antigens Assessed by Deep Sequencing. Front Immunol. 2018;9:280.

    6. Bettens F, Calderin Sollet Z, Buhler S, Villard J. CD8+ T-Cell Repertoire in Human Leukocyte Antigen Class I-Mismatched Alloreactive Immune Response. Frontiers in Immunology 2021;11:3440.

    7. Chang L-W, Doan LT, Fields P, Vignali M, Akilov OE. The Utility of T-Cell Clonality in Differential Diagnostics of Acute Graft-versus-Host Disease from Drug Hypersensitivity Reaction. J Invest Dermatol. 2020;140(6):1282–5.

    8. Chapuis AG, Egan DN, Bar M, Schmitt TM, McAfee MS, Paulson KG, et al. T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant. Nat Med. 2019;25(7):1064–72.

    9. Smith C, Corvino D, Beagley L, Rehan S, Neller MA, Crooks P, et al. T cell repertoire remodeling following post-transplant T cell therapy coincides with clinical response. J Clin Invest. 2019;129(11):5020–32.

    10. Smith C, Beagley L, Rehan S, Neller MA, Crooks P, Solomon M, et al. Autologous Adoptive T-cell Therapy for Recurrent or Drug-resistant Cytomegalovirus Complications in Solid Organ Transplant Recipients: A Single-arm Open-label Phase I Clinical Trial. Clin Infect Dis. 2019;68(4):632–40.

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