DISCOVERY AND VALIDATION OF A URINARY EXOSOME MRNA SIGNATURE FOR THE DIAGNOSIS OF HUMAN KIDNEY TRANSPLANT REJECTION
Background: The traditional biomarkers currently used to monitor a kidney allograft for rejection are late markers of injury and they lack sensitivity and specificity. Allograft biopsies on the other hand, are invasive and costly. The authors describe the discovery and validation of two urinary exosomal mRNA multigene signatures for the diagnosis of acute T cell–mediated and antibody-mediated rejection and chronic, active antibody-mediated rejection in recipients of kidney transplant. Using a clinically validated platform for exosome isolation and analysis, they demonstrated the high stability of urinary exosomes and the reliability of this approach in monitoring patients for allograft rejection. One gene signature for all-cause rejection and another for discriminating T cell–mediated rejection from antibody-mediated rejection showed high predictive performances and offer clinicians the possibility of new tools for monitoring emergence of rejection in kidney allografts.
Rania El Fekih, James Hurley, Vasisht Tadigotla, Areej Alghamdi, Anand Srivastava, Christine Coticchia, John Choi, Hazim Allos, Karim Yatim, Juliano Alhaddad, Siawosh Eskandari, Philip Chu, Albana B. Mihali, Isadora T. Lape, Mauricio P. Lima Filho, Bruno T. Aoyama, Anil Chandraker, Kassem Safa, James F. Markmann, Leonardo V. Riella, Richard N. Formica, Johan Skog and Jamil R. Azzi
CyTOF-Enabled Analysis Identifies Class-Switched B Cells as the Main Lymphocyte Subset Associated With Disease Relapse in Children With Idiopathic Nephrotic Syndrome
Background: B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid–dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.
Miguel Fribourg1†, Michela Cioni2†, GianMarco Ghiggeri2, Chiara Cantarelli1,3, Jeremy S. Leventhal4, Kelly Budge1, Sofia Bin1, Leonardo V. Riella5, Manuela Colucci6, Marina Vivarelli7, Andrea Angeletti2, Laura Perin8‡ and Paolo Cravedi1*‡
A Comprehensive Phenotypic and Functional Immune Analysis Unravels Circulating Anti-Phospholipase A2 Receptor Antibody Secreting Cells in Membranous Nephropathy Patients
Background: Primary membranous nephropathy (MN) is characterized by the presence of antipodocyte antibodies, but studies describing phenotypic and functional abnormalities in circulating lymphocytes are limited. After adjusting for multiple testing, plasma cells and regulatory B cells (BREG) were significantly higher (P < 0.05) in MN patients compared with both control groups. The percentages of circulating plasma cells correlated with serum anti-PLA2R antibody levels (P = 0.042) and were associated with disease activity. Ex vivo-expanded PLA2R-specific IgG-producing plasmablasts generated from circulating PLA2R-specific memory B cells (mBCs) correlated with serum anti-PLA2R IgG antibodies (P < 0.001) in MN patients. Tumor necrosis factor-α (TNF-α) was the only significantly increased cytokine in MN patients (P < 0.05), whereas there was no significant difference across study groups in the autoantibody and antiviral antibody repertoire.
Chiara Cantarelli 1 2, Marta Jarque 3, Andrea Angeletti 4, Joaquin Manrique 5, Susan Hartzell 1, Timothy O'Donnell 6, Elliot Merritt 6, Uri Laserson 6, Laura Perin 7, Chiara Donadei 4, Lisa Anderson 1, Clara Fischman 1, Emilie Chan 1, Juliana Draibe 3, Xavier Fulladosa 3, Joan Torras 3, Leonardo V Riella 8 9, Gaetano La Manna 4, Enrico Fiaccadori 2, Umberto Maggiore 2, Oriol Bestard 3, Paolo Cravedi 1
REGULATORY T CELLS ENGINEERED WITH TCR SIGNALING–RESPONSIVE IL-2 NANOGELS SUPPRESS ALLOIMMUNITY IN SITES OF ANTIGEN ENCOUNTER
Background: Adoptive cell transfer of ex vivo expanded regulatory T cells (Tregs) has shown immense potential in animal models of auto- and alloimmunity. However, the effective translation of such Treg therapies to the clinic has been slow. Because Treg homeostasis is known to require continuous T cell receptor (TCR) ligation and exogenous interleukin-2 (IL-2), some investigators have explored the use of low-dose IL-2 injections to increase endogenous Treg responses. Systemic IL-2 immunotherapy, however, can also lead to the activation of cytotoxic T lymphocytes and natural killer cells, causing adverse therapeutic outcomes. Here, we describe a drug delivery platform, which can be engineered to autostimulate Tregs with IL-2 in response to TCR-dependent activation, and thus activate these cells in sites of antigen encounter. To this end, protein nanogels (NGs) were synthesized with cleavable bis(N-hydroxysuccinimide) cross-linkers and IL-2/Fc fusion (IL-2) proteins to form particles that release IL-2 under reducing conditions, as found at the surface of T cells receiving stimulation through the TCR. Tregs surface-conjugated with IL-2 NGs were found to have preferential, allograft-protective effects relative to unmodified Tregs or Tregs stimulated with systemic IL-2. We demonstrate that murine and human NG–modified Tregs carrying an IL-2 cargo perform better than conventional Tregs in suppressing alloimmunity in murine and humanized mouse allotransplantation models. In all, the technology presented in this study has the potential to improve Treg transfer therapy by enabling the regulated spatiotemporal provision of IL-2 to antigen-primed Tregs.
Siawosh K Eskandari, Ina Sulkaj, Mariane B Melo, Na Li, Hazim Allos, Juliano B Alhaddad, Branislav Kollar, Thiago J Borges, Arach S Eskandari, Max A Zinter, Songjie Cai, Jean Pierre Assaker, John Y Choi, Basmah S Al Dulaijan, Amr Mansouri, Yousef Haik, Bakhos A Tannous, Willem J van Son, Henri G D Leuvenink, Bohdan Pomahac, Leonardo V Riella , Li Tang, Marc A J Seelen, Darrell J Irvine, Jamil R Azzi
Science translational medicine vol. 12,569 (2020): eaaw4744. doi:10.1126/scitranslmed.aaw4744
HUMAN REGULATORY T CELLS UNDERGO SELF-INFLICTED DAMAGE VIA GRANZYME PATHWAYS UPON ACTIVATION
Background: Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show – by using cytometry by time of flight (CYTOF) – an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non-GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.
Esilida Sula Karreci, Siawosh K. Eskandari, Farokh Dotiwala, Sujit K. Routray, Ahmed T. Kurdi, Jean Pierre Assaker, Pavlo Luckyanchykov, Albana B. Mihali, Omar Maarouf, Thiago J. Borges, Abdullah Alkhudhayri, Kruti R. Patel, Amr Radwan, Irene Ghobrial, Martina McGrath, Anil Chandraker, Leonardo V. Riella, Wassim Elyaman, Reza Abdi, Judy Lieberman, and Jamil Azzi1
INTEGRATED KIDNEY EXOSOME ANALYSIS FOR THE DETECTION OF KIDNEY TRANSPLANT REJECTION
Background: Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.
Jongmin Park, Hsing-Ying Lin, Jean Pierre Assaker, Sangmoo Jeong, Chen-Han Huang, A Kurdi, Kyungheon Lee, Kyle Fraser, Changwook Min, Siawosh Eskandari, Sujit Routray, Bakhos Tannous, Reza Abdi, Leonardo Riella, Anil Chandraker, Cesar M Castro, Ralph Weissleder, Hakho Lee, Jamil R Azzi
MICRONEEDLE-BASED LOCAL DELIVERY OF CCL22 AND IL-2 ENRICHES TREG HOMING TO THE SKIN ALLOGRAFT AND ENABLES TEMPORAL MONITORING OF IMMUNOTHERAPY EFFICACY
Background: Skin allografts only serve as temporary dressing for patients suffering major burns due to their high immunogenicity and rejection by the immune system, requiring systemic immunosuppressive therapies that lead to deleterious side effects. Microneedle arrays composed of hyaluronic acid (HA) and placed on skin allografts can locally deliver immunomodulators and simultaneously sample immune cells in interstitial fluid to monitor the response to the therapy. The cells can be retrieved from the microneedles for downstream analysis by degrading the HA using a reducing agent. Using an allogeneic skin transplantation model, it is shown that the microneedle-mediated local delivery of the chemokine CCL22 (to attract Tregs) and the cytokine IL-2 (to promote their expansion) increases the local immune suppression in the allograft. Moreover, immune cell population in the allograft correlates with that seen in the microneedles. The delivery and sampling functions of the microneedle arrays can help regulate the immune system locally, without inducing systemic immune suppression, and facilitate the monitoring of the response to the therapy following skin transplantation.
Núria Puigmal, Pere Dosta, Zhabiz Solhjou, Karim Yatim, Cynthia Ramírez, John Y. Choi, Juliano B. Alhaddad, Ana Paula Cosme, Jamil Azzi,* and Natalie Artzi*
Adv. Funct. Mater. 2021, 31, 2100128. https://doi.org/10.1002/adfm.202100128
DONOR MYELOID DERIVED SUPPRESSOR CELLS (MDSCS) PROLONG ALLOGENEIC CARDIAC GRAFT SURVIVAL THROUGH PROGRAMMING OF RECIPIENT MYELOID CELLS IN VIVO
Background: Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2Kb, I-Ab) recipients pre-treated with BALB/c MDSCs were transplanted with either donor-type (BALB/c, H2Kd, I-Ad) or third-party (C3H, H2Kk, I-Ak) cardiac grafts. Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b+Gr1+ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b+Gr1+ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient’s endogenous MDSCs.
Songjie Cai, John Y. Choi, Thiago J. Borges, Hengcheng Zhang, Ji Miao, Takaharu Ichimura, Xiaofei Li, Simiao Xu, Philip Chu, Siawosh K. Eskandari, Hazim Allos, Juliano B. Alhaddad, Saif A. Muhsin, Karim Yatim, Leonardo V. Riella, Peter T. Sage, Anil K. Chandraker & Jamil R. Azzi
Sci Rep 10, 14249 (2020). https://doi.org/10.1038/s41598-020-71289-z
REGULATORY CD8 T CELLS THAT RECOGNIZE QA-1 EXPRESSED BY CD4 T-HELPER CELLS INHIBIT REJECTION OF HEART ALLOGRAFTS
Background: Induction of longstanding immunologic tolerance is essential for the survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.
John Y Choi, Siawosh K Eskandari, Songjie Cai, Ina Sulkaj, Jean Pierre Assaker, Hazim Allos, Juliano AlHaddad, Saif A Muhsin, Eman Alhussain, Amr Mansouri, Melissa Y Yeung, Marc A J Seelen, Hye-Jung Kim, Harvey Cantor, Jamil R Azzi