Isolation of human antigen‐specific regulatory T cells with high suppressive function

Abstract

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition,we show that the depletion of CD154 + cells from LAP + or GARP + Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4 + CD25 high or CD4 + CD25 high CD127 low Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.Keywords: Antigen-specific r CD154 r GARP r LAP r Regulatory T (Treg) cells Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIn organ transplantation, acute rejection can be prevented by a combination of nonspecific immunosuppressive drugs and immunomodulatory induction therapies, yet chronic rejection and chronic graft dysfunction remain a clinical problem [1]. In addition, chronic nonspecific immunosuppression causes renal dysfunction and predisposes patients to infections and increased Correspondence: Dr. Elmar Jaeckel e-mail: Jaeckel.Elmar@mh-hannover.de risk of malignancies [1]. Innate and adaptive immune responses can be controlled by Treg cells, thereby offering new therapeutic options after organ transplantation [2][3][4][5][6]. CD4 + CD25 high FOXP3 + Treg cells have been shown to be of major importance in allospecific tolerance in animal models and to influence donor-specific immune responses in transplant patients [7][8][9][10]. After allogeneic hematopoietic stem cell transplantation, polyspecific Treg cells can prevent GVH disease without influencing * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 2592-2602 Cellular immune response 2593 graft-versus-leukemia responses [11,12]. Therefore, polyspecific CD4 + CD25 high cells are currently being evaluated after stem cell transplantation [13]. However, under nonlymphopenic conditions as seen in patients with autoimmune diseases or in patients after organ transplantation, polyspecific Treg cells have so far been largely ineffective in controlling immune responses [5,14,15] [24], and CD137 (4-1BB) [25] were described as Treg-cell activation markers after polyspecific Treg-cell activation.In this manuscript, we demonstrated for the first time that LAP and GARP can be use...

Transcriptome Analysis of Mycobacteria-Specific CD4+ T Cells Identified by Activation-Induced Expression of CD154

“…The expression of IL-10 transcript, together with the relative increase in Lrrc32 and Foxp3 transcripts in the CD154 − population compared to the CD154 + population (Figure 3D and 4C), was consistent with previous reports showing that regulatory T cells lack pre-formed CD154 and that induced regulatory T cells are unlikely to be included in the CD154 + population [43–45]. …”

“…Expression microarray analysis performed on mycobacteria-specific CD4 + T cells further supported the validity of using CD154 expression as a surrogate marker for antigen-specificity. In addition, microarray analysis revealed that while multiple functional T helper subsets were present in the CD154 + population, antigen-specific regulatory T cells were likely to be excluded, consistent with previous reports in the literature [43–45]. …”

Combined PI3K/Akt and Hsp90 targeting synergistically suppresses essential functions of alloreactive T cells and increases Tregs

“…Furthermore, it has been demonstrated that Hsp90-mediated Hsp70 induction is necessary for promoting T reg survival and suppressive function. Strikingly, CD4 + CD25 + LAP + iT regs demonstrated high suppressive and antigen-specific capacity, substantiating a recent report describing LAP + expression on CD4 T cells as a reliable marker for the identification of antigen-specific T regs [28]. Furthermore, Mahalingam et al [67] demonstrated that isolated CD4 + CD25 + LAP + showed higher suppressive activity than CD4 + CD25 + LAP 2 , underscoring the use of LAP expression for identification of a potent, suppressive subgroup of T regs .…”

GARP: a surface molecule of regulatory T cells that is involved in the regulatory function and TGF-β releasing

“…In recent years, GARP is discovered to express highly on the surface of activated human Tregs in vitro [36]. Freshly isolated human Tregs and Tregs clones express a low level of surface GARP.…”