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. 2021 Mar;203(3):480-492.
doi: 10.1111/cei.13537. Epub 2020 Dec 28.

CD40L-stimulated B cells for ex-vivo expansion of polyspecific non-human primate regulatory T cells for translational studies

P Alonso-Guallart  1 , N Llore  1 , E Lopes  1 , S-B Kofman  1 , S-H Ho  1 , J Stern  1 , G Pierre  1 , K Bruestle  1 , Q Tang  2 , M Sykes  1   3   4 , A Griesemer  1   4
Affiliations

CD40L-stimulated B cells for ex-vivo expansion of polyspecific non-human primate regulatory T cells for translational studies

P Alonso-Guallart et al. Clin Exp Immunol. 2021 Mar.

Abstract

The therapeutic applications of regulatory T cells (Tregs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific Tregs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded Tregs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of Treg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific Tregs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.

Keywords: CD40L-stimulated B cells; non-human primates; regulatory T cells; tolerance.

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Conflict of interest statement

Q. T. is a co‐inventor on two patents on regulatory T cell therapy and a cofounder of Sonoma Biotherapeutics. The rest of the authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Expansion and activation of cynomolgus macaque (MCM) B cells for regulatory T cell (Treg) expansion. B cell expansion from fresh or cryopreserved peripheral blood mononuclear cells (PBMCs) or splenocytes. Protocol 1 includes 10 lines, protocol 2 includes 14 lines and protocol 3 includes 18 lines from nine animals. (a) Schema for the expansion and activation of MCM B cells. (b) B cell growth (mean and standard error of the mean (s.e.m.) from day 0 until the harvest for each of the B cell expansion protocols. (c) Average cell number at the end of the B cell culture for each protocol. Average and s.e.m. are detailed at the top of each bar. (d) Fold expansion for each B cell protocol. (e) Expression of B cell characteristic markers [major histocompatibility complex (MHC) II, CD80, CD20, CD40] and assessment of T cell contamination for each B cell expansion protocol at different time‐points (day 0, restimulation and harvest).
Fig. 2
Fig. 2
Expansion of cynomolgus macaque (MCM) regulatory T cells (Tregs) with CD40L‐sBc. Expansion of MCM Tregs with CD40L‐sBc stimulation. (a) Treg numbers at each restimulation and take‐down (n = 57). (b) Mean and standard error of the mean (s.e.m.) of the fold expansion of the Treg lines at each restimulation until the take‐down. ‘R’ represents restimulation number. (c) Percentage of CD25+FoxP3+ cells within CD3+CD4+ cells at the take‐down. (d) Representative Helios expression on cultured Tregs (red) (n = 5). (e) Representative cytotoxic T lymphocyte antigen‐4 (CTLA‐4) expression on cultured Tregs (red) and cultured Tregs activated with phorbol myristate acetate (PMA)/ionomycin for 4 h (blue) (n = 5). (f) Percentage of suppressive capacity of the Treg lines (n = 57). The dotted line represents 50% suppression as reference. Mean of the suppressive capacity is represented with a black line for each ratio of Treg : peripheral blood mononuclear cells (PBMCs). (g) Correlation between CD45RA and forkhead box protein 3 (FoxP3) expression. Correlation between the percentage of Treg‐specific demethylated region (TSDR) demethylation and the (h) expression of FoxP3, (i) suppressive capacity (the Treg : responder ratio that achieves 50% suppression of the positive control) and (j) days of culture. Each color represents a different Treg line that is the same throughout Fig. 2h–j (n = 15).
Fig. 3
Fig. 3
Forkhead box protein 3 (FoxP3) expression and suppressive capacity of CD4+CD8+ DP T cells. (a) Comparison between the percentage of CD3+CD4‐CD8+ SP and CD3+CD4+CD8+ double‐positive (DP) T cells present in the regulatory T cell (Treg) cultures at the harvest time‐point (days 14, 21 or 28) (n = 57). Study of the (b) FoxP3, (c) Helios expression and (d) suppressive capacity [mean and standard error of the mean (s.e.m.)] of total CD3+ T cells (black), CD3+CD4+CD8− SP T cells (blue) and CD3+CD4+CD8+ DP T cells (red) (n = 2). (e) Correlation between the percentage of Treg‐specific demethylated region (TSDR) demethylation in total CD3+ cells and the percentage of CD8+ single‐positive (SP) and CD4+CD8+ (DP) T cells present in culture. Each color represents a different Treg line (n = 15). (f) Assessment of the secretion of interferon (IFN)‐γ, interleukin (IL)‐13 and IL‐17A in peripheral blood mononuclear cells (PBMCs) and cultured sBc‐Tregs (n = 5).
Fig. 4
Fig. 4
Difference in suppression based on the responder‐type cells. (a) Schema for the expansion protocol of cynomolgus macaque (MCM) regulatory T cells (Tregs) with CD40L‐sBc. (b) Representative figure of a specificity assay with peripheral blood mononuclear cells (PBMCs) as responders. (c) Representative figure of before (left panel) and after (right panel) magnetic‐activated cell sorted (MACS)‐isolated T cells. (d) Representative figure of a specificity assay with MACS‐isolated T cells as responders.
Fig. 5
Fig. 5
Specificity data for cynomolgus macaque (MCM) CD40L‐sBc‐stimulated regulatory T cells (Tregs). Mean and standard error of the mean (SEM) of the suppressive capacity of CD40L‐sBc Tregs to responders that were (a,b) autologous to Tregs (n = 5, n = 3), (c,d) major histocompatibility complex (MHC)‐matched (n = 4, n = 2) or (e,f) MHC‐mismatched to CD40L‐sBc used for Treg expansion (n = 3, n = 4) with a variety of stimulators. Responders were MACS‐isolated T cells from peripheral blood mononuclear cells (PBMCs) or splenocytes and stimulators were PBMCs.
Fig. 6
Fig. 6
Restimulation of sBc‐regulatory T cells (Tregs) after cryopreservation. Study of cryopreserved and re‐expanded CD40L‐sBc Tregs. Representation of two Treg lines from two different animals recultured under three different conditions. (a) Treg growth after thawing. (b) Fold expansion after 9 days of culture. (c) CD25+forkhead box protein 3 (FoxP3)+ expression in CD3+CD4+ cells. (d) Percentage of CD4+CD8+ T cells. (e) Suppressive capacity of the Treg lines cultured under each condition at the thawing day and days 2, 3, 4, 7 and 9 after reculture.
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