This application claims the benefit of Mexican Patent Application No. MX/a/2020/011355, filed Oct. 26, 2020, the contents of which are incorporated herein by reference.
The present invention provides a new method for the generation and expansion of allospecific human Type 1 regulatory T cells to induce tolerance to transplants and their use as an alternative or complementary therapy to conventional immunosuppression.
Transplantation is considered the most effective therapy for patients with advanced chronic injury in some organs. In the last 20 years, the quality and life expectancy of transplanted patients has increased due to the use of immunosuppressive drug-based therapy, whose purpose is to prevent allograft rejection development mediated by the adaptive and innate response from the patient immune system [1]. Currently, the survival rate of kidney transplants is above 90% at 5 years. However, long-term use of immunosuppressive drugs transplanted patients results in adverse events, including the development of cardiovascular diseases, increased susceptibility to infections, and neoplasia emergence, due to the widespread effect on the immunological system [2] [3] [4].
One of the most promising alternatives for tolerance induction to the allograft is Treg-based therapy, which includes the use of CD4+ T cells, aiming to specifically suppress inflammatory responses towards the graft and to maintain immune cell homeostasis [5] [6] [7].
The majority of Treg cells that have been used in immunotherapy for transplantation are denominated thymic regulatory T cells (tTreg), characterized by the expression of surface CD25, and the transcription factor Foxp3 [8]. These cells have been shown to be effective in the context of bone marrow allograft transplant to avoid graft versus host disease (GVDH). However, Foxp3+ Tregs have the disadvantage that they do not have a superficial specific marker that allows their purification prior to their transfusion into the patients [9] [10] [11]. Thus, in the last years, several research groups have proposed alternative therapies with another type of regulatory T cells denominated Type 1 regulatory T lymphocytes (Tr1).
Tr1 cells are CD4+ T lymphocytes capable of suppressing immune effector responses. They were shown to play an important role in oral tolerance and the intestinal microenvironment [12] [13]. Tr1 cells can be in vitro differentiated from CD4+ naïve T cells under suboptimal activating signals in the presence of anti-inflammatory cytokines [14] [15] [16]. This subpopulation is characterized by high production of IL-10, as well as TGF-β and a low or null IL-4 and IL-2 production [17] [18].
Tr1 lymphocytes were described for the first time in patients with Severe Combined Immunodeficiency (SCID) that had developed tolerance to allograft after allogeneic hematopoietic stem cell transplantation. These patients presented higher concentration levels of IL-10 in the serum, compared to those that had not developed allograft tolerance [19]. Later, it was demonstrated the presence of an alloreactive T lymphocyte subpopulation from which it was possible to identify, purify and clone the IL-10 coding gene, suggesting the development of tolerance mechanisms mediated by T lymphocytes [20].
Subsequent studies by Baccheta and coworkers described that this T CD4+ subpopulation had a low proliferative capability and high production of IL-10, when activated in vitro [21]. Further, it was demonstrated in a murine preclinical model of Inflammatory Bowel Disease (IBD), that in vitro stimulation of CD4+ T lymphocytes in the presence of exogenous IL-10, identified a T cell subpopulation that, when infused to IBD mice, was capable to resolve colitis, confirming the great potential of Tr1 cells in the regulation of inflammatory processes [20].
Importantly, Gagliani and coworkers reported the coexpression of the surface molecules CD49b and LAG-3 as specific markers of Tr1 subpopulation, both in murine and human CD4+T cells [22], allowing the identification and isolation of this T cell subset, which greatly contributed to the development of new protocols for their use in cellular therapy.
One of the first developed methodologies for the in vitro obtention of Tr1 lymphocytes was based on the coculture between peripheral blood mononuclear cells (PBMCs) or isolated CD4+ cells and monocytes (CD14+) obtained from different donors, in the presence of exogenous IL-10. The result was an heterogeneous population of anergic cells with suppressive capability against several antigens that contained Tr1 lymphocytes [14] [23].
Another developed methodology based on the coculture of isolated CD3+ and total PBMCs from 2 different individuals in the presence of IL-10 for 10 days generated a cellular product, which consisted of an heterogeneous population of anergic cells with suppressive capability, containing a low proportion of CD49b+LAG-3+ Tr1 lymphocytes (˜6% of the total population) and were named “IL-10 DLI” [24]. In the same work, it was reported clinical trial results from a cohort of 12 total patients with hematopoietic stem cell transplantation, using the IL-10 DLI population, to prevent the development of graft versus host disease (GVHD). Of note, four of those patients were able to avoid GVHD.
One of the most efficient methodologies for the generation of allospecific Tr1 cells is the use of tolerogenic DCs denominated DC10. These cells are characterized by the high production of IL-10, the expression of CD14, and the inhibitory receptors HLA-G and ILT4, which are crucial to allow the differentiation of allospecific Tr1 [25].
The tolerogenic capacity of DC10 was considered for the generation of regulatory T cells that were used in a large-scale collaborative international study for solid organ transplantation, named “the ONE Study” [26]. In this context, coculture between in vitro differentiated DC10 from monocytes (CD14+) and isolated CD4+ T lymphocytes in the presence of exogenous IL-10 during 10 days, induces an heterogeneous population of anergic cells with suppressive capability that includes a percentage of Tr1 lymphocytes (6-12%), named “T10” was obtained [27]. This cellular product has been used for immunotherapy in patients with renal transplant [27].
Despite the progress reached in the design of new methodologies aimed to obtain Tr1 cells with therapeutic potential, the main focus of theses studies has been the large-scale obtention of anergic cells with suppressive capability with the goal to reach the requested number of cells for their infusion in patients (approximately 5×105 to 2×106 cells/kg), thereby sacrificing the purity of the Tr1 population transferred. Moreover, these studies have not included a detailed phenotypic characterization from their cellular products to ensure the obtention of an homogeneous population with stable regulatory functions. Moreover the reported methodologies have not achieved the sufficient numbers to represent a feasible method for the obtention of Tr1 on a large scale. In this context, recent studies have evidenced great heterogeneity among Tr1 cells (CD4+CD49b+LAG-3+), based on their IL-10 production, as well as differential co inhibitory molecule expression, including PD-1, TIM-3, CD39, CTLA-4 y TIGIT (“CIR phenotype”). Interestingly, IL-10+ CIR+ cells represent a subpopulation with the highest suppressive potential both in murine models and humans [28].
With the invention described here, we describe a new in vitro protocol to obtain a high yield of allospecific Tr1 lymphocytes from isolated naive CD4+ T lymphocytes, expressing an enriched CIR+ phenotype and which can be isolated and expanded on a large scale, maintaining their phenotype and suppressive function under proinflammatory environment.
The present invention describes a new method for large-scale production of Tr1 cells (1000 times the initial number, reaching >5×108 cells) for immunotherapy in transplantation, with high purity (>80%) through the isolation and in vitro expansion of allospecific Tr1 lymphocytes enriched in CIR phenotype and with stable suppressive function.
Based on the purity and yield obtained, our invention constitutes a feasible alternative to previously described methodologies for transplantation cell therapy. Also, with the established experimental conditions in the present invention, it is possible to obtain a population of human allospecific Tr1 lymphocytes with a phenotype CD49b+ LAG-3+ IL-10+ PD1+ CD39+ CTLA-4+ TIM-3+ TIGIT+, with a stable suppressive function in the presence of proinflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α.
For the present invention, the terms “donor 1 and 2” refer to healthy donors, not genetically related (allogenic), so the compatibility grade among them is low or null. It is considered as “donor 3”, a non related individual different from donors 1 and 2.
The method to generate and expand in vitro allospecific Tr1 lymphocytes in the present invention considers four stages:
DC10 differentiation is made from monocytes (CD14+), obtained from peripheral blood mononuclear cells (PBMCs) of donor 1 cultured for 7-8 days in RPMI medium with 10% of human serum (HS) AB (Gemini Bio Product) in the presence of human recombinant (hr) GM-CSF, hrIL-4 (50 ng/mL) and hrIL-10 (10 ng/mL). At this time, non-adherent cells, with large size and several projections in their membranes, can be identified, which express the surface molecules characteristic of DC10 populations (CD14, HLA-G and ILT4).
The obtention of allospecific Tr1 lymphocytes was performed by co-culturing DC10 from donor 1 and naive T lymphocytes from donor 2 which, in the context of transplantation, correspond to donor and recipient, respectively, in a 1:5 to 1:10 proportion. Naïve T cells were stained with the vital dye CellTrace™ Violet (CTV, Invitrogen), to evaluate T cell proliferation based on fluorescence loss. The differentiation culture is carried out in the presence of rhIL-10 (10 ng/mL) during 14 days, followed by a restimulation with rhIL-10 (10 ng/mL) and rhIL-2 (20-50 U/mL) on day 7 of culture. These conditions promote the differentiation and proliferation of allospecific Tr1 lymphocytes.
Isolation of Tr1 lymphocytes is performed based on the expression of CD4, CD49b, and LAG-3 from the proliferated allospecific CTV− population. Tr1 cells are purified by FACS sorting and then lymphocytes are expanded during 3 consecutive cycles, each one including an activation and resting phase. During the activation phase, Tr1 lymphocytes are cultured in the presence of anti-CD3/CD28-coupled beads in a proportion of 1:5 (beads:Tr1) in the presence of rhIL-10 (10 ng/mL) and hrIL-2 (200-250 U/mL) for 4 days. During the resting phase, the polyclonal activation stimulus is removed and Tr1 lymphocytes are cultured and maintained with rhIL-2 (20-50 U/mL) for 3 days to prevent induction of anergy (a hyporesponsive state) or activation-induced cell death. During the expansion cycles >80% of allospecific Tr1 cells maintain the expression of CD49b and LAG-3 and >90% show high production of IL-10, as well as expression of co-inhibitory receptors (>80%) involved in Treg suppressive function (CTLA-4, PD-1, CD39, TIGIT y TIM-3).
Tr1 cells were harvested in RPMI 1640 medium supplemented with FBS (20%), cells were centrifuged and the sample supernatant was discarded. Tr1 lymphocytes were resuspended in expansion medium supplemented with 10% of AB human serum (40-60×103 per each 150-200 μL) in the presence of rhIL-2 (20-50 U/mL) and they were cultured in “U” bottom 96 well plates for 2-3 days.
The methodology developed in the present invention, allows the differentiation of up to 60% of allospecific Tr1 lymphocytes, surpassing the percentages reported by previously reported methodologies (˜6-12%). Furthermore, our protocol includes high purification of allospecific Tr1 lymphocytes (CD49b+LAG-3+) by flow cytometry, eliminating the potential heterogeneity in the cellular product.
The suppressive function of the cellular population obtained with the established conditions of the present invention, was evaluated by suppression assays which measure the capacity of allo-Tr1, to inhibit the proliferation of CD3+ T cells from donor 2, towards dendritic cells from donor 1 or from individuals genetically different to donors 1 and 2.
Notably, this invention allows the obtention of higher numbers of allospecific Tr1 lymphocytes, than those reported to be required in previous clinical assays (5×105 to 2×106 cells per kilogram patient), of which only ˜6-12% were Tr1[24] [27].
The present invention describes, for the first time, a methodology for obtaining Tr1 with 80% purity, which will allow reducing the number of cells required per kilogram body weight due to the cellular product characteristics.
Finally, in the present invention, allospecific Tr1 lymphocytes with a high phenotypic and functional stability have been obtained, even after an expansion in the presence of an inflammatory environment, thus providing greater safety for their use as cellular therapy.
In
Buffy coat was obtained from blood samples of the National Institute of Respiratory Diseases blood bank (donor 1), and PBMCs were isolated by Ficoll-Paque™ Plus (GE Healthcare) density gradient, following the manufacturer's instructions. For all assays, PBMCs were cryopreserved in a solution containing 40% of RPMI 1640 (Gibco) media, 50% of fetal bovine serum (FBS, Bioswest), and 10% of Dimethyl Sulfoxide (DMSO, Gibco), in a cellular density of 107 PBMCs/mL, for 24 hours at −70° C., and later they were transferred to liquid nitrogen for their long-term storage. For the functional assays, PBMCs were thawed in a 37° C. water bath, harvested in RPMI media supplemented with FBS 10% (37° C.), washed twice with culture media and resuspended in culture media. The cellular viability was determined with trypan blue dye.
Next, CD14+ monocytes were isolated from donor 1 PBMCs by positive selection, using Human CD14 MicroBeads kit (Miltenyi Biotec) following the manufacturer's instructions. Purified CD14+ cells were cultured in RPMI media supplemented with 10% HS AB, 50 ng/mL of hrGM-CSF (Peprotech) and 50 ng/mL of rhIL-4 (Peprotech), in the absence (DCs) or presence (DC10) of 10 ng/mL of rhIL-10 (Peprotech), placed into a 48-well plate with 5×105 cells per well. Cells were incubated for 7-8 days, and media with cytokines was exchanged on days 3 and 5. The DCs cultures were activated with 50 mg/ml rhTNF-α (Peprotech) on day 5. Finally, cells were harvested, washed with RPMI media, and resuspended in CTS™ OpTmizer™ T Cell Expansion SFM medium (Gibco) (expansion media), for T cell cocultures. To characterize the DCs, an aliquot of them was stained with anti-HLA-G, anti-ILT4 (
To isolate naive CD4+ T cells, 108 PBMCs from donor 2 were incubated with monoclonal antibodies anti-CD4, anti-CD25, and anti CD45RA for 20 min at 4° C. in dark; after that, cells were washed and resuspended in 3 mL of phosphate buffer saline (PBS) 1×. Then naive T cells (CD4+CD25-CD45RA+) were isolated by flow cytometry using FACsAria I (BD Biosciences). Purified cells were harvested in RPMI media supplemented with FBS 20%, centrifuged and washed with PBS 1×. At last, cells were resuspended in PBS 1× and counted for the co-cultures (viability was determined by trypan blue exclusion).
To determine cell proliferation, CD4+CD25−CD45RA+ cells were stained with the vital dye CellTrace™ Violet (CTV, Invitrogen) following the manufacturer's instructions. CD4+CD25−CD45RA+ cells from donor 2 were stained, washed, resuspended in media expansion supplemented with HS-AB 10% and cocultured with DC10 o DCs from example 1 (donor 1) in a proportion of 1:10 (DC:CD4+CD25−CD45RA+ y DC10:CD4+CD25−CD45RA+), using “U” bottom 96-well plates. Cocultures between DC10 and CD4+CD25−CD45RA+ were supplemented with 10 ng/mL of hrIL-10, incubated for 14 days (37° C./5% CO2) and re-stimulated at day 7 with hrIL-2 (20-50 U/mL) and hrIL-10 (10 ng/mL).
On day 14, cells were stained with the monoclonal antibodies anti-CD4, anti-CD49b, anti-LAG-3 and anti-IL-10, and the samples were acquired in the Attune NxT (Thermo Fisher) cytometer. The percentage of differentiated Tr1 lymphocytes were determined by the co-expression of the surface markers CD49b and LAG-3 (
On day 14 of differentiation cultures from example 2, allospecific Tr1 lymphocytes were purified from proliferative cells (CTV−). For this, differentiation co-cultures were stained with anti-CD4, anti-CD49b, anti-LAG-3 monoclonal antibodies, as well as a viability dye, and allospecific Tr1 lymphocytes were purified as live CD4+CD49b+LAG-3+CTV− population by flow cytometry in MoFlo XDP Cell Sorter (Beckman Coulter). Allospecific Tr1 were harvested in RPMI 1640 media supplemented with 20% of FBS and cells were centrifuged discarding the sample supernatant. Tr1 lymphocytes were resuspended in expansion media supplemented with 10% of HS-AB (40-60×103 per well) in the presence of rhIL-2 (20-50 U/mL) and they were cultured in “U” bottom 96-well plates for 2-3 days. Next, Tr1 lymphocytes were harvested in expansion media, centrifuged and the supernatant was discarded. Then, lymphocytes were resuspended in expansion media supplemented with 10% HS-AB (10-30×103 cells per well).
Purified Tr1 lymphocytes were stimulated with anti-CD3/anti-CD28 coupled beads (at a proportion 1:5 bead:Tr1), IL-2 (200-250 U/mL) and IL-10 (10 ng/mL). After 4 days of expansion, beads were removed using DynaMag™ (Invitrogen), cells were washed with culture media and cultured again in expansion media with 10% HS-AB supplemented with rhIL-2 (20-50 U/mL) in a “U” bottom 96-well plate for 3 more days (resting phase).
Following this initial expansion scheme, we performed 2-3 more expansion-resting cycles.
As shown in
To phenotypically characterize the Tr1 expanded lymphocytes, expression of CD49b, LAG-3, PD-1, TIM-3, CD39, CTLA-4, TIGIT, as well as IL-10 production, were evaluated by flow cytometry. Tr1 lymphocytes maintained a high co-expression of CD49b and LAG-3 molecules (>80%) (
In conclusion, our results demonstrate that with the present invention it is possible to isolate and increase the number of allospecific Tr1 lymphocytes with a highly suppressive phenotype.
To evaluate the function of expanded allospecific Tr1 lymphocytes in vitro suppressive assays were carried out. In these studies DC were differentiated from CD14+ monocytes (allogeneic) from donor 1 and from donor 3, a donor different from donor 1 and 2, following the conditions from example 1. Responder CD3+ T cells (R.C.) were obtained from donor 2 PBMCs, after isolation using the Pan T Cell Isolation Kit (Miltenyi Biotec), following the manufacturer instructions.
The expanded allospecific Tr1 lymphocytes, stained with CTV and R.C. cells, stained with CFSE, were co-cultured in several proportions Tr1:R.C. (0:1, 1:1, 1:3, 1:9, 1:27). The Tr1:R.C. lymphocytes (donor 2) cultures were stimulated with allogeneic DCs (donor 1 or donor 3) in expansion media with 10% HS-AB. On day 4 a surface staining was carried out with anti-CD4, anti-CD8 and anti-CD3 for 20 minutes in the dark, samples were washed with FACS buffer and acquired in the Attune NxT flow cytometer to determine the proliferation of CD8+ and CD4+ T cells by CFSE dilution. To calculate the suppression percentage we used the formula: [(% Tconv proliferation without Tr1−% R.C. in the presence of Tr1)/% R.C. proliferation without Tr1]×100.
To evaluate the phenotypic stability under an inflammatory microenvironment, allospecific Tr1 lymphocytes, expanded during 3 weeks, were cultured for 2 additional cycles (following the scheme from example 3) in the presence or absence of IL-6 (10 ng/mL), IL-1β (10 ng/mL), IFN-γ (10 ng/mL) and TNF-α (50 ng/mL). At the end of these cycles, expression of CD4, CD49b, LAG-3, TIM-3 and TIGIT was evaluated, together with IL-10 production by flow cytometry. Interestingly, Tr1 lymphocytes maintained the CD49b and LAG-3 (11a-b) coexpression, as well as co-inhibitory molecules TIM-3 (
Finally, following the stimulation scheme from Example 5, function stability was evaluated in Tr1 lymphocytes in the presence of proinflammatory cytokines. The results showed that Tr1 cells from donor 2 inhibited the proliferation of alloreactive CD4+ responder T cells (R.C.) at 1:1 ratio (
Evaluation of phenotypic and functional stability in the presence of inflammatory environments is of vital importance for the cellular products used in transplantation immunotherapy, given the potential inflammatory responses elicited against the graft, in order to provide greater safety to the patients.
Number | Date | Country | Kind |
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MX/A/2020/011355 | Oct 2020 | MX | national |