METHOD FOR THE IDENTIFICATION OF TRANSIENTLY FOXP3 NEGATIVE REGULATORY T-CELLS FROM HUMAN PERIPHERAL BLOOD

Abstract

The invention relates to a method for determination of the frequency of regulatory T-cells in samples obtained from human blood and to methods for the preparation of compositions comprising predetermined amount of regulatory T-cells. The invention is based on the conception that a large fraction of regulatory T-cells present in human peripheral blood do not express detectable amounts of Foxp3, the master transcription factor used for identification of regulatory T-cells, as a result of cytokine deprivation outside of the tissue context.

Claims

1) Method for the in-vitro determination of regulatory T-cells (Treg cells) in a sample obtained from human blood with the following steps: A1) said sample is supplemented with a compound capable of activating the Signal Transducer and Activator of Transcription 5 (STAT5) and in an amount capable for such activation, and cultured for a predetermined cultivation duration under conditions which keep T-cells comprised in said sample viable, wherein the cultivation is carried out either during said supplementing or after said supplementing, A2) after cultivation of said sample an analytical compound specific for CD25 and/or Forkhead-Box-Protein P3 (Foxp3) is added to said sample and the cell frequency of CD25 and/or Foxp3 positive cells is determined in said sample, in particular by determining the ratio of the amount of CD25 and/or Foxp3 positive cells to the amount of CD4 positive cells.

2) Method for preparing a composition, in particular a pharmaceutical composition, comprising a predetermined amount of viable Treg cells with the following steps: B1) a sample is obtained from human blood and subjected to an in-vitro cultivation under conditions which keep T-cells comprised in said sample viable, said cultivation being performed during or after supplementing said sample with a compound capable of activating the Signal Transducer and Activator of Transcription 5 (STAT5), B2) then an analytic compound specific for CD25 is added and the frequency of CD25 positive cells is determined, in particular by determining the ratio of the amount of CD25 positive cells to the amount of CD4 positive cells. B3) then a fraction of said sample is taken, wherein the fraction is calculated from the frequency of CD25 positive cells such that the fraction comprises the predetermined amount of viable Treg cells, and said fraction is optionally prepared for administration.

3) Method for preparing a composition, in particular a pharmaceutical composition, comprising a predetermined amount of viable Treg cells with the following steps: C1) a sample is obtained from human blood and subjected to an in-vitro cultivation under conditions which keep T-cells comprised in said sample viable, said cultivation being performed during or after supplementing said sample with a compound capable of activating the Signal Transducer and Activator of Transcription 5 (STAT5), C2) then an analytic compound specific for Foxp3 is added to a first fraction of said sample and the frequency of Foxp3 positive cells is determined, in particular by determining the ratio of the amount of Foxp3 positive cells to the amount of CD4 positive cells. C3) then a second fraction of said sample is taken, wherein the amount of the second fraction is calculated from the frequency of Foxp3 positive cells in the first fraction such that the second fraction comprises the predetermined amount of viable Treg cells, and said second fraction is optionally prepared for administration.

4) Method according to one of the claims 1 to 3, wherein said sample is untreated human blood or obtained from the human blood by isolation of peripheral blood mononuclear cells (PBMC) from the human blood.

5) Method according to one of the claims 1 to 4, wherein the analytic compound specific for Forkhead-Box-Protein P3 (Foxp3) is a monoclonal antibody comprising a marker, preferably a fluorochrome.

6) Method according to one of the claims 1 to 5, wherein additionally cells, which are CD25 and/or CD4 positive, are identified and/or isolated before, at the same time, or after step A2), B2), or C2).

7) Method according to one of the claims 1 to 6, wherein the compound capable of activating STAT5 is selected from the group consisting of IL-2, IL-7 and IL-15, in particular is IL-2.

8) Method according to one of the claims 1 to 7, wherein CD25 positive, and optionally CD4 positive, Treg are separated from said sample.

9) Method according to one of the claims 1 to 8, wherein the cultivation is carried out for at least 1 h, preferably at least 6 h, more preferably at least 16 h, and up to 48 hours or longer, most preferably for 16 to 24 hours.

10) Method according to one of the claims 1 to 9, wherein the compound capable of activating STAT5 is added at a dose of at least 1 U/ml, preferably at least 5 U/ml, more preferably at least 10 U/ml, even more preferably at least 50 U/ml, most preferably at least 200 U/ml.

11) Composition obtained with a method according to one of the claims 2 to 10.

12) Composition according to claim 11 for the treatment of a condition induced by too low levels of Treg in an organism.

Description

[0035] In the following the invention is explained in more detail by way of non-limiting figures and examples. The figures show:

[0036] FIG. 1: Expression levels of Foxp3 and CD25 and frequencies of Treg after stimulation of freshly prepared PBMC cultures with IL-2. The effect of overnight stimulation of freshly prepared PBMC cultures with IL-2 on the frequencies of Treg cells and the expression levels of the Treg markers Foxp3 and CD25 is shown. PBMC from a healthy donor were either analyzed immediately (A) or after 6 (B) or 20 hours (C) with or without 200/ml IL-2, for cell surface expression of CD4 and CD25, and for intracellular expression of Foxp3. Numbers represent percentages in the respective fields. Numbers in brackets represent frequencies of Foxp3 positive cells among CD4 T-cells. (D) Compiled data from 27 healthy individuals illustrates robustness of ability of IL-2 to increase the frequency of detectable Treg cells (p<0.0001). (E) Kinetics of increase in detectable CD25+Foxp3+ Treg cells. Culture conditions as in (A-C).

[0037] FIG. 2: Up regulation of Foxp3 in a subset of CD4 positive T-cells after stimulation with STAT5-activating cytokines. STAT5-activating cytokines induce upregulation of Foxp3 directly in a subset of CD4 T-cells. Unseparated PBMC (A) or purified CD4 T-cells were cultured overnight with 200 U/ml IL-2, or with 20 ng of IL-7 or IL-15.

[0038] FIG. 3: Dose dependent induction of Foxp3. The dose-dependent increase in visible Treg cells is shown. PBMC were cultured overnight in the presence of graded concentrations of IL-2, followed by flow-cytometric analysis of cell surface expression of CD4 and CD25, and for intracellular expression of Foxp3.

[0039] FIG. 4: Data showing that the increase in Foxp3 positive cells is not due to cell division. PBMC were labeled with the covalent dye CFSE, and cultured overnight with or without 200 U/ml IL-2. They were stained for CD4 and Foxp3. CFSE fluorescence is shown for Foxp3-negative (left) and Foxp3-positive CD4 T-cells (right). As a control for the functionality of the assay, PBMC were also stimulated for 72 hours with anti-CD3, which is known to induce T-cell proliferation. Every peak to the left of the peak seen in unstimulated cultures indicates one cell division. MFI=mean fluorescence intensity.

[0040] FIG. 5: Data showing that the CD4 positive cells responding to IL-2, IL-7, or IL-15 treatment with Foxp3 up regulation express CD25, i.e. are Treg cells. PBMC were depleted of CD25 expressing cells by magnetic separation (or not), and cultured overnight with 200 U/ml IL-2, IL-7 or IL-15 before analysis as in FIG. 1. Numbers in brackets are percent Foxp3+ or all CD4+ T-cells.

[0041] FIG. 6: Analysis of previously frozen PBMC. The experiment was performed as in FIG. 1, but with previously frozen PBMC.

[0042] FIG. 7: Data showing that the inclusion of IL-2 during transport of heparinized blood at RT (20° C.) increases detectable Treg cells. Freshly drawn heparinized venous blood was either used for immediate PBMC preparation, or was gently rocked at RT with or without 400 U/ml of IL-2 for 22 hrs before PBMC preparation. Dot plots show raw data, the bar graph summarizes results. Triplicates were performed for the 22 hour rocked groups, of which means and standard deviations are shown.

[0043] FIG. 8: The increase in detectable Treg cells after culture in the presence of IL-2 with three different staining protocols. PBMC were stained as described in Example 1, using Alexa-647-conjugated anti-Foxp3 (clone 259D, Biolegend) (top row), APC-conjugated anti-Foxp3 (clone 236/A/E7, eBioscience) (center row), or PE conjugated anti-Foxp3 (clone 236A/E7, eBioscience) for Foxp3 detection. Dot plots were gated on CD4+ lymphocytes.

[0044] FIG. 9: Data showing that no increase in detectable Treg cells after culture in the presence of IL-2 is observed in lymph node cells. Lymph node cells from the same donor whose PBMC were analyzed in FIG. 8 were stained as in FIG. 8, center and bottom rows. Dot plots were gated on CD4+ lymphocytes.

EXAMPLE 1: COMPARATIVE EXAMPLE USING CONVENTIONAL TREG ANALYSIS AND THE PROTOCOL OF THE INVENTION STARTING WITH FRESHLY ISOLATED PBMC

[0045] For the determination of Treg frequencies, the present invention as well as conventional state-of-the-art measurements use PBMC isolated from heparinized venous blood by centrifugation over a density gradient (lymphocyte separation medium Pancoll human, PAN-BIOTECH GmbH, Aidenbach, Germany) following the manufacturer's instructions.

[0046] PBMCs were cultured in 96-, 48-, or 24-well tissue culture plates (Greiner bio-one, Frickenhausen, Germany), in which 0.2, 0.5, or 1 ml of cells adjusted to 1 Mio/ml were cultured per well in the three types of wells mentioned, using enriched RPMI 1640 culture medium (GIBCO/Invitrogen, Long Island, N.Y., USA) supplemented with 10% autologous serum or commercially available pooled human AB serum (Sigma-Aldrich), with essentially the same results.

[0047] The frequency of Treg cells was determined by 3-colour immunofluorescence and flow cytometry, using fluorochrome-conjugated mAb specific for CD4, CD25 and Foxp3. For the staining procedure, the cells were suspended at 1×10.sup.6/ml in staining buffer (PBS, 0.1% BSA, 0.2% NaN.sub.3), and the CD4 and CD25 specific mAb were added to 0.2 ml of this suspension. After 20 min. on ice, cells were washed with staining buffer by centrifugation (4° C., 1600 rpm). For intracellular staining of Foxp3, the cells were then suspended in Fix/Perm (eBioscience), and stained with the appropriate antibody diluted in Perm/Wash (eBioscience).

[0048] The following fluorochrome-conjugated mAb were used: CD4 (clone RPA-T4, PECy5, 1:300, BioLegend), CD25 (clone M-A251, PE, 1:25, -BD), FoxP3 (clone 259D), Alexa647, 1:50, BioLegend).

[0049] After the final wash, PBMC were resuspended in 0.1 ml staining buffer and analyzed on a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, Calif.). Data were then analyzed using FlowJo software (TreeStar). They were displayed as dot plots wherein each cell is represented by a dot, and logarithmic fluorescence intensity of two markers (e.g. CD4 and Foxp3) defines the position of each dot. Horizontal and vertical lines divide the dot plot into four quadrants, with lower left containing cells expressing neither marker, upper right both markers, and upper left and lower right only one of the two markers studied. Marker-positive cell populations can also be defined by a window encompassing a “cloud” of dots.

[0050] Analysis of PBMC prepared from venous blood from a healthy donor is shown in FIG. 1. Staining was performed directly after isolation of PBMC (FIG. 1A), and after six (FIG. 1B) or 20 hours (FIG. 1C) of incubation in the absence or in the presence of 200 U/ml recombinant human IL-2 (Novartis). Numbers represent percentages in the respective fields. Numbers in brackets represent frequencies of Foxp3 positive cells among CD4 positive T-cells.

[0051] As is readily seen, the frequency of Foxp3 expressing and of Foxp3 and CD25 coexpressing cells within the CD4 T-cell subset, i.e. cells identified as Treg cells, almost doubled after overnight culture in the presence, but further decreased in the absence of IL-2. This positive effect became first apparent after 6 hours of culture. In addition to frequencies of cell populations, also the mean fluorescence intensities (MFI) observed for Foxp3 and CD25 in the positive populations are shown. These are proportional to the amount of the respective proteins expressed by the marker-positive cells. It is obvious that not only the frequencies, but also the expression levels of Foxp3 and CD25 expression per cell were increased as a result of IL-2 stimulation.

[0052] Compiled data from 27 randomly chosen healthy donors obtained in an identical fashion are summarized in FIG. 1D. They illustrate a robust increase in the frequency of detectable Treg cells after 20 hour incubation with 200 U/ml of IL-2.

EXAMPLE 2: CELLULAR AND CYTOKINE REQUIREMENTS FOR THE RECOVERY OF FOXP3 EXPRESSION

[0053] The experiment shown in FIG. 2 asked the question whether the recovery of Foxp3 expression requires additional cell types such as monocytes contained within the PBMC preparation, and whether additional cytokines beyond IL-2, which activate the STAT5 pathway of signal transduction, would be able to promote recovery of Foxp3 expression. FIG. 2A shows results obtained with unseparated PBMC and FIG. 2B results from purified CD4 positive cells. CD4 T-cells were purified by using a CD4 T-cell purification kit (CD4+ T Cell Isolation Kit II, Miltenyl Biotec) and were then cultured as given in previous experiments for 20 hours in the presence of 200 U/ml IL-2, 20 pg/ml IL-7, or 20 pg/ml IL-15, before determining the frequency of Foxp3+ cells among CD4 T-cells. It is apparent that Foxp3 negative Treg cells contained within the CD4 T-cell population are able to respond to IL-2 with re-expression of Foxp3. Surprisingly, IL-7 and IL-15 are also highly efficient in providing this effect.

EXAMPLE 3: DOSE-RESPONSE RELATIONSHIP

[0054] PBMC were cultured with titrated concentrations of IL-2 for 20 hours as in Example 1. Frequencies of Foxp3+CD25+ cells within the CD4 T-cell compartment were determined as in example 1, and are graphically displayed in FIG. 3. It is found that an increase in detectable Treg cells is apparent with as little as 6.25 U/ml of IL-2, followed by a dose-dependent further increase.

EXAMPLE 4: KINETICS OF THE RESPONSE

[0055] To determine the length of time required for an optimal effect, purified CD4 T-cells were incubated with or without 200 U/ml of IL-2 for various lengths of time (FIG. 1E). While in the absence of IL-2, the frequency of Foxp3 positive cells declined, it increased in its presence, reaching a plateau after 20 hours. Extending the culture period to 46 hours did not show any further increase over the overnight incubation time, as evident from Table I, above (donor D267).

EXAMPLE 5: INCREASE IN FOXP3+ CELLS AFTER INCUBATION WITH IL-2 IS NOT DUE TO CELL DIVISION

[0056] The method of carboxyfluorescein succinimidyl ester (CFSE) dye dilution (see e.g. Tabares, P. et al., Human regulatory T cells are selectively activated by low-dose application of the CD28 superagonist TGN1412/TAB08, Eur J Immunol 2014. 44: 1225-:1236) was employed to determine the proliferation history of Foxp3+ cells recovered from IL-2 stimulated overnight cultures. In this method, which is well known to skilled immunologists, individual cell divisions are easily visualized by flow cytometry as a 50% reduction of fluorescence intensity when the label is distributed to the two daughter cells, as is illustrated by the PBMC stimulated for 3 days with a mitogenic monoclonal antibody to verify the method. In this experiment, which is shown in FIG. 4, the frequency of Foxp3+ cells among CD4 T-cells was 1.5 fold higher in 20 hour cultures containing 200 U/ml IL-2 as compared to cultures without IL-2. Nevertheless, the cells display a single peak of CFSE Label of the same MFI under both conditions, indicating that no cell divisions had taken place during the culture period.

[0057] Thus, this experiment reveals no cell division at all in the increased population of Treg cells identifiable in IL-2 treated cultures, thereby establishing that the effect of IL-2, i.e. doubling in the frequency of Foxp3+ cells, is by up-regulation of Foxp3 expression in previously “invisible” Treg cells rather than by stimulation of Treg cell proliferation.

[0058] For the results in FIG. 4 the PBMC were stained for CD4 and Foxp3. The left hand CFSE diagrams are for Foxp3 negative and the right hand diagrams for Foxp3 positive CD4 T-cells.

EXAMPLE 6: INCREASE IN FOXP3+ CELLS AFTER INCUBATION WITH IL-2, IL-7, OR IL-15 IS NOT DUE TO CONVERSION OF CONVENTIONAL CD4 T-CELLS

[0059] To remove Treg cells which had transiently down-regulated Foxp3 from PBMC, cells expressing CD25 (which is expressed by all Treg cells and a few activated conventional CD4 T-cells) were removed by mAb-mediated magnetic depletion. As seen in FIG. 5, this eliminated the capacity of IL-2 and, to a large extent, of IL-7 or IL-15 to increase the frequency of Foxp3+ cells, indicating that culture in the presence of these cytokines, increases the frequency of pre-existing but previously “invisible” Foxp3+ CD25+ cells, by allowing re-expression of Foxp3 in Treg cells which had transiently lost Foxp3 expression but still expressed sufficient CD25 to allow their depletion with CD25-specific mAb. Culturing was carried out overnight with 200 U/ml IL-2, IL-7 or IL-15 before analysis as in FIG. 1.

EXAMPLE 7: CULTURE WITH IL-2 INCREASES DETECTABLE TREG CELLS IN FROZEN/THAWED PBMC

[0060] PBMC were stored frozen in DMSO containing medium at −80° C., and thawed prior to analysis. This procedure is routinely applied by researchers working with PBMC, which are aliquoted and frozen for later analysis. As can be seen in FIG. 6, the detectable Treg frequency in these cells was also considerably enhanced by culture with IL-2. The experiments were performed as in FIG. 1, but with previously frozen PBMC.

EXAMPLE 8: INCLUSION OF IL-2 DURING TRANSPORT UP-REGULATES CD25 AND FOXP3 EXPRESSION

[0061] Freshly drawn heparinized venous blood was either used for immediate PBMC preparation, or transport at 20° C. was mimicked by gently rocking the heparinized blood with or without 400 U/ml of IL-2 for 22 h before PBMC preparation. As can be seen in FIG. 7, this procedure also increased the frequency of detectable Treg cells. The dot plots show raw data and the bar graph summarized results.

EXAMPLE 9: THE INCREASE IN DETECTABLE TREG FREQUENCY IS INDEPENDENT OF THE STAINING PROTOCOL EMPLOYED

[0062] PBMC were stained without pre-culture, or after 20 h culture in the presence or absence of 200 U/ml of IL-2. In addition to the Alexa647 conjugate of the anti-Foxp3 mAb clone 259B (Biolegend, top row in FIG. 8) used so far, two different antibody-fluorochrome conjugates of an alternative clone were used for the detection of Foxp3: Clone 236A/E7 conjugated with phycoerythrin (PE, bottom row in FIG. 8) or with allophycocyanin (APC, both from eBioscience, center row in FIG. 8). FIG. 8 shows that while the latter two mAb yielded superior results in the detection of Foxp3, the phenomenon that culture with IL-2 increases the frequency of detectable Treg among CD4 T-cells, whereas culture without IL-2 leads to a further decrease was clearly visible with all three staining procedures. The dot plots were gated on CD4+ lymphocytes.

EXAMPLE 10: TREG CELLS IN BLOOD, BUT NOT IN LYMPH NODES ARE “INVISIBLE” BECAUSE OF CYTOKINE DEPRIVATION

[0063] Lymph nodes were collected from the para-iliac region of renal transplant recipients at the Academic Medical Center, Amsterdam, The Netherlands. Paired peripheral blood samples were collected before the transplantation procedure. Cells were frozen in IMDM supplemented with 10% DMSO, 20% FCS, penicillin, streptomycin, and 0.00036% mercaptoethanol. The study was approved by the local ethics committee at the Academic Center at the University of Amsterdam.

[0064] Cells shown in FIG. 9 were stained without pre-culture, or after 20 h culture in the presence or the absence of 200 U/ml of IL-2, using the anti-Foxp3 mAb clone 236A/E7 conjugated with phycoerythrin (PE) or with allophycocyanin (APC, both from eBioscience). The lymph node cells were from the same individual as the PBMC in FIG. 8. It is noted that in contrast to these, culture with IL-2 did not lead to a further increase in Treg frequency, whereas as predicted, culture without IL-2 led to their apparent reduction.