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Podsumowanie

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

Streszczenie

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

Wprowadzenie

Cardiac allograft in rat is a reliable organ transplant model to assess tolerance induction treatments, to decipher the mechanisms of tolerance induction and maintenance, and has the potential to induce functionally competent and dominant regulatory cells. The protocols below describe a fully mismatch heterotopic cardiac graft from a Lewis 1W donor rat (LEW.1W, RT1u) into a Lewis 1A recipient rat (LEW.1A, RT1a). In this graft combination, acute rejection occurs rapidly (in about 7 days) and can be easily assessed by graft beating measurement through palpation of the abdomen. Here we propose three protocols to induce tolerance to the cardiac allograft in rat. In these models, tolerance is induced and/or maintained by different regulatory cell types. First, the blocking of CD40-CD40L interactions with an adenovirus encoding CD40Ig (AdCD40Ig) induced the generation of CD8+ Tregs capable of inducing tolerance when adoptively transferred to secondary grafted recipients1. Furthermore, depletion of CD8+ cells (with anti-CD8α antibodies) in AdCD40Ig-treated recipients generated Bregs and RegMCs2. Deep analysis of CD8+ Tregs properties highlighted the key role of several immunoregulatory molecules defined as interleukin-34 (IL-34) and Fibroleukin-2 (FGL-2)3,4,5,6. Whereas overexpression of IL-34 (with an AAV vector) induced Tregs through generation of RegMCs, overexpression of FGL-2 induced Bregs, underlying the complex network of regulatory cells.

Because chronic rejection develops slowly and is long-term, an in-depth analysis is required to distinguish tolerance versus chronic rejection. Graft is usually assessed for cell infiltration, fibrosis, thickening of vascular wall and complement C4d deposition by immunohistology7. While histology methods require animal sacrifice or graft biopsy, here we describe a simple method to assess different features of tolerated allograft: the emergence and function of regulatory cells and the anti-donor specific antibody responses from blood sample by flow cytometry (here, we used fluorescence-activated cell sorting (FACS)).

Maintenance of tolerance to the allograft after arrest of the treatment is generally associated with the induction of regulatory cells8. In the last decades, studies focused on CD4+ Tregs unanimously characterized them by the key markers Foxp3+, CD25high, and CD127-9,10,11. Similarly, several markers were attributed to CD8+ Tregs, like CD122+, CD28-, CD45RClow, PD1+, and Helios+1,12,13,14,15,16,17. Over the years, expression of GITR, CTLA4, and cytokines (IL-10, TGFβ, IL-34, IL-35, FGL-2) were additionally associated to a Treg profile3,4,6,13,18,19,20,21. However, emerging regulatory cell populations, such as Bregs, RegMCs, or NKTregs, lack relevant specific markers. Indeed, Bregs are mostly reported as immature CD24+ cells, with ambiguous CD27 expression and sometimes production of IL-10, TGFβ, or granzyme B22,23,24. The complexity of the myeloid cell lineage requires a combination of several markers to define their regulatory or proinflammatory profile such as CD14, CD16, CD80, CD86, CD40, CD209a, or CD16325,26. Finally, some markers have been reported to identify NKTregs such as CD11b+, CD27+, TGFβ+, but more studies are needed to further phenotypically describe them27,28,29,30,31,32. Thus, evidences of suppressive activity are required to legitimize further phenotypic description for the identification of new biomarkers, new immunoregulatory mediators, and to extend the scope to new cell therapies.

We propose two complementary methods to evaluate the suppressive activity of cells. First, the in vitro method consists of culturing suppressive cells with labeled effector T cells stimulated by allogeneic donor antigen presenting cells (APCs) at different ratios over 6 days, and analyzing the effector T cell proliferation that reflects donor-directed immune suppression. Cells from treated rats can be compared directly to cells from naive rats and non-treated grafted rats for suppressive activity (or to any other regulatory cell population), in a range of suppressor:effector ratios. Furthermore, this method does not require any transplantation, and results are obtained within 6 days. Second, the in vivo method consists of transferring the intended regulatory cells from a treated rat to a newly irradiated grafted recipient. While B cells, myeloid cells or T cells from non-treated naive rats are usually unable to inhibit acute rejection and to prolong graft survival upon adoptive transfer, cells with potentiated suppressive activity from treated-recipients have these attributes1,2,3,4,33. Lymphopenia induced by irradiation of the recipient is recommended to allow adoptively transferred cells to remain unaffected by blood homeostasis and to master more easily the anti-donor immune responses. For both methods, the in vitro utilization of allogeneic third party APCs or in vivo adoptive transfer of suppressive cells into recipients grafted with a third-party heart allow analysis of the anti-donor specificity. Whereas the in vivo method requires a substantial number of cells, poorly represented cell subpopulations can be more easily assessed for suppressive activity in vitro33.

Humoral responses can also be measured to assess the state of tolerance and the control of directed antibody responses to donor antigens. Indeed, tolerance can be characterized by the absence of humoral response toward the donor but conservation of the capacity for the recipients to develop humoral response to new antigens and preservation of memory responses. First, the principle of alloantibody detection is based on recognition of the donor cells by recipient antibodies following incubation of donor cell type with serum from a grafted recipient. Second, humoral responses directed to exogenous antigens can be assessed following stimulation of long-term tolerant recipients with Keyhole Limpet Hemocyanin (KLH) emulsified with complete Freund's adjuvant. The presence of specific IgM and IgG antibodies against antigens can be detected 4 and 13 days, respectively, following immunization, with Enzyme Linked ImmunoSorbent Assay (ELISA)34. Third, the preservation of immune memory responses can be assessed by injection of xenogeneic red blood cells (RBCs) at days -7 and +3 of transplantation and RBCs staining with recipient serum collected at days +8 and +17 following transplantation. All these methods allow for the identification of immunoglobulin subtypes by using specific secondary antibodies, and rapid acquisition of results in less than 1.5 h by FACS staining or a few hours by ELISA.

Finally, these protocols are designed for characterization of transplantation models, and can be, to some extent, applied to autoimmune disease models. The principles of the method can be transposed to all species.

Protokół

Note: All protocols here have been approved by an ethical committee and should be performed in a sterile manner.

1.Generation of Tolerance in a Model of Cardiac Allograft in Rat

  1. LEW.1W to LEW.1A allograft procedure
    1. Anesthetize a donor male LEW.1W rat using isoflurane-O2 inhalation, supplemented with 1% N2O after 5 min. Place the animal in dorsal decubitus, and disinfect the abdomen with betadine to perform a thoracotomy (i.e., incision into the pleural space of the chest).
      1. Clamp the inferior and superior venae cavae, ligature them, and cut them. Then cut the pulmonary artery and the aorta and save the graft in cold 0.9% NaCl.
    2. Anesthetize a 250 g male LEW.1A recipient rat (of 8-12 weeks) using isoflurane-O2 inhalation, supplemented with 1% N2O after 5 min. Place the animal in dorsal decubitus, and disinfect the abdomen with betadine to perform a xyphopubic laparotomy (i.e., large incision from the xiphoid process to pubic symphysis).
      1. Externalize the intestines, clamp the abdominal blood vessels, perform a termino-lateral anastomosis (i.e., connection between the end of one channel and the wall of the other) between the graft aorta and the abdominal aorta and between the pulmonary artery and the abdominal vena cava, and remove clamps.
    3. Suture the muscular plane and skin, and disinfect with betadine.
    4. Inject nalbuphine (analgesic) 6 mg/kg subcutaneously (s.c.) and oxytetracycline (antibiotic) intramuscularly (i.m.), and place the animal under a heat lamp until the animal awakens. Inject buprenorphine (opioid) (50 µg/kg) i.m. and meloxicam (nonsteroidal anti-inflammatory drug) (0.3 mg/kg) s.c. the day of the transplantation and one day after.
  2. Induction of tolerance by blockade of costimulatory interactions
    1. Follow steps 1.1.1. to 1.1.2.
    2. In a classified A2 area of the animal facility, dilute 2 x 1010 infectious particles of AdCD40Ig (an adenovirus encoding CD40Ig, a chimeric molecule that blocks the CD40-CD40L interactions) in Ringer's lactate solution to reach a final volume of 150 µL and inject in 3 points (3 x 50 µL) in the ventricular wall of the apex of the graft.
    3. Follow steps 1.1.3 to 1.1.4.
      NOTE: AdCD40Ig treatment can be associated with anti-CD8α, anti-ICOS, or anti-CD28 antibody injections2,35,36.
  3. Induction of tolerance by overexpression of a recombinant protein
    1. Dilute 1 x 1012 viral genome of rat IL34-recombinant AAV in Ringer's lactate solution to reach a final volume of 100 µL. Anesthetize a 150 g LEW.1A rat with isoflurane-O2 inhalation and inject intravenously (i.v.) in the penile vein.
    2. One month following AAV-IL34 injection, perform a LEW.1W graft on the LEW.1A recipient according to protocol 1.1.

2. In Vitro Assessment of Cells Suppressive Activity by Mixed Lymphocytes Reactions (MLRs)

Note: Suppressive activity of cells from treated tolerant rats should be compared with the equivalent population from syngeneic grafted recipients or naive rats.

  1. Isolation of allogeneic APCs: plasmacytoid dendritic cells (pDCs)
    1. Anesthetize a male LEW.1W naive donor rat using isoflurane-O2 inhalation, supplemented with 1% N2O after 5 min. Place the animal in dorsal decubitus, and disinfect the abdomen with betadine to perform a splenectomy. Remove the spleen by transecting the vessels, save the spleen in cold 1X phosphate-buffered saline (PBS) and suture the animal.
    2. Transfer the spleen in a dish, remove 1X PBS and perfuse with 5 mL of 0.2% collagenase D. To improve the enzyme digestion, cut the spleen into small pieces and incubate 15 min at 37 °C.
    3. Add 500 µL of 0.1 M ethylenediaminetetraacetic acid (EDTA), transfer the spleen pieces into a sieve and crush the spleen with a syringe's piston to dissociate the cells. Transfer into a tube and wash the cells with 15 mL of 1X PBS. Centrifuge 10 min at 430 x g. Discard the supernatant.
    4. To eliminate RBCs and platelets, resuspend the splenocyte pellet in 10 mL hypotonic solution and incubate 5 min at room temperature (RT). Wash with 1X PBS and centrifuge 10 min at 190 x g. Discard the supernatant.
    5. Remove collagen fibers by filtering on a 100 µm tissue filter. Count the cells to adjust cell concentration to 5 x 107 cells/mL in 1X PBS/2% fetal calf serum (FCS)/0.5 mM EDTA.
      Note: The number of splenocytes should be between 4 x 108 and 7 x 108 cells.
    6. Enrich for cells of interest by negative selection before cell sorting. Incubate 15 min at 4 °C with 2 µg/mL purified antibodies specific to T cells (anti-TCRαβ, R7/3 clone, and anti-TCRγδ, V65 clone), B cells (anti-CD45RA, OX33 clone), and NK cells (anti CD161, 3.2.3 clone), wash with 1X PBS/2% FCS/0.5 mM EDTA and centrifuge 10 min at 430 x g. Discard the supernatant.
    7. Wash with magnetic beads 3 times with 1X PBS/2% FCS/0.5 mM EDTA. Use 3.5 µL beads/106 splenocytes, wash by adding 30 mL 1X PBS/2% FCS/0.5 mM EDTA, place for 1 min on the magnet, and discard the supernatant. Repeat twice. Resuspend the beads in 10 volumes of 1X PBS/2% FCS/0.5 mM EDTA (for example, 35 µL beads is diluted in 350 µL 1X PBS/2% FCS/0.5 mM EDTA).
    8. Remove unwanted cells by mixing the splenocytes with the magnetic beads for 10 min at 4 °C under agitation, place the tube on the magnet for 1 min and transfer the supernatant to a new tube. Repeat twice. Count the cells and adjust the cell concentration to 5 x 107 cells/mL in 1X PBS/2% FCS/0.5 mM EDTA.
      NOTE: The enriched splenocytes number should range between 5 x 107 and 9 x 107.
    9. Stain the cells using labeled antibodies for further sorting of pDCs: exclude remaining T cells (anti-TCRαβ, R7/3 clone) or B cells (anti-CD45RA, OX33 clone), and sort CD4+ (anti-CD4, OX35 clone) CD45R+ cells (anti-CD45R, His24 clone). Label beads with each Ab to establish a compensation matrix on FACS. Incubate 15 min at 4 °C and wash with 1X PBS/2% FCS/0.5 mM EDTA.
    10. Resuspend the cells at a concentration of 5 x 107 cells/mL, filter on a 60 µm tissue filter, and label dead cells by adding DAPI at a final concentration of 0.1 µg/mL. Sort cells with a 70 µm nozzle cell sorter, by gating on DAPI-TCR-CD45RA-CD4+CD45R+ as shown in Figure 1.
  2. Isolation of responder effector T cells (CD4 + CD25 - T cells)
    1. Harvest the spleen from a non-treated non-grafted naive LEW.1A rat and save it in cold 1X PBS.
    2. Transfer the spleen in a sieve placed in a dish and add 10 mL of 1X PBS. Crush the spleen with a syringe's piston to dissociate the cells. Transfer the cells into a 50 mL tube, wash with 1X PBS and centrifuge 10 min at 430 x g. Discard the supernatant.
    3. To eliminate RBCs and platelets, resuspend the splenocyte pellet in 10 mL hypotonic solution for 5 min at RT, then wash in 1X PBS and centrifuge at 10 min 190 x g. Discard the supernatant.
    4. Remove the collagen fibers by filtering on a 100 µm tissue filter. Count the cells to adjust the cell concentration to 5 x 107 cells/mL in 1X PBS/2% FCS/0.5 mM EDTA.
      NOTE: The number of splenocytes should be between 3 x 108 and 5 x 108 cells.
    5. Enrich the cells of interest before sorting. Incubate 15 min at 4 °C with 2 µg/mL purified antibodies specific to CD8 cells (anti-CD8α, OX8 clone), B cells (anti-CD45RA, OX33 clone), NK cells (anti CD161, 3.2.3 clone), myeloid cells (anti-CD11b/c, OX42 clone), gamma delta T cells (anti-TCRγδ, V65 clone). Then wash with 1X PBS/2% FCS/0.5 mM EDTA and centrifuge 10 min at 430 x g. Discard the supernatant.
      NOTE: To sort natural CD8+ Tregs from naive rats at the same time as CD4+ effector T cells, do not add anti-CD8α Ab during the depletion.
    6. Wash the magnetic beads 3 times with 1X PBS/2% FCS/0.5 mM EDTA as described in step 2.1.7.
    7. Remove the unwanted cells by mixing splenocytes with the magnetic beads for 10 min at 4 °C under agitation, then place the tube on the magnet for 1 min and transfer the supernatant to a new tube. Repeat twice. Count the cells and adjust the cell concentration to 5 x 107 cells/mL in 1X PBS/2% FCS/0.5 mM EDTA.
      NOTE: The splenocytes number should range between 5 x 107 and 8 x 107 cells.
    8. Add labeled antibodies to sort CD4+CD25- T cells: anti-TCRαβ (R7/3 clone), anti CD4 (OX35 clone), anti-CD25 (OX39 clone) and incubate 15 min at 4 °C. To simultaneously sort CD8+CD45RClow Tregs, add anti-CD45RC Ab (OX22 clone). Label beads with each Ab to set up a compensation matrix for further processing on flow cytometry. Wash the cells with 1X PBS/2% FCS/0.5 mM EDTA and discard the supernatant.
    9. Resuspend cells to 5 x 107 cells/mL, filter on a 60 µm tissue filter, and label dead cells by adding DAPI at a final concentration of 0.1 µg/mL. Sort the cells with a 70 µm nozzle cell sorter by gating on DAPI -TCR+CD4+CD25- cells as responder cells (and if needed DAPI -TCR+CD4-CD45RClow as CD8+ Tregs suppressive cells) as shown in Figure 2.
  3. Isolation of suppressive cells
    Note: Divide the spleen in two aliquots: sort one on B cells, myeloid cells, and NK cells, and the other on CD4+ and CD8+ CD45RClow T cells, to assess their suppressive function.
    NOTE: For adoptive cell transfer, save 1 x 108 splenocytes to serve as the positive control of tolerance by adoptive transfer, if needed.
    1. Sorting of B cells, myeloid cells, and NK cells
      1. Follow protocol 2.1.1 to 2.1.5.
      2. Incubate 15 min at 4 °C with 2 µg/mL T cell-specific unlabeled antibodies (anti-TCRαβ, R7/3 clone, and anti-TCRγδ, V65 clone), wash with 1X PBS/2% FCS/0.5 mM EDTA and centrifuge 10 min at 430 x g. Discard the supernatant.
      3. Wash with magnetic beads 3 times with 1X PBS/2% FCS/0.5 mM EDTA as described in step 2.1.7.
      4. Mix the splenocytes with the magnetic beads to remove unwanted cells and incubate for 10 min at 4 °C under agitation, then place the tube on the magnet for 1 min and transfer the supernatant to a new tube. Repeat twice. Count the cells and adjust the cell concentration to 5 x 107 cells/mL in 1X PBS/2% FCS/0.5 mM EDTA.
      5. Add labeled antibodies to the splenocytes to sort cells with suspected suppressive activity such as myeloid cells (anti-CD11b/c, OX42 clone), B cells (anti-CD45RA, OX33 clone), or NK cells (anti-CD161, 3.2.3 clone). Label the beads with each Ab to establish a compensation matrix on FACS. Incubate 15 min at 4 °C and wash with 1X PBS/2% FCS/0.5 mM EDTA.
      6. Resuspend the cells at a concentration of 5 x 107 cells/mL, filter on a 60 µm tissue filter, and label dead cells by adding DAPI at a final concentration of 0.1 µg/mL. Sort the cells with a 70 µm nozzle cell sorter by gating on DAPI-CD11b/c+ for myeloid cells, CD45RA+ for B cells, and CD161+ for NK cells as shown in Figure 3.
    2. Sorting of CD4 + and CD8 + CD45RC low T cells
      1. Follow protocol 2.2.1 to 2.2.4.
      2. For negative selection on a magnetic column, incubate the cells 15 min at 4 °C with 2 µg/mL purified antibodies specific to B cells (anti-CD45RA, OX33 clone), NK cells (anti CD161, 3.2.3 clone), myeloid cells (anti-CD11b/c, OX42 clone), gamma delta T cells (anti-TCRγδ, V65 clone), wash them with 1X PBS/2% FCS/0.5 mM EDTA and centrifuge 10 min at 430 x g. Discard the supernatant.
      3. Wash the magnetic beads 3 times with 1X PBS/2% FCS/0.5 mM EDTA as described in step 2.1.7.
      4. Remove the unwanted cells by mixing the splenocytes with the magnetic beads for 10 min at 4 °C under agitation, then place the tube on the magnet for 1 min and transfer the supernatant to a new tube. Repeat twice. Count the cells and adjust the cell concentration to 5 x 107 cells/mL in 1X PBS/2% FCS/0.5 mM EDTA.
      5. Add labeled antibodies to the cells to sort Tregs: anti-TCRαβ (R7/3 clone), anti-CD4 (OX35 clone), anti-CD45RC (OX22 clone). Label the beads with each Ab to establish a compensation matrix on FACS. Incubate 15 min at 4 °C and wash with 1X PBS/2% FCS/0.5 mM EDTA.
      6. Resuspend the cells at a concentration of 5 x 107 cells/mL, filter on a 60 µm tissue, and label dead cells by adding DAPI at a final concentration of 0.1 µg/mL. Sort the cells with a 70 µm nozzle cell sorter by gating on DAPI -TCR+CD4+CD45RClow as CD4+ Tregs and DAPI -TCR+CD4-CD45RClow as CD8+ Tregs ( Figure 4).
        NOTE: The anti-CD8α Ab (OX8 clone) can be used instead of the anti-CD4 Ab if T cells are discriminated from CD4+ non-T cells by anti-TCR labeling. An excess of CD4+ Tregs should be sorted in anticipation of cell death due to the Cell Proliferation Dye (CPD) labeling.
  4. Carboxyfluorescein succinimidyl ester (CFSE) labeling of responder cells to measure proliferation
    1. After responder cell sorting, wash twice the cells with 1X PBS.
    2. Resuspend the cells at a concentration of 1 x 107 cells/mL in 1X PBS and incubate with 0.5 µM CFSE for 5 min at RT in the dark. Stop the reaction by adding FCS at 1.5X the volume and centrifuge 10 min at 430 x g at 4 °C. Discard the supernatant.
    3. Wash twice the cells with complete medium, and count the cells.
      Note: Expect 30% of cells death at this step.
  5. CPD labeling of CD4 + Tregs for suppressive assay on CD4 + effector cells
    NOTE: This step is required to discriminate CFSE-CD4+ Tregs from CFSE- proliferating responder CD4+ T cells at day 6 of coculture by gating on CPD- cells.
    1. After CD4+ Tregs cell sorting, wash twice the cells with 1X PBS.
    2. Resuspend the cells at a concentration of 1 x 107 cells/mL in 1X PBS and incubate with 10 µM of CPD V450 for 20 min at RT in the dark.
    3. Wash twice the cells with complete medium, and count the cells.
      NOTE: Expect 30% of cells death at this step.
  6. Advice for coculture
    1. Use culture in medium comprising: RPMI 1640 medium supplemented with beta-mercaptoethanol (5 x 10-5 M), penicillin (100 U/mL), streptomycin (0.1 mg/mL), sodium pyruvate (1 mM), glutamine (2 mM), HEPES buffer (1 mM), non-essential amino acids (1X), FCS (10%).
    2. Culture cells in 96-well U bottom plates.
    3. Add cells in the following order: suppressive cells, responder cells, and allogeneic cells.
    4. Keep constant the number of responder T cells and allogeneic APCs and test a range of Tregs number. For example, ratio 4:4:1, 5 x 104 suppressive cells, 5 x 104 responder cells, and 1.25 x 104 allogeneic cells, and ratio 2:4:1, 2.5 x 104 suppressive cells, 5 x 104 responder cells, and 1.25 x 104 allogeneic cells.
    5. Maintain the proportion of 5 x 104 responder cells for 200 µL medium/well.
    6. For controls, include a few wells with responder T cells without allogeneic APCs (negative control) and responder T cells with allogeneic APCs without regulatory cells (positive control) for CFSE gating at day 6 (see step 2.7.6.).
  7. FACS staining and analysis after 6 days of coculture
    NOTE: The proliferation of effector cells can be assessed at several time points. Note that proliferation is exponential: as cell division increases, more differences between the suppressive conditions and negative control can be observed.
    1. Transfer the cells from the 96-well U bottom plates to 96-well V bottom plates and centrifuge 1 min at 1,200 x g at 4 °C.
    2. Save the supernatant in a new plate at -20 °C for measuring cytokine levels, if needed.
    3. Wash the cells with 200 µL 1X PBS/2% FCS/0.5 mM EDTA per well and centrifuge 1 min at 1,200 x g at 4 °C.
    4. Add antibodies to the cells to further gate on responder T cells: anti-TCRab (R7/3 clone) and anti-CD4 (OX35 clone). Incubate 15 min at 4 °C and wash twice with 200 µL 1X PBS/2% FCS/0.5 mM EDTA per well. Discard the supernatant.
    5. Add 100 µL of DAPI diluted at 0.1 µg/mL in 1X PBS and read the plate with a FACS analyzer.
    6. Analyze the CFSE profile by gating on DAPI negative (live cells) CPD negative (non-CD4+ Tregs) TCR+CD4+ cells. Unstimulated responder cells have maximal CFSE brightness as shown in Figure 5 bottom left histogram. In the presence of allogeneic APCs, the responder cells have maximal proliferation and minimal CFSE brightness (bottom, middle). In the presence of allogeneic APCs and regulatory cells, the effector cells have medium proliferation and CFSE brightness (bottom, right).

3. In Vivo Assessment of the Cells Suppressive Activity by Adoptive Cell Transfer in a Heart Grafted Recipient

NOTE: Irradiation is needed to eliminate the host cells and favor donor cell engraftment and proliferation.

  1. Irradiate the graft recipients early on the day before the graft to precondition the recipient. Anesthetize rats with i.m. injection of xylazine (8 mg/kg)-ketamine (80 mg/kg) and irradiate them at a dose of 4.5 Gy with X rays.
  2. Follow protocol 2.3 to isolate cells of interest.
    NOTE: Save 1 x 108 splenocytes to serve as a positive control of tolerance by adoptive transfer, if needed.
  3. 12 h after irradiation (the day before the graft, in the evening), anesthetize the rats by inhalation of 4% isoflurane-O2 and infuse the cells (5.0 x 107 T cells, 3.0 x 107 B cells, 3.0 x 107 myeloid cells, or 1.50 x 108 splenocytes as the positive control of tolerance by adoptive transfer) i.v. in the penile vein.
    NOTE: The number of cells required for tolerance by adoptive transfer depends on the suppressive activity of the cells.
  4. The next day, follow protocol 1.1 to perform the allograft. Follow graft evolution by palpation through the abdomen.

4. Donor Specific Antibody Detection

NOTE: IgG responses directed toward the graft donor are measured by incubating the cells from the donor with serum of the recipient. Subtract the background induced by direct staining of LEW.1W B cells by incubating the cells with syngeneic LEW.1W serum.

  1. Harvest blood from the rats and centrifuge 15 min at 1,200 x g at 4 °C. Save the serum. Serum can be stored at -20 °C or used immediately. Inactivate the complement in the sera by incubating for 30 min at 56 °C.
  2. Harvest the spleen from a donor LEW.1W rat and save it in cold 1X PBS. Follow the steps 2.2.1. to 2.2.4. Add 2 x 105 cells/well in a 96-well V bottom plate. Centrifuge 1 min at 1,200 x g at 4 °C and discard the supernatant.
  3. Dilute the sera serially from 1/10 to 1/270 in 1X PBS (4 dilutions/sample). Incubate the cells for 1 h at 4 °C with 25 µL of diluted serum/well. Anticipate the number of donor-specific IgG subtypes (IgG, IgG1, IgG2a, IgG2b) immune responses to analyze per sample (1 serum x 4 dilutions x 4 IgG subtypes). Wash the cells with 1X PBS/2% FCS/0.5 mM EDTA and discard the supernatant.
  4. Incubate the cells with each mouse anti-rat IgG subtype Ab fluorochrome-labeled for 30 min at 4 °C, one subtype/well.
    Note: If fluorochrome-labeled anti-rat Ig Ab are unavailable, it is possible to use purified unlabeled mouse anti-rat IgG subtype Ab and a fluorochrome-labeled secondary anti-mouse Ab. Depending on the available fluorochromes and cytometer configuration, several anti-rat IgG subtypes can be tested in one well with appropriate settings.
  5. Wash the cells with 1X PBS/2% FCS/0.5 mM EDTA twice and discard the supernatant.
  6. Add 100 µL of DAPI diluted at 0.1 µg/mL in PBS and read the plate with a FACS analyzer.
  7. Read the mean fluorescence intensity (MFI) of anti-rat IgG subtype Ab labeled with fluorochrome on all DAPI- cells. Subtract the MFI obtained with naive LEW.1A serum on the LEW.1W cells (background staining, bottom left) from the MFI obtained with the sera from each recipient on the LEW.1W cells at the corresponding dilution (bottom middle for untreated rejecting recipients that display maximal MFI and bottom right for treated tolerant recipients that display intermediate MFI) (Figure 6).

5. Assessment of Humoral Responses to Exogenous Antigens (Naive and Memory)

NOTE: Serum from rats before transplantation, treatment, and immunization should be used as negative controls of humoral responses. Otherwise, non-immunized naive rats can be used. Serum from immunocompetent recipients, i.e., transplanted exoantigen-immunized and rejecting recipients, are used as positive controls of humoral responses.

  1. Capacity to develop humoral response to a new antigen (Figure 7)
    NOTE: This method is a relative quantification of humoral responses as it does not include a standard. An absolute Ig quantification would be possible by adding a range of dilutions of rat IgG or IgM anti-KLH Ab with known concentration in step 5.1.6.
    1. Emulsify 50 µg of KLH in 200 µL of complete Freund's adjuvant and inject in the tail of long-surviving/tolerant recipients, control untreated rejecting recipients, or naive rats as positive control of humoral responses.
    2. Harvest blood samples at 4 and 13 days after immunization, and centrifuge 15 min at 1,200 x g at 4 °C. Save the upper phase that is the serum. Harvest the serum from non-immunized rats for a negative control. Serum can be frozen at -20 °C until the ELISA assay.
    3. Coat ELISA plates (flat bottom) with 50 µL/well KLH diluted at 10 µg/mL in 1X PBS overnight at 4 °C. Make sure the coating solution covers all the wells.
    4. Remove the excess uncoated KLH by washing. To wash, add 150 µL/well of wash buffer (1X PBS containing 0.1% Tween) and flick.
    5. Block unspecific binding of recipient's Ig by incubating for 1 h at 37 °C with 100 µL/well of wash buffer containing 10% FCS. Wash once.
    6. Serially dilute the recipient sera in wash buffer starting from 1/40 to 1/512, add 50 µL/well in duplicates of the serial dilution to the coated plate, and incubate for 2 h at 37 °C. Keep some wells free of serum for a negative control. Wash twice.
    7. Add 50 µL of 1 µg/mL of biotin-conjugated anti-rat IgM Ab in wells containing the serum harvested at day 4 in recipients, or 50 µL of biotin-conjugated anti-rat IgG in wells containing the serum harvested at day 13, and incubate 1 h at 37 °C. Wash twice.
    8. Add 50 µL/well of HRP-conjugated streptavidin at 1 µg/mL for 45 min at 37 °C. Wash 3 times.
    9. Add 50 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for < 30 min at RT and stop the reaction with 25 µL of 2 M sulfuric acid when positive controls are saturated.
    10. Read the absorbance at 405 nm. Compare the optical density obtained with the recipient's serum to the one obtained with the naive rat control serum.
  2. Assessment of memory humoral response capacity (Figure 8)
    1. 7 days before transplantation, inject i.v. 109 of xenogeneic RBCs diluted in 800 µL of 1X PBS into the naive rats. RBCs can be from sheep, horse or any other species.
    2. Perform the transplantation and administer the tolerogenic treatment.
    3. 3 days after the graft, inject again i.v.109 RBC diluted in 800 µL of 1X PBS in the graft recipients and non-grafted rats.
    4. Harvest blood samples 5 and 14 days after the second immunization and centrifuge 15 min at 1,200 x g at 4 °C to recover the upper serum phase. Serum can be stored at -20 °C or used immediately. Inactivate the complement in rat sera by incubating 30 min at 56 °C before use.
    5. Add 2 x 105 RBC/well of a 96-well V bottom plate. Centrifuge 1 min at 1,200 x g at 4 °C and discard the supernatant carefully by aspiration.
    6. Serially dilute the sera 2-fold from 1/10 to 1/1280 in 1X PBS (8 dilutions/sample). Incubate the cells for 1 h at 4 °C with 25 µL of diluted serum/well. Wash the cells with 1X PBS/2% FCS/0.5 mM EDTA and discard the supernatant.
    7. Incubate the cells with the RBC species-adsorbed anti-rat IgG or IgM subtypes labeled with fluorochrome for 30 min at 4 °C, one subtype/well. Assess the rat IgM and IgG anti-RBC in serum harvested 5 and 14 days after immunization, respectively. Wash the cells with 1X PBS/2% FCS/0.5 mM EDTA twice and discard the supernatant.
      NOTE: If fluorochrome-labeled anti-rat Ig Ab are unavailable, it is possible to use purified unlabeled mouse anti-rat IgG subtype Ab and a fluorochrome-labeled secondary anti-mouse Ab.
    8. Add 100 µL of DAPI diluted at 0.1 µg/mL in 1X PBS and analyze the cells with a FACS analyzer.
    9. Represent the MFI as a function of the dilution for each group.

Wyniki

The assessment of suppressive activity following sorting of the APCs (Figure 1), responder cells and Tregs simultaneously (Figure 2), or individually (Figure 4), and any other putative regulatory cells (Figure 3), can be done in vivo by direct injection of the regulatory cells and in vitro by measurement of CFSE brightness (Figure 5...

Dyskusje

Adoptive transfer of total splenocytes into a newly grafted recipient is an efficient way to detect the presence of regulatory cells induced or potentiated by a treatment. Host irradiation-induced transient lymphopenia promotes cell survival after transfer and establishment of tolerance. Moreover, sub-lethal irradiation leaves time for cells with tolerogenic properties to convert to new regulatory cells during immune reconstitution, a phenomenon called infectious tolerance34. Usually, well-describ...

Ujawnienia

The authors declare that they have no competing financial interests.

Podziękowania

This work was realized in the context of the Labex IGO project (n°ANR-11-LABX-0016-01) which is part of the "Investissements d'Avenir" French Government program managed by the ANR (ANR-11-LABX-0016-01) and by the IHU-Cesti project funded also by the "Investissements d'Avenir" French Government program, managed by the French National Research Agency (ANR) (ANR-10-IBHU-005). The IHU-Cesti project is also supported by Nantes Métropole and Région Pays de la Loire.

Materiały

NameCompanyCatalog NumberComments
animals
LEW.1W and LEW.1A ratsJanvier Labs, France8 weeks old, 
BN third party donor ratsJanvier Labs, France8 weeks old, 
namecompanycatalogue numbercomments
reagents
AdCD40IgViral Vector Core, INSERM UMR 1089, Nantes, Francehome made plasmids
IL34-AAVViral Vector Core, INSERM UMR 1089, Nantes, Francehome made plasmids
FGL2-AAVViral Vector Core, INSERM UMR 1089, Nantes, Francehome made plasmids
anti-TCRabHybridoma from European Collection of Cell Culture, Salisbury, U.KR7/3 cloneHome made culture, purification and fluororophore coupling
anti-CD25Hybridoma from European Collection of Cell Culture, Salisbury, U.KOX39 cloneHome made culture, purification and fluororophore coupling
anti-CD8Hybridoma from European Collection of Cell Culture, Salisbury, U.KOX8 cloneHome made culture, purification and fluororophore coupling
anti-CD45RAHybridoma from European Collection of Cell Culture, Salisbury, U.KOX33 cloneHome made culture, purification and fluororophore coupling
anti-CD161Hybridoma from European Collection of Cell Culture, Salisbury, U.K3.2.3 cloneHome made culture, purification and fluororophore coupling
anti-CD11b/cHybridoma from European Collection of Cell Culture, Salisbury, U.KOX42 cloneHome made culture, purification and fluororophore coupling
anti-TCRgdHybridoma from European Collection of Cell Culture, Salisbury, U.KV65 cloneHome made culture, purification and fluororophore coupling
anti-CD45RCHybridoma from European Collection of Cell Culture, Salisbury, U.KOX22 cloneHome made culture, purification and fluororophore coupling
anti-CD4Hybridoma from European Collection of Cell Culture, Salisbury, U.KOX35 cloneHome made culture, purification and fluororophore coupling
anti-CD45RBD Biosciences, Mountain View, CA#554881, His24 clone
anti-rat IgG-FITCJackson ImmunoResearch Laboratories, INC, Baltimore, USA#112-096-071
anti-rat IgG1Serotec#MCA 194
anti-rat IgG2aSerotec#MCA 278
anti-rat IgG2bSerotec#MCA 195
anti-rat IgM-FITCJackson ImmunoResearch Laboratories, INC, Baltimore, USA#115-095-164
streptavidin HRPBD Biosciences, Mountain View, CA#554066
KLHSigma Aldrich, St. Louis, USA#9013-72-3
PBS 1XThermo Fisher Scientific Inc, USAPhosphate Buffer Solution without calcium and magnesium, 
Tween 20Sigma, Saint-Louis, USA#9005-64-5
TMB substrate reagent kitBD Biosciences, Mountain View, CA#555214
CellTraceTM CFSE cell proliferation kitThermo Fisher Scientific Inc, USA#C34554
RPMI 1640 medium 1XThermo Fisher Scientific Inc, USA#31870-025
penicilline streptomycineThermo Fisher Scientific Inc, USA#15140-122
Hepes BufferThermo Fisher Scientific Inc, USA#15630-056
non essential amino acidsThermo Fisher Scientific Inc, USA#11140-035
Sodium pyruvateThermo Fisher Scientific Inc, USA#11360-039
2 beta mercaptoethanolSigma, Saint-Louis, USA#M3148
Cell Proliferation Dye eFluor® 450 CellThermo Fisher Scientific Inc, USA#65-0842-85
GlutamineSigma, Saint-Louis, USA#G3126
DAPIThermo Fisher Scientific Inc, USA#D1306
Collagenase DRoche Diagnostics, Germany#11088882001
EDTASigma, Saint-Louis, USA#E5134
NaCl 0.9%Fresenius Kabi#B230561
Magnetic dynabeadsDynal, Invitrogen#11033Goat anti-mouse IgG
One Comp eBeadsEbiosciences, San Diego, USA#01-1111-42
BetadineRefer to the institutional guidelines
IsofluraneRefer to the institutional guidelines
NaplbuphineRefer to the institutional guidelines
TerramycineRefer to the institutional guidelines
BuprenorphineRefer to the institutional guidelines
MeloxicamRefer to the institutional guidelines
Complete Freund's adjuvant
RompunRefer to the institutional guidelines
Ringer lactateRefer to the institutional guidelines
KetamineRefer to the institutional guidelines
Red blood cell lysis solutionDilute 8,29g NH4Cl (Sigma, Saint-Louis, USA A-9434), 1g KHCO3 (Prolabo 26 733.292) and 37.2mg EDTA (Sigma, Saint-Louis, USA E5134) in 800ml H2O. Adjust pH to 7.2-7.4 and complete to 1L with H2O.
Collagenase DDilute 1g collagenase in 500 ml RPMI-1640 + 5 ml Hepes + 2% FCS
PBS-FCS (2%)-EDTA (0.5%)Add 5 mL EDTA 0,1M (Sigma, Saint-Louis, USA E5134) and 20ml FCS to 1ml PBS 1X
CFSE (Vybrant CFDA SE Cell Tracer Kit Invitrogen)Dilute 50µg (=1 vial) of CFDA SE (component A) in 90μl DMSO (component B) solution to obtain a 10mM stock solution. Then, dilute stock solution at 1/20 000 in PBS 1X to obtain a 0.5μM solution
complete medium for coculture500ml complete RPMI-1640 medium with 5 ml Penicillin (80 unit/ml)-Steptomycin (80 mg/ml), 5 ml L-Glutamine, 5 ml Non Essential Amino Acids (100X), 5ml Pyruvate Sodium (100mM), 5 ml HEPES buffer (1M), 2.5 ml b mercaptomethanol (7 ml of 2-bmercaptoethanol stock diluted in 10 ml RPMI), 10% FCS
namecompanycatalog numbercomments
equipments
falcon 50mlBD Biosciences, Mountain View, CA#227261
falcon 15mlBD Biosciences, Mountain View, CA#188271
sieve
Corning plastic culture dishesVWR, Pessac#391-0439
100µm and 60µm tissue filtersSefar NITEX, Heiden, Switzerland#03-100/44 and #03-60/35
96 wells U bottom plates for coculture Falcon U-bottom Tissue Culture plate, sterile, Corning#353077
96 wells V bottom plates for FACS stainingThermoScientifique, Danemark#249570
96 wells flat bottom ELISA platesNunc Maxisorb
seringue for spleen crushBD Biosciences, Mountain View, CA#309649
ELISA readerSPARK 10M, Tecan, SwitzerlandSPARK 10M, Tecan, Switzerland
centrifuge
bain marie 
X rays irradiatorLincolshire, EnglandFaxitron CP160
solar agitator
FACS Canto IIBD Biosciences, Mountain View, CA
FACS Aria IIBD Biosciences, Mountain View, CA
magnetThermo Fisher Scientific Inc, USA12302D

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