This work was realized in the context of the Labex IGO project (n&#176;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 M&#233;tropole and R&#233;gion Pays de la Loire.

The authors declare that they have no competing financial interests.

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-described CD4+CD25highFoxp3+CD127low Tregs are first studied when tolerance is observed. However, transfer of the negative fraction (splenocytes depleted in CD4+ Tregs) allows sometimes extension beyond the already known regulatory cells and identification of new cell populations1,2,3,4. In AdCD40Ig-induced tolerance, the transfer of CD4+ T cells-depleted splenocytes revealed the discovery of CD8+CD45RClow Tregs1. Furthermore, successive transfer of total CD8+ T cells, and then restricted to CD45RClow cells, showed the potential of such a method to progressively identify a new population of regulatory cells. Moreover, this strategy allows analysis of all populations. Indeed, we have shown that many regulatory cells can coexist and are even probable, and that compensatory mechanisms exists2,4. In rats treated with CD40Ig, depletion of CD8+ cells allowed the emergence of B cells and myeloid cells with regulatory properties, and transferred tolerance to the cardiac allograft in rat according to the protocol described above2. In a model of tolerance induced by overexpression of IL34, both CD4+ and CD8+Tregs were able to transfer tolerance4.
Donor-directed specificity of treatment-induced tolerance can be assessed at cellular and humoral levels. First, adoptive transfer of regulatory cells into recipients of a third party graft will not succeed in transferring tolerance if the cells are specific to the first donor graft33. Second, suppressive cells should efficiently inhibit the proliferation of responder cells in response to stimulation by the APCs from the first donor of the graft but not from a third party donor2. Finally, humoral responses against the donor of the graft can be distinguished from total Ig production in the recipient by using the protocol described above. Furthermore, in a case of tolerance specific to the first graft donor, recipient should be able to develop a new humoral response against a secondary third-party graft, a new antigen, or a former known antigen16.
Collagenase D treatment of the spleen is preferred and advisable to extract all cell populations from the organ. Finally, when the phenotype of regulatory cells is so tight that the cells are poorly represented (&#60; 1% of splenocytes), transfer of the negative fraction compared to the transfer of the total population can help distinguish the suppressive activity of this small population. For example, CD40Ig-induced CD8+ Tregs specific to an allogeneic peptide could be FACS stained using tetramers but their low number did not allow positive adoptive transfer33,38. However, adoptive transfer of the tetramer-depleted CD8+CD45RClow T cells did not transfer tolerance compared to total Tregs, highlighting the potential of this subpopulation. This result was confirmed by an in vitro suppressive experiment. Indeed, the suppressive experiment was also a convincing way to show the capacity of Du51-specific Tregs to suppress immune responses33.
The suppressive protocol described above is restricted to recipient effector CD4+ T cell responses to pDCs from the donor. This protocol can be adapted by replacing pDCs by cDCs or total APCs, but ensuring that the effector:stimulator ratios are appropriate to achieve a moderate proliferation manageable by the suppressive cells. Indeed, a ratio of CD4+CD25-T cells:pDCs should be 4:1, whereas a ratio of CD4+CD25- T cells:cDCs should be 8:1, and CD4+CD25- T cells:APCs, 1:1 or 1:2. The ratios depend on the alloreactivity between the donor and recipient; the above ratios are appropriate for a LEW.1W:LEW.1A combination with an acute rejection occurring at day 7, but the range of responder:stimulator ratios should be tested before the sacrifice of animals. Finally, CFSE is preferred to thymidine to analyze suppressive activity, for safety and reliability concerns. However, ensure the possible distinction between suppressive cells and responder cells for the CFSE analysis at day 6 if the effector CD4+CD25- T cells are replaced by total splenocytes. Similarly, ensure that the remaining T cells among stimulator cells cannot proliferate following 35 Gy irradiation.
Design of the FACS staining is based on the availability of antibodies listed in Table 3. Similar protocols can be adapted for mice models, ensuring target denomination (for example myeloid cells are differentially described in mouse and rat).
The heterotopic cardiac allograft in rat is a solid organ transplantation model with great opportunities for graft outcome monitoring. While renal allograft model are characterized by a sudden death of the recipient, palpation of the beat strength of the cardiac graft through the abdominal wall informs of the graft evolution39,40. A concomitant skin graft or a second cardiac graft can be realized on the cardiac allograft recipient to study memory responses41. Furthermore, biomolecular engineering and biological or chemical tools for depletion of specific cell types like B cells (IgM KO), CD8 cells (anti-CD8&#945; antibodies), or myeloid cells (clodronate liposomes), are useful tools to measure the importance of such cell populations in induction or maintenance of the tolerance depending on the time post transplantation where depleting treatment is administered to the recipient1,2,3,4.
To date, rat Bregs, myeloid cells, and CD8+ Tregs are not well described. The protocols of tolerance induction above are proposed as reference for the characterization of rat regulatory cells1,2,3,4. Further phenotypic description of these cells and comparison to other models of allotransplantation and other species could help to discriminate markers of great interest. Indeed, comparison of Bregs from mouse models of tolerance and operationally tolerant patients highlight predominance of CD5 or CD24 marker expression as biomarkers of Bregs3,23,24,42,43,44,45. In our model of tolerance induced by AdCD40Ig combined with CD8&#945; depletion, CD24 is overexpressed compared to B cells lacking suppressive activity2. High throughput digital gene expression RNA sequencing recently emerged as an innovative tool to further characterize regulatory cells46.
Finally, protocols to detect the presence of regulatory cells induced by a tolerogenic treatment can be adapted to other models. Here we used LEW.1W into LEW.1A combination of allograft, characterized by a graft rejection in about 7 days. The invert combination, which has been described as more stringent and where acute rejection happens faster and stronger, can be used. Autoimmune disease models can also benefit from our experience in transplantation for deciphering the mechanisms of tolerance induction with treatments.

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-CD8&#945; 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, TGF&#946;, 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, TGF&#946;, 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+, TGF&#946;+, 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&#39;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.

<table><tbody><tr><td>&lt;strong&gt;animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LEW.1W and LEW.1A rats</td><td>Janvier Labs, France</td><td></td><td>8 weeks old,&amp;nbsp;</td></tr><tr><td>BN third party donor rats</td><td>Janvier Labs, France</td><td></td><td>8 weeks old,&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalogue number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AdCD40Ig</td><td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td><td></td><td>home made plasmids</td></tr><tr><td>IL34-AAV</td><td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td><td></td><td>home made plasmids</td></tr><tr><td>FGL2-AAV</td><td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td><td></td><td>home made plasmids</td></tr><tr><td>anti-TCRab</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>R7/3 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD25</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX39 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD8</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX8 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD45RA</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX33 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD161</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>3.2.3 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD11b/c</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX42 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-TCRgd</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>V65 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD45RC</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX22 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD4</td><td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td><td>OX35 clone</td><td>Home made culture, purification and fluororophore coupling</td></tr><tr><td>anti-CD45R</td><td>BD Biosciences, Mountain View, CA</td><td>#554881, His24 clone</td><td></td></tr><tr><td>anti-rat IgG-FITC</td><td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td><td>#112-096-071</td><td></td></tr><tr><td>anti-rat IgG1</td><td>Serotec</td><td>#MCA 194</td><td></td></tr><tr><td>anti-rat IgG2a</td><td>Serotec</td><td>#MCA 278</td><td></td></tr><tr><td>anti-rat IgG2b</td><td>Serotec</td><td>#MCA 195</td><td></td></tr><tr><td>anti-rat IgM-FITC</td><td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td><td>#115-095-164</td><td></td></tr><tr><td>streptavidin HRP</td><td>BD Biosciences, Mountain View, CA</td><td>#554066</td><td></td></tr><tr><td>KLH</td><td>Sigma Aldrich, St. Louis, USA</td><td>#9013-72-3</td><td></td></tr><tr><td>PBS 1X</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td></td><td>Phosphate Buffer Solution without calcium and magnesium,&amp;nbsp;</td></tr><tr><td>Tween 20</td><td>Sigma, Saint-Louis, USA</td><td>#9005-64-5</td><td></td></tr><tr><td>TMB substrate reagent kit</td><td>BD Biosciences, Mountain View, CA</td><td>#555214</td><td></td></tr><tr><td>CellTrace&lt;sup&gt;TM&lt;/sup&gt; CFSE cell proliferation kit</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#C34554</td><td></td></tr><tr><td>RPMI 1640 medium 1X</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#31870-025</td><td></td></tr><tr><td>penicilline streptomycine</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15140-122</td><td></td></tr><tr><td>Hepes Buffer</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15630-056</td><td></td></tr><tr><td>non essential amino acids</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#11140-035</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#11360-039</td><td></td></tr><tr><td>2 beta mercaptoethanol</td><td>Sigma, Saint-Louis, USA</td><td>#M3148</td><td></td></tr><tr><td>Cell Proliferation Dye eFluor&amp;reg; 450 Cell</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#65-0842-85</td><td></td></tr><tr><td>Glutamine</td><td>Sigma, Saint-Louis, USA</td><td>#G3126</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#D1306</td><td></td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics, Germany</td><td>#11088882001</td><td></td></tr><tr><td>EDTA</td><td>Sigma, Saint-Louis, USA</td><td>#E5134</td><td></td></tr><tr><td>NaCl 0.9%</td><td>Fresenius Kabi</td><td>#B230561</td><td></td></tr><tr><td>Magnetic dynabeads</td><td>Dynal, Invitrogen</td><td>#11033</td><td>Goat anti-mouse IgG</td></tr><tr><td>One Comp eBeads</td><td>Ebiosciences, San Diego, USA</td><td>#01-1111-42</td><td></td></tr><tr><td>Betadine</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Naplbuphine</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Terramycine</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Buprenorphine</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Meloxicam</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Complete Freund&#039;s adjuvant</td><td></td><td></td><td></td></tr><tr><td>Rompun</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Ringer lactate</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Ketamine</td><td>Refer to the institutional guidelines</td><td></td><td></td></tr><tr><td>Red blood cell lysis solution</td><td></td><td></td><td>Dilute 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.</td></tr><tr><td>Collagenase D</td><td></td><td></td><td>Dilute 1g collagenase in 500 ml RPMI-1640 + 5 ml Hepes + 2% FCS</td></tr><tr><td>PBS-FCS (2%)-EDTA (0.5%)</td><td></td><td></td><td>Add 5 mL EDTA 0,1M (Sigma, Saint-Louis, USA E5134) and 20ml FCS to 1ml PBS 1X</td></tr><tr><td>CFSE (Vybrant CFDA SE Cell Tracer Kit Invitrogen)</td><td></td><td></td><td>Dilute 50&amp;micro;g (=1 vial) of CFDA SE (component A) in 90&amp;mu;l DMSO (component B) solution to obtain a 10mM stock solution. Then, dilute stock solution at 1/20 000 in PBS 1X to obtain a 0.5&amp;mu;M solution</td></tr><tr><td>complete medium for coculture</td><td></td><td></td><td>500ml 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</td></tr><tr><td>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>falcon 50ml</td><td>BD Biosciences, Mountain View, CA</td><td>#227261</td><td></td></tr><tr><td>falcon 15ml</td><td>BD Biosciences, Mountain View, CA</td><td>#188271</td><td></td></tr><tr><td>sieve</td><td></td><td></td><td></td></tr><tr><td>Corning plastic culture dishes</td><td>VWR, Pessac</td><td>#391-0439</td><td></td></tr><tr><td>100&amp;micro;m and 60&amp;micro;m tissue filters</td><td>Sefar NITEX, Heiden, Switzerland</td><td>#03-100/44 and #03-60/35</td><td></td></tr><tr><td>96 wells U bottom plates for coculture</td><td>&amp;nbsp;Falcon U-bottom Tissue Culture plate, sterile, Corning</td><td>#353077</td><td></td></tr><tr><td>96 wells V bottom plates for FACS staining</td><td>ThermoScientifique, Danemark</td><td>#249570</td><td></td></tr><tr><td>96 wells flat bottom ELISA plates</td><td>Nunc Maxisorb</td><td></td><td></td></tr><tr><td>seringue for spleen crush</td><td>BD Biosciences, Mountain View, CA</td><td>#309649</td><td></td></tr><tr><td>ELISA reader</td><td>SPARK 10M, Tecan, Switzerland</td><td>SPARK 10M, Tecan, Switzerland</td><td></td></tr><tr><td>centrifuge</td><td></td><td></td><td></td></tr><tr><td>bain marie&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>X rays irradiator</td><td>Lincolshire, England</td><td>Faxitron CP160</td><td></td></tr><tr><td>solar agitator</td><td></td><td></td><td></td></tr><tr><td>FACS Canto II</td><td>BD Biosciences, Mountain View, CA</td><td></td><td></td></tr><tr><td>FACS Aria II</td><td>BD Biosciences, Mountain View, CA</td><td></td><td></td></tr><tr><td>magnet</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>12302D</td><td></td></tr></tbody></table>

in vitro and in vivo assessment of t, b and myeloid cells suppressive activity and humoral responses from transplant recipients

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.

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). The status of the humoral response of the recipient toward the donor (Figure 6) or exogenous antigens (Figures 7 and 8) can also be assessed in vitro following in vivo immunization as depicted.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55510/55510fig1.jpg" /> 
Figure 1. Gating strategy to sort pDCs. 
Cells were selected on morphology size and granularity (SSC-A/FSC-A), doublets were excluded by FSC-W/FSC-H and SSC-W/SSC-H parameters, live cells were selected by gating on DAPI- cells, and pDCs were selected on TCR-CD45RA-CD4+CD45R+ expression. Purity was assessed by adding DAPI to sorted cells and running on the cell sorter with the sorting parameters. Purity of a rare sorted population (less than 2%) should be greater than 95%. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55510/55510fig2.jpg" /> 
Figure 2. Gating strategy to sort CD4+CD25- responder T cells and CD8+CD45RClow Tregs. 
Cells were selected on morphology, i.e., size and granularity (SSC-A/FSC-A), doublets were excluded by FSC-W/FSC-H and SSC-W/SSC-H parameters, live cells were selected by gating on DAPI- cells, and responder cells were selected on TCR+CD4+CD25- expression. CD8+ Tregs have been simultaneously sorted by gating on TCR+CD4-CD45RClow cells. Purity was assessed by adding DAPI to sorted cells and running on the cell sorter with the sorting parameters. Purity should be greater than 95%. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55510/55510fig3.jpg" /> 
Figure 3: Gating strategy to sort B cells, myeloid cells, and NK cells. 
Cells were selected on morphology size and granularity (SSC-A/FSC-A), doublets were excluded by FSC-W/FSC-H and SSC-W/SSC-H parameters, living cells were selected by gating on DAPI- cells, and B cells were selected on CD45RA+ expression, myeloid cells on CD11b/c+ expression, and NK cells on CD45RA-CD11b/c-CD161high expression. Purity was assessed by adding DAPI to sorted cells and running on the cell sorter with the sorting parameters. Purity should be greater than 95%. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55510/55510fig4.jpg" /> 
Figure 4. Gating strategy to sort CD8+CD45RClow and CD4+CD45RClow Tregs. 
Cells were selected on morphology size and granularity (SSC-A/FSC-A), doublets were excluded by FSC-W/FSC-H and SSC-W/SSC-H parameters, lived cells were selected by gating on DAPI- cells, and CD4+ Tregs were selected on TCR+CD4+CD45RClow expression and CD8+ Tregs on TCR+CD4-CD45RClow expression. Purity was assessed by adding DAPI to sorted cells and running on the cell sorter with the sorting parameters. Purity should be greater than 95%. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55510/55510fig5.jpg" /> 
Figure 5. Representative analysis of the in vitro suppressive assay. 
The responder cell proliferation was analyzed by selection on morphology size and granularity (SSC-A/FSC-A), exclusion of doublets by FSC-W/FSC-H and SSC-W/SSC-H parameters, exclusion of dead cells and CD4+ Tregs by gating on DAPI-CPDV450- cells, selection of TCR+CD4+ cells, and CFSE profile analysis. The CFSE gate was based on unstimulated CFSE-labeled responder cells. CFSEhigh cells are non-proliferating cells and CFSElow are cells with &#8805; 1 division. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55510/55510fig6v3.jpg" /> 
Figure 6. Representative analysis of the donor specific alloantibody response. 
Splenocytes from the donor rat were incubated with a range of diluted heat-inactivated serum from treated or untreated recipients or from naive rat, and then with anti-rat IgG-FITC. Alloantibodies are detected by analyzing MFI of the FITC on DAPI- z cells after SSC and FSC doublets exclusion. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55510/55510fig7.jpg" /> 
Figure 7. Principle of the anti-KLH detection method. 
Recipients were immunized 120 days after transplantation with KLH supplemented with CFA, and blood samples were harvested 4 and 13 days after immunization. KLH proteins were coated on 96-flat bottom wells plates, and incubated with serum samples from immunized rats. Biotinylated goat anti-rat IgG or IgM allowed detection of anti-KLH Ab present in serum harvested 4 or 13 days after immunization. HRP-coupled streptavidin transformed the TMB substrate to a blue colored product that turns yellow when the reaction was stopped with sulfuric acid. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55510/55510fig8.jpg" /> 
Figure 8. Scheme of the anti-RBC detection method. 
Recipients were immunized 7 days before and 3 days after transplantation with xenogeneic red blood cells (RBCs), and blood samples were harvested from immunized recipients 5 and 14 days after the last immunization. RBCs were incubated with serum samples from immunized rats. Presence of anti-RBCs antibodies on the RBCs was detected by staining with labeled goat anti-rat IgG or IgM antibodies and FACS Canto analysis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55510/55510fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients at JoVE.com

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

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.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

in-vitro-vivo-assessment-t-b-myeloid-cells-suppressive-activity

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.3K Views.  INSERM, Université de Nantes. Hier presenteren we een protocol bij het opwekken van tolerantie in transplantatie, en beoordelen van in vitro en in vivo de onderdrukkende capaciteit van afzonderlijke cel subsets van de ontvanger en de immuunstatus van de ontvanger naar donor of exogene antigenen.

Video: In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cancer Research

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

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