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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>animals</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,&#160;</td>
		</tr>
		<tr>
			<td>BN third party donor rats</td>
			<td>Janvier Labs, France</td>
			<td></td>
			<td>8 weeks old,&#160;</td>
		</tr>
		<tr>
			<td>name</td>
			<td>company</td>
			<td>catalogue number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>reagents</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-TCR</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&#160;Scientific Inc, USA</td>
			<td></td>
			<td>Phosphate Buffer Solution without calcium and magnesium,&#160;</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>CellTraceTM CFSE cell proliferation kit</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium 1X</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#31870-025</td>
			<td></td>
		</tr>
		<tr>
			<td>penicilline streptomycine</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes Buffer</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>non essential amino acids</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher&#160;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&#174; 450 Cell</td>
			<td>Thermo Fisher&#160;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&#160;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&#39;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&#181;g (=1 vial) of CFDA SE (component A) in 90&#956;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&#956;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>name</td>
			<td>company</td>
			<td>catalog number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>equipments</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&#181;m and 60&#181;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>&#160;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&#160;</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&#160;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

14.1K Views.  INSERM, Université de Nantes. 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.

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

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...