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

Summary

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.

Abstract

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.

Introduction

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, RT1^u) into a Lewis 1A recipient rat (LEW.1A, RT1^a). In this graft combination, acute rejection occurs rapidly (in about 7 days) and can be easily assessed by graft beating measurement through palpation of the abdomen. Here we propose three protocols to induce tolerance to the cardiac allograft in rat. In these models, tolerance is induced and/or maintained by different regulatory cell types. First, the blocking of CD40-CD40L interactions with an adenovirus encoding CD40Ig (AdCD40Ig) induced the generation of CD8⁺ Tregs capable of inducing tolerance when adoptively transferred to secondary grafted recipients¹. Furthermore, depletion of CD8⁺ cells (with anti-CD8α antibodies) in AdCD40Ig-treated recipients generated Bregs and RegMCs². Deep analysis of CD8⁺ Tregs properties highlighted the key role of several immunoregulatory molecules defined as interleukin-34 (IL-34) and Fibroleukin-2 (FGL-2)^3,4,5,6. Whereas overexpression of IL-34 (with an AAV vector) induced Tregs through generation of RegMCs, overexpression of FGL-2 induced Bregs, underlying the complex network of regulatory cells.

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

Maintenance of tolerance to the allograft after arrest of the treatment is generally associated with the induction of regulatory cells⁸. In the last decades, studies focused on CD4⁺ Tregs unanimously characterized them by the key markers Foxp3⁺, CD25^high, and CD127^–9,10,11. Similarly, several markers were attributed to CD8⁺ Tregs, like CD122⁺, CD28^–, CD45RC^low, PD1⁺, and Helios^{+1,12,13,14,15,16,17}. Over the years, expression of GITR, CTLA4, and cytokines (IL-10, TGFβ, IL-34, IL-35, FGL-2) were additionally associated to a Treg profile^{3,4,6,13,18,19,20,21}. However, emerging regulatory cell populations, such as Bregs, RegMCs, or NKTregs, lack relevant specific markers. Indeed, Bregs are mostly reported as immature CD24⁺ cells, with ambiguous CD27 expression and sometimes production of IL-10, TGFβ, or granzyme B^22,23,24. 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 CD163^25,26. Finally, some markers have been reported to identify NKTregs such as CD11b⁺, CD27⁺, TGFβ⁺, but more studies are needed to further phenotypically describe them^{27,28,29,30,31,32}. 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 attributes^1,2,3,4,33. Lymphopenia induced by irradiation of the recipient is recommended to allow adoptively transferred cells to remain unaffected by blood homeostasis and to master more easily the anti-donor immune responses. For both methods, the in vitro utilization of allogeneic third party APCs or in vivo adoptive transfer of suppressive cells into recipients grafted with a third-party heart allow analysis of the anti-donor specificity. Whereas the in vivo method requires a substantial number of cells, poorly represented cell subpopulations can be more easily assessed for suppressive activity in vitro³³.

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

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

Protocol

Note: All protocols here have been approved by an ethical committee and should be performed in a sterile manner. 1.Generation of Tolerance in a Model of Cardiac Allograft in Rat LEW.1W to LEW.1A allograft procedure Anesthetize a donor male LEW.1W rat using isoflurane-O2 inhalation, supplemented with 1% N2O after 5 min. Place the animal in dorsal decubitus, and disinfect the abdomen with betadine to perform a thoracotomy (i.e.</…

Representative Results

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…

Discussion

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

Materials

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