15. GVHD Model

UNIT 4.3Animal Models of Acute and ChronicGraft-Versus-Host DiseaseGraft-versus-host disease (GVHD) represents a special situation in transplantation im-munology in which immunocompetent donor cells are engrafted into recipients that areincapable of rejecting them due to tolerance (parent offspring, or P F1), immaturity(adult neonate), or radiation- or chemotherapy-induced immune deficiency (donor irradiated host). Donor T cells encountering allogeneic stimulators become activated,secrete cytokines, proliferate, and differentiate into effectors; this in vivo immuneresponse is known as the graft-versus-host reaction (GVHR). The systemic effects of thisinitial donor anti-host reaction comprise a multiorgan syndrome, graft-versus-host disease(GVHD). Murine GVHD experiments have been utilized to model the clinical disordersof acute and chronic GVHD (AGVHD and CGVHD) that occur after allogeneic bonemarrow transplantation, and also to study T cell regulation, induction of tolerance, andautoimmune diseases.Presented in this unit are methods for generating and assessing both AGVHD and CGVHDin mice. While the two syndromes differ markedly in immunopathogenesis (see Com-mentary), both can be induced by the two main methods presented: transfer of allogenicdonor lymphocytes and stem cells into irradiated hosts (see Basic Protocol 1) and transferof parental strain lymphocytes and stem cells into unirradiated, immune-competent F1strain hosts (see Basic Protocol 2). The key factors in determining outcome (i.e., inductionof acute versus chronic GVHD) are the selection of donor and host strains and the T cellnumber and subsets injected (see Tables 4.3.1 and 4.3.2).Several endpoints of AGVHD and CGVHD should be evaluated in experimental mice,with comparisons made to the syngeneic transplant control or the T celldepletedallogeneic control. These include assessment of survival rates (see Support Protocol 1),weight loss (see Support Protocol 2), chimerism (see Support Protocol 3), donor-hostcytotoxicity (see Support Protocol 4), and cytokine and proliferative responses to mito-genic or allogeneic stimuli (see Support Protocol 5). Histopathology (see SupportProtocol 6) and assays of B cell immune function (see Support Protocol 7) can also beused to evaluate the pathogenesis of GVHD.

BASICPROTOCOL 1

INDUCTION OF GRAFT-VERSUS-HOST DISEASE IN IRRADIATED HOSTSThe closest approximation of clinical AGVHD is obtained by transplanting bone marrowand lymphocytes from appropriate donor mice (see Table 4.3.1) into irradiated allogeneichosts. This method has been extensively used to study the role of T cell subsets in reactivityto major and minor histocompatibility loci, the involvement of inflammatory cytokines(cytokine storm) in AGVHD, and the efficacy of T cell populations in generating graftversus leukemia effects. As noted in the Commentary and Table 4.3.2, few irradiated hostmodels have been useful for studying CGVHD. Each experiment should include irradiatedcontrol mice that have been reconstituted either with syngeneic bone marrow or Tcelldepleted allogeneic bone marrow.

MaterialsAppropriate allogeneic donor and host mice (see Table 4.3.1)70% ethanolHanks balanced salt solution (APPENDIX 2) containing 5 mM HEPES

(HBSS/HEPES), sterileAntiThy 1.2 antibody (clone HO-13.4; ATCC) and complement (C rabbit

low-tox-M, Accurate Chemical), for ex vivo depletion of T cells in donor bonemarrow (optional)

Supplement 27

Contributed by Frances Hakim, Daniel H. Fowler, Gene M. Shearer, and Ronald E. GressCurrent Protocols in Immunology (1998) 4.3.1-4.3.21Copyright 1998 by John Wiley & Sons, Inc.

4.3.1

In Vivo Assays forLymphocyteFunction

Heparin (optional)Anti TCR antibodies, anti-CD4 (clone GK1.5; ATCC) or anti-CD8 (clone

2.43; ATCC) antibodies, or anti-NK1.1 or anti-asialo-GM1 antibodies (optional,for in vivo T cell depletion of host animals; see Table 3.4.1 for further listings ofT cellspecific antibodies)

Sets of dissection tools containing fine scissors and forceps, autoclavedPetri dishesFine mesh (autoclaved) or filtration cups (Falcon sterile cell strainer)3-cc syringes with Luer-Lok tip27-G needlesCesium source for -irradiation of whole animals (e.g., Gammacell 40 irradiator,

MDS Nordion)Heat lampAnimal restrainer (for injections)Additional reagents and equipment for animal euthanasia (UNIT 1.8), removal of

spleen and lymph nodes (UNIT 1.9), preparation of bone marrow cells (UNIT 6.4),preparation of cell suspensions (UNIT 3.1), depletion of T cells and T cell subsets(UNIT 3.4; optional), flow cytometry (UNITS 5.3 & 5.4; optional), T cell quantitationby limiting dilution (UNIT 3.15; optional), cell counting (APPENDIX 3A), intravenousinjection (UNIT 1.6), and in vivo T cell depletion (UNIT 4.1; optional)

NOTE: Keep cells on ice throughout the preparation procedures.

Table 4.3.1 Models of Acute GVHD

Hostpreparation Experimental readouts Genetic disparity Common strain combinations

T cell subsetdependenceb

Irradiateda Death; weight loss; delayed B cellrepopulation; tissue pathology(intestine, liver, skin); increasedinflammatory cytokines (IFN-,TNF-, IL-1, as indicated bytissue-based RT-PCRmeasurements); deficient T and Bcell proliferation

MHC I and II B6 B6C3F1B6 B6D2F1B6 B6AF1B10 B10.BRB10 B10.D2

CD4+ orCD8+

MHC I B6 B6.bm1B6 (B6 B6.bm1)F1

CD8+

MHC II B6 B6.bm12B6 (B6 B6.bm12)F1

CD4+

Minor histo-compatability

B10.BR AKR or CBAB10.D2 DBA/2B6 C3H.SW

CD8+CD4+, CD8+CD8+

Unirradiated Donor/host chimerism (donor T cellexpansion, depletion of host B andT cells, donor lymphocyterepopulation); anti-host CTLreactivity; immune deficiency (in Tand B cell proliferation, cytokineproduction); tissue pathology(intestine, liver); LPS-inducedlethalityc

MHC I andII (P F1)

B6 B6C3F1B6 B6D2F1B6 B6AF1B6 (B6.bm1 B6.bm12)F1

CD4+ andCD8+

aIrradiation dose typically ranges from 900 to 1150 rad of total body irradiation.bWhen a given T cell subset is depleted, GVHD severity is greatly attenuated.cLethality is not observed in unirradiated acute GVHD models unless LPS are administered exogenously (10 to 25 g i.v.).

Supplement 27 Current Protocols in Immunology

4.3.2

Animal Models ofAcute and

ChronicGraft-Versus-Host

Disease

Collect and prepare bone marrow1. Euthanize donor mice by cervical dislocation or CO2 asphyxiation (UNIT 1.8). Spray

mice with 70% ethanol to minimize contamination of tissues by hair. If syngeneicdonors are used as a control, collect tissues as with allogeneic donors.

2. If spleens or lymph nodes are to be used, collect aseptically (UNIT 1.9) into sterileHBSS/HEPES in a petri dish.

3. Prepare bone marrow from donor mice as described in UNIT 6.4.The total yield from the four leg bones of a 6- to 8-week-old donor mouse should be 23 107 cells. Older mice, particularly from large-boned strains like C3H/HeN, usually havemuch less recoverable marrow. C57BL/6 and C57BL/10 mice, however, often contain 5 107 cells and provide a good source of marrow even at 4 months of age.

Deplete T cells4. Deplete T cells from marrow by treatment with antiThy 1.2 MAb (clone HO-13.4)

and complement (C; UNIT 3.4). Wash cells thoroughly in HBSS/HEPES to removeFBS used during depletion.

T cell depletion of donor bone marrow is desirable to provide experimental control hosts(receiving only T celldepleted marrow) and to regulate the T cell number and subsetinjected (murine marrow contains 1% to 5% T cells when collected as described). Oneround of MAb + C treatment provides an 1-log T cell depletion. A more thoroughdepletion can be obtained by the further addition of anti-CD4 MAb (clone GK1.5),anti-CD8 MAb (clone 2.43), or rabbit antimouse brain antiserum (which binds to Thy 1on T cells).T cell depletion can be assessed by fluorescence-based cell sorting analysis (e.g., theBecton Dickinson FACS, or fluorescence-activated cell sorting, methodology) using fluoro-chrome-conjugated antibodies to T cells such as antiThy l (see UNITS 5.3 & 5.4), or bylimiting dilution assay (UNIT 3.15).

Prepare donor T lymphocytes5. Tease lymph nodes (from step 2) with sterile needles to break the capsules. Mash

spleens and lymph nodes with the flat end of a sterile syringe, and filter the cellsuspension. Wash cells by adding 15 to 20 ml HBSS/HEPES, then centrifuging 10min at 400 g, 10C, discarding supernatant, and resuspending pellet in the sameamount of HBSS/HEPES. Repeat wash, ending with resuspension in freshHBSS/HEPES.

Either spleen or lymph nodes can be used as a donor T cell source. An 8- to 12-week-oldmouse will have 108 spleen cells and 12 107 axilliary and inguinal lymph node cells.The advantage of using lymph nodes as a T cell source is the high percentage of T cells(sometimes >80%) and absence of stem cells; spleens contain only 25% to 35% T cells.To prevent a significant loss of viability in the lymph node cell suspension, it is importantto exclude adherent fat from the lymph nodes during the dissection or prior to separatingthe cells.

For general discussion and tips concerning preparation of cell suspensions, see UNIT 3.1.6. If desired, to evaluate the effect of T cell subsets on GVHD models, deplete CD4+ or

CD8+ cells by antibody/complement procedures (UNIT 3.4) using anti-CD4 (cloneGK1.5) or anti-CD8 (clone 2.43) Mab.

7. Count cells (APPENDIX 3A) and adjust to appropriate concentration (see step 9) inHBSS/HEPES.

Current Protocols in Immunology Supplement 27

4.3.3


If cell suspensions must be held prior to injection, store on ice and refilter immediatelybefore injection to break up clumped cells, which may cause embolism.

Transplant bone marrow/T cells into host mice8. Within 24 hr prior to injection, lethally irradiate host mice with 900 to 1100 rads (e.g.,

using a Gammacell 40 137Cs -irradiation source dispensing 100 rad/min of wholebody irradiation).

The optimal radiation dose to induce a lethal AGVHD will vary according to the straincombination utilized, the age of the host mice (younger mice, especially 8 weeks of age,are more sensitive to radiation), T cell dose administered, and the cleanliness of the colony.If radiation doses >1000 rad are used, gut toxicity can be minimized by splitting the dosein half, with 4 hr between treatments.Sublethal irradiation (600 rad) has been used effectively in one CGVHD model. Injectionof B10.D2 donor cells into sublethally irradiated BALB/c hosts results in a CGVHD withsymptoms of scleroderma; lethal irradiation of the same host results in an AGVHD.

9. For animals in the experimental group, inject a combination of 107 T celldepletedbone marrow and 0.55 106 lymph node cells, in a volume of 0.5 to 1.0 ml, into thetail vein of each host mouse using a 3-cc syringe and 27-G needle. For controlanimals, inject comparable numbers of either syngeneic cells or only T celldepletedallogeneic donor bone marrow.

See UNIT 1.6 for injection procedure. Heating adult mice under a heat lamp can distend thetail veins and increase the ease of injection. When high concentrations of cells are injected,the risk of embolism can be reduced by i.p. injection of 50 USP units of heparin in 0.05 ml10 to 20 min before injecting cells; however, this is generally less of a problem with thismethod than with the unirradiated model because the number of cells injected is so muchlower.

The number of lymphocytes needed to induce AGVHD is dependent upon the geneticdisparities between donor and host. To induce a lethal AGVHD in MHC-disparateirradiated hosts, 0.10.5 106 donor lymph node cells are sufficient; however, in modelsutilizing MHC-matched, minor antigendisparate combinations, 15 106 lymph nodecells are needed for lethality. For studies of immune dysfunction during AGVHD (nonlethalregimens), use one-fifth the lethal dose of lymph node cells. When whole splenic populationsare used as T cell sources, larger doses of 29 107 donor spleen cells are typically requiredto induce lethal AGVHD. The reasons for this difference have not been adequatelyexplained or investigated.

Regulate GVHD post induction by manipulation of donor T cells (optional)10. If desired, deplete T cells (using antiThy 1.2 or anti TCR antibodies), T cell

subsets (using anti-CD4 or anti-CD8 antibodies), or NK cells (using anti-NK1.1 oranti-asialo-GM1 antibodies) in vivo during the course of GVHD (UNIT 4.1) by injectinghost mice daily with the antibodies for the first 8 days post-transplant.

T cell depletion is discussed by Blazar et al. (1994) and Johnson et al. (1995) and describedin UNIT 4.1. When assessing the effect of a novel antibody-based treatment on the inductionof GVHD, treatment with antiT cell antibody in vivo can provide an appropriate efficacycontrol for comparison with the treatment. Injection of anti- TCR has been the mosteffective regimen in blocking T cell function in GVHD.


4.3.4



Disease

BASICPROTOCOL 2

INDUCTION OF GRAFT-VERSUS-HOST DISEASE IN UNIRRADIATED,IMMUNOCOMPETENT ADULT F1 HOST MICEAn alternative model involves injection of spleen cells from parental-strain (P) donorsinto unirradiated immune-competent adult F1 hosts. By proper selection of strain combi-nations (see Tables 4.3.1 and 4.3.2), P F1 models have been used to study T cellfunctions in both acute and chronic GVHD models. Unlike in AGVHD models, where F1host mice must be disparate from the donor at both class I and class II MHC loci, inCGVHD models a donor/host disparity at only the class II locus is effective. When fullMHC disparity exists, either donor CD8+ cells must be depleted, or a donor strain withdiminished cell-mediated immunity (DBA/2 or BALB/c) must be selected to produce theautoimmune phenotype of CGVHD.

Additional Materials (also see Basic Protocol 1)Appropriate allogeneic donor (parental) and host (F1) mice (see Table 4.3.2)Interleukin 2 (IL-2; Chiron)Interleukin 4 (IL-4; Peprotech)

1a. Prepare single-cell suspension of spleen or lymph node lymphocytes in HBSS fromdonor mice as described for the irradiated model (see Basic Protocol 1, steps 1, 2, 5,and 6).

Many laboratories inject only splenocytes; others prefer a 2:1 mixture of spleen to lymphnode cells to increase the percentage of T cells and reduce the number of donor miceneeded.

To create a model of transfusion-induced AGVHD, inject only parental donor lymph nodecells and no spleen cells. In the absence of repopulating donor hematopoietic stem cells(present in spleen but not in lymph nodes), mice die of pancytopenia in 3 to 5 weeks.If using spleen cells, RBC can be removed from the preparation by ammonium chloridelysis (UNIT 3.1) at this point; however, this may not be desirable in all cases as splenocytesfrom some strains, such as A/J, are themselves extremely sensitive to ammonium chloridelysis.

1b. To evaluate the effect of donor T cell cytokine phenotype on GVHD (optional):Pretreat donor mice with a combination of 500 ng IL-4 i.p. and 25,000 Cetus unitsIL-2 i.p. (twice daily for 5 days), or culture in vitro to generate allospecific (donoranti-host) T cells of defined TH1 or TH2 cytokine profile, before preparing suspension(step 1a).

Pretreatment with IL-2 and IL-4 shifts the CD4+ T cell cytokine profile of donor cells to aTH2 phenotype (Fowler et al., 1994). See Krenger et al. (1995) and Fowler and Gress (1996)for details of the in vitro culture method.

2. To evaluate the effect of T cell subsets on GVHD models (optional): Deplete subsetsby antibody-complement procedures: e.g., deplete CD4+ or CD8+ cells (see BasicProtocol 1, step 4) and/or deplete NK cells utilizing monoclonal (e.g., anti-NK1.1,clone PK136) or polyclonal (e.g., rabbit anti-asialo-GM1 serum; Wako Bioproducts)antibodies.

Depletion of donor CD8+ cells prior to injection of the donor inoculum results in CGVHDin strain combinations that would otherwise produce AGVHD (e.g., C57BL/6 B6D2F1).CD8 depletion can also increase the severity of the autoimmune pathology (Saitoh et al.,1991).See Ghayur et al. (1991) for details of the anti-asialo-GM1 depletion method.

3. Count the cells and resuspend in HBSS/HEPES at 510 107/ml. Keep at 4C untilhost animals are ready for injection.


4.3.5


4. Immediately before injection, refilter suspension to break up clumped cells.Cell clumps may cause embolism if injected.

5. For animals in the experimental group, inject donor spleen cells into the tail vein ofhost mice in a volume of 0.5 to 1.0 ml using a 3-cc syringe and 27-G needle. Forcontrol animals, inject either syngeneic donor cells or T celldepleted allogeneicdonor cells (the same number of cells as for recipients in the experimental group).

Heating adult mice under a heat lamp can distend the tail veins and increase the ease ofinjection. When high concentrations of cells are injected, the risk of embolism can bereduced by i.p. injection of 50 USP units of heparin in 0.05 ml 10 to 20 min before injectingcells.The number of lymphocytes needed to induce AGVHD is strain dependent. In P F1AGVHD with unirradiated hosts, there is a strong host resistance against parental lympho-cytes expressing H-2b (Hh-1) or H-2d (Hh-2; Davenport et al., 1995). A dose of 57.5 107 spleen cells is thus necessary for C57BL/10 or C57BL/6 donors, whereas only 2.53.5 107 A strain spleen cells are needed.In CGVHD strain combinations 57.5 107 spleen cells are typically used; injecting >108donor spleen cells may result in AGVHD.For transfusion-induced GVHD model, inject 1.52 107 lymph node cells (the T cellequivalent of 6 107 spleen cells).

Table 4.3.2 Models of Chronic GVHD

Hostpreparation

Experimentalreadouts Genetic disparity

Common straincombinations

Organ-specificpathology

Autoimmune diseasemodel (reference)

Lethallyirradiateda

TH2 clones Minorhistocompatibility antigens

LP B6a Skin Scleroderma(DeClerck et al., 1986)

Sublethallyirradiated

Minorhistocompatibility antigens

B10.D2 BALB/c Skin Scleroderma(Claman et al., 1985)

Unirradiated TH2 cytokines;serum IgE; lackof anti-host CTL;rheumatoidfactor; antibodiesto ssDNA, ribo-nucleoprotein,renal tubularantigens, andlaminin

MHC class I andclass II (P F1)

DBA/2 B6D2F1 Kidneys Glomerulonephritis(Kuppers et al., 1988)

BALB/c (BALB/c A)F1

Systemic lupus(Kuppers et al., 1988)

Salivary glands Sjogrens syndrome(Fujiwara et al., 1991)

Muscles Myositis(Gelpi et al., 1994)

Intestines Enteropathy(de Geus et al., 1993)

Joints Rheumatoid arthritis(Pals et al., 1985)

Nails Scleroderma(Fujiwara et al., 1991)

MHC class II(P F1)

B6 (6 B6.bm12)F1

Liver Primary biliary cirrhosis(Saitoh et al., 1991)

Kidneys Lupus nephritis(Ito et al., 1992)

aLow doses (2 107) of splenic T cells produce a CGVHD in the LP B6 strain combination; larger T cell inocula (5 107) result in lethal AGVHD.bSublethal irradiation (600 rad) is necessary to generate chronic GVHD in the B10.D2 BALB/c strain combination. Higher doses of irradiationproduce lethal AGVHD.


4.3.6



Disease

SUPPORTPROTOCOL 1

ASSESSMENT OF ACUTE AND CHRONIC GVHD BASED ON SURVIVALOUTCOMESurvival has long been the most common means of assessing AGVHD in irradiatedtransplantation models (mortality is uncommon in P F1 unirradiated host models). Themean survival time will be dependent upon several factors, but primarily upon geneticdisparity between donor and host, and donor lymphocyte dose. Note that contemporarystandards for animal care require euthanasia for suffering or moribund animals (UNIT 1.1).It is important to establish appropriate criteria for euthanasia and apply these uniformlyto all experimental groups to prevent skewing of survival data (UNIT 1.8). Such criteria mayinclude loss of >30% of initial body weight or severe systemic inflammatory symptoms(such as ruffled fur, hunched posture, shivering, and severe edema). It is important to notethat irradiated animals typically will undergo an initial transient weight loss of 10% to15% and may have some degree of diarrhea.

1. Set up experimental groups containing five to ten animals per group, includingnon-GVHD (syngeneic transplant or T celldepleted transplant) animals. Determinethe number of mice that survive each day following induction of AGVHD. Convertto percentages of original number in the group.

2. Plot animal survival data as a stepped function relative to time, with the percentageof surviving hosts decreasing on each day that animals in the group die (Fig. 4.3.1).Compare experimental and control groups based on mean survival time usingnonparametric rank sum analyses (such as the Wilcoxon test).

Computer software for analyzing survival data is available in Statview version 4.5 (SASInstitute), or in the Survival Tools module available for earlier versions of the program.

SUPPORTPROTOCOL 2

ASSESSMENT OF ACUTE GVHD BASED ON WEIGHT LOSSDifferences in the degree of weight loss (or the time course of weight recovery) cansometimes discriminate between treatment groups in nonlethal AGVHD models.1. Mark individual animals with ear punches or tags (UNIT 1.5). Weigh all animals and

assign animals so that the average weights of different groups are comparable andthe variation in weight within groups is minimized.

Days

% s

urvi

val

control

AGVHD

0 14 280

20

40

60

80

100

Figure 4.3.1 Survival curve of AGVHD.


4.3.7


2. Weigh animals on alternate days in the first week and twice weekly thereafter.Calculate mean weights and graph data as mean weight per group against time (Fig.4.3.2).

Experimental conditions resulting in loss of all animals in a treatment group are depictedas a line ending on the date of the last animals death.Control mice receiving lethal total body irradiation and a T celldepleted or syngeneicmarrow transplant will lose 10% to 20% of total body weight within the first 4 or 5 days,but then recover. Mice undergoing AGVHD will have a similar initial drop in weight butwill recover more slowly or will undergo a continuing decline in weight.

3. At an endpoint appropriate to the experiment (typically 6 to 12 weeks), compareweights of mice in the experimental groups with pretreatment weights and with thoseof control animals.

One problem with weight curves is that the mean weight of a small group may fall and riseerratically as lower-weight mice die, leaving heavier survivors. It is thus necessary to havesufficient numbers of hosts in each group to minimize such shifts.

SUPPORTPROTOCOL 3

ASSESSMENT OF DONOR/HOST CHIMERISM IN MICE WITH ACUTE ORCHRONIC GVHD BY FLOW CYTOMETRYFlow cytometric analysis of chimerism in lymphocyte populations is a particularlyeffective method for tracking the progress of the anti-host attack in unirradiated P F1AGVHD, as well as distinguishing AGVHD from CGVHD. Lymphocyte populationlevels and donor/host chimerism change radically during the first few weeks after parentalspleen injection (Hakim et al., 1991, 1995). Donor CD4+ and CD8+ populations constitute10% to 25% of the total CD4+ and CD8+ populations within the first week; by the secondweek, donor CD8+ cells comprise the dominant T cell population. Between 2 and 3 weeks,the host B cell population is completely ablated, and after 4 to 6 weeks most hostpopulations have been eliminated. In comparison, in unirradiated P F1 CGVHDmodels, donor CD4+ cells expand to become only 15% to 25% of the total CD4+ cellspresent, and few donor CD8+ cells are present. Host B cells increase in frequency andexpress elevated levels of MHC class II (Ia) antigen as a result of cytokine-induced

42352821147010

20

30

Days

Mea

n w

eigh

t (g)

AGVHD

control

Figure 4.3.2 Weight curve of AGVHD.


4.3.8



Disease

activation. These population shifts can be observed by two-parameter flow cytometricanalysis of host splenic lymphocytes by combining a class I (or for B cells, class II) MHCmarker specific for the host and a lymphocyte population marker (Fig. 4.3.3). The protocolthat follows describes the use of fluorescein isothiocyanate (FITC)labeled antibodiesand biotin-labeled antibodies specific for MHC followed by streptavidin-phycoerythrin.Phycoerythrin-labeled antibodies, or other antibody/dye combinations, may be usedinstead. Several analytical instruments, such as the Becton Dickinson 9BD FACScan (UNIT5.4) or FACSORT with CellQuest software, may be used for this purpose.Flow cytometry can also provide a particularly sensitive measurement of AGVHD inirradiated allogeneic transplant models (at nonlethal doses of donor cells) by distinguish-ing a delay in recovery of B cell populations in the bone marrow, spleen, or peripheralblood (as assessed by anti-B220 or anti-IgM staining compared with hosts receiving Tcelldepleted donor or syngeneic grafts). The best time to evaluate B cell recoverydepends upon the strain disparity (MHC mismatched or matched) and the tissue assayed(evaluate bone marrow pre/pro B cells 2 weeks before spleen or blood). Flow cytometrymay also be used to distinguish CGVHD from AGVHD in unirradiated P F1 models.For example, in CGVHD, the donor CD8+ expansion is minor or absent and the host Band T cell populations remain intact. In addition, IL-4 production during CGVHD resultsin elevated class II antigen expression on B cells; thus, the two-color combination of a Bcellspecific marker (B220) and class II is again useful to assess the presence of activatedB cell populations.

MaterialsMice with AGVHD or CGVHD (see Basic Protocols 1 and 2)Untreated donor- and host-strain miceAntibody to block Fc receptor binding (clone 24G-2, Pharmingen), diluted 1/10 in

FACS bufferFITC-labeled antibodies against CD4, CD8, and B220 (e.g., Pharmingen or Caltag)

CD4

CD8

IAd

donor host AGVHR 1 week AGVHR 2 weeksAGVHR 6 weeks

H-2Kd

H-2Kd

B220

Figure 4.3.3 Flow cytometry of AGVHR.


4.3.9


Biotin-labeled antibodies against host MHC class I and class II antigens(Pharmingen)

FACS buffer (see recipe)Streptavidin-phycoerythrin (streptavidin-PE; Caltag)20 g/ml propidium iodide (Sigma) or 7-aminoactinomycin D (Sigma) in FACS

bufferAdditional reagents and equipment for ammonium chloride lysis of erythrocytes

(UNIT 3.1), cell counting with trypan blue exclusion (APPENDIX 3B), and flowcytometry (UNITS 5.3 & 5.4)

1. Collect spleens from AGVHD or CGVHD mice, as well as control untreated donor-and host-strain mice, and prepare single-cell suspensions, as described for GVHDinduction procedures (see Basic Protocol 1, steps 1, 2, 5, and 6).

2. Perform ammonium chloride lysis to deplete erythrocytes (UNIT 3.1), then count usingtrypan blue to distinguish dead cells (APPENDIX 3B).

During the first few weeks of AGVHD, spleens will be large and bloody, with much fibrotictissue left after the cell suspension is dispersed. In CGVHD splenomegaly is common.

3. Distribute splenic single-cell suspensions into individual tubes at 1 106 cells/tubein HBSS/HEPES. Add 5 l/tube of antibody to block Fc receptor binding.

4. Stain cells with relevant isotype controls; FITC-labeled antibodies against CD4, CD8,and B220; and biotin-labeled antibodies specific for host MHC class I and class IIantigens for each mouse spleen.

For example, in C57BL/6 B6D2F1 AGVHD, use anti-H-2Kd or -H-2Dd, and anti-H-2IAd.The authors have used Pharmingen antibody clones SF1-1.1, 34-2-12, and AMS-32.1,respectively. The following combinations of antibodies comprise a suitable set: anti-H-2Khost-FITC/anti-CD4-biotin; anti-H-2Khost-FITC/anti-CD8-biotin; anti-B220-FITC/anti-H-2IAhost-biotin.B cell chimerism can be monitored by staining for B220 (the CD45 isoform specific to Bcells) versus a host class II antigen haplotype. Anti-B220 produces a more clear-cutidentification of B cells than anti-IgG, which may bind to FcR-bearing cells.

5. Incubate 30 min at 4C.

6. Wash cells twice by adding 2 ml FACS buffer, then centrifuging 5 min at 175 g,10C, decanting supernatant, blotting, and resuspending pellet in the same amountof buffer.

7. Incubate with streptavidin-PE for 10 min at 4C.The appropriate concentration of streptavidin-PE to use should be determined by titration.One author (FH) uses 10 l/tube of a 1/10 or 1/20 dilution of Pharmingen antibody or ofa 1/40 dilution of Caltag antibody.

8. Wash twice and resuspend in 200 to 500 l FACS buffer.

9. Add 10 l/tube of 20 g/ml propidium iodide or 7-aminoactinomycin D to identifydead cells.

10. Analyze by flow cytometry, acquiring only live-cell events.Exclusion of dead cells is particularly important given that as many as 50% of the spleencells may be dead after 2 weeks of AGVHD.Because dramatic shifts in donor and host lymphocyte populations occur in the first weeksof unirradiated P F1 AGVHD, the expected outcomes depend upon the time at which


4.3.10



Disease

populations are assessed. In the first week, the main findings of AGVHD are presence ofdonor CD4+ and CD8+ T cells. During weeks 2 to 3, donor CD4+ and CD8+ cellspredominate and host B cell populations are lost. In weeks 4 to 6, few donor or hostlymphocytes are present; after 6 weeks, donor-derived lymphocytes expand.

SUPPORTPROTOCOL 4

ASSESSMENT OF ACUTE GVHD BY MEASUREMENT OF DONORANTI-HOST CYTOTOXICITYDuring the first 4 to 6 weeks after induction of unirradiated P F1 AGVHD, anti-hostcytotoxic effectors eliminate host populations. To measure anti-host cytotoxic effectorsduring AGVHD, spleen cells from transplanted mice can be utilized as effectors in directcytotoxicity assays using tumor cell lines bearing the F1 other parent haplotype. Forexample, for the C57BL/6 B6D2F1 AGVHD model, use an H-2d tumor cell line suchas P815. It should be noted that cytotoxic effectors are not observed identified in CGHVDmodels.

MaterialsMice with PF1 AGVHD (see Basic Protocol 1)Donor-strain miceF1 host-strain mice, untreated or injected with F1 spleen cellsHBSS (APPENDIX 2)Complete RPMI-10 (APPENDIX 2)Triton X-100U-bottom 96-well microtiter plate scintillation counterAdditional reagents and equipment for counting cells (APPENDIX 3A) and measuring

cytotoxic T lymphocyte activity (UNIT 3.11)NOTE: All cell culture incubations should be performed in a humidified 37C, 5% CO2incubator.

1. Collect spleens from AGVHD mice, as well as control untreated donor-strain and F1host-strain mice (or host F1 mice injected with F1 spleen cells), and prepare single-cellsuspensions, as described for GVHD induction procedures (see Basic Protocol 1,steps 1, 2, and 5).

2. Count cells (APPENDIX 3A) and resuspend in complete RPMI-10 at 5 106 cells/ ml.3. Distribute cells into three replicate wells of a U-bottom 96-well plate at 200 l/well.

Serially dilute through three 2-fold dilutions, resulting in 100 l diluted effector cellsper well. Set up four to six replicate wells for maximum release (50 l of 0.05%Triton X-100) and spontaneous release (100 l medium).

4. Label tumor cell targets with 51Cr (UNIT 3.11).5. Wash cells twice and resuspend in complete RPMI-10 at 5 104 cells/ml.

6. Add 100 l of target cells to each experimental well and to maximum-release andspontaneous-release wells.

The serial dilutions produce effector/target cell ratios of 100:1, 50:1, 25:1, and 12.5:1.7. Incubate plates overnight at 37C.

8. Harvest supernatants and count in a -scintillation counter.Expect 40% to 60% cytotoxicity on target cells after 2 weeks of AGVHD. Cytotoxic effectorsare still measurable as late as 6 weeks. Normal donor cells will be negative (

cytotoxicity) because of lack of priming with host-strain stimulators; untreated host cellswill be negative because of tolerance.Sample results of this assay are shown in Figure 4.3.4.

SUPPORTPROTOCOL 5

ASSESSMENT OF CYTOKINE PRODUCTION AND PROLIFERATIVERESPONSES IN ACUTE AND CHRONIC GVHDCytokine production in the autologous mixed lymphocyte reaction and in response toalloantigens is severely reduced in mice undergoing AGVHR and CGVHR. Thus immunefunctional changes in GVHD can be broadly assessed with assays for specific cytokinessuch as IL-2 and IL-4 (UNIT 6.3), IL-3 (UNIT 6.4), IL-5 (UNIT 6.5), interferon (IFN-; UNIT6.8), granulocyte/macrophage colony-stimulating factor (GM-CSF), and tumor necrosisfactor (TNF; UNIT 6.10). This protocol can be used to assess IL-2 production and prolifera-tive responses to mitogenic stimuli, which are also severely reduced in GVHD. If desired,other cytokines mentioned above can be assayed with the modifications in incubationperiod noted below.

MaterialsMice with CGVHD (see Basic Protocol 2)Untreated donor- and host-strain miceComplete RPMI-10 (APPENDIX 2)Mitomycin C (optional)Concanavalin A (Con A; Sigma)Antimouse CD25 MAb (clone 7D4; Pharmingen)Lipopolysaccharides (LPS; Sigma)[3H]ThymidineCesium source for -irradiation of whole animals capable of delivering 2500 rad24-well and flat-bottomed 96-well microtiter plates

Figure 4.3.4 Anti-hostcytotoxicity in AGVHR.

100:1 50:1 25:1 12.5:10

10

20

30

40

50

60

AGVHR

donor

UNTRT F1

Effector/target ratio

% c

ytot

oxici

ty


4.3.12



Disease

Additional equipment and reagents for counting cells (APPENDIX 3A), IL-2 ELISA orCTLL bioassay (UNIT 6.3), blocking of cellular division of stimulator cells (UNIT3.12), and cell labeling and harvesting (APPENDIX 3D)

NOTE: All cell culture incubations should be performed in a humidified 37C, 5% CO2incubator.

Prepare cells1. Collect spleens from GVHD and control mice, untreated donor- and host-strain mice,

and an allogeneic-strain mouse, and prepare single-cell suspensions, as described forGVHD induction procedures (see Basic Protocol 1, steps 1, 2, and 5).

2. Wash cells and count (APPENDIX 3A). Resuspend allogeneic cells at 2 106 cells/mland other cells at 5 106 cells/ml in complete RPMI-10.

3. Irradiate the allogeneic cells at 2500 rad (or treat with mitomycin C; see UNIT 3.12).

Measure spontaneous and concanavalin Astimulated cytokine production4. Distribute 10 106 cells/well (2 ml) into 2 wells on a 24-well culture plate. Add

nothing to one well (medium-alone control); add 5 g Con A/ml to the second well.5. Culture plates 24 hr, then collect supernatants and assay for IL-2, IL-3, IL-4,

GM-CSF, and/or TNF.IL-5 and IFN- can be assayed after 72 hr.In AGVHD, TH1 cytokines may be detected in the first week in the unstimulated wells; levelsof IFN- may exceed 50% of the Con Astimulated normal control supernatant. ConAstimulated cytokines will be depressed to very low levels within 1 week and will remaindepressed for several weeks. In CGVHD in unirradiated P F1 models, immune deficitsin mitogen-stimulated cytokines are minimal.

Measure AMLR- and MLR-stimulated IL-2 production6. Distribute 5 106 cells/well (1 ml) into 2 wells on a 24-well culture plate. Add 1 ml

of medium to the first well (autologous mixed-lymphocyte reaction) and 2 106irradiated allogeneic cells to the second well (mixed-lymphocyte reaction). Addanti-CD25 antibody to both wells to block consumption of IL-2 (converting this assayinto a cumulative production assay).

Addition of an antibody blocking IL-2 consumption is crucial to this assay. The antibodymust be titered to determine the proper concentration to maximize IL-2 content in theculture supernatant from normal cells. If a CTLL proliferation assay is used to measureIL-2, then the effect of carryover of the antibody onto CTLL cells must also be tested.

7. Culture plates 5 days, then collect supernatants and assay for IL-2 by ELISA or CTLLproliferation assay (UNIT 6.3).

Some IL-2 will be present in the unstimulated (medium) supernatant from spleens in thefirst 2 to 3 days after induction of AGVHD. Subsequently, severely reduced levels of IL-2will be found in all AGVHD splenic culture supernatants. Culture supernatants fromCGVHD spleens will have near-normal levels of IL-2 in the mixed lymphocyte reactionculture, but severely reduced levels in the autologous mixed-lymphocyte reaction culture.

Perform proliferation assays8. Dilute cell suspension to 2.5 106/ml. Distribute 100 l of cells/well in twelve wells

on a flat-bottomed 96-well plate.


4.3.13


9. To replicates of four wells, add 100 l medium, 2.5 g/ml concanavalin A, or 100ng/ml LPS. Culture 3 days, adding 25 l of [3H]thymidine (40 Ci/ml) during thelast 16 hr (APPENDIX 3D).

10. Harvest cells and determine proliferation (APPENDIX 3D).AGVHD cultures will have markedly reduced proliferation in response to Con A or LPS.

SUPPORTPROTOCOL 6

HISTOPATHOLOGICAL ASSESSMENT OF ACUTE AND CHRONIC GVHDHistopathology can be an important element in the analysis of GVHD. In irradiatedAGVHD models, the main tissues affected are the liver, intestinal tract, and skin; inunirradiated P F1 AGVHD models, the skin is comparatively unaffected. Changes inthe liver include lymphocytic infiltrates in the peribiliary areas, intraepithelial infiltratesin the bile ducts and ductules, and degeneration of hepatocytes (acidophilic bodies) andbiliary epithelial cells. In the intestine, lesions consist of shortening and loss of crypts anddegeneration of crypt epithelial cells, infiltraton of lymphocytes into the lamina propria,and erosion of the mucosal epithelia. Skin lesions affect primarily the epidermis, includingvacuolar degeneration of basal epithelial cells, dyskeratotic squamous epithelial cells, andepidermal clefts. In CGVHD, the skin is again a major target organ, but the changes differfrom those of AGVHD; CGHVD skin lesions are characterized by dermal fibrosis andimmunoglobulin deposits (and in scleroderma models, increased numbers of degranulatedmast cells). Lymphocytic infiltrates in specific organs are common (see Table 4.3.2). Inthe kidneys, immunoglobulin deposits in the glomeruli and glomerular nephritis arecommonly observed. These characteristic pathological changes can be converted intonumerical indices to compare GVHD and control mice or, more commonly, treatedGVHD mice. The steps that follow are general guidelines for performing histologicalassessment of GVHD. Specific steps for fixing tissues, embedding in paraffin, sectioning,and preparing slides for examination are provided in UNIT 21.4.

1. Select the organs to be assayed, and collect and fix in formalin.The small intestine can be cut at the pyloric and ileal-caecal valves, removed from themesenteric membranes, rinsed with formalin, and coiled in a fixation/embedding chamberso that the entire structure can be sectioned fully and evaluated. Some laboratories haveused the tongue to assess epidermal changes.

2. Establish a numerical rating scale based on organ-specific histologic characteristicsand the percentage of total area occupied by these characteristics within a field ofview.

3. Assess blinded slides from all groups in mixed order. Examine ten fields of view inat least three different planes of section of each organ.

4. Sum the ratings from all organs in each mouse. Average the ratings per experimentalgroup and compare by nonparametric statistics.

SUPPORTPROTOCOL 7

ASSESSMENT OF SERUM ANTIBODY LEVELS (HUMORAL IMMUNITY)IN CHRONIC GVHDAssays of humoral immunity are primarily informative in CGVHD but not in AGVHDmodels. In AGVHD models, the immune system is profoundly suppressed for severalweeks. The serum is hypogammaglobulinemic due to destruction and delayed recoveryof B cells. In contrast, in CGVHD models, the B cells are chronically stimulated andautoantibody production in commonly observed. Elevated levels of IgG1 and IgE can bemeasured in the serum by ELISA (UNIT 2.1) or immunodiffusion (UNIT 2.3). A variety ofautoantibodies are produced. In the common DBA/2 B6D2F1 combination, antibodies


4.3.14



Disease

against ssDNA are produced that can be assessed by ELISA assay. Anti-ssDNA activitywill be detectable in CGVHD sera after 2 weeks and peak at 4 to 6 weeks.

MaterialsMethylated bovine serum albumin (MBSA; see recipe)Dulbeccos PBS/Tween (DPBS/Tween; see recipe)10 g/ml denatured calf thymus DNA (working solution; see recipe)Dulbeccos PBS containing 5% FBS (DPBS/5% FBS)Positive reference control: e.g., serum from an autoimmune strain of mice, such as

MRL/lprSerum samples from mice with CGVHDHorseradish peroxidaseconjugated goat antimouse IgG (HRP-g-anti-mIg; see

recipe)96-well flat-bottomed microtiter plateELISA microtiter plate reader

1. Pretreat microtiter plate by adding 50 l/well of 10 g/ml MBSA. Incubate 60 to 90min at room temperature.

2. Wash plates three times with PBS/Tween, then rinse once with deionized H2O. Blotdry by repeatedly slapping the plate, open face down, onto a pad of absorbent paper.

3. Add 50 l/well of 10 g/ml denatured calf thymus DNA. Incubate 90 to 120 min atroom temperature, then repeat washing (step 2).

4. Add 50 l DPBS/5% FBS per well as a blocking agent. Cover and incubate overnightat 4C, then repeat step 2.

5. Add fresh DPBS/5% FBS as a diluent to wells as follows:10 l to well A190 l to each remaining well in row A50 l to each well in rows B-H.

The first column will be used as a blank to determine the background OD.6. Add 10 l of a positive reference control to well A2, and add 10 l of serum samples

to wells A3-A12.

7. Perform 2-fold serial dilutions along each column by transferring 50 l from row Ato row B, and so on, discarding the extra 50 l in row H.

These volumes result in serial dilutions of serum from 1:10 to 1:1280. Adjust the volumesof serum or diluent, or amount transferred, to alter the dilution series, if necessary. Thefinal volume in the wells after dilution is 50 l.

8. Incubate 2 to 3 hr at room temperature, then repeat step 2.

9. Add 50 l/well of HRP-goat-anti-murine Ig and incubate 60 min at room temperature.Repeat step 2.

10. Add 100 l/well of ABTS peroxidase substrate mixture. Incubate at room tempera-ture until developed (positive wells turn green; background and negative wells shouldremain clear); this is usually 2.0; blanks should be

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 5.Denatured calf thymus DNA solution, 10 g/ml

Concentrated stock: Dissolve calf thymus DNA to 1 mg/ml in Dulbeccos PBS(DPBS); the DNA will go into solution slowly. Divide into 250-l aliquots and storeindefinitely at 20 or 70C.Working solution: Shortly before use, thaw stock at 37C and dilute 1:100 in DPBS.Heat 15 min at >80C while stirring to denature DNA, then put beaker immediatelyinto ice to cool. Reconstitute with distilled water to replace any lost volume.

DPBS/Tween, 102.5 ml Tween 20500 ml 10 DPBSStore up to 6 months at room temperatureDilute 1/10 in H2O before useDo not store diluted solution

FACS bufferHanks balanced salt solution without phenol red (APPENDIX 2)0.2% BSA0.1% sodium azideStore up to 3 months at 4C

Horseradish peroxidaseconjugated goat antimouse IgG (HRP-g-anti-mIg)Dilute HRP-g-anti-mIg (Fc portion, heavy chain specific) 1:250 in DPBS/Tween(see recipe). Run a titration assay to determine the proper dilution of each new lotof antibody. Store up to 6 months at 4C.

Methylated bovine serum albumin (MBSA), 10 g/mlDilute MBSA to 1 mg/ml in water. Divide into 250-l aliquots and store indefinitelyat 20 or 70C. Dilute stock 1:100 in DPBS before use.

COMMENTARYBackground Information

Two distinctive forms of GVHD occur:acute and chronic GVHD. These disorders dif-fer in that acute GVHD (AGVHD) predomi-nately involves a T cellmediated attack on thehost, with subsequent inflammatory cytokine-induced systemic effects. Chronic GVHD, incontrast, is an autoimmune disorder charac-terized by chronic B cell stimulation, andautoantibody production. The basic experi-mental methods utilized for establishing bothacute and chronic GVHD models are similar;variables such as donor/host strain combinationand transplant preparative regimen can be indi-vidualized to induce the different disease states.

Models of AGVHDThe main model for induction of AGVHD

(see Basic Protocol 1) involves transplantationof donor bone marrow and lymphocytes into

lethally irradiated allogeneic hosts. Thismethod results in the complete expression ofthe clinical syndrome, including inflammatorypathology in the liver, skin, and gut, markedweight loss, and significant mortality. Trans-plantation of donor cells into hosts differing atmajor or minor histocompatibility loci can pro-duce severe, lethal AGVHD by this method (seeTable 4.3.1).

An alternative model (P F1 AGVHD),extensively used to study mechanisms of T celldysfunction, involves injection of spleen cellsfrom parental strain (P) donors into unirradi-ated immune-competent adult F1 hosts. Unlikein irradiated models, the donor must differ fromthe host at both class I and class II majorhistocompatibility (MHC) loci to initiate a re-action, and the parental inocula must includeboth CD4+ and CD8+ T cells. In the F1 hosts,parental donor T cells expand and produce


4.3.16



Disease

cytotoxic effectors, resulting in the eliminationof the semiallogeneic host lymphohematopoie-tic system (Hakim et al., 1991). Subsequently,the donor/host chimera is repopulated by donorcells derived from stem cells in the originalsplenic inoculum. A severe deficiency in bothT and B cell function is observed for severalweeks. In contrast to irradiated AGVHD mod-els, however, skin pathology and systemicweight loss are minimal, and death is uncom-mon. Hence, the main parameters studied arethe generation of anti-host cytotoxic effectors,level of donor/host chimerism, and immunedeficiency.

In irradiated AGVHD models, hosts receiveboth donor bone marrow (as a source of stemcells for hematopoietic reconstitution), andlymphocytes from donor spleen or lymphnodes (as a source of T cells for induction ofGHVD). In severe cases, such transplants resultin rapid weight loss and death; in less severeGVHD, transient weight loss, delayed immunereconstitution, and prolonged immune defi-ciency are the only observed abnormalities. Theseverity of AGVHD in any given model isdependent upon the genetic disparity betweendonor and host and the T cell subsets present inthe allogeneic inocula (Korngold and Sprent,1991). Donor CD4+ T cells are necessary forlethal GVHD in MHC-disparate or class IIantigendisparate combinations, whereasCD8+ T cells are both necessary and sufficientto generate lethality in class I antigendisparatecombinations. In most MHC-matched, minorantigenmismatched combinations, CD8+ cellsare sufficient to induce lethality, although CD4+cells can increase the severity in many combi-nations and can produce lethality in some(Korngold, 1992). Table 4.3.1 outlines the com-mon strain combinations utilized for AGVHDmodels.

Much of the pathogenesis of AGVHD hasbeen attributed to a cytokine storm (Ferraraet al., 1993). The irradiation utilized in acuteGVHD models damages the gut and skin, re-sulting in systemic exposure to bacterial-de-rived toxins, and subsequent inflammatory cy-tokine release (primarily TNF- and IL-1) andgeneralized immune activation. Donor T cellsreacting to host alloantigens produce TH1 cy-tokines, especially IFN- (Troutt and Kelso,1992), which activate proinflammatory macro-phages; these activated macrophages are thentriggered by bacterial products such as LPS torelease high levels of IL-1, TNF-, and nitricoxide, resulting in weight loss and death bytoxic shock (Nestel et al., 1992). Thus, three

primary factors influence the severity ofAGVHD: the conditioning regimen (radiationdose), donor T cell response, and environ-mental pathogens (as a source for LPS endo-toxin). Many laboratories have observed that asanimal colony conditions have become cleaneror colonies have been converted to a specific-pathogen-free status, the dose of radiationand/or donor T cells needed to induce lethalAGVHD has increased. The importance of LPSin triggering the cytokine storm has also beendemonstrated in P F1, unirradiated AGVHDmodels: injection of low, normally nonlethaldoses of LPS to AGVHD recipients results inacute lethality 8 to 12 hr post LPS administra-tion (Nestel et al., 1992).

Models of CGVHDWhereas AGVHD models emphasize cell-

mediated immune processes, chronic GVHD(CGVHD) models focus upon the developmentof chronic, B cellstimulatory autoimmune dis-orders (Goldman et al., 1991); AGVHD ap-pears to be an inflammatory process mediatedby TH1 cytokines, whereas CGVHD is medi-ated by TH2 cytokines. CGVHD is charac-terized by prolonged splenomegaly and lym-phadenopathy. Mice develop symptoms of sys-temic autoimmune disorders, includingimmune complex glomerulonephritis (Ito et al.,1992), primary biliary cirrhosis (Saitoh et al.,1991), and Sjgren-syndrome- or scleroderma-like lesions (Fujiwara et al., 1991). Elevatedlevels of immunoglobulins, particularly IgG1and IgE, and a variety of autoantibodies (in-cluding anti-DNA) are present in the serum. Ingeneral, the autoimmune syndrome of CGVHDis lethal within 6 months, due to developmentof immune complexmediated glomerular ne-phritis. Murine CGVHD models share many ofthe pathologic features of the clinical disorderof chronic graft-versus-host disease that occursafter allogeneic bone marrow transplantation inman. It is important to note, however, that thehuman syndrome develops several months aftermarrow transplant, whereas murine CGVHDdevelops within weeks. Additionally, humanCGVHD occurs after an acute suppressiveGVHD, and may require ongoing donor anti-host reactivity and aberrant T cell maturationin the post-transplant thymic environment. Themurine disorder, in contrast, is usually basedon an immediate and continuing donor T cellreaction to allogeneic antigens in a mixedchimeric host.

Murine modeling of clinical CGVHD by useof irradiated hosts has generally been ineffec-


4.3.17


tive; only one strain combination (LP C57BL/6) has been reported to produce theautoimmune symptoms of clinical CGVHD(see Table 4.3.2). LP/J spleen and bone marrowcells are injected into lethally irradiated B6hosts (the reverse combination of strains isineffective). These strains are MHC matched,but differ at multiple minor antigen loci. De-pending on the dose of LP spleen cells injected,the host develops either a rapidly lethalAGVHD (50 106 cell inocula) or a slowlydeveloping chronic GVHD (20 106 cell in-ocula). The CGVHD observed in this model ischaracterized primarily by dermal sclerosis; Tcell clones derived from these mice are primar-ily CD4+, and secrete primarily IL-4 and afibroblast growth factor (DeClerck et al., 1986).Although this represents the closest approxi-mation of clinical CGVHD, much is still un-known concerning B cell function and auto-an-tibody production in this model.

With the exception of this one strain combi-nation, animal research in CGVHD has utilizednonablative transplant regimens (which resultin mixed donor/host chimeras) to replicateautoimmune symptoms. The other twoCGVHD models in use involve the intentionalgeneration of a mixed donor/host chimera inwhich alloactivated T cells produce B cellstimulatory cytokines, which in turn trigger Bcell hyperplasia, elevated immunoglobulin lev-els, autoantibody production, and pathologicchanges consistent with autoimmunity (see Ta-ble 4.3.2).

In the first of these models, B10.D2 cells aretransplanted into sublethally irradiated MHC-matched BALB/c hosts , resul t ing inscleroderma-like skin pathology. The use ofsublethal irradiation (600 rad) is critical for thesuccessful generation of this form of CGVHD:higher doses produce lethal AGVHD. Strainsutilized are MHC matched (H-2d), but differ atmultiple minor loci, including mlsc linked toMTV-6 expression. In these models, donorCD4+ T cells responsive to host antigens releaseTH2 cytokines, inducing a proliferation of hostmast cells (Claman et al., 1985). Such mast cellsdegranulate, resulting in extensive cutaneousand vascular fibrosis analogous to changes ob-served in the human autoimmune diseasescleroderma.

In the second model, MHC-disparate donor(parental strain) spleen and/or lymph node cellsare injected into unirradiated semiallogeneicadult F1 hosts; to avoid the AGVHD observedin most P F1 strain combinations, donorstrains with compromised cellmediated func-

tion (DBA/2 or BALB/c) are utilized, or donorCD8+ cells are deleted (as described in this unit;see Basic Protocol 2). Alternatively, strain com-binations with only class II MHC disparitiesare used (see Table 4.3.2). These unirradiatedmodels have been extensively used to studydonor T cell function, autoimmune pathology,and autoantibody production in CGVHD. Thedisease process involves the continuous directinteraction of donor CD4+ T cells with disparateclass II MHC antigen present on host B cells.Three lines of evidence support this pathoge-netic mechanism in this model: first, CD4+donor T cells are both necessary and sufficientto induce the disease; second, only those hostB cells disparate at class II are stimulated toproduce antibody; and third, the disorder isdependent on the continued presence of donorT cells and remains transferable with donor Tcells even after several weeks (Moser et al.,1988; Morris et al., 1990).

Because the generation of CGVHD resultsfrom the interaction of donor CD4+ cells andhost class II antigens, the injection of donor B6spleen cells into B6 B6bm12-F1 mice (B6bm12has a mutation in I-Ab) produces a severeCGVHD, with characteristic splenomegaly andmultiorgan lymphocytic infiltrates (Saitoh etal., 1991). Donor CD8+ T cells are not neededin this strain combination; indeed, CD8+ celldepletion increases the severity of the his-topathology. Moreover, donor/host strain com-binations with both class I and class II antigenicdisparities, which would produce AGVHDwith an intact donor T cell complement, pro-duce CGVHD when donor CD8+ cells are de-pleted. For example, B6 donor cells induceAGVHD in B6D2F1 hosts, but CD8-depletedB6 splenocytes produce a CGVHD in the samehosts. Finally, the best-studied CGVHD straincombination involves injection of DBA/2spleen cells into unirradiated (C57BL/6 DBA/2)F1 (B6D2F1) or (C57BL/10 DBA/2)F1 (BDF1) hosts. Although these straincombinations involve both MHC class I andclass II antigenic disparities, donor spleen cellinjection results in a CGVHD. Interestingly, notonly do DBA/2 CD8+ T cells occur at a lowerfrequency than CD8+ cells in other strains, butthe frequency of precursor anti-B6 pCTL is also10-fold lower (Via and Shearer, 1988). Thus, Tcellreplete transplants utilizing this strain arecomparable to CD8-depleted transplants inother strain combinations. The DBA B6D2F1 combination has been extensivelystudied as a model of multiorgan systemicautoimmune disease: production of autoanti-


4.3.18



Disease

bodies is extensive (including anti-DNA), re-sulting in the development of immune-com-plex-mediated glomerulonephritis (Kuppers etal., 1988). Lymphocytic infiltrates can also bedetected in salivary glands (analogous to thehuman disease Sjgrens syndrome; Fujiwaraet al., 1991) and muscle (analogous to autoim-mune myositis; Gelpi et al., 1994).

The main methods of characterizingCGVHD models evaluate both pathologicaland immune changes. Spleen and lymph nodesare enlarged severalfold, primarily by expan-sion of F1 host B cells. Lymphocytic infiltratesin liver and salivary glands are evident on rou-tine pathologic evaluation. Serum IgE may beelevated; specific autoantibodies can be readilyassessed by ELISA (Aoki et al., 1992). B cellexpression of class II antigen is elevated, andcan be detected by two-color flow cytometryusing an antiB cell marker (such as anti-B220)and an anti-I-A marker of the F1 host (forexample, in the DBA/2 B6D2F1 model, onewould utilize anti-I-Ab). Commonly utilizedtests of immune dysfunction (such as totalsplenic IL-2 production or mitogen prolifera-tion responses) often show little deficit. Morereliably, a significant deficit is observed in testsof IL-2 production in autologous mixed-lym-phocyte reaction (UNIT 3.12), or tests of the gen-eration of cytotoxic effectors against TNP-modified donor cells (UNIT 3.11). The deficit inthese responses is consistent with a defect inthe production of the TH1 cytokine IL-2. Toreliably demonstrate production of TH2 cyto-kines, it is necessary to enrich for donor CD4+T cells (which may represent only 25% of thetotal CD4+ population or

survival time in lethal models, the degree ofweight loss, and the duration of immune defi-ciency all depend on the strain combination, Tcell subsets injected, and T cell dose.

The classic assay for GVHD was histori-cally the splenic enlargement assay. Althoughsplenic enlargement is a characteristic of bothAGVHD and CGVHD in unirradiated P F1models, splenic size is not particularly infor-mative compared to other techniques. CGVHDspleens enlarge 2- to 4-fold and remain en-larged for months; AGVHD spleens may en-large 2- to 3-fold within 1 week, with the totalcell number decreasing thereafter. Evaluationof a GVHD model is best approached throughselection of multiple endpoints. UnirradiatedAGVHD models can be assessed by fluores-cence-based cell sorting (FACS; for chimer-ism), anti-host cytotoxicity assays, immune-function assays, and histopathology. The defin-ing studies for CGVHD are often autoantibodyassays and histopathology. Whereas AGVHDin irradiated hosts is frequently assessed byevaluation of survival curves, in less lethaldoses weight loss, immune-function assays,histopathology (liver and gut), and fluores-cence-based cell sorting (delayed B cell re-population) may be more informative.

TroubleshootingA list of common problems with these pro-

cedures and their possible solutions is providedin Table 4.3.3.

Anticipated ResultsSurvival of irradiated GVHD hosts will vary

from 10 to 100 days depending upon the straincombination and T cell dose. Even long-termsurvivors may demonstrate prolonged weightloss or immune dysfunction.

Immune dysfunction will be evident in mostP F1 unirradiated AGVHD hosts within thefirst week and will continue for several weeks.The donor and host populations will changemarkedly during this period as donor cells at-tack and replace host lymphohematopoieticpopulations.

In CGVHD models, some immune dysfunc-tion (elevated levels of class II antigens on Bcells, reduced AMLR, absence of TNP-modi-fied syngeneic CTL) will be evident within 2weeks. Autoantibodies appear within 2 to 4weeks, as will lymphocytic infiltrates in targetorgans.

Literature CitedAoki, I., Aoki, A., Otani, M., Miyagi, Y., Misugi, K.,

Ishii, N., Hagiwara, E., Tani, K., Okubo, T., andIshigatsubo, Y. 1992. A correlation between IgGclass antibody production and glomerulonephri-tis in the murine chronic graft-versus-host reac-tion. Clin. Immunol. Immunopathol. 63:34-38.

Blazar, B.R., Taylor, P.A., and Vallera, D.A. 1994.In vivo or in vitro anti-CD3 epsilon chain mono-clonal antibody therapy for the prevention oflethal murine graft-versus-host disease acrossthe major histocompatibility barrier in mice. J.Immunol. 152:3665-3674.

Claman, H.N., Jaffee, B.D., Huff, J.C., and Clark,R.A. 1985. Chronic graft-versus-host disease asa model for scleroderma. II. Mast cell depletionwith deposition of immunoglobulins in the skinand fibrosis. Cell. Immunol. 94:73-84.

Table 4.3.3 Troubleshooting Problems in the Induction and Assessment of GVHD

Problem Possible cause Check

All mice, including syngeneictransplants and T cellde-pleted marrow recipients, dieas a result of the inductionprocedure.

Mouse strain is particularlysensitive to irradiation(sensitivity varies betweenstrains).

Try lowering radiation doseor splitting dose into twoexposures 4 hr apart.Use hosts that are older than10 weeks.Assess cleanliness of colony.

No evidence of AGVHDimmune deficiency is seen inP F1 model.

F1 host may be resistant toengraftment of H-2b donorcells. Limiting numbers ofdonor CD8+ may result in aCGVHD.

Increase dose of T cells,especially CD8 +cells.Assess CGVHD parameters.

All (or no) irradiatedAGVHD mice die too early(or too late).

Strain combination and T celldose are not optimal.

Titer dose of T cells (monitordonor inoculum with FACS).


4.3.20



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Davenport, C., Haile, A., Kumar, V., and Bennett, M.1995. Hybrid and allogeneic resistance to T cellgrafts mediated by murine NK and CD8+ T cells.J. Immunol. 154:2568-2577.

DeClerk, Y., Draper, V., and Parkman, R. 1986.Clonal analysis of murine graft-vs-host disease.II. Leukokines that stimulate fibroblast prolifera-tion and collagen synthesis in graft-vs-host dis-ease. J. Immunol. 136:3549-3552.

de Geus, B., Hogenesch, H., de Heer, E., Bruijn,J.A., van den Enden, M., and Rozing, J. 1993.Effect of chronic graft-versus-host disease on theintestine in adult BDF1 mice. Int. J. Exp. Pathol.74:371-377.

Ferrara, J.L., Abhyankar, S., and Gilliland, D.G..1993. Cytokine storm of graft-versus-host dis-ease: A critical effector role for interleukin-1.Transplant. Proc. 25:1216-1217.

Fowler, D.H. and Gress, R.E. 1996. GVHD as aTh1-type process: Regulation by cells of Th2cytokine phenotype. In Graft-vs.-Host Disease:Immunology, Pathophysiology, and Treatment,2nd ed. (S. Burakoff, H.J. Deeg, J. Ferrara, andK. Atkinson, eds.) pp. 479-500. Marcel Decker,New York.

Fowler, D.H., Kurasawa, K., Husebekk, A., Cohen,P.A., and Gress, R.E. 1994. Cells of Th2 cytokinephenotype prevent LPS-induced lethality duringmurine graft-versus-host reaction. Regulation ofcytokines and CD8+ lymphoid engraftment. J.Immunol. 152:1004-1013.

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Contributed by Frances Hakim, Daniel H. Fowler, Gene M. Shearer, and Ronald E. GressNational Cancer Institute, NIHBethesda, Maryland


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15. GVHD Model