Allogeneic T cells impair engraftment andhematopoiesis after stem cell transplantationAntonia M. S. Müllera,b, Jessica A. Lindermana, Mareike Floreka, David Miklosa, and Judith A. Shizurua,1

aDepartment of Medicine, Division of Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA 94305; and bDepartment ofHematology/Oncology, University Medical Center Freiburg, D-79106 Freiburg, Germany

Communicated by Irving L. Weissman, Stanford University, Palo Alto, CA, June 30, 2010 (received for review May 31, 2010)

Because of the perception that depleting hematopoietic grafts ofT cells will result in poorer immune recovery and in increased risk ofgraft rejection, purehematopoietic stemcells (HSC),whichavoid thepotentially lethal complication of graft-versus-host disease (GVHD),have not been used for allogeneic hematopoietic cell transplanta-tion (HCT) in humans. Ideal grafts should contain HSC plus maturecells that confer only the benefits of protection frompathogens andsuppression of malignancies. This goal requires better understand-ing of the effects of each blood cell type and its interactions duringengraftment and immune regeneration. Here, we studied hemato-poietic reconstitution post-HCT, comparing grafts of purified HSCwith grafts supplemented with T cells in a minor histocompatibilityantigen (mHA)-mismatchedmousemodel. Cell counts, composition,and chimerism of blood and lymphoid organs were evaluated andfollowed intensively through the first month, and then subse-quently for up to 1 yr. Throughout this period, recipients of pureHSC demonstrated superior total cell recovery and lymphoid re-constitution comparedwith recipients of T cell-containing grafts. Inthe latter, rapid expansion of T cells occurred, and suppression ofhematopoiesis derived fromdonor HSCwas observed. Ourfindingsdemonstrate that even early post-HCT, T cells retard donor HSCengraftment and immune recovery. These observations contradictthe postulation that mature donor T cells provide important tran-sient immunity and facilitate HSC engraftment.

immune reconstitution | graft composition | mice

Allogeneic hematopoietic cell transplantation (HCT) com-prises the only curative treatment option for a spectrum of

fatal malignancies and insufficiency syndromes of the blood (1).Furthermore, the replacement of the immune system by HCT hasthe potential to cure nonmalignant disorders, such as severe au-toimmune diseases (2), and to induce immune tolerance to trans-planted organs (3). However, the high treatment-relatedmorbidityand mortality continue to limit the application of this powerfulcellular therapy toabroaderpatient base.Themainobstacles to thesuccess of HCT are graft-versus-host disease (GVHD), infections,and relapse, all ofwhich are critically influencedby the compositionof the graft. In adults, the most commonly used sources of alloge-neic hematopoietic cells aremobilized peripheral blood (MPB), orbone marrow (BM). Both graft types contain mixtures of hemato-poietic stem cells (HSC), progenitors, lineage-committed cells, andmature cells.DonorT cells are themainmediators of acuteGVHDbut are also credited with conferring beneficial graft-versus-tumor(GVT) effects, are thought to enhance engraftment, and provideprotective immunity post-HCT. Indeed, early attempts to reduceGVHDbyT-cell depletion (TCD)were hamperedbyunacceptablerates of engraftment failure (4–7). In retrospect, these results mayhave been due to an insufficient stem/progenitor cell content of themanipulated BM products (8), as later trials revealed significantlyimproved rates ofGVHDand similar engraftment in patients givenTCD as compared with whole BM grafts (9–12).Although there are serious concerns that TCD results in signif-

icantly increased infectious complications, GVHD itself is knownto impair immune functionpost-HCT (3–5, 13).GVHD-associatedlymphoid hypoplasia has been well described (6–8, 14–17). Mye-

losuppression, the most telling manifestation of graft-versus-hostreactions (GVHR) directed against hematopoietic elements hasalso been reported. It appears that the hematopoietic system is themost sensitive target of donor immunity, as relatively low numbersof T cells that do not damage other target organs can nonethelessinduce marrow aplasia (13, 18–20). Given the important con-sequences that attack of the BM and lymphoid organs have on theoutcomes of clinical allogeneic HCT, surprisingly little is knownaboutGVHRas it pertains to hematopoiesis and the establishmentof donor chimerism.In this study, we sought to better delineate the impact of graft

content on hematopoiesis and immune recovery in an mHA-mismatched murine GVHD model. Transplants of grafts com-posed of purifiedHSC versusHSC plus unfractionated splenocytesor splenic T-cell subsets were compared. As early as day (d) 7 post-HCT, marked differences were noted in hematopoietic recon-stitution in the blood, BMand lymphoid organs between recipientsof purifiedHSC andHSCplus lymphocytes.Mice givenHSC alonehad faster recovery of blood counts, prompt production of B lym-phocytes, and faster normalization of themyeloid to lymphoid ratiocompared with recipients of HSC plus lymphocytes. In this lattergroup, donor lymphocytes mediated early infiltration of themarrowand spleen, resulting in elimination of host cells and suppression ofHSC-derived hematopoiesis. In fact, retardation of engraftmentrather than facilitation was observed. These studies highlight the ra-pidity by which de novo hematolymphoid reconstitution can occurfollowing transplantation of purified HSC while underscoring thedeleterious effects that accompanying lymphoid cells have on HSCengraftment and hematopoiesis.

ResultsGVHD and Survival. To study the association between graftcomposition, GVHD, and immune reconstitution in mHA-mismatched mice, lethally irradiated BALB.B mice received pu-rified HSC or HSC plus mature lymphoid cells, including wholesplenocytes (wSP), total T cells (CD4 and CD8; ToTC), CD4, orCD8 cells from C57BL/6 (B6) donors (Fig. S1). Fig. 1A shows thesurvival and Fig. 1B the weight curves for these groups. Asexpected, recipients of HSC only showed no signs of GVHD,whereas differential effects were observed depending upon thetype of graft supplement. GVHDwas pronounced in recipients ofToTC,wSP andCD4 cells, as 32%, 30%, and 25%died before d40,respectively. In contrast, CD8 cells caused only mild signs ofGVHD, and only one of 23 mice died on d53 post-HCT (Fig. 1A).The severity ofGVHDsymptomswas also reflected by their weightcourse (Fig. 1B) and organ histology, which confirmed the clinicalfindings (Fig. 1C). The weight curves are censored to include only

Author contributions: A.M.S.M. and J.A.S. designed research; A.M.S.M., J.A.L., and M.F.performed research; A.M.S.M., D.M., and J.A.S. analyzed data; and A.M.S.M. and J.A.S.wrote the paper.

The authors declare no conflict of interest.1To whom correspondence should be addressed. E-mail: Jshizuru@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1009220107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1009220107 PNAS | August 17, 2010 | vol. 107 | no. 33 | 14721–14726

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0

mice surviving >4 wk, as only those animals were available forchimerism analysis.

Reconstitution of Blood. Absolute cell content. Hematopoietic re-constitution was assessed by absolute WBC counts. Immediatelypost-HCT, at 2–3 wk, a marked drop of the absolute WBC num-bers in all groupswas noted that persisted until 6 wk post-HCT andstabilized thereafter. Recipients of pure HSC recovered WBCmore rapidly than all other groups and achieved significantlyhigher total cell counts by 7wk (Fig. 2A), whereasWBC levels werelowest for mice that received ToTC. Evidence that mature donorlymphocytes suppressed donor HSC contribution to hematopoi-esis is shown in Fig. 2 B and C, which compare spleen-derived vs.HSC-derived blood elements, respectively, as determined by thepercentage and absolute number of WBC. For recipients of HSConly, most WBC were generated from the infused HSC. In con-trast, only∼30%ofWBCwere donorHSC derived in recipients ofwSP or ToTC. The addition of either CD4 or CD8 cells also re-duced the HSC contribution to recipient WBC. Thus, comparedwith mice that received lymphocyte-replete grafts, HSC recipientshad superior recovery of WBC established on the basis of trueHSC-derived hematopoiesis.Blood composition and GVHD. The blood of WT mice consists of 30–55% B cells (B220+), 30–50% T cells (TCRβ+), and 10–20%Mac1+ granulocytes/monocytes. Fig. 3 shows that graft contentsubstantially affected regeneration of specific WBC lineages. Thiseffect was most pronounced for B cells, which promptly recoveredto normal levels in recipients of pureHSC (median 38%), but wereseverely suppressed in mice given HSC plus lymphocytes. In thelatter groups, B cells comprised only amedian 0.75–6%of live cellsat 1 mo (p < 0.0001). B cell levels did not significantly differ withthe type of lymphocyte supplement, except that B lymphopenia

was most pronounced in recipients of CD4 cells. The degree ofB lymphopenia correlated with GVHD severity as mice with moreextreme weight loss had proportionally lower B cell levels (Fig.3B). B cell recovery occurred slowly as GVHD symptoms resolvedand reached normal levels by 3 mo (Fig. S2A). There was alsoa clear predominance of myeloid (Mac1+) cells in mice givenlymphocyte-replete grafts. Mac1+ cells accounted for a median of63–76%ofblood cells at 1mo in thesemice, as comparedwith 46%forHSC recipients (Fig. 3A). The degree of acute GVHDwas alsoassociated with this shift to myelopoiesis (Fig. 3B), as greaterweight loss correlated with higherMac1+ cell levels. In contrast, inall groups, T cells were reduced for several months, with medianlevels ranging from 10% to 16% in the first 3 mo (Figs. 3A andS2B). These reduced T-cell levels were independent of graftcontent, and there was no obvious correlation with symptoms ofacute GVHD. However, the proportion of donor spleen-derivedT cells was higher in mice with greater weight loss (Fig. 3B).Chimerism and competitive reconstitution. The origins of the threedifferent hematopoietic sources (HSC- vs. spleen-derived donorvs. residual host) were determined by expression of CD45.1/2,Thy1.1/2 alleles andGFP (Fig. 4A). In this way “nascent”HSC- vs.lymphocyte-derived donor cells were identified. In all recipients,lethal irradiation eliminated host myeloid and B cells, whereasT cells were not eradicated unlessmature donorT cells were given.Accordingly, in HSC recipients, myeloid and B cell lineages werepromptly replaced by donor cells, whereas the T cells were derivedfrom a mixture of donor and host (Fig. 4 B and C). Recipients ofToTC, CD4, or CD8 cells were also mixed T-cell chimeras butderived from both HSC and splenocyte donor sources (Fig. 4 Aand C). As primarily T cells were infused in these groups, B andmyeloid cells were mainly donor HSC derived, although minorcontributions from donor spleen were detected. However, as com-pared with HSC recipients, the origin of hematopoiesis was dis-

0 20 40 60 80 1000

20

40

60

80

100

70

75

80

85

90

95

100

0 10 20 30 40 50

A B %

Su

rviv

al

% o

f B

L w

eig

ht

Days post-HCT Days post-HCT

HSC HSC+CD8 HSC+CD4 HSC+wSPHSC+ToTC

HSC CD8 ToTC

wSPCD4

HS

C

C

HS

C+w

SP

Liver (20x) Large bowel (10x)

n=27 n=23 n=24 n=45 n=19

Liver (20x) Large bowel (10x)

Fig. 1. GVHD in B6 into BALB.B recipients. (A) Kaplan–Meier curves showingpercent survival and (B) median percentage of baseline (BL) weight of BALB.B mice given B6 HSC alone or HSCwSP, ToTC, CD4, or CD8 cells. Data werefrom six independent experiments. (C) H&E staining of GVHD target tissuesfrom representative recipients of HSC (Upper) or HSC+wSP (Lower) at 4 wkpost-HCT. Liver and bowel architecture were normal in HSC recipients,but had lymphocyte infiltrates and parenchymal cell damage in mice givenHSC+wSP.

02468

1012

0

20

40

60

80

100A

Total WBC count HSC-derived WBC count

HSC n=5

wSPn=8

ToTCn=7/4

CD4 n=5

CD8 n=5

HSC wSP ToTC CD4 CD8

% H

SC-d

erive

d ce

lls

C

HSC n=5

wSPn=8

ToTCn=4

CD4 n=5

CD8 n=5

0

2

4

6

8

10

CBC

(x10

6 cell

s/L)

CB

C (x

106 c

ells/

L)

B

Fig. 2. White blood cell counts. (A) WBC counts of BALB.B mice given B6HSC alone or HSC+wSP, ToTC, CD4, or CD8 measured by Coulter Counter at 4wk and 7 wk post-HCT. Control WBC values for age adjusted (12 wk) WTBALB.B mice are shaded in gray. At 7 wk, recipients of HSC alone had higherWBC counts (median 10 × 106/μL) than mice given HSC+wSP (median 5.7 ×106/μL; P = 0.003), HSC+ToTC (median 3.7 × 106/μL; P < 0.001), HSC+CD4(median 7 × 106/μL; P = 0.03), or HSC+CD8 T cells (median 4.6 × 106/μL;P = 0.002). (B) Proportion of HSC-derived donor cells in the blood at 7 wkpost-HCT. HSC recipients had a significantly higher proportion (median 93%)compared with HSC+wSP (median 29%; P < 0.001), ToTC (median 31%;P = 0.07), or CD4 (median 61%; P = 0.02), but not CD8 (median 82%; P = 0.2)groups. (C) Absolute number of donor HSC-derived WBC 7 wk post-HCT.Recipients of HSC only had significantly higher absolute numbers of WBCderived from donor HSC (9 × 106/μL) compared with mice given HSC+wSP(median 1.6 × 106/μL; P < 0.001), HSC+ToTC (median 1.1 × 106/μL; P = 0.002),HSC+CD4 (4.2 × 106/μL; P = 0.002), or HSC+CD8 T cells (median 3.2 × 106/μL;P = 0.001). B and C display the mean and SEM for each respective group,derived from one experiment with five to eight mice per group.

14722 | www.pnas.org/cgi/doi/10.1073/pnas.1009220107 Müller et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0

tinctly different in mice given HSC+wSP. Spleen-derived donorcells not only eradicated the residual host but permanently (>1 y)dominated lymphoid and myeloid (Mac1) production (Figs. 4Dand S3). The preponderance of spleen-derived myeloid cells wassurprising, given the low proportion of myeloid cells and rareHSCcontained in spleens (Fig. S4). Nonetheless, these data show thatsplenocyte-derived hematopoietic stem/progenitor cells effec-tively contribute and compete with the infused BM-derived puri-fied HSC, and that the ratio between donor spleen- and HSC-derived cells, once established, remained stable.Influence of CD45 allelic differences. The dominance of transferredwSP over HSC in hematopoieisis led us to ask whether CD45allelic differences permitted immune recognition of HSC and,hence, suppression of HSC blood formation. CD45.1 and CD45.2are congenic markers commonly used in mice to distinguish theorigins of hematopoiesis. For our initial studies, HSC were fromB6.CD45.2 and wSP from B6.CD45.1 mice, whereas recipientswere BALB.B.CD45.2. Similarly, reversal of the allele markers,such that HSC were from B6.CD45.1 and wSP from B6.CD45.2mice, showed hematopoiesis dominated by donor splenocytes(Fig. 5A). However, when all blood-forming sources were iden-tical at the CD45.2 allele, a markedly higher percentage of bloodcells originated from donor HSC and from residual host cells (Fig.5B). In addition, the severity of GVHD, as assessed by weight loss,was attenuated compared with transplants involving CD45 dis-parities (Fig. S5C). These data suggest that CD45 allelic dif-ferences between hematolymphoid sources can elicit immune

responses, and the data highlight the importance of minor anti-gens expressed on hematopoietic cells as targets of mature lym-phoid cells.

A

0

10

20

30

40

50

0

10

20

30

40

50

0

20

40

60

80

100

HSC n=25

wSP n=35

ToTC n=16

CD4 n=20

CD8 n=22

HSC n=23

wSP n=32

ToTC n=16

CD4 n=16

CD8 n=18

HSC n=25

wSP n=33

ToTC n=16

CD4 n=20

CD8 n=22

B cells T cells Mac1 cells

% o

f liv

e ce

lls

0

10

20

30

40

0

20

40

60

80

100

n=13

n=

13

n=7

n=9

n=4

n=4

n=3

n=6

n=13

n=14

n=

6

n=25

HSC wSP ToTC CD4 CD8

0

10

20

30

40

0

20

40

60

80

100

T cells

SP-derived donor TC

Dead or <80% of BL weight 80-90% of BL weight >90% of BL weight

B

HSC wSP ToTC CD4 CD8

wSP ToTC CD4 CD8 HSC wSP ToTC CD4 CD8

B cells Mac1 cells

Fig. 3. Effects of graft composition on hematopoiesis at 1 mo post-HCT.(A) Contribution of cells by lineage determined by FACS analysis. HSCrecipients had normal B cell levels (median 38%/live cells). B cell levels weresignificantly reduced in mice given HSC+wSP (median 6%; P < 0.0001),HSC+ToTC (median 2%; P < 0.0001), HSC+CD4 (median 0.75%; P < 0.0001),or HSC+CD8 cells (median 4%; P < 0.0001). Recovery of Mac1 cells occurredrapidly in all groups, although levels were significantly lower in recipientsof HSC (median 46% of live cells) versus mice given HSC+wSP (median 73%;P < 0.0001), ToTC (median 66%; P < 0.001), CD4 (median 74%; P < 0.0001),or CD8 T cells (median 63%; P < 0.01). T-cell levels reached a median of10%/live cells in recipients of HSC or HSC+wSP, and were slightly higher(16–17%) in recipients of HSC+ToTC, CD4 or CD8 (P < 0.01 for differencesbetween HSC only and HSC+ToTC, CD4 or CD8). (B) Severity of GVHD wasclassified according to the following criteria: death from acute GVHD orweight <80%, 80–90%, or >90% of BL weight. Shown are the contri-butions by lineage as %/live cells for mice stratified into these groups.Weight loss correlated with B lymphopenia and predominance of Mac1cells in the blood. There was no obvious correlation between T-cell levelsand GVHD; however, higher levels of donor spleen-derived T cells corre-sponded with greater weight loss. Data for these figures were derivedfrom same mice as studied in Fig. 1.

13 35.11.42 46.92.42 47.80.51 24.29.56

0.23

HSC wSP CD4 CD8

GF

P

SSC

Spleen- derived (CD45.1+)

CD45.1- HSC-donor or host

TC

R

A

0 97

30

ToTC

0

20

40

60

80

100

T cells

0

20

40

60

80

100Mac1 cells

0

20

40

60

80

100

HSC wSP ToTC CD4 CD8

B B cells

% d

onor

HSC wSP ToTC CD4 CD8 HSC wSP ToTC CD4 CD8

0102030405060708090

100

0102030405060708090

100

0102030405060708090

100

0102030405060708090

100HSC ToTC CD4 CD8 C

1m 2m 3m 1m 2m 3m 1m 2m 3m 1m 2m 3m

0102030405060708090

100

0102030405060708090

100

0102030405060708090

100

D T cells B cells

Donor HSC-derived Donor SP-derived Host

1m 2m 3m 1m 2m 3m 1m 2m 3m

0 55.7

44.30

0 98.7

1.320

0 98.9

1.130

0 42

580

HSC donor- derived

residual host

CD45.1

Mac1 cells

% o

f ce

lls

% o

f ce

lls

Fig. 4. Effects of graft composition on chimerism. (A) Delineation of T-cellsource by FACS analyses of blood 4 wk post-HCT from representative mice ineach transplant group. T cells from BALB.B aremarked by Thy1.2, CD45.2; fromB6 HSC by Thy1.1, CD45.2, GFP; and from B6 splenic cells by Thy1.1 CD45.1.Recipients of HSC remained mixed donor/host T-cell chimeras. Recipients ofHSC+wSP converted to full donor type, and all T cells were derived from donorsplenocytes. T cells in recipients of HSC+ToTC, CD4, or CD8 cells were derivedfrom donor splenocytes and donor HSC with residual host in some mice. (B–D)Summary of FACS results obtained frommice studied in Figs. 1 and 3. (B) Donor/host chimerism of B, Mac1, and T cells 4 wk post-HCT. All groups converted tofull donor type in the B cell and myeloid lineages. T-cell origins were mixed inrecipients of HSC, whereas T cells were largely donor derived in mice givenlymphocyte-replete grafts. (C) Origins of T cells as percentage of T cells fromeach source relative to total T cells. Gray bars indicate nascent T cells fromdonorHSC, blackbars indicate T cells cotransferred fromdonor spleencells, andwhite bars represent host T cells, all at 1, 2, and 3 mo post-HCT. HSC recipientsremained mixed T-cell chimeras and the proportion of HSC-derived T cells in-creased over time. Inmice givenHSC+ToTC or HSC+CD8 cells, the proportion ofnascent HSC-derived donor T cells increased whereas spleen-derived donorT cells decreased, and residual host cells were largely eliminated. In recipientsof HSC+CD4 cells, spleen-derived T cells persistently predominated, and elimi-nation of host T cells was incomplete. (D) Origins of cells from the differentlineages at 1, 2, and 3 mo post-HCT for recipients of HSC+wSP. Residual hostcells were readily cleared, and spleen-derived hematopoiesis was superiorcompared with lymphoid and myeloid cell production from donor HSC.

Müller et al. PNAS | August 17, 2010 | vol. 107 | no. 33 | 14723

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0

GVHR in BM and Lymphoid Organs. The skewing of WBC awayfrom normal values caused by mature donor T cells involvednot only blood but all hematolymphoid organs. Similar to theblood, B lymphopenia was the most notable perturbation in theBM, spleen, and lymph nodes of mice with GVHD, whereasrecipients of purified HSC had unimpaired and rapid recovery ofB cells. Shown in Fig. 5 are FACS plots of BM and spleen fromrepresentative recipients of HSC alone (Fig. 6A) or HSC+wSP(Fig. 6B). Two weeks after transplantation, mice given pure HSChad 30–65%Bcells in BMand spleen that were largely donor type;and as in the blood, T cells originated from both donor and host(Fig. 5A). In contrast, B lymphopoiesis was markedly suppressedin recipients of HSC+wSP, and all B cells were donor spleen de-rived. At the same time, these tissues were heavily infiltrated withdonor splenic T cells, the majority of which were CD8 T cells.Antihost alloreactivity of these donor CD8 T cells was verified bystaining with a tetramer against H60, a known immunodominantminor antigen, expressed by hematopoietic cells of BALB.B hostbut not B6 donormice.Of theCD8T cells in theBMofHSC+wSPrecipients, 10–25% were H60-tetramer positive (Fig. 6B), whichsuggested that recognition of H60 contributed importantly tothe eradication of residual host BM cells. FACS analyses of thehematolymphoid organs were repeated on d28 and d50, andrevealed that donor T-cell infiltration peaked at 2 wk post-HCTand decreased thereafter. The results for lymph nodes resembledthose of the spleens. Of note, lymph nodes frommice that receivedHSC+wSP were smaller and had markedly reduced cellularity ascompared with organs from mice that received HSC only, again

implying that the mature donor cells retarded rather than aug-mented immune recovery.

Hematopoietic Reconstitution Early Post-HCT. To better character-ize early hematolymphoid regeneration, BM and spleens of re-cipients of HSC vs. HSC+wSP were examined for total cellnumbers, composition, and chimerism on d4, 7, 11, 14, 17, and 21post-HCT. During this period, extreme hypocellularity wasnoted, and cell numbers in BM and spleen did not differ betweenthe HSC and HSC+wSP groups (Fig. S5A), suggesting that en-vironmental factors may limit cell expansion. For both groups, byd14, the donor cell levels were uniformly high (Fig. S5B). How-ever, substantial differences in the donor source and cell com-position were noted. At d4, donor cells were already present inthe spleens of mice given HSC+wSP, whereas the spleens ofHSC recipients were mainly of host origin (Fig. S5B). The wSP-derived cells dominated total spleen cellularity in HSC+wSPrecipients and persisted in the BM. Cell lineage representationalso varied depending upon the graft type (Fig. S5C). Recipientsof HSC alone recovered normal lineage proportions in the BMby d14, and spleens recovered by d21, albeit with persistentT lymphopenia. In contrast, the BM and spleen of mice infusedwith HSC+wSP contained high numbers of donor wSP-derived Tcells, relatively few B cells, and granulocytes/monocytes (Mac1+)dominated the cellularity.Analyses of the relative contribution of cells derived from

the three different blood forming sources by lineage (Fig. S6A)showed that for HSC recipients donor B cells arose early (d7) andreplaced the host B cells by d21. In contrast, the small numbers ofB cells present in HSC+wSP recipients originated largely from

A B

C

Fig. 5. Influence of CD45.1/2 allele differences on engraftment and GVHD.Blood T-cell chimerism is shown as the median percentage of T cells derivedfrom donor HSC (gray) and spleen (black) vs. host (white) at 1, 2, and 3 mopost-HCT. (A) CD45 allele disparity of HSC and wSP B6 donors. Regeneratingblood T cells were mainly donor spleen-derived (median 84% vs. 15% donorHSC derived at 1 mo post-HCT), and host T cells were eliminated (<1%). (B)CD45.2 allele identity of HSC and wSP B6 donors. HSC were from B6.Thy1.1and wSP were from B6.Thy-1.1.GFP+ mice. At 1 mo post-HCT, a median 33%and 58% of T cells were donor spleen vs. HSC derived, respectively. Residualhost T cells (median 12.5%) persisted. A and B show means with SEM. (C)Median weights of BALB.B (CD45.2) that received HSC+wSP grafts from CD45disparate donors (A and B) vs. HSC+wSP from CD45.2 identical donors thatalso shared the CD45.2 allele with recipients (C, all CD45.2). Weight loss wasmore pronounced for CD45 allele disparity vs. identity between donor andhost. Data for these figures were derived from same mice as studied in Fig. 1.

A

B

Fig. 6. Lymphoid lineage and chimerism as determined by FACS analyses ofBM and spleen 2 wk post-HCT. (A) Recipients of HSC only showed promptreconstitution of the lymphoid cells (SSC/FSC plot), which were pre-dominantly B cells (B220) derived from the GFP+ HSC donor. T cells in BMoriginated mainly from the host, whereas splenic T cells were derived equallyfrom donor HSC and the host. (B) Recipients of HSC+wSP displayed markedlymphopenia (SSC/FSC plot) secondary to marked reduction of B cells. BMand spleen were heavily infiltrated by donor T cells (spleen derived). Little tono hematopoiesis originated from donor HSC or residual host cells. Themajority of donor T cells were CD8+, a substantial proportion of which weretetramer reactive against H60. Representative FACS plots from mice 2 wkpost-HCT are shown. Data were confirmed in three independent experi-ments, with three to four animals per group.

14724 | www.pnas.org/cgi/doi/10.1073/pnas.1009220107 Müller et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0

wSP (Figs. S5C and S6B). The dynamics of T-cell regenerationalso differed markedly between HSC and HSC+wSP recipients.For HSC recipients, a high proportion of T cells were of host type,as T cells are known to be more radiation resistant than B andMac1 cells. In these mice, the earliest TCRβ+ cells originatingfrom HSC were detected by d11. In mice given HSC+wSP, earlyexpansion of wSP-derived donor T cells occurred, which resultedin both rapid eradication of residual host T cells and suppressionof T lymphopoiesis from donor HSC (Fig. S6B). For both grafttypes, by d7, the radiation-sensitive host myeloid cells wereeliminated and replaced by donor cells (Fig. S6 A and B).Recipients of HSC+wSP demonstrated increasing myelopoiesisoriginating from the donor splenocytes, which again shows thatsplenocytes contain HSC and progenitors that efficiently competewith HSC from the BM.

DiscussionAn ultimate goal for the field of HCT is the engineering ofgrafts tailored to individual disease requirements. Purified HSCprovide the platform upon which other defined mature pop-ulations (i.e., antigen-specific T cells) can be added. However,the implementation of this strategy requires better understand-ing of the biology of engraftment and hematopoiesis post-HCT and, in particular, of how graft composition affects theseprocesses.Although methods to purify HSC from human sources were

developed more than a decade ago (21), concerns remain thatgrafts depleted of T cells will engraft poorly and result in deficientimmunity, discouraging translation of this technology to patienttrials. However, the earlier clinical studies from the 1980s of TCDversus nonmanipulated BM grafts that reported increased rates ofengraftment failure in the TCD group (4–7) were subsequentlycountered by trials showing equivalence in engraftment andoverall survival with TCD, and variability regarding loss of GVTeffects (9–12). Similarly, the direct correlation of TCD with in-fectious complications is questionable. In the largest randomizedtrial, in which patients on the TCD arm had a 1-log10 reductionof T cells, TCD was associated with an increased risk of seriousinfections, specifically CMV and Aspergillus (11). However, amore recent prospective trial using rigorous TCD (5-log10) andinfusion of grafts with doses of CD34+ cells at levels comparableto MPB demonstrated engraftment in all evaluable patients anddeath from opportunistic infection in only 2% (12). Taken in ag-gregate, it is clear that heterogenous factors, such as the methodof host conditioning, method of TCD, and post-HCT immunesuppression, influence the outcomes. Yet, the perception that do-nor T cells improve hematopoietic and immune recovery per-sists and is used to justify the continued use of grafts that carrysignificant risk of GVHD (cumulative risk for grade 2–4 acuteGVHD 30–50%) (22, 23) and a consequent transplant-relatedmortality of 10–25% (24).The data presented here and elsewhere (25) contradict the

conventional view that grafts of rigorously purified stem/pro-genitor cells will produce inferior immunity, and suggest that suchconclusions should be re-examined. The body of data demon-strating the deleterious effects of GVHR on BM and lymphoidfunction (13–20, 26–28) is often overlooked or underestimated.We previously reported that even low amounts of T cells causesubclinical, yet lympho-depleting, GVHR (25). Here, we focusedon an mHA-antigen–mismatched strain combination and usedgrafts that allowed us to distinguish between “new” HSC-derivedhematopoiesis versus expansion of donor lymphocytes. Any ex-pectation that posttransplantation hematopoiesis and immunerecovery, even in the earliest days after graft infusion, is enhancedby addition of donor lymphoid cells (in the absence of pharma-cologic immune suppression) was refuted in our model system.Blood production, as measured by absolute cell numbers andrestoration of normalized ratios of myeloid to lymphoid elements

in hematolymphoid tissues, was consistently superior for recipi-ents of HSC alone as compared with HSC plus lymphocytes.Rather than promote HSC proliferation and differentiation, ma-ture donor T cells suppressed their activities. The heavy infiltrationby T andMac1+ cells observed in the BM and lymphoid organs ledus to conclude that suppression ofHSC-derived hematopoiesis wasmediated by alloreactive T cells that homeostatically expandedand induced a proinflammatory environment. It has been shownthat cytokines, such as IL-6, TGFβ, TNFα, and IFNγ, suppresshematopoiesis by blocking lineage commitment (29), impairingcell division (30), and inducing programmed cell death (31). In-flammation can also damage the host cellular components of HSCniches (13), making the marrow inhospitable for blood cell pro-duction. Thus, the way that donor lymphocytes exert negativeeffects on hematopoiesis likely involves both direct and indirectperturbation of HSC function, survival, and engraftment.Our data further highlight the differences in the origins of

T cells that repopulate recipients of HSC as compared with HSCplus lymphoid cells. Recipients of pure HSC retained a largepercentage of host T cells, and the levels of HSC-derived T cellsrose gradually over time. In contrast, addition of splenocytesresulted in rapid elimination of host T and suppression of donorHSC-derived T lymphopoiesis. Spleen-derived CD8 T cells pre-dominated in the BM early after transplantation, and a substan-tial proportion were reactive against the minor alloantigen H60,suggesting that CD8 T cells were responsible for clearance of thehost lymphoid cells. CD4 T cells appeared to mediate GVHR anddamage BM, as recipients of HSC plus CD4 T cells developed thefull picture of B lymphopenia; however, incomplete eradication ofhost cells was observed more often. The pattern of host T-celleradication by mature donor lymphocytes and expansion of thedonor peripheral pool mirrors the clinical studies that have ex-amined T-cell reconstitution posttransplantation (32, 33). Formany months post-HCT, the T-cell compartment is abnormal,largely comprising activated T cells and few naive cells. This ab-errant skewing is thought to occur because T cells expand home-ostatically, and clones that recognize antigens present in the hostin the peri-transplantation period (i.e., mismatched histocom-patibility antigens or herpes viruses) can dominate whereas othersare lost. Although early reconstitution of the T-cell compartmentoccurs by oligoclonal expansion, resulting in the loss of manyantigen specifications, a complete T-cell repertoire can comeonly from naive T cells arising from stem/progenitors by way ofthe thymus.The profound and lasting dominance of spleen- over HSC-

derived lymphoid cells led us question whether CD45 allelic dif-ferences between the two donor parties contributed to this phe-nomenon. Historically, CD45 was thought to be nonantigenic(34), and CD45 congenic strains have been extensively used todetermine engraftment and chimerism in congenic and MHC-matched HCT models. However, our data and others suggestthat polymorphic CD45 expressed on hematopoietic cells is im-munogenic (35–37). In our system, if mature donor lymphoid cellswere CD45 disparate with the HSC-donor, complete eliminationof host elements, domination of hematopoiesis by the donor lym-phoid cells over the congenic HSC, and aggravated GVHD wasobserved. When all parties were CD45 identical, higher levels ofresidual host cells persisted, and the contribution of donor HSCto hematopoiesis was significantly increased.In summary, our studies were motivated to better elucidate

how donor immune cells influence HSC engraftment, hema-tolymphoid recovery, and establishment of donor chimerism. Weexpected that despite development of GVHD, grafts with maturedonor T cells would augment HSC-derived hematopoiesis andwe would observe quantitatively better lymphoid recovery com-pared with grafts composed solely of HSC, at least in the earlyphase post-HCT; rather, transplantations of pure HSC were uni-formly superior in these regards. Achievement of doses of HSC

Müller et al. PNAS | August 17, 2010 | vol. 107 | no. 33 | 14725

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0

relevant to clinical transplantation has already been proved. Ona per-kilogram basis, the dose of HSC used in our studies (1.2 ×105 HSC/kg) that provided rapid and sustained hematopoieticrecovery is less than the dose of homologous human CD34+Thy1+

HSCused in a clinical trial of autologousHCT (38), and is less thanthat contained in megadose CD34+ enriched grafts used in haplo-identical trials. Finally, although treatment of malignancies mayrequire complete donor chimerism to achieve full GVT potency,other disease states, such as immune deficiencies and autoimmunedisorders, do not require full conversion or may even benefit frommixed chimerism. Our findings on the superior effects on bloodand lymphoid recovery after transplantation of pure HSC give usconfidence that hematopoietic graft engineering will be possible,and that someday the problems of GVHD will be obsolete.

MethodsMice. Congenic C57BL/6 (B6) mice (H2b; Thy1.1; B6.CD45.1, B6.CD45.2, and B6.GFP) were used as donors of HSC and lymphocytes for BALB.B hosts (H2b,Thy1.2; CD45.2).

Hematopoietic Cell Transplantation. BALB.B mice underwent lethal 800 cGytotal body gamma irradiation before infusion of hematopoietic grafts. KTLSHSCwere isolated by amodified procedure described by Spangrude et al. (39),which selects c-Kit+ Thy1.1lo-int Sca-1+ Lin− (CD3ε, CD4, CD5, CD8α, B220, Gr1,Mac1, TER-119) from cKit-enriched BMby FACS. In cotransfer experiments, 1×107 wSP, 8 × 106 ToTC, 4–5 × 106 CD4, or 2.5–3 × 106 CD8 T cells enriched fromsplenocytes were injected simultaneously with the HSC for GVHD induction.

Engraftment and Chimerism. Donor engraftment was assessed in the blood ond30, 60, and 90 post-HCT. BM and spleens were harvested at different timepoints between d4 and 14 mo and analyzed by FACS.

Detailed descriptions of mice, hematopoietic stem cell isolation andtransplantation, GVHD assessment, histology, WBC, chimerism, staining forflow cytometry, and statistical methods are provided in SI Methods.

ACKNOWLEDGMENTS. We thank Kathryn Logronio for laboratory manage-ment; Joel Dollaga, Diosdado Escoto, and Ronald Mendoza for animal care;and the National Institutes of Health tetramer facility for kindly providingthe customized H60 tetramers. This work was supported by a GermanResearch Foundation (DFG) Postdoctoral Fellowship (to A.M.S.M.); a StanfordDARE Doctoral Fellowship (to J.A.L.); National Institutes of Health GrantsRO1 HL087240 and PO1 CA049605; and a grant from the Snyder Foundation(to J.A.S).

1. Horowitz M (2009) Uses and growth of hematopoietic cell transplantation. Thomas’Hematopoietic Cell Transplantation, eds Appelbaum FR, Forman SJ, Negrin RS,Blume KG (Wiley-Blackwell, Oxford, UK), 4th Ed, pp 15–21.

2. Sullivan KM, Muraro P, Tyndall A (2010) Hematopoietic cell transplantation forautoimmune disease: Updates from Europe and the United States. Biol Blood MarrowTransplant 16 (1, Suppl):S48–S56.

3. Scandling JD, et al. (2008) Tolerance and chimerism after renal and hematopoietic-celltransplantation. N Engl J Med 358:362–368.

4. Martin PJ, et al. (1988) Graft failure in patients receiving T cell-depleted HLA-identicalallogeneic marrow transplants. Bone Marrow Transplant 3:445–456.

5. Marmont AM, et al. (1991) T-cell depletion of HLA-identical transplants in leukemia.Blood 78:2120–2130.

6. Kernan NA, et al. (1989) Graft failure after T-cell-depleted human leukocyte antigenidentical marrow transplants for leukemia: I. Analysis of risk factors and results ofsecondary transplants. Blood 74:2227–2236.

7. Schmeiser T, et al. (1989) Infectious complications after allogeneic bone marrowtransplantation with and without T-cell depletion of donor marrow. Infection 17:124–130.

8. Shizuru JA, Negrin RS, Weissman IL (2005) Hematopoietic stem and progenitor cells:Clinical and preclinical regeneration of the hematolymphoid system. Annu Rev Med56:509–538.

9. Soiffer RJ, et al. (1992) Prevention of graft-versus-host disease by selective depletionof CD6-positive T lymphocytes from donor bone marrow. J Clin Oncol 10:1191–1200.

10. Keever CA, et al. (1989) Immune reconstitution following bone marrow trans-plantation: Comparison of recipients of T-cell depleted marrow with recipients ofconventional marrow grafts. Blood 73:1340–1350.

11. Wagner JE, Thompson JS, Carter SL, Kernan NA; Unrelated Donor Marrow Trans-plantation Trial (2005) Effect of graft-versus-host disease prophylaxis on 3-yeardisease-free survival in recipients of unrelated donor bone marrow (T-cell DepletionTrial): A multi-centre, randomised phase II-III trial. Lancet 366:733–741.

12. Jakubowski AA, et al. (2007) T cell depleted stem-cell transplantation for adults withhematologic malignancies: Sustained engraftment of HLA-matched related donorgrafts without the use of antithymocyte globulin. Blood 110:4552–4559.

13. Shono Y, et al. (2010) Bone marrow graft-versus-host disease: Early destruction ofhematopoietic niche following MHC-mismatched hematopoietic stem cell trans-plantation. Blood 115:5401–5411.

14. Dulude G, Roy DC, Perreault C (1999) The effect of graft-versus-host disease on T cellproduction and homeostasis. J Exp Med 189:1329–1342.

15. Lapp WS, Ghayur T, Mendes M, Seddik M, Seemayer TA (1985) The functional andhistological basis for graft-versus-host-induced immunosuppression. Immunol Rev 88:107–133.

16. Fukushi N, et al. (1990) Thymus: A direct target tissue in graft-versus-host reactionafter allogeneic bone marrow transplantation that results in abrogation of inductionof self-tolerance. Proc Natl Acad Sci USA 87:6301–6305.

17. Holländer GA, Widmer B, Burakoff SJ (1994) Loss of normal thymic repertoireselection and persistence of autoreactive T cells in graft vs host disease. J Immunol152:1609–1617.

18. Mori T, et al. (1998) Involvement of Fas-mediated apoptosis in the hematopoieticprogenitor cells of graft-versus-host reaction-associated myelosuppression. Blood 92:101–107.

19. Piguet PF (1985) GVHR elicited by products of class I or class II loci of the MHC: Analysisof the response of mouse T lymphocytes to products of class I and class II loci ofthe MHC in correlation with GVHR-induced mortality, medullary aplasia, and enter-opathy. J Immunol 135:1637–1643.

20. Sprent J, et al. (1994) Profound atrophy of the bone marrow reflecting majorhistocompatibility complex class II-restricted destruction of stem cells by CD4+ cells. JExp Med 180:307–317.

21. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B (1992) Isolation ofa candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 89:2804–2808.

22. Weisdorf D (2007) GVHD the nuts and bolts. Hematology (Am Soc Hematol EducProgram) 2007:62–67.

23. Hahn T, et al. (2008) Risk factors for acute graft-versus-host disease after humanleukocyte antigen-identical sibling transplants for adults with leukemia. J Clin Oncol26:5728–5734.

24. Lin MT, et al. (2003) Relation of an interleukin-10 promoter polymorphism to graft-versus-host disease and survival after hematopoietic-cell transplantation. N Engl JMed 349:2201–2210.

25. Tsao GJ, Allen JA, Logronio KA, Lazzeroni LC, Shizuru JA (2009) Purified hema-topoietic stem cell allografts reconstitute immunity superior to bone marrow. ProcNatl Acad Sci USA 106:3288–3293.

26. Baker MB, Riley RL, Podack ER, Levy RB (1997) Graft-versus-host-disease-associatedlymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediatedFas-ligand function, but not perforin function. Proc Natl Acad Sci USA 94:1366–1371.

27. Sakoda Y, et al. (2007) Donor-derived thymic-dependent T cells cause chronic graft-versus-host disease. Blood 109:1756–1764.

28. Nguyen VH, et al. (2008) The impact of regulatory T cells on T-cell immunity followinghematopoietic cell transplantation. Blood 111:945–953.

29. Maeda K, et al. (2005) IL-6 blocks a discrete early step in lymphopoiesis. Blood 106:879–885.

30. Sitnicka E, Ruscetti FW, Priestley GV, Wolf NS, Bartelmez SH (1996) Transforminggrowth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells. Blood 88:82–88.

31. Selleri C, Sato T, Anderson S, Young NS, Maciejewski JP (1995) Interferon-gamma andtumor necrosis factor-alpha suppress both early and late stages of hematopoiesis andinduce programmed cell death. J Cell Physiol 165:538–546.

32. Storek J, et al. (2008) Reconstitution of the immune system after hematopoietic stemcell transplantation in humans. Semin Immunopathol 30:425–437.

33. O’keefe CL, et al. (2004) Molecular TCR diagnostics can be used to identify sharedclonotypes after allogeneic hematopoietic stem cell transplantation. Exp Hematol 32:1010–1022.

34. Sykes M, et al. (1989) Effects of T cell depletion in radiation bone marrow chimeras. III.Characterization of allogeneic bone marrow cell populations that increase allogeneicchimerism independently of graft-vs-host disease in mixed marrow recipients. JImmunol 143:3503–3511.

35. Bhattacharya D, Rossi DJ, Bryder D, Weissman IL (2006) Purified hematopoietic stemcell engraftment of rare niches corrects severe lymphoid deficiencies without hostconditioning. J Exp Med 203:73–85.

36. Xu H, Exner BG, Chilton PM, Schanie C, Ildstad ST (2004) CD45 congenic bone marrowtransplantation: Evidence for T cell-mediated immunity. Stem Cells 22:1039–1048.

37. van Os R, et al. (2001) Immunogenicity of Ly5 (CD45)-antigens hampers long-termengraftment following minimal conditioning in a murine bone marrow transplant-ation model. Stem Cells 19:80–87.

38. Negrin RS, et al. (2000) Transplantation of highly purified CD34+Thy-1+ hema-topoietic stem cells in patients with metastatic breast cancer. Biol Blood MarrowTransplant 6:262–271.

39. Spangrude GJ, Heimfeld S, Weissman IL (1988) Purification and characterization ofmouse hematopoietic stem cells. Science 241:58–62.

14726 | www.pnas.org/cgi/doi/10.1073/pnas.1009220107 Müller et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

, 202

0