Bone Marrow Transplantation (2011) 46, 586–596 & 2011 Macmillan Publishers Limited All rights reserved 0268-3369/11 www.nature.com/bmt

ORIGINAL ARTICLE Subclinical GvHD in non-irradiated F1 hybrids: severe lymphoid-tissue GvHD causing prolonged immune dysfunction

B Sprangers1,5, B Van Wijmeersch1,2,5, A Luyckx1,5, X Sagaert3, B Verbinnen4, O Rutgeerts1, C Lenaerts1, T Tousseyn3, B Dubois2, M Waer1 and AD Billiau1

1Laboratory of Experimental Transplantation, University of Leuven, Leuven, Belgium; 2Laboratory of Neuroimmunology, University of Leuven, Leuven, Belgium; 3Department of Pathology, University of Leuven, Leuven, Belgium and 4Laboratory of Experimental Immunology, University of Leuven, Leuven, Belgium

GvHD is an important complication of allogeneic Introduction hematopoietic SCT. Parent-in-F1 models are frequently used to study GvHD immunobiology; the characteristics GvHD remains an important complication after allogeneic of parent-in-F1 GvHD vary between strain combinations hematopoietic SCT, in particular in the context of donor and induction protocols. Here, we observed that a lymphocyte infusion (DLI) therapy for induction of GvL high-dose challenge of non-irradiated B6DBA2F1 and effects.1 In recent years, substantial progress has been B6SJLF1 recipients with C57BL/6 splenocytes left the made in the understanding of the immunobiology of DLI- majority of recipients clinically healthy, while inducing associated GvHD and GvL effects. Mouse studies have progressive high-grade donor T-cell chimerism. We shown that recipient APCs residing in epithelial tissues are investigated this previously undescribed pattern of critical targets for alloreactive T cells in the induction of parent-in-F1 T-cell alloreactivity and studied the effect GvHD,2 and that local inflammation, such as that resulting of serial parental splenocyte infusions on epithelial and from pretransplant conditioning, is a prerequisite for lymphohematopoietic tissues. The majority of recipients the migration of alloreactive T cells into these GvHD of 4 weekly splenocyte infusions showed long-term target tissues.3 Indeed, abatement of conditioning-induced survival with gradual establishment of high-grade donor inflammation is thought to contribute to the observed chimerism and without any signs of epithelial-tissue reduced GvHD risk when DLI is delayed with several GvHD. A minority of recipients showed BM failure type weeks or months after a transplant.4–9 In contrast, the GvL of GvHD and, respectively, graft rejection. Moreover, response of DLI has been shown in mice to result from the long-term F1 chimeras showed protracted pancytopenia, interaction between donor T cells and recipient APC in the and in peripheral lymphoid tissues severe lymphopenia and lymphohematopoietic tissues, resulting in a lymphohema- near-complete eradication of APCs and dysfunction in topoietic GvH reaction (LHGvHR).2 In mice, mixed antigen-presenting capacity in remaining APC. Hemato- chimerism and a LHGvHR with conversion of mixed poiesis and lymphoid tissue composition recovered only to full donor chimerism have been shown essential for after multilineage donor chimerism had established. GvL responses after DLI.8,9 Moreover in the clinical In conclusion, we report on a novel type of parent-in-F1 setting, antitumor responses after DLI frequently occur in hybrid GvHD, where a cumulative high dose of C57BL/6 association with conversion of mixed to full donor T-cell parental splenocytes in non-irradiated F1 mice induces chimerism.10,11 subclinical but severe hematolymphoid-tissue GvHD, Parent-in-F1 models are frequently used to study the causing prolonged immuno-incompetence. immunobiology of GvHD.12,13 Earlier studies in the Bone Marrow Transplantation (2011) 46, 586–596; C57BL/6-B6DBA2F1 model showed that high-dose doi:10.1038/bmt.2010.162; published online 5 July 2010 C57BL/6 splenocytes induce acute epithelial-tissue GvHD Keywords: GvHD; mouse; APC; parental splenocyte that is lethal over several months.14 In contrast, high-dose infusion C57BL/6 lymph node (LN) cells induces a BM failure type of GvHD (BMGvHD), which is rapidly lethal;15–17 BMGvHD is also seen if recipients are irradiated, in which case lower quantities of C57BL/6 T cells are required. The characteristics of parent-in-F1 GvHD is equally known to Correspondence: Dr AD Billiau, Laboratory of Experimental Trans- vary between strain combinations, and to depend on strain- plantation, University of Leuven, Campus Gasthuisberg, CDG box 811, specific alloreactive T-cell-precursor frequency.18 We here Herestraat 49, Leuven B-3000, Belgium. made the interesting observation in the C57BL6- E-mail: [email protected] B6DBA2F1 model that a high-dose challenge with parental 5These authors contributed equally to this work. Received 15 March 2010; revised 14 May 2010; accepted 23 May 2010; splenocytes in non-irradiated recipients caused typical published online 5 July 2010 BMGvHD in only 1/3 of recipient mice. The majority of P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 587 recipient mice remained clinically healthy, while they paraffin-embedded sections were prepared and stained showed progressive evolution towards complete donor with hematoxylin-eosin H&E. Pictures were taken with a T-cell chimerism. A similar evolution was seen in C57BL/ Zeiss Axioplan-2 microscope (Carl Zeiss AG, Go¨ttingen, 6-B6SJLF1 mice. This pattern of parent-in-F1 alloreac- Germany) equipped with a cooled charge-coupled device tivity has so far not been reported on, and we document camera COHU 4910 (Diagnostic Instruments, Detroit, MI, here that this represents a form of subclinical GvHD with USA) or a Leica DM LB2 microscope (Leica Microsystem, severe lymphoid-tissue GvH reactivity causing prolonged Groot Bijgaarden, Belgium) equipped with a Nicon HC immuno-incompetence. These data may hold implications L3TP camera (Nikon Belux, Brussels, Belgium). for DLI strategies in non-myeloablative transplant regi- mens, which aim at inducing strong lymphohematopoietic Cell phenotypic studies. Cellular compositions of BM, GvH reactivity in the absence of conditioning-related spleen and LNs were studied by flowcytometry inflammation. (as described earlier). Total cell counts were enumerated manually using Trypan Blue Exclusion. Complete blood counts (CBCs) were determined using a Cell Dyn 3500R Materials and methods (Abbott Diagnostics, Ottignies/Louvain-La-Neuve, Bel- gium). The normal range of CBC values was determined Animals in a group of 5–7 age-matched naive F1 mice. Values in B6DBA2F1 (H-2b/d), B6SJLF1 (H-2b/s) or B6C3HF1 (H-2b/k) experimental mice were considered abnormal if lower or female mice were used as recipients and C57BL/6 (H-2b) higher than the mean±2 s.d. from this control group. female mice as donors, and purchased from Janvier (Le Genest Saint Isle, France). Recipients were housed in Induction of experimental autoimmune encephalomyelitis individually ventilated cages; during procedures, animals and in vitro stimulation assays were kept under laminar flow. Diet consisted of standar- Induction of experimental autoimmune encephalomyelitis. dized pellet chow and UV-decontaminated water. All Experimental autoimmune encephalomyelitis (EAE) was experiments were approved by the local ethical committee. induced by bilateral hind-footpad injection of myelin

oligodendrocyte glycoprotein35–55 (MOG35–55) peptide BM transplantation and parental splenocyte infusion (Sigma-Aldrich, Bornem, Belgium), heat-killed Mycobac- In the serial parental splenocyte infusion (PSI) model, terium Tuberculosis in CFA (BD Biosciences) and IV B6DBA2F1 or B6SJLF1 animals were used as recipients injection of Bordetella Pertussis toxin (Sigma-Aldrich), as and given 4 weekly IV injections of 25 Â 106, respectively, described earlier.22 50 Â 106 C57BL/6 splenocytes. In the allogeneic BM trans- plant (BMT) model, B6SJLF1 mice were given 9.5 Gy TBI In vitro stimulation assays. EAE-induced chimeras were (linear accelerator 18 MEV photons (General Electric, killed on day 10. Popliteal LN cells (LNc) were stimulated Baden, Germany), dose rate 3.9 Gy/min with focus-to- 22 with MOG35–55, as described earlier. In brief, LNc were 6 midbody distance 100 cm) and IV infusion of 5 Â 10 plated (2 Â 105 cells/well, quadruplicate) in stimulation T-cell-depleted (described earlier19) C57BL/6 BM cells. medium (RPMI containing Na pyruvate; NEAA; 5 L-glutamine; 2-ME; FCS) together with 2 Â 10 Mitomycin- Leukemia challenge C-treated (Kyowa Hakko Kogyo, Tokyo, Japan) host-type For GvL studies, the P815 cell line (DBA2 mouse splenocytes (as APC source). MOG35–55 was added at mastocytoma, ATCC, H2Kd) was used. Cells from frozen 10 mg/mL, or ConA at 6 mg/mL ConA. After 4-day culture, stock were maintained in vitro for a limited number of [3H]thymidine-incorporation was determined (c.p.m.), as passages. For each experiment, the in vivo behavior was described earlier.22 Results are expressed as stimulation verified by inoculating naive host-type mice. BM chimeras index ¼ c.p.m. of stimulated/c.p.m. of non-stimulated cells. were challenged IV with 5 Â 105 leukemia cells 7 days after the first PSI. MOG35–55-specific T-cell proliferation assay. B6SJLF1 mice were immunized as described and killed on day 10. 6 Animal follow-up Popliteal LNc were stimulated for 72 h at 4 Â 10 cells/mL Clinical monitoring. Animals were inspected daily for in stimulation medium containing 20 mg/mL MOG35–55. weight loss, signs of acute GvHD affecting general condition T cells were subsequently expanded in RPMI containing 4 and epithelial organs,20 leukemic disease and mortality. 10% FCS and 20 U/mL rIL-2 for 5–7 days. Finally, 4 Â 10 T cells were re-stimulated with 10 mg/mL MOG35–55 in the 6 Donor chimerism. presence of 0.8 Â 10 Mitomycin-C-treated splenocytes or Peripheral blood lineage-specific donor 3 chimerism was studied using flowcytometry (FACStarPlus, LNc (from chimeric or naive mice). [ H]Thymidine FACSCanto, BD Biosciences, Erembodegem, Belgium), as incorporation was determined as described above. described earlier,21 using mAb against CD3; CD45R/B220; CD11c; CD11b; H-2Kd; H-2Ks; H-2Kk; H-2Kb (BD MLR Biosciences, eBioscience, Hatfield, UK). MLR was performed as described earlier.21 In brief, responder cells were nylon wool-enriched splenocytes of Histopathology. Moribund animals were killed for naive C57BL/6 mice, and stimulator cells Mitomycin- histopathology: following standard fixation procedures, C-treated LNc or splenocytes from chimeras or naive

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 588 host-type mice. After 5-day culture, [3H]Thymidine reaching 490% around day 30 (Figure 1a). This evolution incorporation was determined. Results are expressed as was associated with a mortality rate of 79% by day 42 c.p.m. or stimulation index. (Figure 1b), the animals experienced sudden death without significant weight loss (Figure 1c) or clinical pathological signs. In a separate experiment, moribund animals in Statistical analysis this early phase were killed: peripheral blood CBC showed The Mann–Whitney U test was used to estimate the level of pancytopenia (Table 1); BM histology (Figure 2a) showed statistical significance of difference between groups of data, dilated sinuses, depletion of hematopoiesis with absent and the log-rank test to test the difference in survival erythropoiesis, maturation arrest of the myeloid lineage between groups (Po0.05 was considered as evidence for and dysplastic megakaryocytes, residual dendritic statistical significance). reticulum cell background and presence of lymphocytes. This is consistent with the previously described C57BL/6- B6DBA2F1 form of BM failure type of acute GvHD.15 Results The majority of recipients, however, showed a gradual increase of donor T-cell chimerism (defined as o40% on Repetitive parental C57BL/6 splenocyte infusion day 14), reaching 490% around day 90 (Figure 1a). This in unconditioned B6DBA2F1 recipients can induce was associated with 84% long-term survival (Figure 1b) progressive engraftment without clinical GvHD and a favorable weight evolution (Figure 1c), and none of B6DBA2F1 recipients were given 4 weekly PSIs of 25 Â 106 the animals showed clinical signs of acute epithelial GvHD C57BL/6 splenocytes. Two patterns of clinical outcome and (not shown). CBC analysis in long-term surviving animals chimerism evolution were documented. Approximately 1/3 equally showed pancytopenia, with recovery of cell counts in of recipients exhibited rapidly progressive donor T-cell all lineages after day 61 (Figure 3), suggesting that chimerism (arbitrarily defined as 440% on day 14), low-grade mortality was equally due to BM failure; indeed,

a b 100 100 * 80 80

60 60 F1 rapid (n=37) F1 gradual (n=84) Control F1 (n=76) 40 40 Survival (%) F1 rapid (n=37) 20 F1 gradual (n=84) 20 T cell donorchimerism (%) 0 0 0 20 40 60 80 100 0 20406080100 Days after start PSI regimen Days after start PSI regimen c d 140 100 120 80 100

80 60

60 40 * Survival (%) 40 F1 rapid (n=12) F1 gradual (n=74) F1 chimera (n=27) Control F1 (n=76) 20 20 Control F1(n=13) Weight (% of starting weight) Weight 0 0 0 20 40 60 80 100 0 102030 40506070 Days after start PSI regimen Days after leukemia challenge Figure 1 Evolution of donor T-cell chimerism, weight, survival and survival after leukemia challenge in the C57BL/6-B6DBA2F1 chimeras. B6DBA2F1 recipients given parental splenocyte infusions (PSI) were stratified according to the pattern of donor T-cell chimerism evolution. (a) Evolution of donor T-cell chimerism: animals showing X40% T-cell donor chimerism on day 14 after the first infusion were assigned to the ‘F1 rapid’-group (n ¼ 37) and those showing o40% T-cell donor chimerism were assigned to the ‘F1 gradual’-group (n ¼ 84). (b) Kaplan–Meier survival curves showing the survival of the ‘F1 rapid’- and the ‘F1 gradual’-group, as well as that of a group of naive host-type mice that were included in each experiment as controls (control F1). Results in panels a and b are from 16 similarly designed experiments. *Po0.05 between ‘F1 rapid’ and ‘F1 gradual’ (log-rank). (c) Weight evolution (expressed as mean±s.d. percentage of individual starting weights) in the ‘F1 rapid’-group (n ¼ 12, long-term survivors n ¼ 4), the ‘F1 gradual’-group (n ¼ 74), and a group of host-type control animals (n ¼ 76). Results are from 12 similarly designed experiments. (d) B6DBA2F1 mice given repetitive C57BL/6 infusion (F1 chimera), and age-matched naive B6DBA2F1 mice not given PSI (control F1) were challenged with 5 Â 105 P815 leukemia cells on day 7. Kaplan–Meier survival curve is shown of a total of 27 F1 chimera and 13 control F1 animals, from four identically designed experiments, which included simultaneous testing of all three groups. *Po0.05 for the difference in survival between groups (log-rank).

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 589 Table 1 Peripheral blood CBC values

Day of analysis White blood cell count Red blood cell count Hemoglobin Platelet count (10e3/mL) (10e6/mL) (g/100 mL) (10e3/mL)

1(wD20) D6 3 8.46 14.88 5256 D13 1.34 7.4 11.12 378 2(wD16) D6 3.7 9.6 14.62 1200 D13 1.19 6.46 9.82 728 D16a 0.25 4.87 6.97 34.2 3(wD7) D6 3.46 12.44 18.44 1480 4(wD15) D6 2.1 9.36 14.06 1172 D13 1.19 5.8 8.7 632 5(wD20) D6 4.04 9.96 14.7 1122 D13 0.92 6.68 9.94 310 D20a 1.4 5.39 7.75 33.6 Reference values (range)b 3.29–9.65 8.1–10.16 10.52–16.98 1128–1538

Abbreviation: CBC ¼ complete blood count. aMoribund. bCalculated as detailed in Materials and methods. Bold font indicates cytopenia. wKilled at.

a b

c d

Figure 2 Histological findings and CBC analysis in F1 chimeras. (a–d) Histological findings in F1 chimeras. H&E stain of a BM section of ‘F1 rapid’ B6DBA2F1 chimera demonstrating dilated bone sinuses, a depletion of hematopoiesis with absent erythropoiesis, maturation arrest of the myeloid lineage and dysplastic megakaryocytes, residual dendritic reticulum cell background and presence of lymphocytes (magnification  20). H&E staining of LN of a naive B6DBA2F1 mouse (b) showing the normal architecture with abundance of lymphocytes, a C57BL/6-B6SLJF1 chimera at day 60 (c) showing an effaced architecture of the lymph tissue that is depleted of lymphoid cells but shows an abnormal presence of myeloid precursor cells identifiable by their kidney- to donut-shaped nuclei and granular cytoplasm (detail shown in inset), and a C57BL/6-B6DBA2F1 chimera at day 60 (d) showing an effaced architecture of the lymph tissue depleted of lymphoid cells without an associated expansion of myeloid precursor cells. Pictures were taken with a Zeiss Axioplan 2 microscope (Carl Zeiss AG, Go¨ttingen, Germany) equipped with a cooled charge-coupled device camera COHU 4910 (Diagnostic Instruments, Detroit, MI, USA). Original magnifications  40.

BM histology of moribund mice was similar to that of mori- the first PSI, chimeric B6DBA2F1 recipients were chal- bund animals in the early mortality group (as in Figure 2a). lenged with 5 Â 105 P815 cells. PSI-treated animals These data indicate that strong LHGvH reactivity occurs showed a significant survival benefit over naive host-type with repetitive PSI. As this type of alloreactivity is animals, with long-term survival of 37% (Figure 1d). exploited by DLI strategies to obtain GvL effects, in a Histopathology in moribund animals confirmed tumor next set of experiments we investigated whether repetitive infiltration in LN, liver, spleen, salivary gland, muscle and PSI would allow for a GvL response. One week after BM (not shown).

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 590

8.0 D35 8.0 8.0 D42 7.0 D61 7.0 7.0 l)

µ D77 / 3 6.0 6.0 6.0

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3.0 3.0 3.0

2.0 2.0 2.0 White blood cells (10 1.0 1.0 1.0

0.0 0.0 0.0 0 20406080100 7295473 132328 61 83

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10.0 10.0 10.0 l) µ / 6 8.0 8.0 8.0

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Red blood cells (10 2.0 2.0 2.0

0.0 0.0 0.0 0 20406080100 7 29 54 73 13 23 28 61 83

1600 1600 1600

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1200 1200 1200 l) µ / 3 1000 1000 1000

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Platelets (10 400 400 400

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0 0 0 0 20406080100 7 29 54 73 13 23 28 61 83 % CD3 donor chimerism Days after first PSI Days after first PSI Figure 3 Longitudinal analysis of peripheral blood counts in long-term surviving B6DBA2F1 chimeras, and relation with level of peripheral blood donor T-cell chimerism and effect of BM coinfusion. (a) Analysis of peripheral CBC and donor T-cell chimerism in C57BL/6-B6DBA2F1 chimeras given parental splenocyte infusions, at four different time points: D35(x), D42(,), D61(J), D77(E). Dotted lines indicate the reference range of CBC values as determined in age-matched naive F1 mice (see Materials and methods). The data from 1 out of 4 independent experiments is shown (n ¼ 13 at D35 and D42, n ¼ 12 at D61 and 77). (b and c) Evolution of peripheral complete blood counts in long-term surviving B6DBA2F1 chimeras with (E) or without (J) coinfusion of donor BM cells on day 0 ( ¼ first PSI) (b) and on day 14 ( ¼ third infusion) (c). Dotted lines indicate the reference range of CBC values as determined in age- matched naive F1 mice (see Materials and methods). In both b and c, the results of 1 of 2 representative experiments are shown, n ¼ 7 per group.

Thus, in unconditioned F1 recipients, repetitive PSI donor BM was coinfused with the parental splenocyte can establish strong LHGvHR with a gradual evolution inoculums. F1 recipients were challenged with serial to high-grade donor chimerism, and with a strong PSIs only, or with serial PSIs and 5 Â 106 T-cell-depleted GvL response. Despite the high cumulative T-cell dose, C57BL/6 BM cells at the first or the third PSI. CBC were this occurs without acute epithelial GvHD. However, sequentially determined but showed that coinfusion of BM although rapid institution of donor T-cell chimerism is cells influenced neither the severity nor the duration of associated with rapidly lethal BMGvHD, the gradual pancytopenia (Figures 3b and c). course is equally accompanied by prolonged pancytopenia, indicating the development of subclinical BMGvHD that is severe but that holds a significantly lower mortality risk. The serial PSI regimen induces a disruption of the cellular To determine whether the additional infusion of donor composition of lymphohematopoietic organs stem cells would alter the characteristics of the GvHR, we The pattern of parent-in-F1 alloreactivity that allows determined the effect of serial PSI in F1 recipients where progressive donor chimerism and GvL with good overall

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 591 tolerance and long-term survival has previously not counts beyond day 84, and a temporary decrease in BM cell been reported. The effects on peripheral blood CBC are counts with normalization by day 84 (Figure 4a). Analysis indicative of subclinical GvHD targeting BM function. We of the cell subpopulations in spleens and LN (Figures 4b further explored the extent of this subclinical GvHD and c) showed a 2-week phase of lymphoproliferation and a by evaluating the cellular composition and histological myeloid cell expansion, followed by a progressive decline appearance of lymphoid and hematopoietic tissues, and in these cell lineages. epithelial GvHD target tissues. Groups of three Lymphoproliferation probably is a result of alloreactive B6DBA2F1 mice receiving serial splenocyte infusions were T-cell proliferation in BM and lymphoid organs. The killed at selected time points, as indicated in Figure 4. None expanding CD11b þ population showed a CD11b þ F4/ of the animals showed histological signs of acute epithelial 80ÀGR1hi phenotype, and a morphology typical of myeloid GvHD, or chronic SLE-like GvHD (for example immune- progenitor cells (not shown), similar to those previously complex glomerulonephritis) previously reported in F1 described in BM chimeras after myeloablation.9,21 hybrids23 (not shown). Total cell counts and proportions of We postulate therefore that this peripheral expansion T, B and myeloid cells were determined on spleen, BM and of myeloid progenitor cells occurs as a consequence LN (Figures 4a–c). Results were expressed relative to the of the BM depression caused by alloreactive T cells. In mean value obtained in a group of three naive B6DBA2F1 conclusion, in addition to causing subclinical BMGvHD, mice that were analyzed at each time point. The chimeras the serial PSI regimen induces a profound disruption showed a transient expansion of total cell numbers, of the cellular composition of lymphohematopoietic followed by a progressive decline in spleen and LN cell organs.

600 Spleen 400 Spleen Lymph node Lymph node 500 Bone marrow 350 Bone marrow 300 400 250 300 200 200 150 100 100 to naive B6SJLF1) to naive B6DBA2F1) 0 50 Total cell count (%, relative Total Total cell count (%, relative Total 0 0 20406080100 0 20 40 60 80 100 Days after start PSI regimen Days after start PSI regimen

+ 10000 CD11b cells 10000 CD3+ cells B220+ cells 1000 1000

100 100

10 10 to naive B6SJLF1) +

to naive B6DBA2F1) CD11b cells CD3+ and B220+ cells Total cell count (%, relative Total Total cell count (%, relative Total 1 1 0 20406080100 020406080100 Days after start PSI regimen Days after start PSI regimen

+ 10000 CD11b cells 10000 + + CD11b cells CD3 cells + + CD3 cells 1000 B220 cells + + B220 cells CD11c cells + 1000 CD11c cells 100

10 100

1 to naive B6SJLF1) to naive B6DBA2F1) 10 Total cell count (%, relative Total

0.1 cell count (%, relative Total 0 20406080100 0 20406080100 Days after start PSI regimen Days after start PSI regimen Figure 4 Evolution of total cell counts and cellular composition in spleen, BM and LN of C57BL/6-B6DBA2F1 chimeras and C57BL/6-B6SJL2F1 chimeras. Groups of three B6DBA2F1 chimera (a–c) or B6SJL2F1 mice (d–f) were killed at selected time points. Total cell enumeration was performed on spleen, BM and LN (a, d) and the proportions of lymphocytes (CD3 þ T cells, B220 þ B cells), CD11b þ myeloid cells and/or CD11c þ dendritic cells were determined in spleen (b, e) and LN (c, f). Each test included the analysis of a group of three naive host-type mice and results are expressed relative to the mean value obtained in this group. Data represent mean %±s.e.m. of individual experimental mice.

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 592 The cellular disruption of the lymphoid organs after serial progenitor cells disappear more rapidly (Figures 2d and PSIs includes an APC defect 4c). Spleen cells and LN cells from chimeras challenged Immune function requires a functional APC compartment, with PSIs only, or challenged with PSIs together with BM and host-type APC have a critical role in the development cells were used as stimulator cells for C57BL/6 T cells of GvL and GvHD.2,8,24,25 We specifically investigated the in MLR. In this particular experiment, day 73 chimeras evolution of host-type APC in the C57BL/6-B6DBA2F1 showed persistent cytopenia in LN and spleens (not model, but also in the similar C57BL/6-B6SJLF1 shown), but importantly, the frequencies of CD11b þ and combination. In the latter model, we also recently showed CD11c þ cells had normalized relative to naive F1 mice, that 60% of non-irradiated B6SJLF1 recipients develop whereas host chimerism in CD11c þ cells was still 480% high-grade donor chimerism while remaining clinically (not shown). We found that spleen and LN cells from these healthy, whereas 40% of them rejected the parental chimeras were poor stimulators for naive C57BL/6 splenocyte grafts.22 We also showed that such C57BL/6- responder cells (Figures 5c and d). Although purified B6SJLF1 chimeras become resistant to the development of APC were not tested owing to insufficient cell yield from 22 MOG35–55-induced EAE. Induction of EAE by footpad the severely cytopenic lymphoid organs, the data suggest injection of MOG peptide/CFA relies on the presentation that—in addition to a quantitative defect in APC num- of MOG peptide by APC in the draining popliteal LN. We bers—long-term PSI chimeras may also develop a qualita- hypothesized that—in view of the lymphohematopoietic tive dysfunction of residual APC. changes seen in C57BL/6-B6DBA2F1 chimeras, which included a profound decline in CD11c þ cells (Figure 4c)— Long-term surviving PSIs chimeras do not exhibit general the resistance to EAE induction in long-term C57BL/6- T-cell hyporesponsiveness B6SJLF1 chimeras relates to disappearance of APC in In view of the profound lymphopenia in long-term F1 draining LN. Both C57BL/6-B6SJLF1 and C57BL/6- chimeras induced by PSIs, we examined T-cell function. We B6DBA2F1 models were used to study the evolution previously showed that, in contrast to EAE-induced of APC in more detail. B6SJLF1 control mice, splenocytes from C57BL/6- First, we confirmed that long-term surviving C57BL/6- B6SJLF1 chimeras fail to exhibit an in vitro MOG - B6SJLF1 chimeras exhibit a similar disruption of the 35–55 specific proliferative response.22 Here, we confirmed that lymphohematopoietic system (Figure 4d): spleen and LNc from such chimeras (taken on day 56 after the start of LN equally showed a consecutive expansion and depletion PSIs, 14 days after MOG challenge) also fail to generate a of myeloid and lymphoid elements, and, in particular, MOG-specific proliferative response, but exhibit a vigorous a marked reduction in total CD11c þ cell counts (Figures 4e response to ConA (Figure 5e). Next, we tested splenocytes and f). Of note, a difference in the kinetics was seen in the from C57BL/6-B6SJLF1 chimeras (taken on day 56) in two models, with the myeloid expansion showing a more MLR with splenocytes from naive B6SJLF1 or BALB/c. protracted course in the B6SJLF1 mice (Figure 4). Histo- Splenocytes from naive B6SJLF1 and C57BL/6-B6SJLF1 logy of lymphoid organs on day 60 showed disruption radiation chimeras (alloBMT/F1, see Materials and meth- of the normal architecture of LN (Figure 2c) and spleen ods) were tested as positive and negative controls, (not shown): LN were largely depleted of lymphocytes, respectively. The MLR response against third party was and, in accordance with flowcytometry data, in B6SJLF1 comparable in all groups, whereas only C57BL/6-B6SJLF1 mice they showed a prominent presence of myeloid splenocyte infusion chimeras showed a significant anti- progenitor cells. B6SJLF1 response (Figure 5f). These data indicate that Next, LNc of these PSI chimeras were used in in vitro T cells from PSI chimeras readily respond to ConA, assays to document the functional presence or absence of allogeneic and third-party targets, and that—despite severe APC. LNc derived from C57BL/6-B6SJLF1 chimeras lymphopenia—the disruptive effect on the lymphoid organs (on day 56 after the first splenocyte infusion) and does not include a state of general T-cell hyporesponsiveness. from naive B6SJLF1 mice were used as APC source in a

MOG35–55-specific B6SJLF1 T-cell proliferation assay or as stimulators in allogeneic MLR. In contrast to naive Lymphohematopoietic recovery in long-term PSI chimeras B6SJLF1 LNc, LNc from C57BL/6-B6SJLF1 chimeras parallels establishment of multilineage donor chimerism supported neither the MOG-specific proliferative T-cell Finally, we illustrated that CBC in B6DBA2F1 PSI response (Figure 5a), nor the MLR response of C57BL/6 chimeras evolved in parallel with the level of donor responder cells (Figure 5b). In the latter setting, coculture T-cell chimerism (Figure 3c), as most animals showed with T-cell-depleted B6SJLF1 splenocytes (as an additional recovery of cell counts on day 77 together with near- source of host-type APC) restored the MLR reaction complete donor T-cell chimerism. This suggested (Figure 5b). From the flowcytometric and these functional that recovered lymphohematopoiesis was derived from data, we conclude that the disruption of lymphoid organs in donor-type stem cells. In LN of long-term (day 88) B6SJLF1 chimeras includes a quantitative defect in host-type B6DBA2F1 PSI chimeras, we documented that high-grade APC. However, LN of C57BL/6-B6SJLF1 chimeras con- donor chimerism had established in not only the T-cell tain a considerable proportion of myeloid progenitor cells lineage (94%±0.7 s.e.) but also in B cells (90%±1.3 s.e.) (Figures 2c and 4f), which may act as suppressor cells and and dendritic cells (65%±3.2 s.e.). Similar results were account for the lack of proliferative T-cell responses in vitro.21 obtained in spleen cells, with—in addition—high-grade Therefore, similar MLR experiments were performed donor chimerism in the CD11b þ lineage (80% ±1.6 s.e.) using cells from B6DBA2F1 chimeras, where myeloid (data from one out of two representative experiments).

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 593 5 80000 LNc LNc +host APC 70000 4 60000 3 50000 40000 2 30000 20000 Stimulation index

1 Stimulation index 10000 0 0 F1 chimera B6SJLF1 F1 chimera B6SJLF1 F1 chimera B6SJLF1 control control control

40 30

30 20

20

10 10 Stimulation index Stimulation index

0 0 Naive F1 F1 chimera BM/F1 Naive F1 F1 chimera BM/F1 chimera chimera

100 14000 PSI/F1 chimera (n =11) F1 chimera (n=4) AlloBMT/F1 chimera (n =8) B6SJLF1 EAE control (n=4) 12000 B6SJLF1 control (n =3)

10000 **

8000 10 6000 * 4000 Stimulation index Counts per minute

2000

0 1 B6SJLF1 BALB/c MOG conA

Figure 5 In vitro assays documenting APC and T-cell function in F1 chimeras. (a) MOG35–55-specific T-cell proliferation assay using Mitomycin-treated

LNc of B6SJLF1 chimeras as source of APC. MOG-specific T cells, obtained from EAE-induced control B6SJLF1 mice were stimulated with MOG35–55 in the presence of LNc derived from C57BL/6-B6SJLF1 chimeras on day 56 (‘F1 chimera’, n ¼ 6) or from naive host-type B6SJLF1 mice (‘B6SJLF1 control’, n ¼ 2); scatterplot shows individually tested animals and the mean value, and represent data from two independent experiments. (b) MLR response of C57BL/6 naive splenocytes stimulated with LNc taken from C57BL/6-B6SJLF1 chimeras on day 56 (‘F1 chimera’, n ¼ 5) or from naive B6SJLF1 mice (‘B6SJLF1 control’, n ¼ 2), either alone (left), or with additional LNc from naive B6SJLF1 mice as a source of host-type APC (right); scatterplot shows individually tested animals and the mean value, and 1 of 2 representative experiments is shown. (c) MLR response of C57BL/6 naive splenocytes stimulated with splenocytes from C57BL/6-B6DBA2F1 chimeras (‘F1 chimera’, n ¼ 4 samples, each representing cells of one (one sample) or two mice (three samples)), splenocytes from C57BL/6-B6DBA2F1 chimeras with BM coinfusion at the first PSI (‘BM/F1 chimera’, n ¼ 3 samples, each representing cells from one mouse (one sample) or two mice (two samples)), or splenocytes from naive B6DBA2F1 mice (‘naive F1’, n ¼ 6), all taken on day 73. Scatterplot shows individually tested samples and the mean value, and represent data from one experiment. (d) MLR response of C57BL/6 naive splenocytes stimulated with LNc from C57BL/6-B6DBA2F1 chimeras (‘F1 chimera’, n ¼ 1 sample, representing cells from two mice), splenocytes from C57BL/6-B6DBA2F1 chimeras with BM coinfusion at first PSI (‘BM/F1 chimera’, n ¼ 2 samples, each representing cells from three mice) or splenocytes from naive B6DBA2F1 mice (‘naive F1’, n ¼ 4), all taken on day 73. Scatterplot shows individually tested samples and the mean value, and represents data from one experiment. (e) Proliferative response of LNc from C57BL/6-B6SJLF1 chimeras (n ¼ 4, harvested on day 56 ( ¼ day 14 after EAE induction) and from naive B6SJLF1 control animals (n ¼ 4, harvested 14 days after EAE induction) in response to MOG35–55 or conA. Bars represent mean stimulation index±s.e.m., *Po0.05 (Mann–Whitney U-test). (f) MLR response of spleen cells from C57BL/6-B6SJLF1 PSI chimeras (‘PSI/F1 chimera’, n ¼ 11) on day 56, naive B6SJLF1 control animals (‘B6SJLF1 control’, n ¼ 3) or C57BL/6-B6SJLF1 radiation chimeras (‘alloBMT/F1’, n ¼ 8) on day 56, in response to B6SJLF1 host-type or BALB/c third-party spleen cells. Bars represent mean c.p.m.±s.e.m., **Po0.01 (Mann–Whitney U test).

Discussion cumulative high dose of alloreactive lymphocytes, can lead to a strong LHGvH response resulting in a form We here report on a previously undescribed pattern of of subclinical GvHD with profound perturbation of GvH reactivity in the well-known C57BL/6-B6DBA2F1 lymphohematopoietic tissues, including a depletion of parent-in-F1 hybrid mouse model. We found that peripheral APCs with associated prolonged immune serial infusions of parental splenocytes, establishing a incompetence.

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 594 The C57BL/6-B6DBA2F1 model is considered a well- pression, GvHD, tumors and chronic infections21,29 where established model GvHD,12,13 but GvHD characteristics they have been implicated in tumor-specific and non- vary according to the induction protocol. Reportedly, high- specific T-cell dysfunction.30–32 Our earlier data,21 and more dose C57BL/6 splenocytes induce acute epithelial GvHD recently those of others,33 suggest that these cells may have that is lethal over several months.14 In contrast, high-dose a role in controlling GvH reactivity. Whether they C57BL/6 LN cells induces a BMGvHD, which is rapidly contribute in this model to confining GvH reactivity to lethal;15–17 BMGvHD is also seen if recipients are the lymphohematopoietic system is the subject of ongoing irradiated, in which case lower quantities of C57BL/6 investigations. T cells are required. Here, we found that challenge of Interestingly, Wang et al.34 recently showed in a similar B6DBA2F1 with C57BL/6 splenocytes gave rise to two C57BL/6-B6DBA2F1 model involving recipient irradia- distinct outcome patterns. Only 1/3 of recipients developed tion that IFN-g promotes the allospecific LHGvH lethal BMGvHD; the majority exhibited progressive response, but attenuates the potential of alloreactive T institution of high-grade donor T-cell chimerism with good cells to mediate epithelial-tissue GvHD. Irradiation is clinical tolerance, long-term survival and no histological known to influence the activation/migration of allospecific evidence of acute epithelial-tissue GvHD. However, T cells,3 and the outcome of parent-in-F1 GvHD.35 subclinically these animals showed protracted pancytope- Assuming that also in our irradiation-free model, IFN-g nia, and in lymphoid tissues prolonged lymphopenia and has a critical role in LHGvH, blockade of this pathway a near-complete eradication of APCs. This pattern of would potentially reduce the risk for severe immuno- lymphohematopoietic GvHD has previously not been incompetence. However, such a limitation of LHGvH reported. responses could equally limit GvL effects, which, as in Our recipient mice were housed in semi-SPF conditions; the current model, occur in association with LHGvH hence, variability in intestinal colonization and inflamma- reactivity. tion may be one of the factors determining to which extent Coinfusion of donor BM with parental splenocytes, parental splenocyte inocula produce alloactivated cytotoxic either at the first challenge in naive mice or at the third T cells26,27 and produce either rapidly lethal or subclinical challenge when cytopenia (and therefore space in the GvHD. In this respect, Murai et al.28 showed that C57BL/6 recipient marrow) was already established, influenced splenocyte inoculation in unirradiated B6DBA2F1 resulted neither the severity nor the duration of pancytopenia in in a rapid and preferential accumulation of donor CD8 þ T our model, and flow cytometric studies and in vitro assays cells in Peyer’s patches before initiation of GvHD. We showed that the defect in APC numbers and APC function assume that a reduced inflammatory-prone intestinal was equally profound in these animals. We further showed environment protected some mice from aggressive that in irradiated B6C3HF1 mice, the coinfusion of C57BL/ BMGvHD; however, the resistance of these animals to 6 spleen cells (as a source of hematopoietic stem cells) with cumulative high-dose PSI, while developing high-grade purified C57BL/6 T cells can rescue mice from early death donor chimerism was striking. The data indicate that this and converts BMGvHD into acute epithelial-tissue GvHD relates to a delicate balance between donor T cells and host (not shown); this effect was not seen when F1 BM was APC in a non-inflammatory environment. coinfused, indicating that there was no innocent bystander The pattern of lymphohematopoietic subclinical GvHD destruction of donor-type hematopoietic stem cells. This could be reproducibly obtained in both B6DBA2F1 and had been earlier demonstrated by Rozendaal et al.36 in the B6SJLF1 hybrids challenged with C57BL/6 parental cells, DBA/2-B6D2F1 model. In contrast, Chen et al.16,17 have with a slight difference only in the kinetics of the cellular shown in the C57BL/6-CByB6F1 model that the BM of changes. Both models showed a severe quantitative F1 mice with BMGvHD not only fails to form stromal reduction in dendritic cells in spleens and LN, and ex vivo feeder layers in vitro, but also contains T cells capable of MLR and MOG-specific T-cell assays showed that the destroying fresh donor-type stem cells as well, indicating stimulatory capacity of whole spleen and LN cells of that in this model innocent bystander destruction of both B6SJLF1 mice was defective. Further, the observation that stromal and hematopoietic progenitor cells is taking place. parental T cells failed to proliferate when stimulated with In contrast to these studies, the model we present here does B6D2F1 chimeric LN or spleen cells in which relative not include irradiation, and inflammation may influence the numbers of CD11c þ cells (that were still largely host degree to which bystander destruction is taking place. The derived) had normalized relative to naive F1 mice, suggests data, however, argue against bystander destruction of that lymphohematopoietic alloreactivity may also affect the donor-type stem cells, as the eventual lymphomyeloid function of residual APC. Together, the data indicate that a reconstitution is predominantly donor derived; in contrast, quantitative defect, and possibly also a qualitative defect of the data suggest that repeated challenges with PSIs target APC may underlie the resistance of these chimeras to the the BM stroma, which fails to support effective myelo- GvHD-inducing capacity of repeated splenocyte inocula hematopoiesis until T-cell alloreactivity has subsided. and their resistance to EAE induction.22 These data reveal a delicate balance between donor T cell, In the B6SJLF1 model, where myeloid cell expansion is donor stem cell and BM stromal function. more protracted, an extrinsic regulatory role of these cells Although this experimental model of unidirectional on the observed in vitro T-cell proliferative responses T-cell alloreactivity presents some limitations with regard cannot be excluded. The expansion of myeloid progenitor to extrapolations to the clinical situation, the data may be cells in these chimeras is reminiscent of the myeloid-derived relevant to the development of clinical DLI strategies. They suppressor cells described in association with myelosup- indicate that a challenge with non-tolerant T cells in an

Bone Marrow Transplantation P-in-F1 lymphoid-tissue GvHD with prolonged immuno-incompetence B Sprangers et al 595 inflammation-free tolerant recipient can lead to severe loablative conditioning regimen and donor lymphocyte subclinical lymphoid-tissue GvHD causing prolonged infusions. Blood 2001; 97: 2574–2579. immuno-incompetence. The haploidentical pattern of 8 Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, T-cell alloreactivity is not entirely representative of the Sykes M. Donor lymphocyte infusions mediate superior graft- clinical situation where most patients receive an MHC- versus-leukemia effects in mixed compared to fully allogeneic Blood matched graft. Haploidentical DLI in humans is expected chimeras: a critical role for host antigen-presenting cells. 2002; 100: 1903–1909. to carry a high risk of causing epithelial GvHD, although 9 Billiau AD, Fevery S, Rutgeerts O, Landuyt W, Waer M. our data in this study indicate that this may depend on a Crucial role of timing of donor lymphocyte infusion in delicate balance. Of note, there is a growing interest in generating dissociated graft-versus-host and graft-versus-leu- haploidentical stem cell transplantation,11 and both delayed kemia responses in mice receiving allogeneic bone marrow graft function and delayed immune recovery have been transplants. Blood 2002; 100: 1894–1902. reported as complications after MHC-mismatched BM 10 Dey BR, McAfee S, Colby C, Sackstein R, Saidman S, Tarbell transplantation,37 supporting the potential relevance of N et al. 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