Abstract
In mice, graft-versus-host reactions (GVHR), associated with powerful graft-versus-tumor effects, can be achieved without graft-versus-host disease (GVHD) by delayed administration of donor lymphocyte infusions (DLI) to established mixed chimeras (MCs). However, GVHD sometimes occurrs after DLI in established mixed chimeric patients. In contrast to mice, in which T cell recovery from the thymus occurs prior to DLI administration, human T cell reconstitution following T cell-depleted hematopoietic cell transplantation is slow, resulting in lymphopenia at the time of DLI. We demonstrate here that T cell lymphopenia is an independent risk factor for GVHD following DLI in the absence of known inflammatory stimuli. DLI-induced GVHD was prevented in lymphopenic recipients by prior administration of a small number of non-alloreactive polyclonal T cells, insufficient to prevent lymphopenia-associated expansion of subsequently administered T cells, through a Treg-independent mechanism, but not by T cells with irrelevant specificity. Moreover, administration of antibiotics reduced the severity of GVHD in lymphopenic hosts. Accumulation of DLI-derived effector T cells and host hematopoietic cell elimination were markedly diminished by Treg-depleted, non-alloreactive T cells. Finally, thymectomized mixed chimeras showed increased GVHD following delayed DLI. Collectively, our data demonstrate that in the absence of known conditioning-induced inflammatory stimuli, T cell lymphopenia is a risk factor for GVHD in MCs receiving delayed DLI and suggest that the predisposition to GVHD can at least in part be explained by the presence of occult inflammatory stimuli due to the absence of T cells to control microbial infections.
Introduction
Following the realization that much of the therapeutic benefit of allogeneic hematopoietic cell transplantation (HCT) in the treatment of leukemias and lymphomas is due to immunological graft-vs.-tumor (GVT) effects, many centers attempted to reduce the toxicity of this procedure by developing non-myeloablative conditioning regimens. Non-myeloablative HCT relies upon the alloreactivity of donor T cells to eradicate remaining host malignant cells through a lymphohematopoietic graft-versus-host response (LHGVHR). However, these alloreactive donor T cells are also capable of attacking normal host epithelial tissues, causing graft-versus-host disease (GVHD). More effective separation of LHGVHR from GVHD could expand the clinical application of HCT across extensive HLA barriers and improve outcomes.
We have previously shown that this separation can be achieved in mice through the establishment of mixed chimerism, followed by delayed DLI (1,2) after the recipient has recovered from the inflammation induced by conditioning. Inflammation, such as that induced by toll-like receptor (TLR) stimuli produced by microbial infection, is a critical factor in allowing GVH-reactive T cells to traffic from the lymphohematopoietic system into the epithelial GVHD target tissues. In the absence of such inflammation, the alloreactive T cells do not traffic to GVHD target tissues (3), and thus do not cause GVHD. Rather, they remain confined to the lymphohematopoietic system, where they are able to mediate LHGVHR, including GVT effects (4-6).
This approach to separating GVHD and GVT effects has been translated to clinical trials, permitting remission of otherwise fatal, refractory malignancies without the development of GVHD (7,8). However, some patients in these trials have developed GVHD, even in patients receiving exhaustively T cell-depleted initial allografts, who had no obvious source of ongoing inflammation at the time of DLI (9,10). One potentially relevant difference between humans and the murine models described above is that at the time of DLI mice have largely reconstituted their lymphocytes, while humans remain lymphopenic for many months after conditioning (11-13). This difference suggests several possible mechanisms for the GVHD seen in human but not murine MCs after delayed DLI. First, lymphopenia includes reduced regulatory T cells (Treg), which can modulate GVHD (14,15). Secondly, T cells expanding in a lymphopenic environment develop an effector phenotype and function (16-18) and can potentiate T cell responses (19-22) . Thirdly, decreased immunity against infections may increase susceptibility to infections, resulting in inflammation induced by TLR stimulation, which promotes DLI-induced GVHD (3). Through any or all of these mechanisms, lymphopenia at the time of DLI might promote GVHD despite the presence of a “quiescent” mixed chimeric state.
While studies in humans (23) and mice (24) have previously suggested that lymphopenia is indeed a risk factor for GVHD, the effect of lymphopenia in the absence of other inflammatory stimuli, such as that induced by chemotherapy or irradiation, was not investigated. We have now investigated the impact of lymphopenia on DLI-induced GVHD in unconditioned lymphopenic hosts. We demonstrate that lymphopenia is an independent risk factor for DLI-induced GVHD and show that GVHD can be prevented by polyclonal non-GVH-reactive T cells, but not by “irrelevant” T cells. A series of mechanistic studies suggest that inflammatory stimuli resulting from microbial stimuli promote GVHD in lymphopenic hosts receiving DLI and that T cells in DLI recipients can prevent GVHD by limiting these inflammatory stimuli.
Materials and Methods
Mice
All studies were performed under an institutionally approved animal protocol in accordance with guidelines from the National Institutes of Health (NIH, Bethesda, MD). B6.129S7-Rag1tm1Mom (RAG-1 KO B6: H-2b) mice and C.129S7(B6)-Rag1tm1Mom (BALB/c RAG-1 KO: H-2d) mice were initially purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility B6.OTI T cell receptor (TCR) transgenic (Tg) Thy1.1 RAG KO mice were kindly provided by Dr. Steven Schoenberger at La Jolla Institute for Allergy and Immunology and bred in our facility. B6 RAG KO OTI mice are transgenic for a Class I-restricted TCR that recognizes an ovalbumin peptide, SINFEKL (25). BALB/c Clone6.5 TCR Tg Thy 1.1 mice were kindly provided by Dr. Ephraim Fuchs at Johns Hopkins University and were crossed to BALB/c RAG KO mice in our facility. BALB/c RAG KO Clone 6.5 mice are transgenic for a Class II-restricted TCR that recognizes a hemagglutinin peptide (26). Wild type (WT) B6, WT BALB/c mice were purchased from Taconic Farms, Inc (Germantown, NY). B6.CD45.1 and CBF1 mice were purchased from NCI (Frederick, MD) and Charles River Labs (Wilmington, MA) respectively. CD45.1+ CBF1 mice were bred in our facility by crossing CD45.1+ B6 and WT BALB/c mice. All mice were housed in a specific pathogen-free microisolator environment. Recipient mice were age-matched. Protocols involving the use of animals were approved by the Massachusetts General Hospital and Columbia University Medical Center Subcommittees on Research Animal Care and all of the experiments were performed in accordance with the protocols.
Establishment of MCs
Recipient mice (6-12 wk old) received depleting anti-NK1.1 (PK136, 150mg) mAb i.p. on Day -1 and 3 Gy TBI from a 137Cs irradiator on Day 0 prior to i.v. injection of 10-15×106 T cell depleted (TCD) donor bone marrow cells (BMCs) harvested as described (27). (When required, ex vivo T cell depletion of B6 bone marrow was performed by negative immunomagnetic selection using anti-CD8 and anti-CD4 microbeads (Miltenyi Biotec, Auburn, CA). After 5-10 weeks of recovery from BMT, mice treated with this regimen developed mixed multilineage chimerism, with 40-60% donor chimerism in neutrophil and monocyte lineages. In some studies, recipients underwent thymectomy as described (28) two weeks before induction of mixed chimerism. These recipients then received anti-CD4 (GK1.5, 1.76mg/mouse) and anti-CD8 (2.43, 0.72mg/mouse) antibodies on Day -5, cyclophosphamide (200mg/kg) on Day-1 and anti-CD40L (MR1, 0.5mg/mouse) antibody on Day 0 followed by i.v. injection of 20×106 donor BMCs.
Donor lymphocyte infusion (DLI) and T cell purification
Splenocyte (SPC) suspensions were prepared as described (29,30). 20-30×106 SPC were given intravenously to each recipient as DLI. Where required, CD4 or CD8 cells from donor mice were isolated (purity >93%) by immunomagnetic selection using anti-CD4 or anti-CD8 microbeads (Miltenyi Biotec). Where required, regulatory T cells were isolated using a CD25+CD4+ Regulatory T cell Isolation Kit (Miltenyi Biotec): CD4 cells were isolated by negative selection, incubated with biotinylated anti-CD25, and then positively selected with anti-biotin-conjugated microbeads. The negative fraction was used as Treg-depleted CD4 cells. Depletion of CD4+CD25+ cells was ∼75% complete, with remaining CD4+CD25+ cells staining low for CD25, suggesting activated CD4 cells rather than Tregs.
Flow cytometry
To analyze the development of multilineage chimerism, donor- or recipient-derived cells were identified in the live cell population (propidium iodide negative) using fluorescein isothiocyanate (FITC)–conjugated anti–H-2Dd mAb 34-2-12, as described (31). Anti-mouse FcγRI/III receptor mAb 2.4G2 was used to block nonspecific FcγR binding. Cells were counterstained with phycoerythrin (PE)–conjugated anti-CD4 (Becton Dickinson [BD]/PharMingen, San Diego, CA) or MAC-1 (Caltag, San Francisco, CA) and with allophycocyanin (APC)–conjugated anti-CD8 or anti-B220 mAb (BD/PharMingen), respectively.
Anti-CD25, -CD44 and –CD62L antibodies (all from BD/PharMingen) were used to evaluate effector/memory differentiation and T cell activation. Intracellular staining was performed to determine the expression of granzyme B in CD8 T cells using an anti-human granzyme B mAb (eBioscience). To assay production of interferon-γ (IFN-γ) by CD4 and CD8 T cells, splenocytes were cultured ex vivo for 5 hours in the presence of Brefeldin A (Biolegend). Intracellular staining was then performed using an APC-anti-mouse-IFNγ mAb (Biolegend). CFSE staining was performed using a CFSE-Vybrant staining kit (Invitrogen, Carlsbad, CA).
Negative control mAbs included HOPC1-FITC (prepared in our laboratory) and rat antimouse IgG2a-PE or -APC. Flow cytometry was performed using a FACSCalibur or FACSCantoII flow cytometer (BD Biosciences). Results were analyzed using FlowJo flow cytometry analysis software (Tree Star, Inc.)
Assessment of GVHD
Body weights were measured on the day of splenocyte transfer, twice weekly during the first week after splenocyte transfer, and then weekly. Animals were also scored for clinical evidence of GVHD by assessment of changes in skin (i.e., alopecia, inflamed or scaly skin), generalized signs (fur texture, posture, activity), inflammation of the eyes, and diarrhea. Each parameter was quantified by scoring as follows: 0 = normal, 1 = mild, 2 = moderate, 3 = severe (for scoring of eye inflammation, 4 = eyes severely inflamed and closed). Total clinical GVHD scores were defined as the sum of the scores from each parameter.
Histopathology
For histopathological analysis of GVHD target tissues, samples were collected from large intestine, lung, liver, and skin (from the head), and were fixed in 10% formalin. Formalin-preserved tissue samples were embedded in paraffin, cut into 5-μm-thick sections, and stained with hematoxylin and eosin for histological examination. Slides were coded without reference to mouse type or prior treatment status, and were systematically examined by a pathologist for evidence of histologic GVHD in the following organs: lung, liver, colon, skin. Defined histologic parameters were assessed in each organ (7 parameters each for liver and colon, 2 parameters each for skin and lung), and each parameter was scored according to the following scale: 0=normal, 0.5=focal and rare, 1=focal and mild, 2=diffuse and mild, 3=diffuse and moderate, 4=diffuse and severe). GVHD scores for each organ were defined as the sum of the scores for each parameter, and where applicable, total GVHD scores were defined as the sum of the scores for each organ.
Administration of antibiotics
Antibiotics were administered as described elsewhere, with minor modification (32). Briefly, water containing 1.32mg/ml ciprofloxacin (Sigma) and 5mg/ml metronidazole (Sigma) was given intragastrically to RAG KO mice at a dose of 1ml/mouse/day. Antibiotics were administered from 2 days before DLI to 14 days post-DLI.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software. Survival data were analyzed using the log rank test. Otherwise, statistical analyses were performed using Student's t-test (two-tailed). A p value less than 0.05 was considered to be statistically significant. Unless otherwise noted, data are presented as mean ± SEM.
Results
GVHD develops in unconditioned lymphopenic mice receiving allogeneic splenocytes
To evaluate GVHD susceptibility in lymphopenic animals in the absence of conditioning-induced inflammation, unconditioned B6 RAG-1 knockout (KO) mice were used as recipients of allogeneic (3×107 B10.A SPC) or syngeneic (3×107 B6 SPC) T cells. Because RAG KO mice have increased numbers of NK cells which could reject allogeneic cells, NK cells were depleted with intraperitoneal injection of 150μg depleting anti-NK1.1 mAb on Days -5 and -1 before injection of splenocytes. Because GVH responses in the allogeneic group, if present, would lead to marrow aplasia in the recipient, bone marrow cells (1×107 B10.A or B6, respectively) were injected as well. As shown in Fig. 1, recipients of allogeneic splenocytes developed significant GVHD, in contrast to recipients of syngeneic splenocytes, as evidenced by clinical GVHD scores (Fig. 1A), weight loss (Fig. 1B), and histologic evidence of GVHD (Fig. 1C). While clinical GVHD diminished over time (followed for 153 days), the recipients of allogeneic splenocytes had pathologic GVHD at all time points examined (Days 7, 48, and 153), with the most marked difference on Day 7. These results demonstrate that in the absence of conditioning-induced inflammation, congenitally lymphopenic recipients of allogeneic T cells develop relatively mild, self-limited GVHD in this strain combination. Since GVHD in this model is most prominent on Day 7, animals were followed up to Day 7 in the ensuing studies.
Figure 1.
GVHD occurs following administration of allogeneic splenocytes to unconditioned lymphopenic mice. B6 RAG-1 knockout (KO) mice (n=8/group) received allogeneic (■) B10.A SPC or syngeneic (▲) B6 SPC, depleting anti-NK1.1 antibody (d -5, -1), and B10.A (■) or B6 (▲) BMCs. While animals were followed up to 153 days post-splenocytes transfer, clinical GVHD scores (A), weight loss (B), and histologic GVHD (C) were most prominent on Day 7. Similar results were obtained in 2 repeat experiments. *, p<0.05; ***, p<0.001, compared with recipients of syngeneic cells.
GVHD develops in lymphopenic MCs receiving DLI
We next investigated whether T cell reconstitution in DLI recipients could prevent GVHD. We compared GVHD in B6 RAG-1 KO recipients of allogeneic B10.A splenocytes (and BMCs) to that in B10.A→B6 RAG KO established MCs. The MCs were generated by treating B6 RAG KO mice with sublethal (3Gy) TBI and T cell-depleted allogeneic (B10.A) plus syngeneic bone marrow 28 days prior to administration of allogeneic donor splenocytes. To control for the effects of sublethal irradiation 28 days prior to splenocyte administration, lymphopenic B6 RAG KO recipients were treated simultaneously with the above group with 3Gy TBI without administration of bone marrow, and allowed to recover for 28 days. As shown in Fig. 2, the lymphopenic mice developed significantly more weight loss (Fig. 2A), higher clinical GVHD scores (Fig. 2B) and more histologic GVHD (Fig. 2C) than MC recipients of allogeneic B10.A splenocytes or lymphopenic B6 RAG KO recipients of syngeneic (B6) splenocytes. Non-chimeric recipients of allogeneic lymphocytes and BMCs developed high levels of donor chimerism within 7 days, by which time the level exceeded that in MCs receiving DLI. The latter showed only a small increment in chimerism by Day 7 and further gradual increases over time (Fig. 2D).
Figure 2.
GVHR and GVHD following administration of allogeneic splenocytes to unconditioned lymphopenic mice are attenuated by the presence of mixed chimerism. Lymphopenic B6 RAG-1 KO recipients of allogeneic B10.A splenocytes and BMC (■; “allogeneic”, n=8) developed significantly more weight loss (A), higher clinical GVHD scores (B), more histologic GVHD (C), and more rapid and extensive chimerism conversion (D) than MCs (B10.A→B6 RAG KO) receiving allogeneic B10.A splenocytes (▲; “mixed chimeric”, n=8) or lymphopenic B6 RAG KO recipients of syngeneic (B6) splenocytes (▼; “syngeneic”, n=8). *, p<0.05; **, p<0.001, compared with B6 RAG-1 KO recipients of allogeneic B10.A splenocytes and BMC (■), or as indicated in the figure.
Since the protection from GVHD observed in MCs compared to lymphopenic mice might either reflect either the presence of mixed chimerism or the immune reconstitution in the former group, we next compared GVHD induction by allogeneic splenocytes in lymphopenic MCs with that in non-lymphopenic MCs. This was done by sublethally irradiating (3Gy) B6 RAG KO mice and administering 5×106 B6 RAG KO + 15×106 BALB/c RAG KO BMCs. Control B6 RAG KO mice received 3Gy TBI and were reconstituted with 5×106 T cell depleted WT B6 + 15×106 BALB/c RAG KO bone marrow to generate non-lymphopenic MCs. Mice were allowed to recover from effects of conditioning-induced inflammation for 8 weeks, and were then given WT BALB/c splenocytes as DLI. As shown in Fig. 3, lymphopenic MCs developed significantly more GVHD than non-lymphopenic MCs (Fig. 3A and 3B), and increased the level of donor chimerism more rapidly following DLI (Fig. 3C). These experiments demonstrate that recipient lymphopenia permits more rapid LGVHR and predisposes to GVHD following DLI to established MCs.
Figure 3.
GVHD occurs following administration of allogeneic splenocytes to unconditioned lymphopenic mixed chimeric mice. Sublethally irradiated B6 RAG KO mice were reconstituted with B6 RAG KO + BALB/c RAG KO BMCs to generate lymphopenic MCs (n=4). Control B6 RAG KO mice were sublethally irradiated and reconstituted with T cell depleted WT B6 + BALB/c RAG KO bone marrow to generate non-lymphopenic MCs (n=4). Mice were allowed to recover from effects of conditioning-induced inflammation for 8 weeks, and were then given WT BALB/c splenocytes as DLI. Lymphopenic MC (◆) developed significantly more weight loss (A) and higher clinical GVHD scores (B) than non-lymphopenic MC (▼), and converted to full donor chimerism more rapidly (C). *, p<0.01; **, p<0.001; ***, p<0.0001, compared with non-lymhopenic MCs (▼).
To further simulate the clinical situation where T cell reconstitution in HCT recipients is hampered by involution of the adult thymus and further impaired by chemotherapies and radiation injuries, we evaluated the effect on DLI-induced GVHD susceptibility of thymectomy performed before mixed chimerism induction. Prior to DLI, peripheral blood T cell counts of thymectomized MCs were about one tenth those of control MCs (p<0.05, Fig. 4A), even though T cell counts in these MCs were lower than those in unconditioned naïve BALB/c mice. Following DLI, severe GVHD occurred in thymectomized MCs receiving DLI, as shown by persistent body weight loss, increased GVHD clinical scores and decreased survival compared to control MCs receiving DLI (p<0.05, Fig. 4B-D). Thymectomized and control MCs not receiving DLI did not show any signs of GVHD. These data confirm that T cell reconstitution is an important factor in determining the susceptibility to GVHD in MCs receiving DLI in the absence of overt inflammation and are consistent with those obtained using RAG KO recipients above.
Figure 4.
Thymectomy predisposes to DLI-induced GVHD. Recipients did or did not undergo thymectomy prior to allogeneic bone marrow transplantation for mixed chimerism induction. Six weeks post-transplant, DLI was administered. Peripheral blood T cell numbers were significantly lower in thymectomized MCs than in control MCs prior to DLI (A; p<0.001). Compared to control MCs receiving DLI (Δ, n=5), thymectomized MCs receiving DLI (○, n=6) demonstrated severe body weight loss (B; p<0.05), higher GVHD clinical scores (C; p<0.05) and decreased survival (D; p<0.05) following DLI. Thymectomized (●, n=5) and control (▲, n=4) MCs not receiving DLI did not show evidence of GVHD.
Non-alloreactive T Cells prevent GVHD in lymphopenic hosts through a Treg-independent mechanism
We next directly evaluated whether GVHD in unconditioned lymphopenic mice receiving allogeneic lymphocytes could be blocked with polyclonal non-GVH-reactive T cells. For this purpose, we administered T cells from CBF1 (BALB/c×B6 F1) mice to B6 RAG KO mice. Because they lack T cells, RAG KO mice are unable to reject CBF1 cells, and CBF1 cells are tolerant to both B6 RAG KO recipients and BALB/c donors used to induce GVHD. Thus, the administration of CBF1 T cells allowed us to populate the lymphocyte compartment without alloreactivity in the GVH or host-vs-graft direction. This adoptive transfer approach also allowed us to investigate whether or not natural Tregs were needed to prevent GVHD in T-cell-replete hosts. To this end, B6 RAG KO recipients were first reconstituted with total or Treg-depleted CBF1 T cells to populate the lymphocyte compartment. Each group received 3.6×106 CD8 T cells and 5.4×106 CD4 T cells, which were equal to the number in 3×107 total splenocytes. Seven days after CBF1 T cell transfer, we administered 30×106 BALB/c splenocytes and 5×106 BALB/c bone marrow cells to both groups of recipients, and to a third group of untreated B6 RAG KO mice. All groups were followed for the development of GVHD. Results in Fig.5A show that the presence of polyclonal T cells significantly decreased GVHD-associated weight loss in lymphopenic mice, and decreased the LGVHR, as reflected by the level of donor chimerism 7 days following BALB/c splenocyte infusion (Fig. 5B). Suppression of weight loss and chimerism conversion occurred to a similar extent regardless of whether or not Tregs were present in the previously administered CBF1 T cell population. Similar results were obtained in a repeat experiment (data not shown).
Figure 5.
Reduction of GVHD by administration of polyclonal splenocytes to lymphopenic mice prior to DLI. Polyclonal CBF1 T cells with (■) or without (▲) Tregs were given to B6 RAG KO mice 7 days prior to BALB/c DLI, and allowed to populate the lymphocyte compartment. BALB/c splenocytes and BMCs were given to both groups of recipients on day 7, as well as to a third group of untreated B6 RAG KO mice (▼). All groups were followed for the development of GVHD. (A) Animals receiving polyclonal T cells had significantly decreased GVHD-associated weight loss compared to untreated lymphopenic mice, regardless of whether or not CBF1 Tregs were present. (B) The rate of conversion to full donor chimerism was significantly decreased by the presence of polyclonal T cells, regardless of whether or not CBF1 Tregs were present. Splenic chimerism is shown on day 14 (7 days after BALB/c splenocyte administration). ***, p<0.0001, n=4-5/group.
Non-alloreactive T cell administration prevents GVHD without blocking lymphopenia-driven expansion
Previous studies have shown that in normal animals lymphopenia results in the proliferation of residual T lymphocytes in an effort to restore normal numbers of T lymphocytes. This expression is driven by increased availability of IL-7 and IL-15 resulting from lymphopenia (33-35) and has been associated with potentiated anti-tumor effects, autoimmune diseases and allograft rejection (19-22). Moreover, IL-7 and IL-15 have been implicated in the increased potency of immunotherapy seen in lymphopenic hosts (36-38)and both IL-7 (39-41) and IL-15 (42-44) have been shown to play critical roles in GVHD. Thus, it was possible that blockade of lymphopenia-driven proliferation (LIP) with reduced availability of these factors could explain the protective effect of non-alloreactive T cells against DLI-induced GVHD. To address this possibility, we asked whether or not reconstituting RAG KO recipients of DLI with CBF1 T cells blocked LIP. To this end, RAG KO B6 recipients were first injected with 3×107 WT CBF1 splenocytes (CD45.2+). Seven days later, the same number of CFSE-labeled congenic CBF1 (CD45.1+) splenocytes was given to reconstituted and non-reconstituted RAG KO B6 recipients and their proliferation was analyzed 7 days later. Evaluation of the T cell compartment of RAG KO mice 7 days after transfer of CD45.2+ CBF1 T cells revealed that although transferred T cells had expanded, the mice remained lymphopenic (mean splenocyte number 2.5×106, vs >30×106 for WT animals). Furthermore, this number of T cells was insufficient to prevent LIP of CD45.1+ CBF1 lymphocytes infused 7 days later (Fig. 6). These results suggest that the ability of the polyclonal non-GVH-reactive T cells to prevent GVHD was not due to blockade of LIP of subsequently administered lymphocytes.
Figure 6.
Reconstitution of RAG KO recipients with CBF1 splenocytes does not block lymphopenia-induced proliferation. Polyclonal CD45.2+ CBF1 T cells were given to B6 Rag KO mice on day 0 and allowed to populate the lymphocyte compartment. CFSE-labeled CD45.1+ CBF1 T cells were given on day 7, and animals were sacrificed on day 14. Expansion of CD45.1+ CBF1 splenocytes as measured by CFSE dilution on day 14 was not affected by the presence of polyclonal CBF1 T cells, as absolute numbers of proliferated CD4 or CD8 T cells were not significantly different between the two groups (n=3-4/group).
Non-alloreactive T cell administration inhibits accumulation of activated DLI-derived effector T cells
To characterize the mechanism by which a lymphopenic environment allows GVH alloresponses to result in GVHD, we analyzed the alloresponses mediated by GVH-reactive donor T cells post-DLI. RAG KO B6 recipients were either untreated or reconstituted with Treg-depleted CBF1 splenocytes one week prior to transfer of allogeneic CFSE-labeled BALB/c splenocytes and BMCs. On Day 6 following donor splenocyte transfer, proliferation and accumulation of donor T cells were determined by CFSE dilution and enumeration of donor T cells in various tissues respectively. Expression of CD25, CD44 and CD62L on donor T cells was assayed to determine the activation of donor T cells and expression of granzyme B and production of IFN-γ were used as readouts of effector differentiation of GVH-reactive DLI T cells. As shown in Fig. 7, on Day 6 post-transfer, a high percentage of CD4 and CD8 T cells had proliferated, as reflected by CFSE dilution, in recipients that did or did not receive CBF1 splenocytes 1 week earlier. The percentages of proliferated DLI CD4 T cells were not significantly different between the two groups. The percentages of proliferated DLI CD8 T cells in recipients without reconstitution were slightly, but significantly higher than those in reconstituted recipients (Fig. 7A and 7B). In contrast, the absolute numbers of DLI CD4 and CD8 T cells in spleen, liver, bone marrow and peripheral blood of non-reconstituted recipients were all significantly higher than those in the reconstituted recipients (p<0.05; Fig. 7C). These data showed that the accumulation, but not the proliferation, of DLI T cells was affected and suggested that the survival of GVH-reactive DLI T cells may be reduced by the presence of non-alloreactive T cells. Significantly higher expression of expression of CD25, higher percentages of CD44highCD62Llow effector memory CD4 and CD8 T cells and lower percentages of both CD44highCD62Lhigh central memory and CD44lowCD62Lhigh naïve CD4 and CD8 T cells were detected in non-reconstituted compared to reconstituted recipients (p<0.05; Fig. 7D and 7E). In line with the enhanced DLI-derived CD8 effector cell accumulation in non-reconstituted lymphopenic hosts, effector functions of DLI CD8 T cells was also enhanced, as reflected by significantly higher expression of granzyme B (p<0.05; Fig. 7F) and production of IFN-γ (p<0.05; Fig. 7G). Collectively, these data show that GVH alloresponses were enhanced in severely lymphopenic RAG KO recipients, and that these were markedly attenuated by the prior administration of non-alloreactive T cells.
Figure 7.
Impact of lymphopenic environment on proliferation, accumulation, activation and effector differentiation of GVH-reactive donor T cells. RAG KO B6 recipients were either reconstituted (open bar) or not reconstituted (filled bar) with Treg-depleted CBF1 splenocytes (3×107/mouse). Seven days later, CFSE-labeled splenocytes from BALB/c mice were given to these recipients (3×107/mouse). Six days after allogeneic splenocyte transfer, proliferation (A and B), accumulation (C), activation (D and E) and effector differentiation (F and G) of donor T cells were analyzed. A. Representative results showing the proliferation of CD4 and CD8 donor T cells in reconstituted and unreconstituted hosts. B. Cumulative data of proliferated CD4 and CD8 donor T cells in reconstituted and unreconstituted hosts. C. Absolute numbers of donor CD4 and CD8 T cells in various tissues. D. Expression of CD25 on donor CD4 and CD8 T cells. E. Expression of CD44 and CD62L on donor CD4 and CD8 T cells. F. Expression of granzyme B on donor CD8 T cells. G. Production of IFN-γ by donor CD4 and CD8 T cells. To determine production of IFN-γ by donor CD4 and CD8 T cells, splenocytes from RAG KO recipients were cultured ex vivo in the presence of Brefeldin A for 5 hours followed by intracellular staining for IFN-γ. *, p<0.05, compared with recipients without reconstitution, n=3/group.
Administration of antibiotics reduces DLI-induced GVHD in lymphopenic hosts
To further elucidate the mechanisms by which lymphopenia predisposed to GVHD, we sought to determine if infection-induced inflammation resulting from lymphopenia might play a role. Given the importance of TLR stimulation in the pathogenesis of GVHD in T cell-replete DLI-recipients (3), we hypothesized that inflammation associated with gut bacterial translocation resulting from lymphopenia might promote GVHD in lymphopenic hosts. To address this possibility, we tested the effect of administering broad-spectrum antibiotics to RAG KO recipients of DLI from 2 days prior to DLI to 14 days post-DLI. As shown in Fig. 8, antibiotic treatment significantly reduced GVHD in RAG KO recipients of DLI as compared to recipients without antibiotic treatment. These data support the hypothesis that in lymphopenic hosts, infection-induced inflammatory stimuli resulting from a lack of T cell function promote GVHD.
Figure 8.
Administration of antibiotics to RAG KO recipients of DLI reduces GVHD. Ciprofloxacin and metronidazole were given to NK1.1+ RAG KO BALB/c mice from 2 day before DLI to 14 days post-DLI. PK136 antibody was given 5 and 1 day before DLI to deplete NK cells. WT B6 splenocytes along with BMCs were given as DLI to RAG KO BALB/c either receiving (●) or not receiving (□) antibiotics and GVHD was monitored. *, p<0.05, compared with recipients not treated with antibiotics, n=5-6/group.
GVHD in lymphopenic recipients of allogeneic splenocytes is not blocked by the presence of a small number of irrelevant T cells
Our observation that antibiotics can reduce DLI-induced GVHD in lymphopenic hosts led us to hypothesize that the ability of CBF1 lymphocytes to block GVHD might reflect rapid T cell expansion in response to endogenous flora, as reported in congenitally immunodeficient mice (45). Such responses might be expected to inhibit the release of TLR ligands by such flora, as TLR ligands have been shown to promote GVHD in DLI recipients (3). If this hypothesis is correct, T cells lacking the potential to respond to microbial flora would not block GVHD induced by DLI. We therefore tested whether or not T cells with irrelevant specificity could inhibit GVHD in lymphopenic mice. As recipients for these experiments we used two lines of RAG KO mice that are transgenic for specific T cell receptors: B6 RAG KO OTI mice that are transgenic for a Class I-restricted TCR recognizing an ovalbumin peptide (25) and BALB/c RAG KO Clone 6.5 mice that are transgenic for a Class II-restricted TCR recognizing a hemagglutinin peptide (26). As neither ovalbumin nor hemagglutinin is present in these recipients, these TCRs do not encounter their cognate ligands and are “irrelevant” in this system. We compared GVHD when allogeneic splenocytes were given to either TCR transgenic RAG KO recipients or to non-transgenic RAG KO recipients. Of note, RAG KO TCR Tg mice generally have low numbers of total lymphocytes (<5×106 lymphocytes/spleen).
The results from these experiments are shown in Fig. 9, which demonstrates that the presence of a small number of irrelevant lymphocytes in the recipient at the time of allogeneic cell transfer did not lead to decreased clinical (Fig. 9A and 9C) or histological (Fig. 9B and 9D) GVHD in either TCR transgenic model. Similar results were obtained in 3 of 3 experiments. Thus, while a small number of polyclonal lymphocytes protects from GVHD, a small number of irrelevant T cells does not. These data further support our hypothesis that non-alloreactive, polyclonal T cells present in the host at the time of DLI prevent GVHD by controlling microbial infections.
Figure 9.
Irrelevant T cells fail to prevent GVHD following administration of allogeneic splenocytes to lymphopenic mice. B6 splenocytes were given to NK cell-depleted BALB/c RAG KO (■) and Clone 6.5 (BALB/c RAG KO Clone 6.5 TCR transgenic, ▲) recipients. Administration of B6 splenocytes to Clone 6.5 recipients was not associated with decreased clinical GVHD scores (A) or histological GVHD (B) compared with BALB/c RAG KO recipients. Each group contained 3-7 animals. BALB/c splenocytes were given to NK cell-depleted B6 RAG KO (■) and OTI (B6 RAG KO OTI TCR transgenic, ▲) recipients. Administration of BALB/c splenocytes to OTI B6 recipients was not associated with decreased clinical GVHD scores (C) or histological GVHD (D) compared with RAG KO B6 recipients. Each group contained 3-4 animals.
Discussion
We demonstrate here that lymphopenia at the time of DLI is an independent risk factor for GVHD in the absence of overt inflammatory stimuli. To identify the mechanisms by which lymphopenia predisposes to GVHD after DLI, we evaluated three possible consequences of lymphopenia that might impact the recipient environment: the absence of regulatory T cells in the host; the potential for lymphopenia-induced proliferation and activation of DLI-derived T cells to enhance GVHD; and the inability of the host to control microbial pathogens. Our data are consistent with a major role for the presence of occult inflammation due to failure to control microbes in promoting GVHD in lymphopenic hosts. We demonstrate that recipient T cells are not uniformly beneficial in preventing GVHD, and that this capacity depends on the specificity of the TCR, as “irrelevant” T cells lacking their peptide ligand in the recipients did not protect from GVHD. Moreover, we show that thymic function at the time of HCT, which is severely lacking in many adult cancer patients, is essential to confer resistance to DLI-induced GVHD in mixed chimeras.
Proliferation of naïve T cells transferred to syngeneic lymphopenic hosts includes slow division that relies on the cytokine IL-7 and interactions with self-MHC (34) as well as rapid proliferation that occurs only upon transfer of naïve T cells to congenitally immunodeficient hosts (45) is IL-7-independent and does not occur in germ-free mice (45). This rapid expansion appears to result from T cell activation by foreign (microbial) peptides and is blocked by polyclonal T cells, but not by irrelevant TCR Tg T cells (45). Polyclonal T cells are able to respond to microbial antigens and eliminate this source of T-cell stimulation, whereas irrelevant TCR-Tg T cells are not. Our observation that polyclonal, non-alloreactive (donor×recipient F1) lymphocytes prevented GVHD induction by DLI, whereas irrelevant TCR-Tg T cells did not, suggests a similar explanation for GVHD protection by polyclonal T cells. The inability of non-GVH-reactive CBF1 splenocytes to block the lymphopenia-driven proliferation of syngeneic CBF1 splenocytes in RAG KO recipients demonstrates that blockade of this actually is not critical for GVHD resistance.
While previous studies have shown that reconstituted T cells are associated with resistance to GVHD (24,46), ours is the first to demonstrate this phenomenon in the absence of overt inflammatory stimuli and hence to demonstrate that lymphopenia is an independent risk factor for GVHD following DLI. Moreover, previous studies have implicated Tregs as the important element resisting GVHD in T cell-reconstituted hosts (46-48), whereas our studies demonstrate that the presence of Tregs is at best a minor component of the protective effect of T cells against GVHD in the absence of overt inflammatory stimuli. The lack of a role for host Tregs in our studies is consistent with previous findings that Tregs of donor origin can control both acute (15), and chronic GVHD (14), whereas Tregs of host origin are only able to regulate chronic GVHD (14). A high dose of Tregs relative to donor T cells was used in these studies (14,15) while the ratio of host Tregs cells to donor T cells may be quite low in our study.
Inflammation has been shown to play a critical role in the development of GVHD. In mice receiving lethal irradiation as conditioning, bacterial translocation due to gastrointestinal damage caused by the conditioning and the ensuing TLR stimulation have been shown to promote GVHD (49-51). In the setting of delayed DLI to established MCs, systemic injection of a TLR agonist to non-inflamed MCs at the time of DLI results in systemic GVHD, and local application of such a stimulus to the skin resulted in GVHD only in the inflamed skin area in DLI recipients (3). Dynamic imaging studies showed that inflammation was required to allow the trafficking of activated and expanded GVH-reactive T cells from the lymphohematopoietic system, where they mediate GVT effects and convert mixed to full chimeras, to the epithelial GVHD target tissue (3). Several lines of evidence support the view that inflammation is present due to microbial stimuli such as TLR ligands in lymphopenic recipients. Studies in humans demonstrate that CD4 T cell lymphopenia resulting from viral infection such as Human Immunodeficiency Virus-1 (HIV-1) (52) or idiopathic CD4 deficiency (53) is associated with increased bacterial translocation characterized by increased serum concentration of bacterial lipopolysaccharide (LPS). In mice with conditional inactivation of the RAG2 gene, increased levels of serum LPS-binding protein, which could be decreased by reconstitution with CD4 T cells, were detected (54). In addition, previous studies in nude mice demonstrated the presence of bacteria in multiple tissues that could be prevented by thymic transplantation resulting in T cell regeneration (55). These data collectively suggest that, even in the absence of gut damage caused by conditioning, intact T cell-mediated immune function is still required to control bacterial translocation from the gut and its deficiency may lead to systemic inflammation due to TLR stimulation by translocated bacterial products. Our observations that polyclonal, non-alloreactive lymphocytes or broad-spectrum antibiotics, but not irrelevant TCR-Tg T cells, can reduce DLI-induced GVHD in lymphopenic hosts is consistent with a role for microbial products in promoting GVHD in this otherwise non-inflamed setting.
The mechanism by which Treg-depleted polyclonal T cells protect against lymphopenia-associated DLI-mediated GVHD involves reduced accumulation and/or differentiation of effector T cells, as evidenced by the marked reduction in the number of CD4 and CD8 effector cells recovered, blockade of conversion to full donor chimerism and reduced expression of cytotoxic effector molecules and cytokines. There are several mechanisms by which control of inflammation induced by microbial stimuli might have such effects, including reduced activation of host- (56) and/or donor-type APCs (57). As the proliferation of donor T cells was not markedly affected by the presence of non-alloreactive polyclonal T cells, the decreased accumulation of donor effector T cells may reflect reduced survival or differentiation of GVH-reactive effector T cells due to the control of microbial stimuli by the reconstituting T cells. These results are in keeping with our previous demonstration in established mixed chimeras that recipient T cells block the effector functions of GVH-reactive effector cells and that this blockade is overcome by providing systemic TLR stimuli or by conditioning-induced inflammation (58). In addition, our results are also consistent with the important role of TLR stimuli in driving the expansion and differentiation of T cells in other settings, such as viral infections (59,60). Although cytokines driving LIP, such as IL-7 and IL-15, have been shown to be important for T cell survival and are more available in lymphopenic hosts (34), LIP was not impeded by prior administration of non-alloreactive polyclonal T cells, arguing against competition for these cytokines as the mechanism for the observed phenomena. Thus, we favor the explanation that innate activation of APCs by microbial stimuli is required for maximal differentiation and/or survival of GVH-reactive effector cells. Additionally, the induction of tissue inflammation by TLR stimuli directly promotes the infiltration of epithelial GVHD target tissues by GVH-reactive T cells (3), providing an explanation for the reduced GVHD pathology in the epithelial target tissues seen in reconstituted compared to lymphopenic mixed chimeras in our study.
Our study demonstrates the importance of restoring T cell immunocompetence before DLI is given to MCs in efforts to achieve GVT effects without GVHD, as decreased T cell immunity against infections may cause not only bacterial infections but also other types of infections, such as viral infections which have been shown to potentiate GVHD experimentally (61-66) and are associated with clinical GVHD (67,68). Thus, enhancing T cell reconstitution post-HCT will be a critical step in building a non-inflammatory environment needed to achieve GVT effects without GVHD following delayed DLI to mixed chimeric patients. Approaches to achieving immune reconstitution that avoid alloreactivity in the early post-HCT, pre-DLI period are therefore needed. Such approaches might include the administration of cytokines, growth factors or T cell progenitors to enhance T cell recovery (69-73). Alternatively, allodepleted donor T cells (74), if truly devoid of GVH reactivity, might achieve sufficient polyclonal T cell reconstitution to prevent microbial infections and allow the administration of delayed DLI for exploitation of the potent GVHR to optimize GVT effects. Our findings are also of importance to the routine use of DLI to treat relapsed hematologic malignancies following HCT, as this treatment is commonly associated with GVHD. Recognition that lymphopenia predisposes to GVHD, and understanding of the mechanisms by which this occurs, is of wide clinical applicability.
Acknowledgments
We thank Dr. Donna Farber for helpful review of the manuscript, Ms. Kate Orf for technical assistance and Ms. Shavree Washington for expert assistance with the manuscript.
1This work was supported by NCI PO1 grant #CA111519.
2. Abbreviations
- BMT
bone marrow transplantation
- DLI
donor lymphocyte infusion
- GVHD
graft-vs-host disease
- LGVHR
lymphohematopoietic graft-vs-host response
- LIP
lymphopenia-driven proliferation
- MCs
mixed chimeras
- TBI
total body irradiation
- TLR
toll-like receptor Introduction
Reference List
- 1.Pelot MR, Pearson DA, Swenson K, Zhao G, Sachs J, Yang YG, Sykes M. Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen. Bio Blood Marrow Transplant. 1999;5:133–143. doi: 10.1053/bbmt.1999.v5.pm10392959. [DOI] [PubMed] [Google Scholar]
- 2.Sykes M, Sheard MA, Sachs DH. Graft-versus-host-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells. J Exp Med. 1988;168:2391–2396. doi: 10.1084/jem.168.6.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Chakraverty R, Cote D, Buchli J, Cotter P, Hsu R, Zhao G, Sachs T, Pitsillides CM, Bronson R, Means T, Lin C, Sykes M. An inflammatory checkpoint regulates recruitment of graft-versus-host reactive T cells to peripheral tissues. J Exp Med. 2006;203:2021–2031. doi: 10.1084/jem.20060376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen presenting cells. Blood. 2002;100:1903–1909. doi: 10.1182/blood-2002-01-0023. [DOI] [PubMed] [Google Scholar]
- 5.Mapara MY, Kim YM, Marx J, Sykes M. Donor lymphocyte infusion-mediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graft-versus-leukemia effects after conversion to full donor chimerism1. Transplantation. 2003;76:297–305. doi: 10.1097/01.TP.0000072014.83469.2D. [DOI] [PubMed] [Google Scholar]
- 6.Sykes M. Graft-versus-leukemia effect using mixed allogeneic bone marrow transplantation. Bone Marrow Transplantation. 1989;4:465–474. [PubMed] [Google Scholar]
- 7.Spitzer TR. Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies. Bio Blood Marrow Transplant. 2000;6:309–320. doi: 10.1016/s1083-8791(00)70056-5. [DOI] [PubMed] [Google Scholar]
- 8.Sykes M, Preffer F, McAfee S, Saidman SL, Weymouth D, Andrews DM, Colby C, Sackstein R, Sachs DH, Spitzer TR. Mixed lymphohaemopoietic chimerism and graft-ver suslymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation. Lancet. 1999;353:1755–1759. doi: 10.1016/S0140-6736(98)11135-2. [DOI] [PubMed] [Google Scholar]
- 9.Bloor AJC, Thomson K, Chowdhry N, Verfuerth S, Ings SJ, Chakraverty R, Linch DC, Goldstone AH, Peggs KS, Mackinnon S. High response rate to donor lymphocyte infusion after allogeneic stem cell transplantation for indolent non-Hodgkin lymphoma. Bio Blood Marrow Transplant. 2008;14:50–58. doi: 10.1016/j.bbmt.2007.04.013. [DOI] [PubMed] [Google Scholar]
- 10.Spitzer TR, McAfee SL, Dey BR, Colby C, Hope J, Grossberg H, Preffer F, Shaffer J, Alexander SI, Sachs DH, Sykes M. Nonmyeloablative haploidentical stem-cell transplantation using anti-CD2 monoclonal antibody (MEDI-507)-based conditioning for refractory hematologic malignancies. Transplantation. 2003;75:1748–1751. doi: 10.1097/01.TP.0000064211.23536.AD. [DOI] [PubMed] [Google Scholar]
- 11.Heining C, Spyridonidis A, Bernhardt E, Schulte-Monting J, Behringer D, Grullich C, Jakob A, Bertz H, Finke J. Lymphocyte reconstitution following allogeneic hematopoietic stem cell transplantation: a retrospective study including 148 patients. Bone Marrow Transplant. 2007;39:613–622. doi: 10.1038/sj.bmt.1705648. [DOI] [PubMed] [Google Scholar]
- 12.Saito T, Kanda Y, Nakai K, Kim SW, Arima F, Kami M, Tanosaki R, Tobinai K, Wakasugi H, Heike Y, Mineishi S, Takaue Y. Immune reconstitution following reduced-intensity transplantation with cladribine, busulfan, and antithymocyte globulin: serial comparison with conventional myeloablative transplantation. Bone Marrow Transplant. 2003;32:601–608. doi: 10.1038/sj.bmt.1704205. [DOI] [PubMed] [Google Scholar]
- 13.Weinberg K. The effect of thymic function on immunocompetence following bone marrow transplantation. Bio Blood Marrow Transplant. 1995;1:18–23. [PubMed] [Google Scholar]
- 14.Anderson BE, McNiff JM, Matte C, Athanasiadis I, Shlomchik WD, Shlomchik MJ. Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease. Blood. 2004;104:1565–1573. doi: 10.1182/blood-2004-01-0328. [DOI] [PubMed] [Google Scholar]
- 15.Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Donor-type CD4+CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med. 2002;196:389–399. doi: 10.1084/jem.20020399. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Murali-Krishna K, Ahmed R. Cutting Edge: Naive T cells masquerading as memory cells. J Immunol. 2000;165:1733–1737. doi: 10.4049/jimmunol.165.4.1733. [DOI] [PubMed] [Google Scholar]
- 17.Gudmundsdottir H, Turka LA. A closer look at homeostatic proliferation of CD4+ T cells: Costimulatory requirements and role in memory formation. J Immunol. 2001;167:3699–3707. doi: 10.4049/jimmunol.167.7.3699. [DOI] [PubMed] [Google Scholar]
- 18.Min B, Yamane H, Hu-Li J, Paul WE. Spontaneous and homeostatic proliferation of CD4 T cells are regulated by different mechanisms. J Immunol. 2005;174:6039–6044. doi: 10.4049/jimmunol.174.10.6039. [DOI] [PubMed] [Google Scholar]
- 19.Bracci L, Moschella F, Sestili P, La Sorsa V, Valentini M, Canini I, Baccarini S, Maccari S, Ramoni C, Belardelli F, Proietti E. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clin Cancer Res. 2007;13:644–653. doi: 10.1158/1078-0432.CCR-06-1209. [DOI] [PubMed] [Google Scholar]
- 20.Brown IE, Blank C, Kline J, Kacha AK, Gajewski TF. Homeostatic proliferation as an isolated variable reverses CD8+ T cell anergy and promotes tumor rejection. J Immunol. 2006;177:4521–4529. doi: 10.4049/jimmunol.177.7.4521. [DOI] [PubMed] [Google Scholar]
- 21.Chang X, Zheng P, Liu Y. Homeostatic proliferation in the mice with germline FoxP3 mutation and its contribution to fatal autoimmunity. J Immunol. 2008;181:2399–2406. doi: 10.4049/jimmunol.181.4.2399. [DOI] [PubMed] [Google Scholar]
- 22.Moxham VF, Karegli J, Phillips RE, Brown KL, Tapmeier TT, Hangartner R, Sacks SH, Wong W. Homeostatic proliferation of lymphocytes results in augmented memory-like function and accelerated allograft rejection. J Immunol. 2008;180:3910–3918. doi: 10.4049/jimmunol.180.6.3910. [DOI] [PubMed] [Google Scholar]
- 23.Miller JS, Weisdorf DJ, Burns LJ, Slungaard A, Wagner JE, Verneris MR, Cooley S, Wangen R, Fautsch SK, Nicklow R, DeFor T, Blazar BR. Lymphodepletion followed by donor lymphocyte infusion (DLI) causes significantly more acute graft-versus-host disease than DLI alone. Blood. 2007;110:2761–2763. doi: 10.1182/blood-2007-05-090340. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Blazar BR, Lees CJ, Martin PJ, Noelle RJ, Kwon B, Murphy W, Taylor PA. Host T cells resist graft-versus-host disease mediated by donor leukocyte infusions. J Immunol. 2000;165:4901–4909. doi: 10.4049/jimmunol.165.9.4901. [DOI] [PubMed] [Google Scholar]
- 25.Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell receptor antagonist peptides induce positive selection. Cell. 1994;76:17–27. doi: 10.1016/0092-8674(94)90169-4. [DOI] [PubMed] [Google Scholar]
- 26.Kirberg J. Thymic selection of CD8 single positive cells with a class II major histocompatibility complex-restricted receptor. J Exp Med. 1994;180:25–34. doi: 10.1084/jem.180.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ildstad ST, Wren SM, Bluestone JA, Barbieri SA, Sachs DH. Characterization of mixed allogeneic chimeras. Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J Exp Med. 1985;162:231–244. doi: 10.1084/jem.162.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Zhao Y, Rodriguez-Barbosa JI, Swenson K, Zhao G, Arn JS, Sykes M. Highly disparate xenogeneic skin graft tolerance induction by fetal pig thymus in thymectomized mice: Conditioning requirements and the role of coimplantation of fetal pig liver. Transplantation. 2001;72:1608–1615. doi: 10.1097/00007890-200111270-00006. [DOI] [PubMed] [Google Scholar]
- 29.Sykes M, Harty MW, Szot GL, Pearson DA. Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects. Blood. 1994;83:2560–2569. [PubMed] [Google Scholar]
- 30.Sykes M, Abraham VS, Harty MW, Pearson DA. IL-2 reduces graft-versus-host disease and preserves a graft-versus- leukemia effect by selectively inhibiting CD4+ T cell activity. J Immunol. 1993;150:197–205. [PubMed] [Google Scholar]
- 31.Tomita Y, Sachs DH, Khan A, Sykes M. Additional monoclonal antibody (mAB) injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mAB and 3-Gy whole body irradiation. Transplantation. 1996;61:469–477. doi: 10.1097/00007890-199602150-00027. [DOI] [PubMed] [Google Scholar]
- 32.Brandon JA, Jennings CD, Kaplan AM, Bryson JS. Murine syngeneic graft-versus-host disease is responsive to broad-spectrum antibiotic therapy. J Immunol. 2011;186:3726–3734. doi: 10.4049/jimmunol.1003343. [DOI] [PubMed] [Google Scholar]
- 33.Sandau MM, Winstead CJ, Jameson SC. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells. J Immunol. 2007;179:120–125. doi: 10.4049/jimmunol.179.1.120. [DOI] [PubMed] [Google Scholar]
- 34.Surh CD, Sprent J. Homeostasis of naive and memory T cells. Immunity. 2008;29:848–862. doi: 10.1016/j.immuni.2008.11.002. [DOI] [PubMed] [Google Scholar]
- 35.Tan JT, Dudl E, LeRoy E, Murray R, Sprent J, Weinberg KI, Surh CD. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc Natl Acad Sci USA. 2001;98:8732–8737. doi: 10.1073/pnas.161126098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, Hwang LN, Yu Z, Wrzesinski C, Heimann DM, Surh CD, Rosenberg SA, Restifo NP. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med. 2005;202:907–912. doi: 10.1084/jem.20050732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol. 2005;26:111–117. doi: 10.1016/j.it.2004.12.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Wang LX, Li R, Yang G, Lim M, O'Hara A, Chu Y, Fox BA, Restifo NP, Urba WJ, Hu HM. Interleukin-7-dependent expansion and persistence of melanoma-specific T cells in lymphodepleted mice lead to tumor regression and editing. Cancer Res. 2005;65:10569–10577. doi: 10.1158/0008-5472.CAN-05-2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Chung B, Dudl EP, Min D, Barsky L, Smiley N, Weinberg KI. Prevention of graft-versus-host disease by anti IL-7Ralpha antibody. Blood. 2007;110:2803–2810. doi: 10.1182/blood-2006-11-055673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Chung B, Dudl E, Toyama A, Barsky L, Weinberg KI. Importance of Interleukin-7 in the Development of Experimental Graft-Versus-Host Disease. Bio Blood Marrow Transplant. 2008;14:16–27. doi: 10.1016/j.bbmt.2007.07.015. [DOI] [PubMed] [Google Scholar]
- 41.Gendelman M, Hecht T, Logan B, Vodanovic-Jankovic S, Komorowski R, Drobyski WR. Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease. J Immunol. 2004;172:3328–3336. doi: 10.4049/jimmunol.172.5.3328. [DOI] [PubMed] [Google Scholar]
- 42.Alpdogan O, Eng JM, Muriglan SJ, Willis LM, Hubbard VM, Tjoe KH, Terwey TH, Kochman A, van den Brink MRM. Interleukin-15 enhances immune reconstitution after allogeneic bone marrow transplantation. Blood. 2005;105:865–873. doi: 10.1182/blood-2003-09-3344. [DOI] [PubMed] [Google Scholar]
- 43.Miyagawa F, Tagaya Y, Kim BS, Patel HJ, Ishida K, Ohteki T, Waldmann TA, Katz SI. IL-15 serves as a costimulator in determining the activity of autoreactive CD8 T cells in an experimental mouse model of graft-versus-host-like disease. J Immunol. 2008;181:1109–1119. doi: 10.4049/jimmunol.181.2.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Roychowdhury S, Blaser BW, Freud AG, Katz K, Bhatt D, Ferketich AK, Bergdall V, Kusewitt D, Baiocchi RA, Caligiuri MA. IL-15 but not IL-2 rapidly induces lethal xenogeneic graft-versus-host disease. Blood. 2005;106:2433–2435. doi: 10.1182/blood-2005-04-1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Kieper WC, Troy A, Burghardt JT, Ramsey C, Lee JY, Jiang HQ, Dummer W, Shen H, Cebra JJ, Surh CD. Cutting Edge: Recent immune status determines the source of antigens that drive homeostatic T cell expansion. J Immunol. 2005;174:3158–3163. doi: 10.4049/jimmunol.174.6.3158. [DOI] [PubMed] [Google Scholar]
- 46.Johnson BD, Konkol MC, Truitt RL. CD25+ immunoregulatory T-cells of donor origin suppress alloreactivity after BMT. Bio Blood Marrow Transplant. 2002;8:525–535. doi: 10.1053/bbmt.2002.v8.pm12434947. [DOI] [PubMed] [Google Scholar]
- 47.Mielke S, Rezvani K, Savani BN, Nunes R, Yong ASM, Schindler J, Kurlander R, Ghetie V, Read EJ, Solomon SR, Vitetta ES, Barrett AJ. Reconstitution of FOXP3+ regulatory T cells (Tregs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease. Blood. 2007;110:1689–1697. doi: 10.1182/blood-2007-03-079160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Murphy KM, Nelson CA, Sedy JR. Balancing co-stimulation and inhibition with BTLA and HVEM. Nat Rev Immunol. 2006;6:671–681. doi: 10.1038/nri1917. [DOI] [PubMed] [Google Scholar]
- 49.Cooke KR, Gerbitz A, Crawford JM, Teshima T, Hill GR, Tesolin A, Rossignol DP, Ferrara JLM. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest. 2001;107:1581–1589. doi: 10.1172/JCI12156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JLM. Total body irradiation and acute graft-versus-host disease: The role of gastrointestinal damage and inflammatory cytokines. Blood. 1997;90:3204–3213. [PubMed] [Google Scholar]
- 51.Hill GR, Ferrara JLM. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood. 2000;95:2754–2759. [PubMed] [Google Scholar]
- 52.Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, Blazar BR, Rodriguez B, Teixeira-Johnson L, Landay A, Martin JN, Hecht FM, Picker LJ, Lederman MM, Deeks SG, Douek DC. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006;12:1365–1371. doi: 10.1038/nm1511. [DOI] [PubMed] [Google Scholar]
- 53.Lee PI, Ciccone EJ, Read SW, Asher A, Pitts R, Douek DC, Brenchley JM, Sereti I. Evidence for translocation of microbial products in patients with idiopathic CD4 lymphocytopenia. J Infect Dis. 2009;199:1664–1670. doi: 10.1086/598953. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Bourgeois C, Hao Z, Rajewsky K, Potocnik AJ, Stockinger B. Ablation of thymic export causes accelerated decay of naive CD4 T cells in the periphery because of activation by environmental antigen. Proc Natl Acad Sci USA. 2008;105:8691–8696. doi: 10.1073/pnas.0803732105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Owens WE, Berg RD. Bacterial translocation from the gastrointestinal tract of athymic (nu/nu) mice. Infect Immun. 1980;27:461–467. doi: 10.1128/iai.27.2.461-467.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Shlomchik WD, Couzens MS, Tang CB, McNiff J, Robert ME, Liu J, Shlomchik MJ, Emerson SG. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science. 1999;285:412–415. doi: 10.1126/science.285.5426.412. [DOI] [PubMed] [Google Scholar]
- 57.Matte CC, Liu J, Cormier J, Anderson BE, Athanasiadis I, Jain D, McNiff J, Shlomchik WD. Donor APCs are required for maximal GVHD but not for GVL. Nat Med. 2004;10:987–992. doi: 10.1038/nm1089. [DOI] [PubMed] [Google Scholar]
- 58.Chakraverty R, Flutter B, Fallah-Arani F, Eom HS, Means T, Andreola G, Schwarte S, Buchli J, Cotter P, Zhao G, Sykes M. The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells. J Immunol. 2008;181:6820–6828. doi: 10.4049/jimmunol.181.10.6820. [DOI] [PubMed] [Google Scholar]
- 59.Ichinohe T, Pang IK, Kumamoto Y, Peaper DR, Ho JH, Murray TS, Iwasaki A. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Pro Natl Acad Sci USA. 2011;108:5354–5359. doi: 10.1073/pnas.1019378108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Gibbert K, Dietze KK, Zelinskyy G, Lang KS, Barchet W, Kirschning CJ, Dittmer U. Polyinosinic-polycytidylic acid treatment of Friend Retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease. J Immunol. 2010;185:6179–6189. doi: 10.4049/jimmunol.1000858. [DOI] [PubMed] [Google Scholar]
- 61.Cray C, Levy RB. CD8+ and CD4+ T cells contribute to the exacerbation of class I MHC disparate graft-vs-host reaction by concurrent murine cytomegalovirus infection. Clin Immunol Immunopathol. 1993;67:84–90. doi: 10.1006/clin.1993.1048. [DOI] [PubMed] [Google Scholar]
- 62.El Amouri IS, Bani-Ahmad M, Tang-Feldman Y, Lin F, Ko C, Pomeroy C, Oakley OR. Increased morbidity and mortality in murine cytomegalovirus-infected mice following allogeneic bone marrow transplant is associated with reduced surface decay accelerating factor expression. Clin Exp Immunol. 2010;162:379–391. doi: 10.1111/j.1365-2249.2010.04241.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Hossain MS, Roback JD, Wang F, Waller EK. Host and donor immune responses contribute to antiviral effects of amotosalen-treated donor lymphocytes following early posttransplant cytomegalovirus infection. J Immunol. 2008;180:6892–6902. doi: 10.4049/jimmunol.180.10.6892. [DOI] [PubMed] [Google Scholar]
- 64.Kinnier CV, Martinu T, Gowdy M, Nugent JL, Kelly FL, Palmer SM. Innate immune activation by the viral PAMP poly I:C potentiates pulmonary graft-versus-host disease after allogeneic hematopoietic cell transplant. Transplant Immunol. 2011;24:83–93. doi: 10.1016/j.trim.2010.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Roback JD, Hossain MS, Lezhava L, Gorechlad JW, Alexander SA, Jaye DL, Mittelstaedt S, Talib S, Hearst JE, Hillyer CD, Waller EK. Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation. J Immunol. 2003;171:6023–6031. doi: 10.4049/jimmunol.171.11.6023. [DOI] [PubMed] [Google Scholar]
- 66.Via CS, Shanley JD, Shearer GM. Synergistic effect of murine cytomegalovirus on the induction of acute graft-vs-host disease involving MHC class I differences only. Analysis of in vitro T cell function. J Immunol. 1990;145:3283–3289. [PubMed] [Google Scholar]
- 67.Olkinuora H, von Willebrand E, Kantele JM, Vainio O, Talvensaari K, Saarinen-Pihkala U, Siitonen S, Vettenranta K. The impact of early viral infections and graft-versus-host disease on immune reconstitution following paediatric stem cell transplantation. Scand J Immunol. 2011;73:586–593. doi: 10.1111/j.1365-3083.2011.02530.x. [DOI] [PubMed] [Google Scholar]
- 68.Versluys AB, Rossen JWA, van Ewijk B, Schuurman R, Bierings MB, Boelens JJ. Strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes. Bio Blood Marrow Transplant. 2010;16:782–791. doi: 10.1016/j.bbmt.2009.12.534. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.Goldberg GL, King CG, Nejat RA, Suh DY, Smith OM, Bretz JC, Samstein RM, Dudakov JA, Chidgey AP, Chen-Kiang S, Boyd RL, van den Brink MRM. Luteinizing hormone-releasing hormone enhances T cell recovery following allogeneic bone marrow transplantation. J Immunol. 2009;182:5846–5854. doi: 10.4049/jimmunol.0801458. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Jenq RR, King CG, Volk C, Suh D, Smith OM, Rao UK, Yim NL, Holland AM, Lu SX, Zakrzewski JL, Goldberg GL, Diab A, Alpdogan O, Penack O, Na IK, Kappel LW, Wolchok JD, Houghton AN, Perales MA, van den Brink MRM. Keratinocyte growth factor enhances DNA plasmid tumor vaccine responses after murine allogeneic bone marrow transplantation. Blood. 2009;113:1574–1580. doi: 10.1182/blood-2008-05-155697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Kelly RM, Highfill SL, Panoskaltsis-Mortari A, Taylor PA, Boyd RL, Hollañder GA, Blazar BR. Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation. Blood. 2008;111:5734–5744. doi: 10.1182/blood-2008-01-136531. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 72.Kenins L, Gill JW, Boyd RL, Holl+ñnder GA, Wodnar-Filipowicz A. Intrathymic expression of Flt3 ligand enhances thymic recovery after irradiation. J Exp Med. 2008;205:523–531. doi: 10.1084/jem.20072065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Min D, Taylor PA, Panoskaltsis-Mortari A, Chung B, Danilenko DM, Farrell C, Lacey DL, Blazar BR, Weinberg KI. Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood. 2002;99:4592–4600. doi: 10.1182/blood.v99.12.4592. [DOI] [PubMed] [Google Scholar]
- 74.Solomon SR, Mielke S, Savani BN, Montero A, Wisch L, Childs R, Hensel N, Schindler J, Ghetie V, Leitman SF, Mai T, Carter CS, Kurlander R, Read EJ, Vitetta ES, Barrett AJ. Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation. Blood. 2005;106:1123–1129. doi: 10.1182/blood-2005-01-0393. [DOI] [PMC free article] [PubMed] [Google Scholar]









