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. Author manuscript; available in PMC: 2009 Jun 20.
Published in final edited form as: Biol Blood Marrow Transplant. 2007 Jul 16;13(9):1022–1030. doi: 10.1016/j.bbmt.2007.05.008

Host T Cells Affect Donor T Cell Engraftment and Graft-versus-Host Disease after Reduced-Intensity Hematopoietic Stem Cell Transplantation

Nancy M Hardy 1, Frances Hakim 1, Seth M Steinberg 2, Michael Krumlauf 1, Romana Cvitkovic 1,3, Rebecca Babb 1, Jeanne Odom 1, Daniel H Fowler 1, Ronald E Gress 1, Michael R Bishop 1
PMCID: PMC2699412  NIHMSID: NIHMS29269  PMID: 17697964

Abstract

Mixed chimerism in the T cell compartment (MCT) after reduced-intensity stem cell transplantation (RIST) may influence immune repopulation with alloreactive donor T cells. We examined effects of host T cell numbers on donor T cell engraftment and recovery and on acute graft-versus-host disease (GVHD) in a relatively homogeneous patient population with respect to residual host T cells through quantified immune depletion prior to RIST and to donor T cells by setting the allograft T cell dose of 1×105 CD3+ cells/kg. In this setting, two patterns of early donor T cell engraftment could be distinguished by Day +42: (1) early and complete donor chimerism in the T cell compartment (FDCT) and (2) persistent MCT. FDCT was associated with lower residual host CD8+ T cell counts prior to transplant and acute GVHD. With persistent MCT, subsequent development of acute GVHD could be predicted by the direction of change in T cell donor chimerism after donor lymphocyte infusion, and no acute GVHD occurred until FDCT was established. MCT did not affect recovery of donor T cell counts. These observations suggest that the relative number and alloreactivity of donor and host T cells are more important than the absolute allograft T cell dose in determining donor engraftment and acute GVHD after RIST.

Introduction

Reduced-intensity allogeneic hematopoietic stem cell transplantation (RIST) is increasingly used to decrease transplant-related complications and broaden the pool of patients who could potentially benefit from this form of therapy. Reports of outcomes after RIST differ widely in the rate and consistency of donor engraftment, depending to a large extent on host immune status prior to transplantation, the intensity of preparative immune depletion, and the post-transplant immune suppression strategy employed.1-7 A period of mixed donor-recipient chimerism is frequently observed after RIST, with delayed graft-versus-tumor (GVT) effects and occasional graft failure described.1,8,9 It has been suggested that full donor chimerism in the T cell compartment (FDCT) is a prerequisite for both GVT and graft-versus-host disease (GVHD), which implies a potential role for host T cells in limiting donor T cell alloreactivity.1,10,11 Attempts have been made to exploit mixed chimerism in the T cell compartment (MCT) as part of a strategy for attaining immunologic tolerance in transplantation for nonmalignant conditions,12,13 in which alloreactivity plays no therapeutic role. In contrast, others have observed both GVT and GVHD in patients with coincident MCT,2 consistent with MCT reflecting bidirectional host-graft reactivity with ongoing potential for clinical manifestation. The ultimate establishment of FDCT or late graft failure suggests that host-graft interactions may be inherently unstable, culminating in deletion of either the host or donor T cell populations. In the treatment of malignant disease, the clinical relevance of MCT remains controversial - not only in its implications for stable donor engraftment, but also in whether ongoing MCT alters potential GVT efficacy or risk for GVHD.

We examined clinical manifestations of donor chimerism progression in a study of allogeneic RIST for patients with metastatic breast cancer(. Two major aims of the protocol were to demonstrate donor engraftment after RIST with a TCD allograft and to define a GVT effect in breast cancer.10 We adopted an approach of targeted host immune depletion prior to RIST with a uniformly TCD allograft; in order to potentiate GVT effects, this was followed by rapid taper of cyclosporine and planned, sequential DLI. This strategy allowed separation of breast cancer responses attributable to allogeneic cell therapy from those due to cytotoxic chemotherapy given as part of the preparative regimen. The intent of allograft TCD with delayed T cell add-back was to permit rigorous evaluation of GVT effects; this is in contrast to other studies that employed similar approaches to prevent GVHD.14-16 While all patients ultimately completed donor engraftment, the degree and duration of MCT was quite variable. Relatively homogeneous populations with respect to both host and donor T cell numbers at the time of transplantation provided a unique opportunity to study dose effects on engraftment. We performed quantitative assessment of circulating donor and host lymphocyte populations before and after RIST to assess the relationship between immune recovery of host and donor T cells and clinical events after transplant. To differentiate the influence of host immune depletion from those of allograft T cell depletion and delayed DLI, donor engraftment and immune recovery after TCD allografting was compared with patients with hematologic malignancies who were treated with unmanipulated allografts after a similar approach of host immune depletion prior to RIST.

Materials and Methods

Patients and treatments

Data for this analysis came from patients with metastatic breast cancer who were enrolled on National Cancer Institute (NCI) protocol 00-C-0119, which evaluated targeted immune depletion prior to RIST with ex-vivo TCD of the allografts followed by sequential DLI. As previously described,10,17 induction consisting of up to three cycles of fludarabine and cyclophosphamide was given at conventional doses for a targeted CD4+ T cell depletion to below 50 cells/μl. This was followed by RIC with fludarabine and cyclophosphamide. Sibling donors were an exact HLA match for HLA-A, -B, -Cw, -DRB1 and -DQB1 at the low-resolution molecular level, and confirmatory types were performed providing information for HLA-DRB1 at the high-resolution molecular level. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood allografts from donors were ex-vivo T cell-depleted with the Isolex 300i immunomagnetic cell selection system (Nexell Therapeutics, Irvine, CA, USA), using the manufacturer's antibody for positive selection of CD34+ cells and a combination of three T cell-specific monoclonal antibodies (CD2, CD6, CD7) for negative selection of T cells (“positive-negative selection”), resulting in a consistent four- to five-log depletion of T cells. Unmanipulated lymphocytes were added back so that allografts contained a final T cell dose of 1×105 CD3+ cells/kg. Patients received GVHD prophylaxis with cyclosporine through Day +28 and rapidly tapered off by Day +42 to potentiate GVT effects against the breast cancer. Scheduled DLI were administered to all patients without evidence of GVHD (irrespective of donor chimerism) on Days 42, 70 and 98 post-transplant (1×106, 5×106, and 1×107 CD3+ cells/kg, respectively). Patients could receive additional DLI beyond Day +98 if clinically indicated.

Comparison was made with patients with hematologic malignancies treated with unmanipulated allografts (also from HLA-identical siblings with typing performed as above) using a similar preparative approach (NCI 99-C-0143), except the induction regimen used for targeted immune depletion was EPOCH-F (fludarabine, etoposide, doxorubicin, vincristine, cyclophosphamide, and prednisone).18,19 The conditioning regimen was identical to 00-C-0119. GVHD prophylaxis also consisted of single-agent cyclosporine, although it was continued through Day +180. The expected differences in diagnoses and prior therapies on patient immune status at the time of enrollment on the two protocols were minimized by administration of induction with similarly immune-depleting regimens and application of identical criteria (CD4+ T cell count < 50 cells/μl, Grade 4 neutropenia or disease progression) for proceeding to the conditioning regimen and allografting.

Lymphocyte population identification and quantification

The absolute numbers of circulating host CD3+ T cells, CD4+ and CD8+ T cells, natural killer cells, and B cells were determined at study entry, post-induction, and at Days 0, +28 and +98 post-transplant. Flow cytometry was performed in a CLIA-certified laboratory (Science Applications International Corporation; Frederick, MD). Complete blood counts and differentials, performed in the NIH Clinical Center Laboratory, were then used to calculate the absolute number of CD3+, CD4+ and CD8+ T cells (and subsets), natural killer cells and B cells per microliter (μl) of blood from the percentage of the CD45+ population. In the case of T cells, estimation of numbers of circulating cells of donor and host origin were derived from calculated absolute T cell counts and measurements of donor chimerism within CD3+ cells obtained from the same blood draws (described below). T cells and their subsets were defined as follows: T cells were CD3+; CD4+ T cells were CD3+CD4+; CD8+ T cells were CD3+CD8+; B cells were as CD19+CD3− cells and natural killer cells were CD3−CD56+CD16+/−.

Assessment of chimerism

Chimerism analysis was performed by PCR-based comparison of variable number tandem repeats in a CLIA-certified laboratory at the Blood Center of Southeastern Wisconsin (Milwaukee, WI).20 Chimerism determinations on samples enriched for T cells (CD3+) were made every 14 days through Day +98, and monthly thereafter. T cell enrichment to greater than 95% purity was performed either by positive selection using magnetic beads (Miltenyi, Inc.; Auburn, CA) or rosette technique (Stem Cell Technologies, Inc.; Vancouver, BC). Establishment of FDCT was defined as the first time-point after which the donor fraction of CD3-enriched cells was consistently greater than 90 percent.

Statistical analysis

Comparisons within the TCD recipients were made with respect to a specified set of dichotomous parameters (defined in Results and in References21,22). In each of these analyses, as well as comparisons of absolute numbers of lymphocyte populations between TCD and TCR recipients, the comparison was performed using an exact Wilcoxon rank sum test. As a very large number of exploratory analyses were performed, with varying degrees of independence and dependence of the parameters being compared, only p-values less than or equal to 0.005 have been interpreted as indicating a statistically significant difference or trend. P-values greater than 0.005 but less than or equal to 0.05 were interpreted as trends toward statistical significance. All p-values presented are two-tailed and without any adjustment for multiple comparisons.

Results

Patient characteristics and transplant outcomes

Characteristics of patients, donors, and clinical outcomes are summarized in Tables 1 and 2. Nineteen patients received allografts on the TCD study and twenty patients received allografts on the TCR study. Patients enrolled on the two protocols were similar in age, CMV status, donor characteristics and CD34+ cell dose received. Median survival at Days +28 and +100 was similar between the two groups. Overall survival rates were markedly lower in among TCD recipients, largely reflecting higher rates of relapse in this patient population with advanced metastatic breast cancer.

Table 1. Patient and Transplant Characteristics and Outcomes.

Protocol Parameter 00-C-0119: TCD Median (range) 99-C-0143: TCR Median (range)
Patient/Donor Characteristics Number and Indication 19 (Metastatic Breast Cancer) 20 (Relapsed or Refractory Hematologic Malignancy)
Median Recipient Age (Range) 43 years (32 – 56) 44 (19 – 67)
Median Donor Age (Range) 44 years (30 - 65) 43 (16 – 74)
Donor Sex M: 12 (63%); F: 7 (37%) M: 11 (55%); F: 9 (45%)
Female Donor/Male Recipient 0 7 (35%)
CMV Risk 14/19 16/20
Transplant Characteristics Induction Regimen Flu/Cy EPOCH-F
Median Number of Induction Cycles 1 (1-2) 3 (1-3)
Post-Induction Cell Counts, cells/μl CD3 86 (1 – 701) 140 (21 – 441)
CD4, p=0.017 44 (1 – 156) 71 (12 – 191)
CD8 34 (0 – 555) 55 (2 – 309)
NK 58 (0 – 376) 88 (3 – 467)
Day 0 Host Cell Counts, cells/μl CD3 1 (1 – 6) 5 (0 – 42)
Allograft Composition Median CD34+ cells/kg 7.75×106 (5.1 – 12.9) 7.68×106 (4.6-18.4)
Median CD3+ cells/kg 1.0×105 (preset) 3.63×108 (1.5 - 8.3)
Transplant Outcomes
Hematopoietic Recovery Neutrophils greater than 1000 cells/μl Day +9 (8-11) Day +9 (7-12)
Platelets greater than 100,000/μl Day +17 (11-40) Day +16 (12-42)
Donor T Cell Engraftment Stable FDCT Day +70 (14 – 180) Day +14 (14 – 100)
Day +28 FDCT, p=0.0044 7/15 (47%) 17/18 (94%)
(25-100%) (85-100%)
Day +100 FDCT, p=0.023 9/13 (69%) 18/18 (100%)
(35-100%)
Clinical Events Grade II-IV acute GVHD 12/17 (71%) 14/20 (70%)
number/evaluable (%), p=1.00 (13/20 by Day +100) (all by Day +100)
Median Survival (Day +28/Day100) 225 days (89%/79%) 1154 days (100%/90%)
(Breast Cancer) (Hematologic Malignancy)
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Table 2. TCD patient clinical outcomes.

UPN* Post-Induction Host CD8+ T Cell Count FDCT (Day) # DLI Prior to Day +100 # DLI Prior to FDCT Maximum Grade acute GVHD Skin/Gut/Liver** Day of Onset acute GVHD (Grade I) Survival (Days)
24 0 28 1 0 3/2/0 47 172
12 10 126 2 3 0/0/0 n/a 562
16 15 14 1 0 3/1/1 66 148
18 18 21 1 0 2/0/0 32 161
6 18 42 0 0 3/1/0 38 278
10 23 14 0 0 2/1/2 23 76
17 26 14 1 0 1/2/0 46 503
4† 32 n/a n/a n/a n/a n/a 2
11 32 126 2 3 0/0/0 (139) 895
20 34 180 2 4 0/0/0 n/a 225
1 47 98 2 2 2/3/4 103 (40) 172
22 53 21 0 0 3/1/0 42 125
23† 58 28 n/a 0 n/a n/a 26
9 154 ≥98§ 2 2 0/2/0 90 421
3 154 84 2 2 0/0/0 n/a 541
5 206 >42 1 1 0/0/0 n/a 76
7 296 126 2 3 2/3/2 180§§ 352
2 335 126 2 3 1/1/0 (54) >1936
25 555 70 1 1 3/1/0 71 290
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*

Unique patient number

**

Glucksberg Criteria22

†

Excluded from analyses due to death prior to Day +28

§

100% donor in peripheral whole blood

§§

Late-acute GVHD after 4th DLI

Targeted host immune depletion achieved similar reduction of circulating lymphocyte counts in the TCD and TCR protocols, except CD4+ T cells which were somewhat lower among TCD recipients (44 vs. 71 cells/ml, p=0.017). At Day 0, prior to allograft infusion, T cell counts were uniformly less than 50 cells/μl in both groups. The overall incidence of Grade II – IV acute GVHD was not statistically different between the two groups (71% vs 70% for TCD vs TCR, p=1.00). Survival between the two groups at Days +28 and +100 was also similar. Consistent with the prognoses of the underlying malignancies in the two studies, overall median survival was significantly different between the two groups.

Delayed T cell engraftment following TCD RIST

While no graft failure was observed on either protocol, establishment of stable FDCT was significantly delayed in the recipients of TCD allografts compared to TCR (median 70 days vs. 14 days, respectively, p=0.0044, Figure 1). The period of MCT following TCD allografting permitted evaluation of donor T cell engraftment kinetics and host T cell recovery that could not be performed in the TCR recipients due to their rapid establishment of FDCT. Following TCD, two patterns of T cell engraftment were observed: some patients demonstrated rapid establishment of stable FDCT prior to scheduled DLI on Day +42 (eight patients, median Day +21), and others demonstrated persistent MCT until after one or more DLI (ten patients, median Day +126, Table 2 and Figure 2). One patient with rapid engraftment died of infection at Day +26 and was omitted from these analyses. One patient with MCT died of progressive disease after the first DLI, prior to demonstration of full engraftment.

Figure 1. The magnitude and duration of mixed chimerism in the T cell compartment is variable after TID-RIST with TCD.

Figure 1

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A. Peripheral blood T cell donor chimerism after TCD (top) and TCR (bottom) RIST. B. Two patterns of donor T cell engraftment are seen after TCD-RIST. (Top) Rapid engraftment was characterized by stable complete donor T cell chimerism by Day +42, prior to DLI. (Bottom) MCT was characterized by variable donor chimerism in the T cell compartment, and FDCT established after administration of DLI. Black bars: median values; shaded bars: first and third quartiles; lines: ranges.

Figure 2. Progression toward FDCT is uneven after TCD RIST.

Figure 2

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Lines connect T cell chimerism data points for individual subjects with MCT; shaded area: donor chimerism > 90%; solid lines: patients with less than Grade II acute GVHD; dotted lines: patients who developed Grades II-IV acute GVHD; open triangle: Cyclosporine taper; arrows: scheduled DLI.

Mixed chimerism of the T cell compartment

Progression of donor chimerism of the T cell compartment among the MCT subset of TCD recipients was variable and often uneven (Figure 2): donor chimerism decreased between Days +14 and +28 in six of seven evaluable patients; upon completion of cyclosporine taper in three of nine patients; and after the first DLI in four of eight evaluable patients. T cell chimerism fell after the first DLI in all patients who did not ultimately develop acute GVHD, regardless of donor or host predominance at Day +42. All of those in whom T cell chimerism was stable or increased after the first DLI eventually developed acute GVHD. Rapid and consistent progression in donor chimerism characterized T cell engraftment of the subset with early FDCT, in which all patients developed acute GVHD.

Acute GVHD

The specific intent of TCD was not GVHD prevention, and we observed nearly identical overall incidences of acute GVHD after TCD and TCR allografting. The onset occurred somewhat later after TCD than TCR (median onset after TCD: 47 days, range 23 – 180 days; after TCR: 23 days, range 11 – 84 days). Among TCD recipients, all seven evaluable patients who established FDCT prior to DLI developed significant (Grade II-IV) acute GVHD, compared to four of the ten patients with initial MCT (p=.035). Notably, none of the patients developed acute GVHD prior to the establishment of FDCT (Figure 3). Clinical acute GVHD often closely followed T cell engraftment, and all in the rapid engraftment group developed acute GVHD at or soon after the completion of FDCT.

Figure 3. Acute GVHD was observed at or after establishment of FDCT irrespective of DLI administration.

Figure 3

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(A) All seven of the patients in the rapid engraftment subgroup developed acute GVHD, including three prior to receiving any scheduled DLI. Acute GVHD occurred in four of ten patients with MCT. The shaded triangle, below the line connecting symmetrical time points, represents the theoretical area in which values would fall if subjects developed acute GVHD at or before the establishment of FDCT. Arrows: scheduled DLI. (B) Incidence and onset of acute GVHD after TCD according to number of scheduled DLI received.

Scheduled DLI were not administered to patients with active acute GVHD, and so the total T cell dose did not correlate with the development of acute GVHD (Figure 3). Three patients developed acute GVHD precluding administration of any DLI; five patients developed acute GVHD after the first DLI (with 1×106 T cells per kilogram) and two patients after the second DLI (total of 6×106 T cells/kg). Only two of the seven patients who received all three of the scheduled DLI (total of 1.6×107 T cells/kg) developed significant acute GVHD.

Host T cell dose effects

Administration of uniformly reduced allograft T cell doses after ex-vivo TCD permitted evaluation of host T cell dose effects on donor T cell engraftment. Quantification of peripheral blood lymphocyte subsets at Day 21 of the final cycle of induction was used to estimate residual host populations at the time of conditioning for transplantation. The median circulating residual host T cell count was significantly lower in the early FDCT group than in the group with MCT (47 vs. 229 cells/μl, p=0.0097). The median CD8+ T cell count was lower as well (18 vs. 154 cells/μl, p=0.0089, Figure 4), while CD4+ T cell counts and natural killer cell counts were not significantly different between the two groups. Although associated with delayed establishment of FDCT, high residual host CD8+ T cell counts did not necessarily protect against the ultimate development of significant acute GVHD. Although the number of subjects is small, differences in residual host CD8+ T cell counts among those who did and did not develop acute GVHD were not statistically significant (29 vs 154 cells/microl; p=0.40, Figure 4), with extensive overlap between the distributions.

Figure 4. While initial host CD8+ T cell counts were associated with MCT there was no direct association with aGVHD.

Figure 4

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Post-induction circulating CD8+ T cell counts in TCD recipients with MCT and rapid FDCT (left panel) and Grade O-I vs. II-IV aGVHD (right panel). Bars: median values.

Donor chimerism in the T cell compartment reflects relative rates of recovery of host and donor T cell numbers - the net result of proliferation and loss.23 Compared to TCR recipients, Day +28 recovery of total numbers of T cells after TCD was not impaired (246 vs 153 T cells/μl, p=.14). However, T cell chimerism analysis revealed that the number of circulating T cells of donor origin was lower following TCD (100 vs 246/μl, p=0.023). The discrepancy between total and donor T cell counts reflects the frequent persistence of circulating host T cells after TCD allografting that was not evident after TCR grafts. By Day +100, total numbers remained similar and the effect of allograft TCD on donor T cell counts was no longer apparent (481 vs 471 donor T cells/μl, p=.36).

To examine whether MCT among TCD recipients reflected host T cell expansion or compromised donor T cell recovery, we compared peripheral T cell counts among the groups with rapid and MCT patterns of donor T cell engraftment. At Days +28 and +100, neither total nor donor T cell counts were significantly different among those with rapid engraftment compared to those with MCT. In contrast, host T cell counts were higher in those with MCT at both time-points, reaching significance at Day +28 (Day +28: 1 vs. 66 cells/μl, p=0.00067; Day +100: 0 vs. 21 cells/μl, p=0.033, Figure 5). Next we evaluated whether clinical evidence of donor-mediated alloreactivity in the form of acute GVHD influenced peripheral T cell counts. As noted above, acute GVHD did not occur in the setting of MCT; thus the development of acute GVHD followed the elimination of host T cells. While no differences could be detected in total or donor T cell counts at Day +28, donor T cell counts at Day +100 were higher in TCD recipients who developed Grade II-IV acute GVHD than in those who did not (579 vs 184 cells/μl, p=0.045).

Figure 5. Patterns of T cell recovery after TCD.

Figure 5

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(A) Recovery of circulating T cells in TCD recipients with MCT and rapid FDCT shows host but not donor T cell counts are affected by engraftment kinetics. (B) Host vs. donor T cell recovery in those with Grade O-I vs. II-IV acute GVHD suggest greater donor T cell expansion in those who develop acute GVHD. Black bars: median values; shaded bars: first and third quartiles, and lines: range.

GVT Effects

As has previously been reported,10 immune-mediated GVT responses were demonstrated in 32% of TCD recipients, and more than 25% had some disease stabilization which was also likely mediated by GVT in this treatment-refractory group of patients. While all tumor responses were seen at or after the time of FDCT, no differences in rates of engraftment, acute GVHD or immune recovery were found comparing those with and without evidence of a GVT effect (data not shown).

Discussion

Host immune depletion with a defined CD4+ T cell-count target plus ex-vivo allograft T cell depletion with relatively uniform donor T cell dose provided a unique opportunity to investigate donor and host T cell dose effects on donor T cell engraftment and recovery and the development of acute GVHD after RIST. Following administration of TCD allografts, we observed delayed donor T cell engraftment and recovery, evidence of host CD8+ T cell dose effect on the kinetics of donor T cell engraftment, and a relatively high incidence of acute GVHD upon establishment of FDCT.

As has been reported after myeloablative HSCT,24ex-vivo TCD results in delayed donor T cell engraftment after RIST. In contrast to the myeloablative setting, however, even after rigorous immune depletion prior to conditioning, persistence of host T cells contributed to T cell recovery early after RIST with TCD such that total numbers of circulating T cells were not different than those observed after TCR allografting. We did not observe significant recovery of circulating host T cell counts after TCR allografting, although comparable levels of host immune depletion were achieved. This suggests that the relative proportion of donor to residual host T cell numbers at the time of transplantation is an important determinant of early recovery of both donor and host T cell counts, and demonstrates the clinical importance of donor cell-mediated deletion of residual host T cells in the establishment of FDCT. This extends the observations made by several authors1,2,25 that the intensity of cytotoxic therapy received prior to RIST accounts for variable kinetics of T cell engraftment by defining a role for host T cell persistence.

Following RIST with TCD, quantification of circulating host T cells prior to conditioning permitted a standardized evaluation of their effects on donor T cell engraftment and acute GVHD. Two patterns of donor T cell engraftment were observed after TCD, based on donor chimerism prior to DLI administration. Rapid establishment of FDCT prior to DLI was associated with both lower residual host CD8+ T cell counts and development of acute GVHD. Residual host CD8+ T cell counts alone did not predict acute GVHD in all patients, as a minority of patients with MCT had very high residual host CD8+ T cell counts, consistent progression toward FDCT with administration of DLI, and ultimately developed acute GVHD. In the patients with MCT who did not develop acute GVHD, loss of donor chimerism in response to the first DLI, which was not predicted by residual host CD8+ T cell counts, is likely a clinical manifestation of host-vs.-graft reactivity. Although the numbers are small, these observations highlight the importance of the intensity of host-graft reactivity separate from donor and host cell dose effects on clinical transplant outcomes.

The similar incidences of acute GVHD observed after RIST with TCD and TCR allografts are explained in part by the early, rapid cyclosporine taper and administration of DLI after TCD allografting. In our TCD recipients, acute GVHD and, as previously reported,10 GVT effect were not seen until at or after the time FDCT was established. Development of acute GVHD and GVT effect in the setting of MCT have been reported after truly nonmyeloablative HSCT2, while others have reported similar dependence upon FDCT using reduced-intensity regimens.1,26 Two factors that may contribute to the apparent protective effect of MCT include: 1) a host T-cell mediated “veto-like” deletion of alloreactive donor T cells; and 2) the effect of competition, with kinetics of donor T cell expansion and anti-host reactivity favoring hematopoietic tissue-reactive clones over donor T cell populations that are reactive to other host tissues. The decrease in chimerism after DLI observed in the TCD recipients that did not develop acute GVHD may reflect expansion of alloreactive host cells in response to a booster effect of the additional donor cells and/or host-mediated deletion of alloreactive donor cells; the latter would suggest that host cells play an active role in controlling GVHD (and potentially abrogating GVT effect) in the setting of MCT. Our observation that circulating donor T cell counts were not affected by MCT could suggest an absence of such host cell-mediated donor T cell interference. No conclusion can be drawn, however, as circulating counts are not likely to be an accurate reflection of cell populations in the tissues. By Day +100, donor T cells were significantly higher in the subset of TCD recipients that developed acute GVHD, perhaps reflecting antigen-driven peripheral expansion27,28 of host-reactive T cell clones that were not deleted in this group of patients.

On the other hand, reports of acute GVHD in the setting of MCT following nonmyeloablative HSCT2 suggest that host cell persistence is insufficient to protect against clinical acute GVHD, and perhaps when relative and absolute donor and host cell doses allow prolonged host-graft competition for engraftment, clones with specificity for less immunogenic GVH target tissues may outpace anti-hematopoietic counterparts, accounting for clinical GVHD and GVT effect appearing prior to FDCT. Factors that modulate host tissue immunogenicity may further explain the appearance of clinical GVHD prior to elimination of host T cells. For example, truly nonmyeloablative regimens may have greater tissue sparing of host antigen-presenting cells, which are key in the pathobiology of acute GVHD29 and may well play a role in GVT as well,30 and total body irradiation may promote GVHD through widespread, subclinical tissue injury.

Together, these observations suggest the importance of the relative numbers of donor and host T cells over the absolute donor T cell dose in determining the kinetics of engraftment and the development of acute GVHD. Severely immune-depleted patients undergoing RIST, including patients who have been heavily pretreated with cytotoxic therapies, may have a lower allograft T cell dose requirement, and benefit from a lower T cell dose. Further, both rapid establishment of FDCT and, in those with MCT, advancing donor chimerism following low-dose DLI appear to predict the risk of acute GVHD. These patterns of engraftment kinetics may prospectively identify patients who need continued prophylactic immune suppression or in whom to avoid full-dose DLI. Conversely, a decline in donor chimerism following DLI in patients with MCT may herald graft failure and identify a group of patients for whom additionally, higher doses of DLI may be both beneficial and relatively safe. The role of host cells in determining clinical outcomes such as GVT responses, GVHD and immune reconstitution remain important issues for further research.

Acknowledgments

The authors gratefully acknowledge the patients and families who participated on these clinical studies, the expert care provided by the clinical staff, and Drs. Joshua Farber, Andreas Klein and Yu-Waye Chu for critical review and helpful discussion. This research was supported by the Intramural National Cancer Institute, Center for Cancer Research, Bethesda, MD.

Footnotes

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References

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