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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Biol Blood Marrow Transplant. Author manuscript; available in PMC 2016 July 5.
Published in final edited form as:
PMCID: PMC4932864
NIHMSID: NIHMS788145

Favorable Outcomes in Patients with High Donor-Derived T Cell Count Following in vivo T Cell Depleted Reduced Intensity Allogeneic Stem Cell Transplantation

Amir A. Toor, M.D.,1 Roy T. Sabo, Ph.D.,2 Harold M. Chung, M.D.,1 Catherine Roberts, Ph.D.,1 Rose H. Manjili, B.S.,1 Shiyu Song, M.D., Ph.D.,3 David C. Williams, Jr, M.D., Ph.D.,4 Wendy Edmiston, B.S., R.N.,1 Mandy L. Gatesman, Pharm.D.,1 Richard W. Edwards, PA-C,1 Andrea Ferreira-Gonzalez, M.D., Ph.D.,4 William B. Clark, M.D.,1 Michael C. Neale, Ph.D.,5 John M. McCarty, M.D.,1 and Masoud H. Manjili, D.V.M., Ph.D.6

Abstract

Patients with hematological malignancies were conditioned using a rabbit anti-thymocyte globulin based reduced intensity conditioning regimen for allogeneic stem cell transplantation (SCT). Donor-derived CD3+ cell count (ddCD3), a product of CD3+ cell chimerism and absolute CD3+ cell count, when less than 110/μL, eight weeks post-transplant, predicted a high risk of sustained mixed chimerism and relapse. Alternatively, patients with a higher ddCD3 developed GVHD more frequently, and when partially chimeric, had higher rates of conversion to full donor chimerism upon withdrawal of immunosuppression. In conclusion, early data from a small cohort of patients indicates that ddCD3+ cell count at 8 weeks may be used to guide the decision-making process regarding withdrawal of immunosuppression and administration of donor lymphocyte infusion in partially T cell depleted reduced intensity regimens.

Keywords: Donor derived T cells, chimerism, ATG, allogeneic stem cell transplant, GVHD

Introduction

Reduced intensity conditioning regimens for allogeneic stem cell transplantation (SCT) are well tolerated, but are characterized by variable immunological recovery, particularly when T cell depletion (TCD) is performed to reduce graft versus host disease (GVHD) risk (1-5). TCD may be performed either in vivo by administration of anti-thymocyte globulin (ATG) during conditioning or ex vivo by a variety of allograft T cell purging techniques. ATG reduces the risk of chronic GVHD and non-relapse mortality in matched related donor (MRD) SCT recipients conditioned with myeloablative regimens (6). Additionally, outcomes in unrelated donor (URD) SCT are improved when either ATG is incorporated in the conditioning regimen (7, 8) or when the allograft is ex vivo T cell depleted with CD6 monoclonal antibodies (9).

However, when T cell depletion is performed in SCT conditioned with reduced intensity regimens, post-transplant outcomes such as GVHD and relapse, are influenced by the level of donor T cell chimerism achieved. Furthermore, mixed donor-recipient chimerism in the T cells often complicates such transplants. In a recently published report, when CD52 monoclonal antibody was used for TCD along with a reduced intensity regimen, a 50% incidence of mixed chimerism (MC) was observed in the T cells at day 100 following SCT. Moreover, declining T cell chimerism was associated with an increased relapse risk (10). Others have found similarly poor outcomes with MC in the T cells in the first month after reduced intensity SCT, particularly when T cell chimerism was <60% (11). Level of T cell chimerism following transplant also affects the response to donor lymphocyte infusions (DLI). Patients conditioned with ATG and reduced intensity allografting had a high rate of graft loss despite prophylactic DLI if T cell chimerism was <20% donor, and high rate of conversion to full donor chimerism (FC) if it was >40% (12). In addition to T cells, NK cell chimerism has also been reported to influence risk for GVHD and graft loss in patients undergoing T cell replete non-myeloablative allografting (13), underscoring the interaction between various effectors of cellular immunity. Generally, the studies incorporating T cell replete allografts report frequent mixed donor-recipient chimerism in the T cells early on after reduced intensity transplantation, which over time converts to full donor chimeric as immunosuppression is withdrawn. Often this shift in chimerism is accompanied by the development of GVHD, potentially compromising outcomes. Conversely, in those undergoing TCD allografts, withdrawal of immuno-suppression results in less precisely predictable outcomes in patients with mixed T cell chimerism, with either maintenance of stable mixed chimerism or occasionally graft loss being observed. Moreover, MC is also accompanied by increased relapse risk (14,15). DLI may be used to convert patients who are mixed chimeric to full donor chimerism and reduce relapse risk, but are complicated by the development of acute or chronic GVHD in as many as 50% of the patients, (16,17) even when CD8 depleted DLI are used (18,19). Alternative strategies in patients with mixed chimerism such as administration of low-dose prophylactic DLI, though less likely to cause GVHD, are ineffective (4).

Because of the unfavorable outcomes associated with the mixed chimeric state, a reliable predictor for the expected evolution of mixed T cell chimerism is needed to help in clinical decision-making regarding withdrawal of immunosuppression and DLI. An alternative immune recovery parameter with prognostic value is T cell recovery post transplant (20, 21). We decided to combine this measure with T cell chimerism and examine the predictive value of a calculated donor-derived T cell count for clinical outcomes following allogeneic SCT conditioned with rabbit ATG and reduced intensity total body irradiation (TBI). This regimen is based on pre-clinical studies in murine transplantation demonstrating engraftment across MHC barrier when T cell antibodies were combined with low dose radiation (22, 23). Feasibility of this approach in human transplantation has been demonstrated in clinical trials, which established a low risk of severe acute GVHD, albeit with high rates of mixed donor-recipient chimerism and occasional patients developing graft loss (1, 3, 24, 25). The current trial examines the effect of two doses of rabbit ATG in recipients of allogeneic stem cell transplantation with post transplant immune reconstitution as the primary endpoint of the trial. (Clinicaltrials.gov identifier: NCT00709592)

Materials and Methods

Patients and eligibility

Consecutive patients were enrolled on a prospective randomized phase II clinical trial, approved by the institutional review board at Virginia Commonwealth University. To be eligible, patients had to be between 18 and 70 years of age, have recurrent or high-risk hematological malignancy, and have adequate end-organ function and performance status. Patients younger than 50 years had to be ineligible for conventional myeloablative conditioning because of comorbidity. The patients were required to have a 7/8 or 8/8 locus matched related (MRD) or unrelated donor (URD), with high-resolution typing performed for HLA-A, B, C and DRB1.

ATG+TBI Conditioning Regimens

The patients were randomized between two different doses of rabbit-anti-thymocyte globulin (ATG 2.5 or 1.7 mg/kg adjusted ideal body weight/day; Thymoglobulin®, Genzyme, Cambridge, MA) given intravenously on day –9 through –7, followed by TBI to a total dose of 4.5 Gray, delivered in three 1.5 Gray fractions, administered twice on day –1, with the final dose on day 0. Methylprednisolone at a dose of 2 mg/kg was given as premedication for ATG. GVHD prophylaxis was with tacrolimus given orally from day –2 with taper commencing around 12 weeks post transplant. Mycophenolate mofetil was given orally at a dose of 15 mg/kg twice daily from day 0 to 28. G-CSF was given at a dose of 5 μg/kg/d from day 4 until myeloid engraftment. Blood stem cells were collected using G-CSF 10 μg/kg/d given subcutaneously on day 1 through 5. Escalating dose DLI was permitted beyond 8 weeks post SCT for the management of declining or persistent mixed chimerism (initial dose 1×106 CD3+cells/kg) and for disease progression (initial dose 5×106 CD3+cells/kg).

T cell engraftment analysis

Donor engraftment was measured using chimerism analyses performed at 4, 8, 12, and 24 weeks following transplant on whole blood, granulocytes, and total T cells. Blood cell separations were accomplished using immuno-magnetic beads (Miltenyi Biotec, Inc.) enriching for CD15 and CD3 expressing cells separated on a Miltenyi AutoMACS Pro Separator (Miltenyi Biotec, Inc.). DNA was isolated using Qiagen EZ1 200 μL Whole Blood Isolation Kit (Qiagen Inc.) on a Qiagen EZ1 Biorobot (Qiagen Inc.); PCR was performed on GeneAmp® PCR System 9700 (Applied Biosystems) with the GenomeLab Human STR Primer Set (Beckman Coulter, Inc.); and capillary electrophoresis of the amplification products was performed on the Beckman Coulter Vidiera NsD Analyzer (Beckman Coulter, Inc.) to determine the short tandem repeat (STR) alleles of the donor and patient. Donor chimerism was calculated for each STR from the areas of unique recipient and donor peaks using the manufacturer's software (Beckman Coulter, Inc.), and averaged over all informative STR alleles. Immuno-phenotypic analysis of the blood for immune reconstitution was performed concomitantly with chimerism analysis, using a dual-platform technique on a Cytomics™ FC500 flow cytometer (Beckman Coulter Inc., Miami, FL). Antibodies to CD3 and CD56 (Beckman Coulter, Inc.) were employed to enumerate T cells and NK cells.

Donor-derived CD3+ T cell count (ddCD3) was calculated by multiplying the T cell chimerism (% recipient DNA, expressed as a fraction) with the absolute blood CD3+ T cell count obtained simultaneously. The resulting absolute recipient derived CD3+ T cell count was then subtracted from the total absolute CD3+ cell count to obtain a donor-derived CD3+ T cell count.

Study Design and Statistical analysis

This randomized phase II study compared two different ATG doses used in conditioning and was designed with immune reconstitution as the primary endpoint, with engraftment, GVHD rate and survival serving as secondary endpoints. Engraftment was defined as sustained hematopoietic recovery after transplant with <5% recipient chimerism in whole blood or relevant cell fraction. Overall survival was taken from the day following transplant to the day of death. GVHD was classified according to consensus criteria. Acute GVHD was graded according to the Glucksberg criteria. Because of the small patient number and relatively low event rate, acute and chronic GVHD data were pooled when analyzing GVHD risk. GVHD observations were censored if this complication developed after DLI. Disease specific criteria were used for diagnosing relapse or progression.

To determine the optimal cut-off to categorize ddCD3 into High and Low groups, we first measured the association between ddCD3 cell count and clinical outcomes of interest to determine the logistic regression for each outcome, and then determined the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for logistic regressions for each of the clinical outcomes of interest against the ddCD3. The cut-off that maximized the sum of ROC-AUC across all measures was selected. The Kruskal-Wallis test was used to test for mean differences in continuous variables between patients in the ddCD3 groups. To further analyze these relationships, an informative normal prior was used for the mean measure in each ddCD3 group (based on the overall sample mean and variance), and is combined with a normal likelihood for the observed data. Fisher's exact test (FET) was used to analyze the relationship between ddCD3 groups and categorical measurements. As an additional analysis, non-informative Beta priors are assumed on the proportions in both ddCD3 groups, and are coupled with binomial likelihoods for the observed data. Kaplan Meier curves were estimated to account for the timing of remission and overall survival, and differences between the low and high ddCD3 groups were tested using the log-rank test. Corroboratory Bayes methods were also used and for both continuous and categorical cases, direct sampling (with 100,000 repetitions) from the conjugate posterior distributions for each ddCD3 group was used to estimate posterior probabilities (PP) of differences between the two ddCD3 means or proportions (26). For all frequentist analyses, a mean or rate difference between ddCD3 groups is considered significant for p-values < 0.05, while for all Bayes analyses, a PP > 0.95 is considered sufficient evidence of a mean or proportion difference between the ddCD3 groups data summaries. Fisher's exact tests and Kruskal-Wallis tests are conducted using the FREQ and NPAR1WAY procedures in the SAS statistical software (version 9.2, Cary, North Carolina, USA), while Bayes analyses are conducted using the R statistical software.

Results

Patients

Between 2008 and 2011, 25 patients were enrolled in this trial, 22 of whom are eligible for this analysis. One patient was excluded because of disease progression and autologous reconstitution at 4 weeks following SCT, while two other patients have not been enrolled long enough to be evaluable for transplant outcomes. Median age of the 22 evaluable patients was 58 years (range 44-68); diagnosis was multiple myeloma in 7, non-Hodgkin lymphoma in 6, chronic lymphocytic leukemia (or pro-lymphocytic leukemia) in 7, myelodysplastic syndrome and acute myeloid leukemia in one each. Most patients had experienced multiple relapses and had been heavily pretreated, having received a median of 4 prior chemotherapy regimens (range 2-10) and 11 patients had undergone prior autologous SCT. Fourteen patients were in complete remission at the time of SCT, whereas 8 patients had a partial remission or persistent, stable disease. Eleven (50%) patients were included in each ATG dose cohort. The conditioning regimen was tolerated well with no day 100 transplant related mortality observed.

Engraftment and Chimerism

Predominantly donor-derived hematopoiesis was promptly established following transplantation. The median whole blood hematopoietic and granulocyte chimerism was 0% recipient derived at all the time points evaluated post transplant in the 22 evaluable patients. Median T cell chimerism measured simultaneously was 5%, 1.5%, 5% and 0% respectively at 4, 8, 12 and 24 weeks post transplant. T cell chimerism could not be measured in 7/22 patients at 4 weeks post transplant because of inadequate DNA yield consequent to low T cell recovery at that time. Nine of the twenty-two patients had mixed T cell chimerism (≥5% recipient DNA) at 8 weeks post transplant, and of these 33% (n=3) went on to become fully donor T cell chimeric after withdrawal of immunosuppression over the ensuing weeks. One of the nine patients had improvement in donor chimerism after DLI. Of the 13 patients who were full donor chimeric in T cells, one patient reverted from full donor to mixed chimerism.

Clinical outcomes with ATG+TBI conditioning

Classical onset acute cutaneous GVHD developed in 3 patients (grade 1-2 in all), and of these patients two evolved to progressive delayed onset acute grade 3 and 4 gastrointestinal GVHD. Two other patients developed delayed onset acute gastrointestinal GVHD (grade 3 and 4) after withdrawal of immunosuppression (Table 1). Chronic GVHD has developed in 5 other patients (4 with classical onset and one with overlap syndrome). All patients who developed GVHD demonstrated complete donor chimerism at the time of GVHD diagnosis. Relapse or persistent disease was observed in 7/22 (32%) patients of whom 5 had mixed chimerism or declining T cell chimerism following initial engraftment. Five patients received DLI at a median of 204 days post transplant (range 99 to 362) for the management of persistent or relapsing disease with (n=4) or without (n=1) mixed chimerism; of these 2 died of progressive disease (one despite developing GVHD), while the other three remain progression free.

Table 1
Patient outcomes according to ddCD3, eight weeks following transplant

Donor-derived CD3+ cell count

T cell chimerism and simultaneously measured absolute CD3+ cell counts were used to calculate donor-derived CD3+ cell count (ddCD3) at 8 weeks following transplant because reliable data were available for all patients for this time. Median ddCD3+ cell count at 8 weeks was 433/μL (range: 3-2464). After calculating the sum of receiver operating characteristic AUCs for each of the potential ddCD3 cut-off values (Figure 1), a ddCD3 of 110/μL was found to be optimal with both the highest AUC sum and the highest AUC for each measure (cumulative GVHD- present or absent: 0.79, remission- present or absent: 0.74, whole blood chimerism- FC vs. MC: 0.88). We therefore used 110/μL as the cut-off value to distinguish between low (< 110) and high (> 110) values of ddCD3. Based on this criterion, there were 8 patients with low ddCD3 and 14 patients with high ddCD3. Notably, based on the cutoff of 110, the median ddCD3 in the corresponding groups were 60 and 855/μL (Table 3) respectively, suggesting a marked difference in T cell reconstitution between the two groups.

Figure 1
Area Under Curve (AUC) for Receiver Operating Characteristic (ROC) curves for GVHD status, remission status, and whole-blood chimerism against ddCD3 group. Table depicting ddCD3 cutoff calculated from sum of AUC for different ROC curves.
Table 3
Summary of Continuous (Sample Size (N), Median and Inter-Quartile Range (IQR)) and Categorical (Sample Size (N), Frequency and Proportion) Measurements

Clinical impact of donor-derived CD3+ cell count

Significant relationships were found between ddCD3 levels and cumulative GVHD status (present or absent), remission status (present or absent) and whole blood chimerism status (FC or MC) reflecting engraftment, at and beyond 12 weeks following SCT (Table 2). Using frequentist methods, the proportion of patients with MC in whole blood in the low-ddCD3 group was significantly higher than in the high-ddCD3 group (FET: p < 0.0001). Likewise the proportion of patients diagnosed with GVHD in the low-ddCD3 group was significantly lower than in the high-ddCD3 group (FET: p = 0.024). Similarly, the proportion of patients in sustained remission in the low-ddCD3 group was marginally lower than in the high-ddCD3 group (FET: p = 0.0524). These results were corroborated by the Bayes methods, which found higher whole blood mixed chimerism rates (PP = 0.99), lower GVHD rates (PP = 0.99), and lower remission rates (PP = 0.99) in the low-ddCD3 group as compared to the high-ddCD3 group. Consequently, with a median follow up of 18 months (range: 4-29 months) in the surviving patients, both overall and progression-free survival (Figure 2A and B) as well as time to relapse (Figure 2C) were favorable in the high-ddCD3 group as compared to the low-ddCD3 group, but while the relationship was significant for relapse ( χ12=7.9, p = 0.005) and progression-free survival (X2 = 5.3, p = 0.02), it was only marginally significant for overall survival ( χ12=3.2, p = 0.07). Correspondingly, one-year Kaplan-Meier estimates for overall survival (84% vs. 54%) and progression-free survival (69% vs. 15%) are superior in the high- compared to the low ddCD3 cohort, with all the relapses occurring in the first year following SCT. Notably, treatment related mortality is similar (16% vs. 15%) in both cohorts.

Figure 2Figure 2
A: Kaplan Meier survival curve for overall survival time, comparing ddCD3 Hi (>110) and Lo (<110) groups.
Table 2
Relative clinical outcomes in patients with low or high ddCD3 recovery

To confirm the utility of ddCD3 cell count in predicting clinical outcomes, additional analyses were undertaken to assess the impact of its factors in isolation. We found no significant associations between absolute T cell count and T cell chimerism at eight weeks post-transplant with mortality (T cell recovery: p = 0.65, % T cell Chimerism: p = 0.57), relapse (T cell recovery: p = 0.51, % T cell chimerism: p = 0.47) and GVHD (T cell recovery: p = 0.35, % T cell Chimerism: p = 0.21).

Kinetics of donor-derived T cell reconstitution

When examined over time, patients who had ddCD3 <110/μL and mixed T cell chimerism had either declining donor or persistent mixed T cell chimerism as immunosuppression was withdrawn, (Fig 3A) whereas those with ddCD3 >110/μL had increasing donor T cell chimerism over time as immunosuppression was tapered off (Fig 3B). This resulted in a progressive rise in the absolute ddCD3 count over time in the patients with a higher ddCD3 (Fig 3C) and was also reflected in the other hematopoietic lineages, as patients with high ddCD3 also demonstrated a progressive increase in whole blood chimerism over time (Fig 3D). Modulation of the effect of immunosuppression withdrawal by the ddCD3 count was best exhibited in the rare patient with mixed granulocyte chimerism (Fig 4). Early DLI arrested the decline of donor hematopoiesis in the patient with low ddCD3 recovery.

Figure 3Figure 3
A: T cell chimerism trend in patients with mixed chimerism and ddCD3 <110/μL.
Figure 4
Effect of immunosuppression withdrawal on granulocyte chimerism in patients with high or low ddCD3.

Effect of pre- & post-transplant variables on ddCD3

No significant relationships were found between ddCD3 levels and donor age, donor type (MRD vs. URD), total ATG dose (5.1 vs. 7.5 mg/kg) and the infused graft CD34+ or CD3+ cell dose. (Table 3) Using frequentist methods, means for donor age ( χ12=0.5, p = 0.49), infused CD3 ( χ12=0.0, p = 0.84) and CD34+ cell dose ( χ12=0.5, p = 0.49), as well as proportions for donor type (FET: p = 0.66) and ATG dose (FET: p = 0.66), were not significantly different between the two ddCD3 categories (high and low). These results were corroborated by the Bayes statistical methods, which showed small posterior probabilities (PP) for mean differences between ddCD3 groups for donor age (PP = 0.83) and CD34+ cell dose (PP = 0.72), as well as proportion differences between ddCD3 groups for ATG dose (PP = 0.80) and donor type (PP = 0.26).

NK cell reconstitution following SCT was examined to determine interrelationship between it and ddCD3 cell count. NK cell counts were higher in the high-ddCD3 group than in the low-ddCD3 group at 8 weeks ( χ12=4.1, p = 0.044; PP = 0.99). However, post-transplant NK cell recovery at 4 and 8 weeks did not influence the ddCD3 count ( χ12=0.8, p = 0.37; PP = 0.88). We analyzed the effect of NK cell recovery on clinical outcomes of interest such as, GVHD ( χ12=0.4, p = 0.55), remission status ( χ12=2.4, p = 0.12), whole blood engraftment ( χ12=3.3, p = 0.07), as well as on time to event measurements of mortality (p = 0.56), relapse (p = 0.10) and GVHD (p = 0.40) and unlike ddCD3 cell count found no measurable independent effect in our patient cohort.

Discussion

Cellular immune reconstitution post transplant is known to be of prognostic relevance, particularly with respect to relapse. In patients undergoing T cell depleted SCT a higher rate of mixed chimerism is seen, and subsequently immune reconstitution is delayed with slower recovery of donor T cells. Neither chimerism analysis nor T cell subset recovery consistently predicts the effect of withdrawing immunosuppression in these patients. Patterns of T cell chimerism trends have been described and generally fall into four broad categories: a) early establishment of stable full donor chimerism; b) early mixed chimerism evolving to full donor with withdrawal of immunosuppression; c) stable mixed chimerism despite withdrawal of immunosuppression; and d) mixed chimerism (or early full chimerism) with late autologous reconstitution and graft loss (27-29). Generally, patients with mixed chimerism have inferior outcomes; especially those with high levels of recipient chimerism in T cells are at a higher risk of eventual graft loss when undergoing T cell depleted allografts. Clearly, early identification of the chimerism pattern patients are likely to experience will allow modification of therapy and reduction of relapse and graft loss risk.

In order to get a more accurate measure of the magnitude of allo-immune reconstitution, and downstream effect, we examined the impact of donor-derived T cell recovery on post-transplant outcomes in patients conditioned with an ATG based preparative regimen. This parameter, donor-derived CD3+ cell count, was calculated from simultaneously measured CD3+ T cell chimerism and absolute CD3+ cell count in circulation. The earliest time point at which ddCD3 cell count could be determined in all our patients was at eight weeks following transplant. This was likely secondary to ATG effect and the extended course of mycophenolate our patients received for GVHD prophylaxis, with the testing schedule being an obvious determinant. Nevertheless, even at this relatively early time point, the ddCD3 cell count has predictive value for clinical outcomes developing later in the course of transplant. This effect was particularly striking for donor engraftment when the ROC curves for ddCD3 cell count effect on common post transplant parameters were determined, and when hematopoietic and T cell chimerism was plotted over time for patients who fell into high and low ddCD3 cell recovery groups at 8 weeks post transplant. The absolute magnitude of donor T cell recovery and its effect in our patients reflects the conditioning regimen used and underlying disease biology, and is thus unique to this patient population and most likely subject to revision with a larger sample size. This is particularly so because of the relatively low value of the ddCD3 cutoff (110/μL) in our patients, likely a manifestation of the T cell depleting regimen. Thus, the timing and magnitude of prognostically important ddCD3 will likely differ with the conditioning regimen, and particularly with different T cell depletion strategies.

Timing and magnitude aside, the most clinically relevant aspect of the ddCD3 cell count is its association with hematopoietic and T cell chimerism, upon withdrawal of immunosuppression. Generally, mixed T cell chimerism following reduced intensity SCT is managed by cessation of immunosuppression in T cell-replete allografts, however this may result in progressive graft loss in T cell depleted allografts, particularly in patients with mixed chimerism. That the patients with a higher ddCD3 have a more readily predictable trajectory in both chimerism and in T cell reconstitution over time is intuitive, and provides a discriminator for decision-making regarding DLI in recipients of T cell depleted reduced intensity allografts. Whether this adds to the general value of T cell chimerism measurement alone is not addressed in our report, however in our small data set it appears to be informative, particularly when the downstream effects of T cell engraftment are examined, i.e., relapse and GVHD. As this measure accounts for both quantitative and qualitative donor T cell recovery post transplant with a simple calculation, the observed association with downstream outcome is likely to be confirmed in larger cohorts.

Similar findings have been reported in patients undergoing transplantation following TCD allogeneic SCT and scheduled DLI. In a cohort of breast cancer patients, relative preponderance of host-derived T cells was observed at day 28 and 100 following transplantation in patients with mixed chimerism. Conversely, patients who developed grade 2-4 GVHD despite TCD, had a significantly higher number of donor-derived T cells in circulation at day 100 (30). These findings are consistent with our observation of a higher rate of GVHD in patients with a higher ddCD3 at eight weeks. In recipients of T cell replete allografts, conditioned with Fludarabine and 2 Gray total body irradiation, and with early discontinuation of immunosuppression, a day 14 donor-derived CD8+ cell count of >0.043*106/ml (>43/μL) was strongly associated with grade 2-4 acute GVHD. Similar to our cohort, no predictors for donor-derived CD8+ cell recovery were seen in this study when donor age and graft composition were analyzed (31). Together with our data these findings suggest that the magnitude of donor derived T cell recovery has a critical role in allo-immune responses following SCT.

Although ddCD3 was largely predictive of relapse (and thus need for DLI) in our patients, there were nonetheless two patients with high ddCD3 cell count who relapsed. Both had heavily pretreated myeloma with adverse cytogenetics, suggesting the primacy of disease biology in prognostic determination. Adverse impact of disease status and cytogenetics in patients undergoing reduced intensity, in vivo T cell depleted, allografts is well reported (32, 33). Therefore ddCD3 cell counts should be interpreted in light of disease biology when used for decision-making about the need for DLI versus ongoing immunosuppression in patients who have undergone T cell depleted, reduced intensity allografts.

Our data set is not large enough to establish whether ddCD3 adds to the value of isolated T cell chimerism and absolute T cell count measurement in most patients undergoing SCT, however in selected patients with poor immune reconstitution this more precise metric does have additional predictive value for post transplant outcomes beyond the factors that determine it. In our estimation donor-derived CD3 cell count appears to be of greatest value in patients who have low lymphocyte count recovery or high levels of recipient chimerism. To better understand its value one may consider, the general nature of biologic dose-response relationships, which often tend to follow a sigmoid or logistic relationship with threshold effects observed. Although this may not be entirely true for T cell mediated responses which are antigen specific, or binary, increasing donor derived T cell counts would increase the likelihood of the presence of relevant recipient or tumor-antigen specific T cell clones, thus the association of higher ddCD3 counts with GVHD and freedom from relapse. We speculate that our data would best define outcomes of the patients on the steep part of the above referred to sigmoid relationships between T cell reconstitution and post transplant outcome likelihood. This is reflected by the ROC-AUC curves for GVHD, relapse and achievement of full chimerism. Additional studies examining the impact of ddCD3 in larger cohorts of patients will be needed before it can be established as a qualitative and quantitative measure to predict onset and magnitude of GVHD and graft versus tumor effects, which are largely determined by minor histo-incompatibilities between HLA identical donors and recipients (34-36). In addition, the likelihood of developing GVHD may eventually be more accurately predicted by assays, such as T cell clonality.

The interaction between hematopoietic lineages and T cells is likely important in determining the rate and durability of T cell engraftment, and also the donor-derived T cell recovery. Investigating patterns in T cells and dendritic cells, mixed chimerism in dendritic cells with full chimerism in T cells predicts the development of allo-immune response (37). NK cell reconstitution following allografting has been studied as a predictor of post transplant outcomes, and has been found to have variable effect on outcomes such as graft rejection and relapse (13, 38, 39). Although we were not able to identify an effect of post transplant NK cell reconstitution on ddCD3 cell count recovery, patients with a high ddCD3 cell count also had higher NK cell counts at the eight-week mark, suggesting interactions between factors which determine cellular immune reconstitution. None of the graft and patient characteristics we studied, such as CD3+ cell dose infused or ATG dose employed in conditioning, were predictive of cellular immune recovery. However, parameters such as ATG levels at the time of graft infusion, tacrolimus and mycophenolate levels post transplant, and minor histocompatibility differences are not accounted for in our analysis and may well have a deterministic effect on ddCD3 cell recovery and subsequently outcomes. It is therefore reasonable to state that clinicians caring for SCT recipients should closely follow cellular immune reconstitution following transplantation, and depending on disease biology, intervene with measures such as DLI if this process is delayed.

Our findings can only be considered preliminary keeping in mind the small sample size of our cohort, and the heterogeneity of disorders for which the transplants were performed. Therefore to increase the confidence in the validity of our findings, Bayes methods are included in addition to the proposed classical methods because they: (a) account for small sample sizes; and (b) yield interpretable inference on the parameters of interest, rate or mean differences, that is often similar to inference from frequentist methods (40). In analyses such as ours, a particular concern is that a substantial sample size may be required to deliver adequate statistical power and yield appropriate asymptotic behavior of the statistics being used (26). However, the posterior probabilities obtained using Bayes methods are valid even for small sample sizes, since those probabilities are conditioned upon the observed data and, by necessity, the sample size. Thus a posterior probability reflects the data at hand, and will require more statistical evidence in order to (e.g.) produce a high probability at smaller sample sizes than at larger sample sizes.

In conclusion, we report the effect of donor T cell reconstitution, easily calculated from routinely measured post-transplant variables, on clinical outcomes, particularly stability of engraftment and disease relapse. Donor-derived CD3+ cell count may help in the decision-making regarding immuno-suppression withdrawal and DLI timing in allogeneic SCT recipients of in vivo T cell depleted allografts. The optimal ddCD3 cell count cutoff will, however, have to be refined by examining this parameter in larger patient cohorts with comparable disease biology, over a range of time and conditioning regimens before it can be widely applied in the clinic.

Acknowledgments

We would like to acknowledge the nurses, nurse practitioners and coordinators in the bone marrow transplant program at Virginia Commonwealth University. Special thanks are extended to Ms. Cheryl Jacocks-Terrell for help in preparing the manuscript, to Mrs. Carol Cole and Laura Couch for protocol management. We also acknowledge Genzyme Corporation for providing an unrestricted research grant.

Footnotes

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