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Biology of Blood and Marrow Transplantation 9:742-752 (2003) 䊚 2003 American Society for Blood and Marrow Transplantation 1083-8791/03/0912-0002$30.00/0 doi:10.1016/j.bbmt.2003.09.007

Selective Elimination of Alloreactive Donor T Cells Attenuates Graft-versus-Host Disease and Enhances T-Cell Reconstitution Maria Gendelman,1 Maryam Yassai,2 Elizabeth Tivol,1 Ashley Krueger,2 Jack Gorski,2 William R. Drobyski1 1

The Bone Marrow Transplant Program and the Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin; 2Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin Correspondence and reprint requests: William R. Drobyski, MD, Bone Marrow Transplant Program, 9200 W. Wisconsin Ave., Milwaukee, WI 53226 (e-mail: [email protected]). Received August 12, 2003; accepted September 8, 2003

ABSTRACT Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host–reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2b] 3 AKR/J [H-2k]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell– depleted bone marrow plus thymidine kinase–positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline–treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell– depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline–treated animals had persistent skewing in most V␤ families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4ⴙ T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted. © 2003 American Society for Blood and Marrow Transplantation

KEY WORDS Alloreactive donor T cells ● bone marrow transplantation

Graft-versus-host disease

INTRODUCTION Graft-versus-host disease (GVHD) is the primary complication associated with allogeneic bone marrow transplantation (BMT). One of the major problems that contributes to mortality in patients with GVHD 742

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T-cell reconstitution

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Allogeneic

is impaired immune reconstitution, which predisposes this patient population to potentially life-threatening opportunistic infections (see review [1]). T-cell immunity, in particular, is often compromised in patients with GVHD. This occurs as a consequence of the immunosuppressive therapy administered to treat this

Preservation of T-Cell Immunity after Allogeneic BMT

complication and as the direct result of the graftversus-host (GVH) reaction that impairs thymic production of new T cells and expansion of mature T cells transferred in the donor marrow graft [2,3]. Decreased thymic production of naive donor T cells occurs as a result of direct epithelial cell damage in the recipient thymus [4,5] along with reduced production of the cytokines necessary for thymopoiesis (eg, interleukin-7 [IL-7] and keratinocyte growth factor) [6,7]. Reconstitution of the mature T-cell compartment is also adversely affected by the propensity of these cells to have shortened survival in the periphery [8] and to have limited T-cell receptor (TCR) repertoire diversity [9]. Clinical strategies to reduce GVHD have focused on the use of immunosuppressive agents that nonspecifically affect T-cell function with the goal of inhibiting GVH reactivity. Unfortunately, these agents often further compromise T-cell immunity by reducing absolute T-cell numbers or inhibiting the function of existing donor T cells [10]. Another approach has been to employ ex vivo T-cell depletion to reduce the severity of GVHD. Although this is effective in preventing GVHD, T-cell immunity is often adversely affected because of the reduced number of transferred mature T cells, which has a corresponding deleterious effect on host immunity [11-13]. An alternative strategy to address the problem of GVHD is to selectively eliminate only those donor T cells that have antihost reactivity while sparing donor T cells that are nonalloreactive. The approach that has been most critically evaluated both in preclinical models [14,15] and in the clinic [16-19] has been incorporation of the thymidine kinase (TK) gene into donor T cells so that these cells can then be targeted for elimination after administration of the antiviral agent ganciclovir (GCV). This strategy is based on the elimination of dividing donor T cells early after transplantation with the premise that most of these cells will be GVH-reactive T cells responding to recipient alloantigens [20]. The success of this approach, however, is contingent on the retention of T-cell immune competency in recipients after elimination of alloreactive donor T cells. The purpose of this study was to address this question in mice that had been protected from lethal GVHD by GCV administration.

MATERIALS AND METHODS Mice

C57BL/6 (B6; H-2b, Thy1.2⫹) and AKR/J (H-2k, Thy 1.1⫹) mice (aged 4-16 weeks) were obtained from The Jackson Laboratories (Bar Harbor, ME). TK transgenic mice on a pure B6 background in which the TK gene is targeted to the T cell by using a CD3 promoter/enhancer construct have been previously described [21]. All animals were housed in the Amer-

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ican Association for Laboratory Animal Care–accredited Animal Resource Center of the Medical College of Wisconsin. Mice received regular mouse chow and acidified tap water ad libitum. Reagents

GCV (Cytovene; Roche Laboratories, Nutley, NJ) was dissolved in distilled water and stored in aliquots at ⫺20°C. GCV was thawed before use and administered to animals intraperitoneally in phosphate-buffered saline (PBS) at 50 mg/kg/d. Human recombinant IL-7 was kindly provided by Dr. Toby Hecht (The National Cancer Institute-Frederick, Frederick, MD). IL-7 was reconstituted in PBS before administration and was administered at 5 ␮g/d to murine transplant recipients. Bone Marrow Transplantation

Bone marrow (BM) was flushed from donor femurs and tibias with Dulbecco modified Eagle medium (DMEM) and passed through sterile mesh filters to obtain single-cell suspensions. BM was T cell– depleted in vitro with anti-Thy1.2 monoclonal antibody (clone 30-H12; rat immunoglobulin [Ig]G2b; American Type Culture Collection, Rockville, MD) plus low-toxicity rabbit complement (C-Six Diagnostics, Mequon, WI). BM cells were washed and resuspended in DMEM before injection. Naive donor T cells were obtained from transgenic animals by passing erythrocyte-depleted spleen cells through nylon wool columns to remove non-T cells. Host mice were conditioned with lethal total body irradiation (1100 cGy) administered as a single exposure at a dose rate of 67 cGy by using a Shepherd Mark I Cesium Irradiator (J.L. Shepherd and Associates, San Fernando, CA). Irradiated recipients received a single intravenous injection of T cell– depleted (TCD) BM (107 cells) with or without added TK⫹ T cells within 24 hours. T-Cell Spectratype Analysis

Total RNA was prepared from mouse blood or splenocytes by using Trizol (Gibco-BRL, Gaithersburg, MD) and converted to complementary DNA (cDNA) as previously described [22]. Rearrangement analysis was performed by polymerase chain reaction (PCR) amplification of the CDR3 by using V␤ and C␤ region-specific primers [22,23]. The C-region primer was labeled with fluorescein, and the PCR products were analyzed on denaturing polyacrylamide gels. The fluorescent PCR products were quantitated by using a FluorImager (Molecular Dynamics, Sunnyvale, CA). The gel data were collected as a 16-bit tagged image file format file, and the intensities were analyzed with ImageQuant software (Molecular Dynamics). The analyses from splenocytes were performed on cDNA samples that were titrated to ensure equal efficiency of 743

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amplification of the TCR ␤-chain cDNA constant region. For calculation of CDR3 length changes, band intensities originally measured as relative fluorescence units by the FluorImager were converted to the relative frequency (RF) of each band over the total band intensity. The relative band intensities correct for minor fluctuations in the data. To represent the large data sets generated, we used a graphic representation of differences between control and experimental spectratypes by generating comparative RF plots [24]. For 2 nearly identical spectratype distributions, a comparative RF plot yields a diagonal. If the distributions are symmetric, the leading points fall close to the trailing points because of the Gaussian distribution. If the distributions are very different, however, the plots will balloon, and the tracings may cross. For each data set, we generated an average diagonal for the control group that underwent transplantation with TCD BM only (no-GVHD group). This was used as 1 dimension in all ensuing plots. The entire plot of an experimental data set was reduced to a point by taking the square root of the sum of the square of the differences between the data set and the BMT control values. This represents the skew of the experimental data set. Ovalbumin Inoculation

Ovalbumin (OVA) was dissolved in PBS, and the concentration was quantitated on an Eppendorf Biophotometer (Brinkmann Instruments, Westbury, NY) at 280 nm. Mice were then inoculated subcutaneously at the base of the tail with 100 ␮g of OVA (Pierce, Rockford, IL) in 100 ␮L of complete Freund adjuvant. Proliferation Assays

Spleen cells were harvested from OVA-inoculated and control animals. Red blood cells were removed by hypotonic lysis. Spleen cells were seeded at a concentration of 2 ⫻ 105 cells per well in complete DMEM and 10% fetal bovine serum in the presence of soluble anti-CD3 antibody (clone 145-2C11; 1 ␮g/mL) or OVA (75 ␮g/mL). Cells were cultured for either 3 days (anti-CD3 antibody) or 5 days (OVA). One microcurie of [3H]thymidine was added to wells for the final 12 to 18 hours before harvest. Cells from triplicate wells were harvested, and proliferation was assessed by using a liquid scintillation counter (Micromedics Systems, Huntsville, AL). Control wells consisted of cells cultured in the absence of either anti-CD3 or OVA. Data are presented as the ⌬ counts per minute (ie, average counts per minute of triplicate experimental wells minus average counts per minute of triplicate control wells) and were normalized for the percentage of CD3⫹ T cells in the spleens of each animal. 744

Flow Cytometric Analysis and Assessment of Chimerism

Monoclonal antibodies conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were used to assess chimerism in marrow transplant recipients. PE anti-CD8 (clone CT-CD8a; rat IgG2a) was obtained from Caltag (San Francisco, CA). PE anti-TCR ␣␤ (clone H57-597; hamster IgG), PE antiCD4 (clone GK1.5; rat IgG2b), FITC anti-Thy1.2 (clone 30-H12; rat IgG2b), FITC anti–Gr-1 (clone RB6-8C5; rat IgG2b), and FITC anti–H-2Kb (clone AF6-88.5; mouse IgG2a) were all purchased from Pharmingen (San Diego, CA). Spleen and thymus cells were obtained from chimeras at defined intervals after transplantation, processed into single-cell suspensions, and stained for 2-color analysis. Red blood cells were removed when necessary by hypotonic lysis. Cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA). At least 10000 cells were analyzed for each determination whenever possible. Statistics

Survival curves were constructed by using the Kaplan-Meier product-limit estimator and compared by using the log-rank rest. Group comparisons of thymidine incorporation in proliferation assays and absolute cell numbers in IL-7 experiments were performed by using the Mann-Whitney U test. A P value ⱕ.05 was deemed to be significant in all experiments. RESULTS TCR Repertoire Analysis in Murine Transplant Recipients

Individual spectratypes derived from tissue samples of mice that have undergone allogeneic marrow transplantation are often analyzed by visual inspection. The lack of quantitation generally associated with this approach can make the comparative assessment of repertoire differences among multiple groups problematic. In an effort to circumvent this problem, we used an approach in which diagonal RF plots were constructed to quantify the RF distributions of individual bands from spectratypes derived from individual mice. To illustrate this approach, lethally irradiated AKR mice underwent transplantation with TCD B6 BM alone. These mice do not develop GVHD because of the absence of mature donor T cells in the marrow graft. Animals were killed on day 37, and spleen cells were obtained for TCR spectratype analysis. The band intensities of 2 spectratype gels generated from cDNA obtained from the spleens of these mice (Figure 1A and C) were converted into an RF, which is the intensity of the band divided by the sum of all intensities. The RF values for equivalent bands

Preservation of T-Cell Immunity after Allogeneic BMT

plus 5 ⫻ 105 TK⫹ T cells. This T-cell dose is sufficient to cause clinically significant GVHD in non– GCV-treated mice [27]. Transplanted animals were bled 37 days after transplantation and then killed within 24 hours to obtain spleen cells for analysis. Spectratype studies revealed 2 different patterns. In some families (Figure 2, panels 1 and 2), significant skewing was visible in the spleen, whereas the repertoire in the blood was relatively normal. Alternatively, in other animals, represented by data shown in panel 3, skewing was visible in both blood and spleen but was much more pronounced in the spleen. Overall, spectratype analysis using blood as the source of the sample tended to underestimate the degree of repertoire skewing. Therefore, in subsequent studies, we performed TCR repertoire analyses on cDNA samples obtained from spleen cells as a more accurate reflection of repertoire complexity. GCV-Treated Mice Are Able to Reconstitute a Complex T-Cell Repertoire Figure 1. Generation of diagonal relative frequency (RF) plots. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD B6 BM and were then killed 37 days after transplantation. RNA was extracted and cDNA generated from spleen cells obtained from 2 chimeras. CDR3 spectratype analysis was then performed on cDNA samples. Data shown in panels A and C represent spectratypes from the V␤5.1 family derived from individual mice. RF values for equivalent bands from the 2 gels are shown in panel B.

from the 2 gels were then plotted as a single point (Figure 1B). These data show a symmetric distribution within the V␤ family, as evidenced by the fact that all points fall on the diagonal. Another indicator of symmetry is the fact that points from the rising part of the distribution overlap with those from the descending part of the distribution because of the normal Gaussian distribution of bands within most V␤ families. This distribution is identical to what is observed in normal nontransplanted B6 mice (data not shown). Therefore, these data indicate that in mice that are free from GVHD, a linear diagonal representation is the expected plot.

Studies were then conducted to assess the effect of GCV administration on reconstitution of the T-cell repertoire in mice that were protected from lethal GVHD. Irradiated AKR mice underwent transplantation with TCD B6 BM alone (no-GVHD control) or together with 5 ⫻ 105 TK⫹ T cells. Mice that underwent transplantation with TK⫹ T cells were treated with either PBS (GVHD control) or GCV on days 1 to 10 after transplantation. Animals from these 3 groups were then killed on either day 37 or day 62 after transplantation. Serial weight curves indicated that GCV-treated mice had higher weights, indicative

Comparative Analysis of T-Cell Repertoires in Blood and Spleen of GVHD Recipients

We next applied this approach to TCR repertoire analysis in mice undergoing GVHD. Prior studies have indicated that the evaluation of repertoire diversity is influenced by the source of tissue [25,26], indicating that spectratypes generated from different tissue sources may not necessarily yield concordant results. To examine this question in our model, we analyzed blood and spleen cell samples that were concurrently obtained from lethally irradiated AKR mice who underwent transplantation with TCD B6 BM

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Figure 2. Analysis of spleen cells is a more sensitive indicator of TCR repertoire complexity than peripheral blood in GVHD mice. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD B6 BM together with 5 ⫻ 105 TK⫹ T cells. Representative mice were bled 37 days after transplantation and then killed within 24 hours to obtain spleen cells for determination of TCR repertoire complexity. Data show repertoire complexity and skewing in representative V␤ families after analysis of peripheral blood (B) and spleen cells (S) obtained from 3 individual mice. Data from individual mice are partitioned by vertical lines. 745

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Figure 3. GCV administration protects animals with transplants from severe GVHD. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD BM alone (E; n ⫽ 3 per group) or together with 5 ⫻ 105 TK⫹ T cells. Animals that underwent transplantation with TK⫹ T cells were then treated with either PBS (䊐; n ⫽ 4-5 per group) or GCV (■; n ⫽ 5-6 per group) for 10 days beginning on day 1 after transplantation. Mean serial weight measurements are shown for the first 37 days (A) and 62 days (B) in 2 separate experiments. All mice in panel A survived until day 37. Two of 5 GVHD and 4 of 6 GCV-treated animals survived until day 62 (B). The mean percentage of donor T cells in the spleens of transplanted animals at the time they were killed is shown in panel C. Actual values ⫾ 1 SEM for percentage of donor T cell engraftment were as follows— day 37: BM, 86 ⫾ 1; GVHD, 100 ⫾ 0; and GCV, 98 ⫾ 1; day 62: BM, 93 ⫾ 1; GVHD, 100 ⫾ 0; and GCV, 100 ⫾ 0.

of protection from GVHD (Figure 3A and B). This was most pronounced at day 62 but was also evident 37 days after BMT. Analysis of spleen cell chimerism demonstrated that mice in all 3 groups were fully engrafted with donor T cells at the time TCR repertoire analysis was performed (Figure 3C), indicating that only donor T cells were being evaluated. The extent of repertoire skewing was analyzed by spectratype analysis. An example of the data obtained is shown in Figure 4A for the V␤5.1 family analyzed in 2 mice. Pronounced skewing in the spleens of GVHD mice (PBS treated) was observed 37 days after transplantation and was also evident at day 62. GCVtreated animals showed less skewing at day 37, and by day 62, the pattern was similar to that of the TCD BM control animals. The data for all the animals in an experimental group are shown in the form of diagonal RF plots for the V␤5.1 family (Figure 4B) and the V␤11 family (Figure 4C). For each animal and each V␤ family, the spectratype band RF is plotted against the average RF of the TCD BM mice (controls). The latter mice generated tight diagonal plots with clustered data points, indicating nearly identical and symmetric distributions. Mice receiving TK⫹ T cells and PBS had skewed repertoires at both time points, as shown by the ballooning and crossing graphs. Although the GCV-treated mice showed some early skewing, by day 62, the graphs were tighter and on the diagonal for both the families. A single measure for the skew was obtained by calculating the deviation of each point in the RF plot from the average of the control (TCD BM) data set. The square root of the sum of the squares of the deviations was used as the skew value. This allowed a composite analysis of all the V␤ families at both time 746

points and under both conditions (Figure 5). The shaded region below a skew of 0.1 indicates the mean (0.051) and 2 SD (0.023) of the control data and represents the background noise in the measurements. The data show that for the untreated mice (Figure 5A), all but 1 of the skew values started above the controls and that the skew increased at day 62 except for 2 families (V␤5.1 and 6), for which the skew stayed the same. For the GCV-treated animals, the skewing at 37 days was reduced relative to the untreated mice (Figure 5B). By day 62, all but 1 of the families showed spectratype patterns that fell in the control range. Collectively, these data indicate that GCV-treated animals were protected from severe GVHD and thus able to reconstitute a normal complex T-cell repertoire. T Cells from GCV-Treated Mice Are Able to Respond to a Nominal Antigen

One of the primary immune deficits in allogeneic marrow transplant recipients is impaired reconstitution of CD4⫹ T cells, which has been shown to correlate with the development of infectious complications [12]. The T-cell response to exogenous administration of the nominal antigen OVA is preferentially mediated by CD4⫹ T cells, and, therefore, this serves as an experimental approach to assess the extent of CD4⫹ T-cell reconstitution in marrow transplant recipients. To examine CD4⫹ T-cell reconstitution, lethally irradiated AKR mice underwent transplantation with either TCD B6 BM alone or together with 5 ⫻ 105 TK⫹ T cells. Mice transplanted with T cells were treated with either PBS or GCV on days 1 to 10, as described previously. Animals in all groups were then immunized with OVA in complete

Preservation of T-Cell Immunity after Allogeneic BMT

Figure 4. Selective elimination of alloreactive donor T cells results in normalization of the T-cell repertoire. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD B6 BM alone or together with 5 ⫻ 105 TK⫹ T cells. Mice that underwent transplantation with TK⫹ T cells were treated with either PBS or GCV on days 1 to 10 after BMT. Cohorts of mice were killed on days 37 and 62 after transplantation, and spleen cells were analyzed by CDR3 spectratyping. A, V␤5.1 spectratypes from 2 of the mice that had received TCD BM only, TCD BM plus TK⫹ T cells (PBS), or TCD BM and TK⫹ T cells followed by GCV administration (GCV), respectively. Repertoire skewing can be easily observed in the experimental animals that had received TK⫹ T cells without GCV. Skewing is graphically visualized in the bottom panels by using diagonal RF plots for the V␤5.1 (B) and the V␤11 (C) families on days 37 and 62 after BMT. The relative frequency (RF) of bands from the individual mice is plotted against the average RF from 3 mice that received TCD BM alone. These latter mice represent mice with normal repertoire development.

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Figure 5. Composite repertoire skew analysis for 20 representative V␤ families in PBS- and GCV-treated mice. Repertoire skew is calculated from diagonal RF plots as described in Materials and Methods. A, Cumulative results from mice (2-3 per group) that underwent transplantation with TCD BM plus TK⫹ T cells and that were treated with PBS for 10 days. B, Results from mice that underwent transplantation with TCD BM plus TK⫹ T cells and that were then administered GCV for 10 days. The shaded values below 0.1 represent the range observed for mice that underwent transplantation with TCD BM alone and served as controls for these experiments.

Freund adjuvant on days 32 to 45 after transplantation. Thirty days after primary inoculation, mice were killed, and harvested spleen cells were rechallenged in vitro with OVA. As a positive control for T-cell proliferation, spleen cells were also stimulated with soluble anti-CD3 antibody. There was no statistically significant difference in the amount of thymidine incorporation after anti-CD3 stimulation between any of the 3 groups of animals (Figure 6), indicating that T cells in all cohorts were not anergic. In contrast, thymidine incorporation after OVA stimulation was significantly lower in PBS-treated mice when compared with either TCD BM control (P ⫽ .0007) or GCV747

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Figure 6. Effect of GCV treatment on the donor T-cell response to posttransplantation immunization with a nominal antigen. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD B6 BM alone (n ⫽ 6) or together with 5 ⫻ 105 B6 TK⫹ T cells. Mice that underwent transplantation with TK⫹ T cells were treated with either PBS (n ⫽ 8) or GCV (50 mg/kg; n ⫽ 8) on days 1 to 10 after BMT. On days 32 to 45, mice were immunized with OVA as described in Materials and Methods. Thirty days after immunization, mice were killed, and spleen cells were stimulated in vitro with either soluble anti-CD3 antibody (CD3) or OVA for 3 or 5 days, respectively. Data are presented as the mean thymidine incorporation counts per minute (cpm) ⫾ 1 SEM from individual animals. Thymidine incorporation was normalized for the percentage of CD3⫹ T cells present in each of the wells at the time of culture. Data are cumulative results derived from 2 independent experiments. Statistics are as follows–CD3: BM versus PBS, P ⫽ .14; BM versus GCV, P ⫽ .49; PBS versus GCV, P ⫽ .38; OVA: BM versus PBS, P ⫽ .0007; BM versus GCV, P ⫽ .75; PBS versus GCV, P ⫽ .038.

treated animals (P ⫽ .038; Figure 6). There was no difference in proliferation to OVA between TCD BM controls and GCV-treated animals (P ⫽ .75). There was also no statistically significant difference in the mean percentage of CD3⫹ CD4⫹ T cells in TCD, GVHD, and GCV-treated animals (18.8%, 18.7%, and 17.4%, respectively), indicating that the lack of responsiveness to OVA in GVHD mice was not due to a quantitative reduction of CD4⫹ T cells in the culture. These studies demonstrated that GCV administration did not compromise the ability of CD4⫹ T cells from GVHD-protected mice to respond to a nominal antigen. IL-7 Augments T- and B-Cell Reconstitution in GCV-Treated Mice

IL-7 is a cytokine that has been shown to enhance immune reconstitution after allogeneic marrow transplantation in mice that have undergone transplantation with TCD BM alone or after syngeneic BM, but it has been ineffectual in animals with GVHD [28,29]. Because GCV administration preferentially spares nonalloreactive donor T cells in mice undergoing GVHD, the administration of IL-7 to GCV-treated mice is a potential strategy to augment T-cell reconstitution in GVHD-protected animals after BMT. Ad748

ditionally, IL-7 has been shown to enhance B-cell proliferation [30] and induce expansion of myeloid progenitor populations [31,32], suggesting that in the absence of GVHD, IL-7 might have other potentially salutary effects with respect to immune reconstitution after BMT. To examine the effects of IL-7 in GVHD versus GCV-treated animals, experiments were performed in which lethally irradiated AKR mice underwent transplantation with TCD B6 BM plus 5 ⫻ 105 TK⫹ T cells and then were administered either PBS or GCV to eliminate alloreactive donor T cells, as described previously. To assess the effect of IL-7 on immune reconstitution, mice in both experimental groups were treated with either PBS or IL-7 (5 ␮g/d) on days 19 to 28 after BMT. Before administration of PBS or IL-7, all mice were bled and confirmed to have complete donor T-cell engraftment (mean of ⱖ93% donor T cells in all groups). GCV-treated mice had significantly higher absolute numbers of spleen cells, donor T cells, B cells, and granulocytes when compared with PBS-treated GVHD control animals (Figure 7). The administration of IL-7 after BMT to GVHD mice, however, resulted in no statistically significant increase in any of these end points when compared with GVHD mice that did not receive IL-7. In contrast, the administration of IL-7 to GCV-

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Figure 7. IL-7 differentially augments immune reconstitution in GCV versus PBS-treated mice. Lethally irradiated (1100 cGy) AKR mice underwent transplantation with TCD B6 BM (107 cells) together with 5 ⫻ 105 TK⫹ T cells. Mice were then treated with either PBS or GCV (50 mg/kg) on days 1 to 10 after BMT. Cohorts of mice from each treatment group were administered either PBS or IL-7 (5 ␮g/d) for 10 days beginning on day 19 after transplantation. Mice were killed on day 30, and spleens were analyzed for overall spleen cellularity and absolute numbers of donor B cells, T cells, and granulocytes. Data are shown as mean values ⫾ 1 SEM and are derived from 2 independent experiments. *P ⬍ .01, GCV versus PBS group; **P ⬍ .05, GCV IL-7 versus GCV group.

treated mice significantly augmented the absolute number of spleen cells, donor T cells, and B cells, but not granulocytes. Collectively, these data indicate that posttransplantation GCV administration augmented immune reconstitution relative to PBS-treated GVHD control mice and that elimination of hostreactive donor T cells by GCV conferred responsiveness to the immune-enhancing effects of IL-7. DISCUSSION The process of T-cell reconstitution after allogeneic BMT can be conceptualized as the engraftment and subsequent expansion of both alloreactive and nonalloreactive donor T cells. During GVHD, there is a dominance of host-reactive donor T cells that can both skew and contract the T-cell repertoire, resulting in limited repertoire diversity [33]. Additionally,

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GVHD results in lymphoid hypoplasia, which significantly reduces the absolute number of B and T cells that are capable of mediating effective immune responses. This combination of events is largely responsible for the increased susceptibility of GVHD patients to opportunistic infections. The challenge in the clinical setting is to reduce the severity of GVHD without simultaneously compromising effective T cell–mediated immune responses. Clinical approaches that have been most extensively examined are the use of nonspecific immunosuppressive agents and ex vivo T-cell depletion. Both, however, suffer from the fact that T-cell immunity is, in most patients, adversely affected because of inhibition of global T-cell function and quantitative reductions in overall T-cell numbers. We have reasoned that an effective strategy to address this clinical problem is one in which alloreactive donor T cells are preferentially eliminated early after transplantation with the intention of sparing nonalloreactive donor T cells that have the capacity to affect other important immune functions. We have previously shown that this approach is an effective way to reduce GVHD severity without compromise of either alloengraftment [21] or graft-versus-leukemia reactivity [34]. In this study, we have extended these observations by examining the effect of this strategy on T-cell reconstitution. Overall, our data demonstrate that host-reactive donor T cells can be eliminated early after BMT without compromising the ability of the recipient to reconstitute functional T-cell immunity. A characteristic of GVHD is that there are typically abnormalities in the T-cell repertoire that manifest as skewing and contraction of specific V␤ families [35,36]. This has been demonstrated both in the peripheral blood [30] and in selected GVHD target organs [26,37]. In some studies, there has also been an association between an abnormal T-cell repertoire and a lack of immune responsiveness to infectious agents [33], suggesting that normalization of the repertoire is one factor that contributes to effective posttransplantation immunity. Comparative assessment of the TCR repertoire, however, can be problematic because of the number of V␤ families and the multiplicity of clonotypes within each family that constitute the full repertoire. Most studies have used qualitative comparisons to assess the repertoire, but this does not always provide a comprehensive assessment of repertoire complexity. To that end, we used a quantitative methodology [22] to assign a value for skewing away from an empirically defined control repertoire (TCD BM alone). The data showed that in the animals treated with PBS in which GVHD was observed, the repertoires were skewed both early and late after transplantation (Figure 5). Mice treated with GCV showed lower levels of skewing at day 37, and for almost all the V␤ families, this decreased to control 749

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levels by day 62. AKR mice delete a number of V␤ families (V␤ 5, 6, 8, 9, 11, and 12) because of thymic responses to viral superantigens, whereas B6 mice do not delete these families. The reconstituted repertoires in the GCV-treated mice showed a B6 pattern of intensities for these V␤ families, indicating that the T cells were of B6 marrow origin and that sufficient donor-derived antigen-presenting cells had entered the thymus to obviate the I-E– dependent deletion that occurs in AKR animals. From this we conclude that the normalization of repertoires in GCV-treated mice was due to the emergence of BM-derived donor T cells that are tolerant to host alloantigens. However, in GVHD animals (PBS treated), thymic damage induced by alloreactive donor T cells hindered the emergence of new T cells, resulting in a perpetuation of repertoire skewing. When tested in functional assays, donor T cells from mice in all groups responded equally well to stimulation with anti-CD3 antibody when normalized for T-cell content in individual wells. These data indicated that T cells from all 3 cohorts were not anergized but remained able to respond to a polyclonal stimulus. A central problem in immunity after BMT, however, is the markedly impaired reconstitution of CD4⫹ T cells, particularly in the setting of GVHD. This results in compromised cellular and humoral immunity. We examined CD4⫹ T-cell function in GCV-treated mice by inoculating with OVA, an exogenous protein presented by antigen-presenting cells to CD4⫹ T cells. We observed that splenic T cells obtained from GCV-treated mice had significantly higher proliferative responses when compared with T cells from mice undergoing GVHD. Because the percentage of CD4⫹ T cells was the same in wells containing spleen cells from GCV-treated and GVHD control animals, this was evidence that CD4⫹ T-cell function was qualitatively impaired in the latter mice. TCR spectratype studies demonstrated that the T-cell repertoire was markedly skewed in GVHD mice during the time that mice were inoculated with OVA (Figures 4 and 5). We infer from these data that the clonotypes represented by the skewed bands were responding to alloantigenic differences and therefore were not capable of responding to a nominal antigen. Conversely, the more diverse repertoire present in GCV-treated mice rendered these mice more competent to respond to a previously unencountered antigenic stimulus. A number of murine studies have demonstrated that administration of IL-7 can enhance lymphocyte recovery in murine recipients undergoing BMT [28,29,38]. This has been attributed to augmentation of thymopoiesis, as well as expansion of mature peripheral T cells [39]. The immune-restorative properties of IL-7, however, have not been observed in mice undergoing GVHD [28,29], indicating that IL-7 750

treatment may not be a viable strategy to augment immune reconstitution in this setting. T-cell reconstitution in animals that underwent transplantation with TCD BM alone is attributable exclusively to the generation of BM-derived donor T cells arising after selection in the thymus. In contrast, both mature donor T cells not eliminated by GCV and BM-derived donor T cells can contribute to host immunity in GCV-treated animals [21]. This distinction is of potential clinical importance. The risk of acquiring an opportunistic infection is highest early in the posttransplantation period. Therefore, the capability of mounting an effective T-cell response may be important in preventing infectious complications during this period [40]. Early after transplantation, T-cell immunity is primarily dependent on mature T cells that are transferred in the marrow graft. The generation of new BM-derived donor T cells, in contrast, occurs over time to supplement T-cell reconstitution, although in adults, this process is retarded and constrained by direct thymic damage from the conditioning regimen, age-related involution, or both [41-43]. In these patients, mature T cells assume the primary role in T-cell immunity because of limited thymic production of new T cells. The ability to attenuate GVHD and preserve mature T cells that can respond to an immunorestorative cytokine, such as IL-7, may therefore be therapeutically advantageous. Our data indicate that post-BMT GCV administration can confer responsiveness to the immune-enhancing effects of IL-7, as evidenced by increased overall spleen cellularity and absolute numbers of donor T and B cells. Therefore, sequential administration of GCV followed by IL-7 may be a viable strategy to augment immune reconstitution after allogeneic marrow transplantation, particularly in older patients in whom thymic production is more limited. The application of this approach to humans, however, may be more challenging because of current technical limitations regarding the efficiency of gene transfer. The use of transgenic TK⫹ T cells ensures that each mature alloreactive T cell is potentially capable of being eliminated by the administration of GCV. In contrast, existing clinical transduction approaches do not result in transfer of the TK gene into all targeted T cells [16-19]. Therefore, control of GVHD may be less effective and immune competency less robust in a setting in which residual alloreactive donor T cells that are not susceptible to elimination by GCV are able to persist in the recipient. The extent to which this may represent a significant clinical limitation remains to be determined and will also be contingent on ongoing technological improvements designed to enhance gene transfer in targeted T-cell populations. In summary, these studies demonstrate that GVHD severity can be substantially attenuated by

Preservation of T-Cell Immunity after Allogeneic BMT

using a genetic approach in which alloactivated donor T cells are selectively eliminated from recipient mice early after transplantation. This strategy results in enhanced T-cell reconstitution, indicating that elimination of GVH-reactive donor T cells allows residual mature and BM-derived donor T-cell populations to emerge and maintain functional T-cell competency. We conclude that, in light of previous studies demonstrating that this strategy can also result in preservation of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.

ACKNOWLEDGMENTS This research was supported by grants from the National Institutes of Health (HL64603 and HL55388) and the Midwest Athletes against Childhood Cancer Fund (Milwaukee, WI).

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