¹The University of Texas Health Science Center, San Antonio, TX, USA

²South Texas Cancer Institute, San Antonio, TX, USA

³VU Academic Hospital, Amsterdam, Netherlands

⁴Aastrom Biosciences, Inc, Ann Arbor, MI, USA

Correspondence to: Dr J M Teale, Department of Microbiology, Mail Code 7758, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA

It is largely unknown whether the immune repertoire can be reconstituted successfully after high-dose chemotherapy and transplantation using ex vivo expanded hematopoietic stem cell (HSC) grafts. It is critically important for the transplant outcome that immune repertoire reconstitution progresses after ex vivo expanded HSC graft transplants at least as efficiently as that seen after conventional HSC transplants. Previously, we showed that the T cell receptor V beta (TCRVB) third complementarity determining region (CDR3) diversification after ex vivo expanded bone marrow (BM) HSC graft transplants was similar to that seen after conventional peripheral blood stem cell transplants (PBSCTs). In the present study, the CDR3 diversity of the six immunoglobulin (Ig) heavy chain variable region gene (V_H) families was examined in five breast cancer patients who were transplanted with ex vivo expanded BM HSCs as the only source of stem cells. For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. Using both CDR3 fingerprinting and single strand conformation polymorphism (SSCP) methodologies, it is shown that the contribution of the V_H families to the overall repertoire among healthy adults is highly variable and not always proportional to V_H family member size. After both ex vivo expanded HSC transplants and conventional PBSCTs, the V_H CDR3 repertoires were limited in size diversity at 6 weeks post transplant. By 6 months, however, V_H families displayed a repertoire diversity that was as complex as that seen in healthy adults. No difference was seen between ex vivo expanded HSC graft transplant recipients and conventional PBSCT recipients in V_H repertoire diversity. In one patient there was a follow-up analysis 12 months after ex vivo expanded graft transplant, and the diversity of the V_H families was maintained. In all patients, the amino acid size of the CDR3 regions fell within adult limits at all time points post transplant. These results indicate that B cell repertoire regeneration after ex vivo expanded hematopoietic cell graft transplants is similar to that seen after conventional PBSCT. Bone Marrow Transplantation (2001) 27, 413-424.

transplantation; B lymphocytes; repertoire; diversity; lymphopoiesis

Hematopoietic stem cells (HSCs) can be collected from bone marrow (BM) or peripheral blood (PB) for use as a graft to reconstitute hematopoiesis after high-dose chemotherapy. However, relatively large volumes of BM or up to several days of blood harvesting is needed to obtain HSCs in optimal quantities for rapid hematologic engraftment. Recently, identification of several hematopoietic growth factors and advances in biotechnology have led to the development of mechanical systems for ex vivo expansion of HSCs.¹ Using these systems, large scale expansion of BM cells can be accomplished ex vivo for potential use as a graft. Early studies showed the feasibility of using these grafts in clinical trials with regard to myeloid reconstitution and tolerability.^2,3,4 However, proof of multilineage engraftment including lymphopoiesis is needed before ex vivo expanded grafts can be widely used in clinical trials.

Previous studies of lymphopoiesis after HSC transplantation have largely focused on numerical and functional recovery of B and T lymphocytes. Diversity of the emergent lymphocyte repertoires, however, has been addressed in only a few published studies (reviewed in Ref. 5). This has largely been due to methodological limitations in repertoire analysis. Quantitative PCR amplification and in situ hybridization have been used to show the relative frequency of variable region gene families as an indicator of repertoire diversity. Further analysis of diversity within variable region gene families has required extensive cloning and sequencing. Recently, however, the third complementarity determining region (CDR3) fingerprinting technology emerged as a simplified approach to further dissect variable region gene family repertoires.^6,7,8 Using fingerprinting, repertoire diversity is assessed based on the CDR3 region size distribution.

To determine the diversity of the emergent lymphocyte repertoires after transplantation using ex vivo expanded HSCs as the only source of graft, we studied five breast cancer patients after such transplants using the CDR3 fingerprinting technology. The T cell receptor (TCR) diversity after ex vivo expanded HSC graft transplants was as complex as that seen after conventional peripheral blood stem cell transplants (PBSCTs).³⁹ In the present study, the B cell repertoire after ex vivo expanded HSC transplantation was examined using CDR3 fingerprinting, as well as single strand conformational polymorphism (SSCP). For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. It is shown that the contribution of the immunoglobulin heavy chain (Ig H) variable region gene (V_H) families to the overall repertoire is variable and not always proportional to their family size in healthy adults. The V_H CDR3 size diversity is limited early post transplant in the recipients of ex vivo expanded HSC transplants and conventional PBSCTs. By 6 months however, repertoires are as diverse as those seen in healthy adults. No difference is seen in the repertoire diversity when ex vivo expanded HSC graft recipients and PBSCT recipients are compared.

Materials and methods

Patients and control subjects

Five patients with breast cancer underwent ex vivo expanded HSC graft transplants under an Institutional Review Board approved protocol. The patient characteristics are shown in Table 1. The patients were conditioned with the STAMP I regimen (cyclophosphamide 1.875 gm/m²/day ´ 3, cisplatinum 55 mg/m²/day ´ 3, carmustine 600 mg/m²/day ´ 1). The BM mononuclear cell (MNC) expansion protocol has been previously described.³ Briefly, approximately 9 ´ 10⁸ BM MNCs purified under Ficoll-Hypaque density gradient were inoculated into the Aastrom's continuous perfusion stromal-coated bioreactors and continuously perfused for 12 days with Iscove's modified Dulbecco's media supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone, PIXY321, glutamine, erythropoietin, Flt3-L, gentamicin and vancomycin. The mean fold expansion of MNCs and CFU-GMs were five and 17, respectively. The CD3⁺ or CD19⁺ lymphocytes determined by flow cytometry comprised less than 1 to 3% of MNCs post expansion.

For comparison, 12 healthy adults and four conventional autologous PBSCT recipients were also studied. The PBSCT recipient characteristics are shown also in Table 1.

Cell collection, separation and RNA isolation

Peripheral blood (PB) samples were collected from patients at 6 weeks and 6 months post transplant. Patient Nos 1 and 3 were also studied at 12 months and 3 months post transplant, respectively. MNCs were separated by Ficoll-Hypaque density gradient (Organon Teknika, Durham, NC, USA), washed twice with phosphate-buffered saline and enriched for B cells by positive selection of CD4⁺ and CD8⁺ cells using immunomagnetic beads (Dynal, Oslo, Norway). RNA was isolated from 1 ´ 10⁷ B cell-enriched MNCs using TRIzol total RNA isolation reagent (Life Technologies, Gaithersburg, MD, USA).

cDNA synthesis and PCR amplifications

One microgram of RNA was used for first strand cDNA synthesis using a combination of oligo(dT) and random hexamers and SuperScript II reverse transcriptase (Life Technologies) in a final volume of 20 ml, as previously described.⁹

As the first step in V_H PCR amplification, the amount of Ig constant region transcripts was estimated for each cDNA sample in semi-quantitative PCRs as previously described.⁹ This step is critical to control for variations among samples in B cell numbers and transcriptional activity and assure that each cDNA template in subsequent V_H PCR amplification contains an equal amount of constant region transcripts. This allows a meaningful comparison between samples.

The V_H family CDR3 repertoires were amplified in nested PCRs as previously described.⁹ Briefly, the cDNA was equally distributed into six PCR tubes for amplification of each of the six V_H family repertoires. The six V_H family specific primers were used in combination with the constant region primers in primary and first nested PCRs. A common 5' pan-V_H primer (FR3 primer) was used in the second nested PCRs instead of the family specific primers. The PCR conditions and primers have been previously described.⁹ The additional V_H family specific primers used in the present study were V_H1 (5'-CCATGGACTG GACCTGGA-3'), V_H2 (5'-ATGGACATACTTTGTTCC AC-3'), V_H4 (5'-ATGAAACACCTGTGGTTCTT-3'), and V_H5 (5'-ATGGGGTCAACCGCCATCCT-3').

IgH CDR3 fingerprinting and SSCP

The IgH CDR3 fingerprinting has been previously described.^7,8,9 Briefly, purified PCR products were run on preheated 5% denaturing polyacrylamide gels for approximately 2 h at 65 W. The Promega silver staining system (Madison, WI, USA) was used to visualize the fingerprinting profiles. For SSCP,⁹ 7% polyacrylamide gels containing 5% ultrapure glycerol (JT Baker, Phillipsburg, NJ, USA) were run at room temperature for approximately 7 h at 25 W while being cooled with an electric fan. SSCP profiles were also obtained using silver staining (Promega).

Because the size of the CDR3 regions differ within a diverse repertoire, PCR products spanning the CDR3 region display a ladder of bands in fingerprinting each corresponding to a certain CDR3 length. The bands are separated from each other by 3 base pairs due to the dominance of in-frame transcripts. The number of bands in fingerprinting directly correlates with the repertoire diversity. The relative intensity of a band is an indirect measure of the number of transcripts that share the corresponding CDR3 amino acid (aa) size.

Flow cytometric analysis of lymphocyte subsets

Numerical recovery of the lymphocyte subsets was monitored post transplant using FACS (Becton Dickinson, Mountain View, CA, USA). Briefly, the fluorochrome-labeled monoclonal antibodies for CD3, CD4, CD8, CD45RA, CD45RO, CD16, CD19 and CD56 (Becton Dickinson) were used in combinations to determine the number of CD4⁺CD45RA⁺, CD4⁺CD45RO⁺, CD8⁺CD45RA⁺ and CD8⁺CD45RO⁺ T cell subsets, NK cells and B cells. Results are shown in Table 2. The serum immunoglobulin levels determined at 5 to 12 months post transplant are also shown in Table 2.

Results

IgM H CDR3 size diversity in healthy adults

As a reference, we first studied the relative contribution of the six V_H families (V_H1 to V_H6) to the PB IgM H CDR3 size diversity in 12 healthy adults. Using fingerprinting, only the V_H3 and V_H6 have been previously studied.⁹ In healthy adults, the IgM CDR3 aa sizes fell within a range of 4 aa to 31 aa for all of the V_H families (Figure 1). The V_H3 family consistently displayed 16 to 20 bands in all of the 12 healthy adults while the other V_H families showed a greater variability in the number of bands displayed among individuals. As an example, V_H4 displayed a single dominant band and a few other faint bands in normal No. 6 and normal No. 12. In other individuals on the other hand, the V_H4 displayed as many bands as did the V_H3. Absence of expression of any band was observed only for the V_H2 family in two of the 12 adults (normals 2 and 5). In another eight individuals, V_H2 either displayed a ladder of faint bands (normals 4, 6, 7, 11 and 12) or a few darker bands (normals 3, 8 and 9). The smaller (single-member) V_H6 family on the other hand, was expressed in all of the individuals studied. With regard to CDR3 size distribution, a smooth Gaussian-like pattern was most frequently observed especially for the larger V_H1, V_H3 and V_H4 families. However, dominant bands disproportionately darker in intensity than expected based on a Gaussian-like distribution could be seen in all of the V_H families. These dominant bands suggest clonal expansions. These results indicate that the V_H families do not contribute equally to the expressed PB repertoire with regard to CDR3 size diversity and that their contribution is not always proportional to their known family member size. Also, there is considerable variability among healthy adults in the contribution of any given V_H family to the overall repertoire at a given time-point.

IgM H CDR3 size diversity after transplantation using ex vivo expanded HSC or conventional PB grafts

The IgM CDR3 fingerprinting profiles were obtained from the five patients (patients 1-5) after transplantation using ex vivo expanded HSC grafts (Figure 2) and from the four patients (patients 6-9) after conventional autologous PBSCTs (Figure 3). Patients were studied at 6 weeks and 6 months post transplant. Patients 1 and 3 were also studied at 12 months and at 3 months, respectively. Samples obtained at 6 weeks post transplant showed fewer overall bands and a CDR3 size distribution that appeared random and restricted in number when compared to those seen in healthy adults. By 6 months, significantly more bands were observed in all patients, and more of the V_H families showed a Gaussian-like distribution resembling the pattern seen in healthy adults. To ensure that the fewer bands observed at 6 weeks post transplant were not simply due to less B cells, samples with an equal amount of amplifiable constant region transcripts were compared at all time-points post transplant and in healthy adults. Therefore, a greater number of bands observed over time post transplant indicates improving CDR3 size diversity after both ex vivo expanded and conventional graft transplants. In patient No. 1, the repertoire diversity was maintained at 12 months after ex vivo expanded HSC graft transplant.

SSCP analysis of the IgM H CDR3 size diversity

In SSCP, a typically diverse repertoire is characterized by a dark smear-like pattern indicating the presence of several CDR3s with different tertiary structures.⁹ In contrast, repertoires limited in CDR3 diversity show a variable number of distinct bands and a less intense background. In healthy adults, SSCP analysis showed a smear-like pattern for most of the V_H families (normals 1, 7 and 12 as examples in Figures 4a and b). A few V_H families (V_H6 in normals 1 and 7, V_H2 in normals 7 and 12, and V_H4 in normal 12) showed a less intense background and a variable number of bands. SSCP results correlated with those of fingerprinting. The V_H families with a diverse CDR3 repertoire based on the number of bands visualized in fingerprinting also showed a dark smear-like pattern in SSCP. Similarly, families exhibiting fewer bands in fingerprinting showed a less intense background and a variable number of distinct bands in SSCP.

SSCP analysis of the patient samples was also performed after ex vivo expanded HSC transplants (patients 3 and 4 in Figure 4a as examples) and after conventional PBSCTs (patients 8 and 9 in Figure 4b as examples). In all patients, samples at 6 weeks post transplant showed a variable number of bands and a less intense background for most of the V_H families while few families showed a smear-like pattern. By 6 months, more of the V_H families showed a smear-like pattern resembling that seen in healthy adults. SSCP analysis further supports that the IgM H CDR3 diversification after ex vivo expanded HSC graft transplants is similar to that seen after conventional PBSCTs.

IgG H CDR3 size diversity in healthy adults and after ex vivo expanded HSC graft transplants and PBSCTs

The IgG fingerprinting profiles were obtained using the c constant region primer instead of the c primer in PCRs. The PB IgG H CDR3 fingerprinting profiles of the 12 healthy adults are shown in Figure 5. Absence of expression of several V_H families was observed in many of the healthy adults studied. Only the largest V_H3 family was consistently expressed in all individuals. The V_H1 and V_H5 were more frequently expressed than the V_H2, V_H4 and V_H6. When compared with the IgM counterparts, the expressed V_H families displayed significantly fewer bands in the IgG fingerprinting profiles. These results indicate that the IgM transcripts dominate over IgG in contribution to steady-state PB repertoire diversity in healthy adults and that IgG transcripts are more likely to reflect clonal expansions.

The IgG fingerprinting profiles obtained from patients 1-5 after ex vivo expanded HSC graft transplants and from patients 6-9 after conventional PBSCTs are shown in Figures 6 and 7, respectively. Both ex vivo expanded HSC graft recipients and conventional PBSCT recipients showed the absence of expression of several V_H families at all time-points studied. Only the largest V_H3 family was consistently expressed in all patients. Unlike IgM, an improvement in IgG diversity was not as appreciable over the follow-up period.

Discussion

In this study, the expressed B cell repertoire diversification is shown to be similar after ex vivo expanded BM HSC graft transplants and conventional PBSCTs. Previously, the T cell repertoire diversification was also shown to be similar after these transplants.³⁹ Together, these studies indicate that lymphoid diversity can be successfully reconstituted after ex vivo expanded HSC transplantation with no further delay when compared to that seen after conventional transplants.

The new technologies of CDR3 fingerprinting and SSCP methods were used for B cell repertoire analysis in this study. The fingerprinting method provides a snapshot of a repertoire in terms of CDR3 size diversity.^6,7,8,9 In healthy adults, only the V_H3 and V_H6 families have been analyzed previously using fingerprinting.⁹ Therefore, as a reference, we first examined the contribution of the six V_H families to the overall expressed repertoire in 12 healthy adults.

The various V_H families differ in number of members.^10,11 V_H3 was shown to be the largest V_H family composed of 19-22 functional genes.^10,11 The number of members found in the other families are as follows: V_H1 (9-11), V_H2 (3), V_H4 (6-11), V_H5 (1-2), V_H6 (1), and V_H7 (1). Whether representation of the V_H families in the human adult repertoire is proportional to their member size has been a matter of debate. Studies of hybridization with V_H family-specific probes have not been conclusive in this regard.^12,13,14,15 Because the hybridization method is not informative with regard to diversity within a given V_H family, results can be biased due to possible clonal expansions. Cloning and sequencing have been used as a more rigorous way to analyze V_H family representation. However, only a few individuals could be studied due to the labor intensity of the method.^{14,16,17,18,19} Therefore, results in sequencing studies could not be generalized. The studies that looked at the relative usage of individual V_H genes in adults have shown preferential utilization of certain genes at different stages of B cell maturation.^{14,16,17,18,19,20,21,22,23,24}

Because of the relative simplicity of the fingerprinting methodology compared to random sequencing, we were able to study a greater number of normal individuals. In this study, there was considerable variability among 12 healthy adults in terms of the contribution of the V_H families to the overall expressed repertoire. With the exception of V_H3, no clear correlation was seen between the V_H family member size and V_H family representation in the overall CDR3 repertoire. A clear bias was observed against the V_H2 family. Although its functional member size is larger than the V_H5 and the V_H6 families, V_H2 was either not expressed or contributed less to the expressed CDR3 repertoire compared with the smaller V_H5 or V_H6 families in 10 out of 12 healthy adults. It is unlikely that this result is due to a bias introduced by PCR because V_H2 was expressed in a size-expectant fashion in normals 1 and 10. One could speculate that there may be a V_H2 specific B cell superantigen common among many healthy individuals. The contribution of the next largest V_H1 and V_H4 families in some healthy adults to the overall CDR3 size diversity was also less than that of some smaller families. Biases towards different families were seen among different individuals suggesting that preferential PCR amplification of certain V_H families is unlikely to be the reason for the results observed. Overall, these results indicate that the contribution of the V_H families to the expressed CDR3 repertoire diversity in healthy adults is not always proportional to their member size and that with the exception of V_H3, there is great variability among individuals in V_H family representation.

The main objective in this study was to analyze the B cell repertoire diversification after high-dose chemotherapy and ex vivo expanded HSC grafting. The capacity of the ex vivo expanded HSC graft in supporting lymphopoiesis post transplant is unknown. In preclinical studies, the colony-forming cell (CFC) and the long-term culture-initiating cell (LTC-IC) assays are used to estimate the hematopoietic progenitor frequencies in the expanded graft. These assays however, are not informative of the lymphoid progenitor frequencies.²⁵ Flow cytometric determination of the CD34⁺ stem cell compartment has shown either a modest expansion or sometimes loss of such cells. These issues have raised concern with regard to lymphoid reconstitution after transplantation using ex vivo expanded HSC grafts.

In this study, it is shown that the B cell repertoire development after ex vivo expanded HSC graft transplants is similar to that seen after conventional PBSCTs. Limitations in CDR3 size diversity was observed early post transplant after both ex vivo expanded HSC transplants and conventional transplants. By 6 months however, all V_H repertoires exhibited diversity that was as complex as in healthy adults. The numerical B cell recovery and the serum Ig levels post transplant in the five patients were also similar to those described after conventional transplants.^5,26,27

Previously, only a few studies have addressed B cell repertoire development after conventional autologous transplants.^9,28,29,30 In two studies where the V_H usage was addressed, alterations in relative usage of the V_H families were shown. In these studies,^28,30 the authors concluded that the post transplant repertoire was fetal-like based on the similarities found in V_H usage between post transplant and fetal repertoires. However, the in situ hybridization and V_H-specific PCR methods used in these studies are not informative about the clonality within the V_H families. Therefore, results obtained with these methods in this regard may be biased because of the clonal expansions that are known to be common post transplant. The most characteristic aspect of the fetal repertoire is the relatively short aa size of the CDR3 regions when compared to those of the adults.^{8,31,32,33,34,35,36,37} When the post-transplant repertoire was analyzed in this regard, the CDR3 aa sizes post transplant were found to be adult-, but not fetal-like.⁹ The results in the present study confirm the adult size of the CDR3s post transplant. The CDR3 regions after both ex vivo expanded HSC transplants and conventional PBSCTs fell within adult limits (4-31 aa) at all time-points and the majority of the CDR3s were longer than the average fetal CDR3 size of approximately 10 aa.^{8,32,33,34,35,36,37}

The results in this study can not be interpreted as proof of the ex vivo expanded HSC graft's true capacity to fully reconstitute lymphopoiesis post transplant. High-dose chemotherapy regimens used in stem cell transplantation may not fully eradicate host lymphopoiesis.³⁸ Therefore, the regeneration of B cells from chemoresistant endogenous stem cells and lymphoid progenitors cannot be excluded in our study. In the autologous transplant setting, this would require gene marking of the graft cells and demonstration of their ontogeny after transplantation. Nevertheless, it is of critical importance for the clinical applicability of the procedure to show that diversification of the B cell repertoire is not delayed or restricted after ex vivo expanded HSC transplants compared with conventional transplants. The findings shown here indicate that the two methods of transplantation are equivalent in terms of B cell recovery with time post transplant. Given the advantages of ex vivo expanded HSC grafts, this is a significant clinical finding. Further studies are needed to examine the functional recovery of lymphocyte subsets after ex vivo expanded HSC transplants.

Acknowledgements

This work was supported by grants AI 19896 and NS 35974 from the National Institutes of Health.

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Figure 1 (a and b). IgM V_H CDR3 fingerprinting repertoires of 12 healthy adults. NL, normal adult; MW, molecular marker. V_H CDR3 repertoires were amplified using nested PCR and the resulting products run on 5% denaturing polyacrylamide gels. Silver staining was used to visualize the fingerprinting profiles as described in Materials and methods. Numbers above lanes indicate V_H families.

Figure 2 (a, b, c) IgM V_H CDR3 fingerprinting repertoires of five ex vivo expanded hematopoietic transplant recipients. Numbers above lanes indicate V_H families.

Figure 3 (a and b) IgM V_H CDR3 fingerprinting repertoires of four conventional PBSCT recipients. Numbers above lanes indicate V_H families.

Figure 4 SSCP analysis of IgM V_H CDR3s. (a) Post ex vivo expanded transplants and in healthy adults; (b) post conventional PBSCT and in healthy adults. For SSCP analyses, PCR products were run on 7% polyacrylamide gels containing ultrapure glycerol followed by silver staining. Numbers above lanes indicate V_H families.

Figure 5 (a and b) IgG V_H CDR3 fingerprinting repertoires of 12 healthy adults. NL, normal adult; MW, molecular marker. Numbers above lanes indicate V_H families.

Figure 6 (a, b, c) IgG V_H CDR3 fingerprinting repertoires of five ex vivo expanded hematopoietic graft transplant recipients. Numbers above lanes indicate V_H families.

Figure 7 (a and b) IgG V_H CDR3 fingerprinting repertoires of four conventional PBSCT recipients. Numbers above lanes indicate V_H families.

Table 1  Characteristics of ex vivo expanded BM MNC graft recipients and conventional PBSCT recipients for breast cancer

Table 2  Serum immunoglobulin levels and numerical lymphoid reconstitution post ex vivo expanded BM MNC transplants