Identical twins discordant for multiple sclerosis have a shift in their T-cell receptor repertoires

CD4 T‐cells have an important role in the autoimmune response in multiple sclerosis (MS). We investigate the possibility that a shift occurs in the T‐cell receptor (TR) repertoire of identical twins discordant for MS. We compare the CDR3 spectratype distributions of 24 different TR V beta (TRBV) segments in naïve CD4 T‐cells from discordant MS twins and from healthy identical twins. We also compare the CDR3 spectratype distributions in unrelated healthy pairs, formed by combining members of different healthy twins, with the CDR3 spectratype distributions in unrelated pairs of MS patients and in unrelated pairs of their apparently healthy cotwins, formed by combining members of different discordant twins. We use the correlation coefficient (r‐value) as a measure of similarity of CDR3 spectratypes in each pair, and we test for the significance of the difference between r‐values from the different pairs. We observe that the r‐value for the CDR3 spectratype distributions among discordant twins differs significantly from the corresponding r‐value for the healthy twins for two TRBV segments. Further, the r‐values, for both the unrelated MS patient pairs and the unrelated pairs of their apparently healthy cotwins, differ significantly from the r‐values for healthy unrelated pairs of individuals. We conclude that both the MS patients and their apparently healthy cotwins have shifts in their CDR3 repertoires. Because we study naïve CD4 T‐cells, we postulate that CDR3 repertoire shifts precede MS and predispose to MS, but are unlikely to be sufficient to cause MS.     

Standardized analysis for the quantification of Vbeta CDR3 T-cell receptor diversity

Assessment of the diversity of the T-cell receptor (TCR) repertoire is often determined by measuring the frequency and distribution of individually rearranged TCRs in a population of T cells. Spectratyping is a common method used to measure TCR repertoire diversity, which examines genetic variation in the third complementarity-determining region (CDR3) region of the TCR Vbeta chain using RT-PCR length-distribution analysis. A variety of methods are currently used to analyze spectratype data including subjective visual measures, qualitative counting measures, and semi-quantitative measures that compare the original data to a standard, control data set. Two major limitations exist for most of these approaches: data files become very wieldy and difficult to manage, and current analytic methods generate data which are difficult to compare between laboratories and across different platforms. Here, we introduce a highly efficient method of analysis that is based upon a normal theoretical Gaussian distribution observed in cord blood and recent thymic emigrants. Using this analysis method, we demonstrate that PBMC obtained from patients with various diseases have skewed TCR repertoire profiles. Upon in vitro activation with anti-CD3 and anti-CD28 coated beads (Xcyte Dynabeads) TCR diversity was restored. Moreover, changes in the TCR repertoire were dynamic in vivo. We demonstrate that use of this streamlined method of analysis in concert with a flexible software package makes quantitative assessment of TCR repertoire diversity straightforward and reproducible, enabling reliable comparisons of diversity values between laboratories and over-time to further collaborative efforts. Analysis of TCR repertoire by such an approach may be valuable in the clinical setting, both for prognostic potential and measuring clinical responses to therapy.     

Longitudinal Analysis of T-Cell Receptor Variable β Chain Repertoire in Patients with Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

T-cell receptor variable β chain (TCRBV) repertoire spectratyping involves the estimation of CDR3 length distributions for monitoring T-cell receptor diversity and has proven useful for analyses of immune reconstitution and T-cell clonal expansions in graft-versus-host disease (GVHD) and graft-versus-leukemia after allogeneic stem cell transplantation. We performed a longitudinal spectratype analysis of 23 TCRBV families in 28 patients who underwent allogeneic T cell–depleted peripheral blood stem cell transplantation. Sixteen patients subsequently developed acute GVHD. We recently developed statistical methods that bring increased power and flexibility to spectratype analysis and allow us to analyze TCRBV repertoire development under appropriately complex statistical models. Applying these methods, we found that patients with acute GVHD demonstrated TCRBV repertoire development statistically distinct from that repertoire development in patients without GVHD. Specifically, GVHD patients showed spectratypes indicative of lower diversity and greater deviation from the spectratypes expected in healthy individuals at intermediate times. Most individual TCRBV subfamilies had spectratypes statistically distinguishable between GVHD and non-GVHD patients at 6 months after transplantation. These results suggest that the T-cell receptor repertoire perturbations associated with acute GVHD are widely spread throughout the TCRBV families.     

Infiltrating T cells during liver graft-versus-host disease show a restricted T-cell repertoire.

Data from animal models have shown that hepatic graft-versus-host disease (GVHD) may be mediated by donor T cells interacting with liver adhesion molecules, other minor histocompatibility antigens, or both. We hypothesized that T-cell infiltrates within a liver biopsy during clinical GVHD would show a restricted T-cell response because the T cells would be responding to a limited number of antigens. We studied the peripheral T-cell repertoire and the liver-infiltrating T-cell repertoire of a patient who developed skin GVHD and subsequent liver GVHD after a matched sibling bone marrow transplantation for acute myeloid leukemia. Spectratype analysis of peripheral blood at the time of liver GVHD revealed that the patient had reconstituted a complex peripheral T-cell repertoire as evidenced by the presence of complementarity-determining region 3 (CDR3) length heterogeneity in most of the T-cell families. The repertoire complexity was skewed in variable gene beta (VB) 5.3, VB4, VB7, VB8, and VB15. Spectratype analysis on the liver biopsy sample revealed a limited infiltrate with an oligoclonal expansion in VBs 4, 7, and 8. We evaluated the T-cell infiltrate in more detail by sequencing the relevant expansions noted by spectratype and developing probes for the predominant CDR3 sequences. These clonotype probes were hybridized to peripheral blood and liver samples from the patient, a T-cell line developed from the patient's peripheral blood at the time of the initial skin GVHD, the donor's blood and marrow, and control samples. The results showed that the T-cell infiltrate during liver GVHD is mediated by a limited number of T cells, and that those cells are mostly different from the ones expanded from the peripheral blood during an acute skin GVHD reaction. These data support the concept that liver GVHD is a response to tissue-specific minor histocompatibility antigens.     

Nonmyeloablative conditioning allows for more rapid T-cell repertoire reconstitution following allogeneic matched unrelated bone marrow transplantation compared to myeloablative approaches.

Nonmyeloablative pretransplantation conditioning regimens have resulted in durable engraftment of allogeneic hematopoietic stem cells. In contrast to conventional fully myeloablative approaches, nonmyeloablative regimens are associated with a marked reduction of morbidity and mortality in the early posttransplantation period. Consequently, such reduced-intensity transplantation approaches can be used in older and frailer patients who would not tolerate fully ablative regimens. However, it is currently unclear how this radically different transplantation strategy affects immunological reconstitution. To address this important issue, we used T-cell receptor Vbeta spectratype analysis to examine the distribution of complementarity-determining region 3 (CDR3)-size bands as a measure of the complexity of the redeveloping T-cell repertoire. For this study, we evaluated the T-cell repertoire of 9 patients receiving T-cell replete, matched unrelated donor transplants following fully ablative or nonmyeloablative conditioning regimens. All 4 of the myeloablative and 2 of the nonmyeloablative patients received bone marrow, whereas 3 other nonmyeloablative patients received peripheral blood stem cells. The results of the spectratype analysis demonstrated that the patients who received nonmyeloablative conditioning together with either bone marrow or peripheral blood stem cells exhibited more rapid reconstitution of T-cell repertoire complexity.     

Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice

Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation.     

Analysis of the interindividual conservation of T cell receptor alpha- and beta-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) alpha- and beta-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR alpha-and beta-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha- and beta-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.     

High‐sequence diversity and structural conservation in the human T‐cell receptor β junctional region during thymic development

The T‐cell repertoire depends on intrathymic genetic rearrangement events in the T‐cell receptor (TCR) locus, followed by positive and negative selection. The repertoire thus generated is highly diverse, but recent data indicate that the recombination of gene segments is less stochastic than previously suggested. Very little is known of the junctional complementarity determining region 3 (CDR3), which is to a large degree not germline encoded. We have analyzed the development of the human TCR β CDR3 repertoire, from the nonselected CD4⁺CD8⁺CD3^? cells up to the fully selected CD4⁺CD8^? thymocytes. In addition to spectratyping, a fraction of the CDR3 repertoire was sequenced and a structural in silico analysis of the CDR3 loop characteristics performed. Our data show that the thymic TCR repertoire is extremely diverse, and the effect of the selection events can be detected as a measurable loss of polyclonality in the CDR3 loop. However, the main physicochemical features of the CDR3 loop were found already at the nonselected repertoire and showed no progressive changes during the selection. Thus, the main structural characteristics of the CDR3 loop were already determined by the recombination process and not significantly affected by the extensive thymocyte death associated with selection in the thymus.     

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.     

Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients.     

T cell repertoire complexity is conserved after LLME treatment of donor lymphocyte infusions.

Slow reconstitution of the T cell repertoire after allogeneic blood or bone marrow stem cell transplantation is a major risk factor for patient mortality. The delivery of immunocompetent T cells as delayed donor lymphocyte infusions (DLIs) is a potential way of counteracting this problem. The development of graft-versus-host disease (GVHD) is a potential complication of this procedure, however. We previously found that in P-->F1 haploidentical murine models, the ex-vivo treatment of donor lymphocytes with L-leucyl-L-leucine methyl ester (LLME) can prevent the onset of GVHD after DLI, likely by inducing cell death in most of the perforin-positive CD8(+) T cells and in a fraction of CD4(+) T cells. Our previous preclinical studies have formed the basis of an ongoing phase I clinical trial in which patients received LLME-treated DLI from their original donor in an attempt to accelerate T cell reconstitution. To understand how this treatment strategy might affect the complexity of the DLI T cell repertoire, we used T cell receptor Vbeta spectratype analysis to evaluate the DLI product pre-LLME and post-LLME treatment. The results indicated that the LLME-treated DLI product exhibited CDR3-size distribution complexities similar to those of its untreated donor sample. In addition, comparisons of the CD4(+) and CD8(+) T cell repertoire from the donor before LLME treatment with that of the recipient post-DLI demonstrated equal complexity for most of the resolvable Vbeta families. Finally, the in vitro proliferative capacity of LLME-treated DLI product in response to allo-stimulation in a one-way mixed lymphocyte reaction was comparable to that of the untreated product.     

VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3- spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).     

VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3 spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).     

Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity

The third complementarity-determining region (CDR3) is the only nongermline-encoded hypervariable region of the T cell receptor β (TCRB) chain, and it is the region that has been predicted to confer fine specificity of the TCR for peptide-MHC complexes. For this reason analysis of TCRB CDR3 heterogeneity may provide insight into immune mechanisms operative in infectious and autoimmune diseases. PBMC stimulated with either mitogen (PHA), superantigen (TSST-1), or nominal antigen (tetanus toxoid) have been compared with unstimulated PBMC using a two-dimensional approach. Analysis of the expressed TCRBV gene repertoire CDR3 length profile coupled with SSCP methodology enabled the discrimination of sequences with the same CDR3 length. For both freshly isolated and PHA- stimulated PBMC, a normally distributed spectrum of CDR3 lengths (five or more products) was observed. These products differed by 3 bp (1 amino acid) due to the strict requirement for in-frame rearrangements in the CDR3 region of TCR. By contrast, tetanus toxoidstimulated PBMC had restricted profiles for most TCRBV families after as few as 7 days of incubation. The oligoclonal nature of samples showing CDR3 length restriction was revealed by SSCP analysis and confirmed by sequence determination. Superantigen stimulation resulted in unique patterns of diversity, which included polyclonal expansion of specific TCRBV families as well as oligoclonal expansion of most other TCRBV families. These data reveal complex yet distinct patterns of TCR diversity in response to different T cell activation stimuli.     

TCRalphabeta repertoire diversity of human naturally occurring CD4+CD25+ regulatory T cells

We examined the alphabeta T cell receptor (TCR) repertoire of naturally occurring CD4+CD25+ regulatory T (Treg) cells isolated from healthy human blood. Three-color FACS analysis demonstrated that the usage of variable region segments of TCRbeta chains by CD4+CD25+ cells did not differ from those of CD4+CD25- cells. Complementarity-determining region 3 (CDR3) size distribution analyses demonstrated that the repertoire diversity of CDR3beta was almost identical between CD4+CD25+ and CD4+CD25- T cell subsets, and that there was no skewing of the CDR3beta repertoire of CD4+CD25+ T cells. In contrast, in vitro activated CD4+CD25+ T cells by cytomegalovirus-derived antigens showed a skewed CDR3 size distribution pattern. These findings support the hypothesis that naturally occurring CD4+CD25+ T cell subset in humans is 1argely composed of a T cell lineage positively selected in the thymus as a consequence of the interaction between self-peptides and TCRs and not derived from recent activation by a limited array of antigens.     

T-Cell receptor Vbeta repertoire CDR3 length diversity differs within CD45RA and CD45RO T-cell subsets in healthy and human immunodeficiency virus-infected children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vbeta gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vbeta families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4(+) and CD8(+) T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8(+) CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4(+) or CD8(+) T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vbeta families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing

Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.     

Reconstitution of T-cell repertoire after autologous stem cell transplantation: influence of CD34 selection and cytomegalovirus infection.

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.