The adaptive immune system uses several strategies to generate a repertoire of T-and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In ␣␤ T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) ␣ and ␤ chains. Although it has been estimated that the adaptive immune system can generate up to 10 16 distinct ␣␤ pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCR␤ genes from T cells of 2 adults. We find that total TCR␤ receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO ؉ antigen-experienced ␣␤ T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of ␣␤ T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging. (Blood. 2009;114:4099-4107)

Risk factors for grades 2-4 acute graftversus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD. (Blood. 2011;117(11):3214-3219) IntroductionDuring the past 3 decades, several studies have identified risk factors associated with the development of acute and chronic graft-versus-host disease (GVHD). 1 In these studies, acute GVHD generally referred to disease manifestations that occurred within the first 100 days after hematopoietic cell transplantation (HCT), [2][3][4] and chronic GVHD referred to disease manifestations that were present after day 100. 5 The most consistently reported factors significantly associated with an increased risk of grades 2-4 acute GVHD were recipient human leukocyte antigen (HLA) mismatching with the donor, 6-8 alloimmunization of the donor, 9-12 the use of a female donor for male recipients, 9,11-13 and older patient age. 11,13,14 Less consistently reported risk factors have included prior cytomegalovirus infection in the recipient, 14,15 higher intensity of the conditioning regimen (irradiation), 12,14 donor age, 16 and grafting with growth factor-mobilized blood cells. 14,17 For chronic GVHD, the most consistently reported risk factors include prior acute GVHD, 18-20 grafting with growth factor-mobilized blood cells, 17,21,22 the use of a female donor for male recipients, 19,20,23 older patient age, 18-20,23 and mismatched and unrelated donors. 20,24 The objective of the current study was to compare risk factor profiles for grades 2-4 acute and chronic GVHD. For this purpose, we used diagnostic criteria recommended by the National Institutes of Health (NIH) Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease. 25 According to these criteria, acute and chronic GVHD are distinguished by differences in clinical manifestations and...

Diversity in T-lymphocyte antigen receptors is generated by somatic rearrangement of T-cell receptor (TCR) genes and is concentrated within the third complementarity-determining region (CDR3) of each chain of the TCR heterodimer. We sequenced the CDR3 regions from millions of rearranged TCR β chain genes in naïve and memory CD8+ T-cells of seven adults. The CDR3 sequence repertoire realized in each individual is strongly biased toward specific Vβ-Jβ pair utilization, dominated by sequences containing few inserted nucleotides, and drawn from an effective sequence space 250-fold smaller than predicted. Surprisingly, the overlap in the naïve CD8+ TCRβ CDR3 sequence repertoires of any two of the individuals is ~1000-fold larger than predicted and essentially independent of the degree of HLA matching.