Immunodominant Clonotypes Research Articles

Polyclonal Immune Response in T-LGL Leads to Clonal Expansions Preceding Occurrence of STAT3 Mutations Further Solidifying Clonal Dominance

T cell large granular lymphocyte leukemia (T-LGLL) characterized by excessive clonal cytotoxic T cell proliferation, is often accompanied by cytopenias and is thought to result from immune-mediated suppression, either by misguided immune antigenic recognition or mimicry triggered by auto-, tumor or viral antigens. Irrespective of the mode of evolution, T-LGLL progresses via: i) purely reactive clonal outgrowth in the context of a polyclonal immune response or: ii) a transforming event such as a STAT3 mutation (STAT3MT) within the immunodominant clone.Studies of TCR rearrangement using DNA based NGS of the TCR Vβ complementarity-determining region 3 (TCR VB CDR3) facilitate analyses of T cell clonality, as the CDR3-based “biological barcodes” allow for clonal quantification. We used serial CDR3 clonotyping accompanied by clonal STAT3 mutant burden measurements to recapitulate the pathological cascade in the course of T-LGLL. We asked if a polyclonal/oligoclonal immune response is the primary process that is clonally “imprinted” by STAT3MT, which would likely arise with the most proliferative clone. We were interested in the dynamics of underlying clonal behavior during treatment.Our initial cohort included 207 well characterized LGL patients. Most presented with anemia (46%), and/or neutropenia (46%). VB expansion was present in 94% of cases, with an average LGL count of 2317k/uL .STAT3MT were found by targeted deep sequencing in 38% of patients in 4 common hotspots: 42% Y640F, 34% D661Y, 11% D661V, and 8% N647I, with an average clonal burden of 28%. Multiple STAT3MT variants were found in some patients. From this cohort, serial samples obtained at presentation and throughout clinical course were obtained from 20 representative cases (10 STAT3MT and 10 STAT3WT), and subjected to analysis of clonal dynamics, including simultaneous deep TCR VB and STAT3 NGS. Patients were sequenced at an average of 4 time points (range 2-8). At least one major clonotype was identified in all patients, and multiple major clonotypes were identified in half.Analyses of clonal architecture revealed that STAT3 clones arose with VB expanded clones. In all cases, the TCR clonal burden was greater than that of STAT3MT demonstrating for the first time that STAT3MT is not the ancestral event for clonal expansion; rather, it evolves within the pre-expanded immunodominant clone.More than half of the patients were treated with immunosuppressive therapy (IST) achieving an approximate 40% response rate. Distinct patterns of clonal dynamics were seen following treatment (see Figure). In some patients, both the STAT3 (if present) and the major TCR clone decreased upon successful therapy. A correlation between a specific IST treatment and a clonal burden decrease was not found. In a subset of patients, the clones persisted despite a hematologic response, suggesting the major clonotype was functionally silenced. We also observed a common phenomenon of TCR “clonotype switching”, whereby therapy contracts one major clonotype, while another previously “minor” clonotype emerges. These newly expanded clones did not harbor STAT3MT, and most patients with “switching” were resistant to IST therapy. Multiple clonotypes were present at initial sampling in a few patients without STAT3MT and persisted at the same rate in subsequent samplings, precluding identification of a clear immunodominant clonotype. A stable or increasing clonal burden of both STAT3MT and VB CDR3 was seen in IST non-responders.In sum, STAT3MT were found to be a secondary event to the clonal expansion of the TCR VB clonotype, as a response within an already expanded clonotype and not the initiator of clonal expansion. The dynamics of both the STAT3MT and the TCR VB clonotype can be assessed over disease course and treatment regimens and demonstrate additional clinical ultility when applied to larger prospective clinical trials. The difficulty in finding a direct correlation between response to specific IST and a decrease in TCR VB clonal burden may be due to variable time frames between samplings, heterogeneity of IST regimens and response assessment, or large asymptomatic clonotypes. Prior to this study, the association of remission and elimination of immunodominant clonotypes was unclear. Our results suggest that clonal elimination is not necessary for complete clinical response; rather, the clone can be contracted to a manageable clonal burden or silenced. DisclosuresMustjoki:Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST).The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients.Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software.Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts.In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1).Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of T cell receptor repertoire in monozygotic twins concordant and discordant for chronic hepatitis B infection

Due to their identical inheritance and shared surroundings, identical twins have been the recommended group for studying the susceptibility and prognosis of diseases. Here, CD8+ T cell receptor beta (TCRβ) chains were analyzed by high-throughput sequencing in three pairs of healthy identical twins and chronic hepatitis B patients. The data showed a high level of similarity in the TCR repertoire of each pair in terms of average TCR Vβ segment expression and frequency of the complementary determining region 3 (CDR3) pattern and skewed or oligoclonal clonotypes. Notably, the level of similarity in TCR Vβ expression between the twins appeared to be independent of the consistency or inconsistency of chronic HBV infection, although the detailed CDR3 pattern and frequency were related to disease prognosis. There were more immunodominant clonotypes in patients with HBV antigen seroconversion, which showed an increased abundance. These immunodominant clonotypes may be used as favorable prognostic biomarkers and potential targets for immunotherapy. Thus, delineating the CD8+ T cell repertoire of identical twins with concordant chronic viral infections provides a promising means to screen protective TCR genes for immunotherapy.

Molecular Immunoprofiling the T Cell Repertoire after Rituximab Administration Reveals Frequent Oligoclonality Albeit with Different Patterns Depending on the Clinical Context

In patients with B lymphoid malignancies, depletion of B cells by Rituximab, an anti-CD20 humanized monoclonal antibody, can induce changes in the subset composition, activation and function of T cells. The spectrum of resultant immune-mediated sequelae encompasses organ-specific manifestations (e.g. pneumonitis, gastritis) as well as Rituximab-associated late onset neutropenia (R-LON). Although the pathogenesis of these clinical syndromes is not fully elucidated, evidence suggests that at least a fraction of cases may develop in a setting of expanded cytotoxic T cell populations with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Similar cytotoxic T cell expansions can be observed after Rituximab administration in other clinical settings e.g. allogeneic transplantation (allo-HCT), where selective restriction of the T-cell receptor (TR) gene repertoire is probably driven by multifactorial mechanisms. Here, we sought to obtain more insight into this phenomenon by molecular immunoprofiling of the TR gene repertoire in two groups of patients who received Rituximab: (i) Group A: patients (n=10) with chronic lymphocytic leukemia (CLL) treated with fludarabine-cyclophosphamide-rituximab (FCR); and, Group B: patients (n=14) who underwent allo-HCT for hematologic malignancies and received Rituximab either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. Each patient included in the study received a mean of 6 cycles of Rituximab (range, 1-14). TR repertoire analysis was performed 11-88 (median, 36) and 5-24 months (median, 5) after the first Rituximab administration in Group A and Group B, respectively. TR beta gene rearrangements were PCR amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data were analyzed using the IMGT/V-QUEST tool. A total of 579 productive TRBV-TRBD-TRBJ rearrangements were analysed, 291 for Group A, 288 for Group B (6-91/case, median=20). Among the 46 TRBV functional genes identified, 3 accounted for >25% of cases in both groups: (i) TRBV27 (13% in both Groups A and B); ii) TRBV19-1 (13% in group A, 7% in group B); and, (iii) TRBV6-1 (7% and 6%, respectively). Clusters of identical (>=2) rearrangements corresponding to clonotypes were identified in all patients. Oligoclonality with immunodominant clonotypes (>12% of the repertoire) accounting for over 30% of the analyzed sequences was more frequent in Group A (7/10 cases) versus Group B (5/10 cases); however, larger clonotype expansions were seen in group B. Longitudinal analysis was performed in 3 patients with oligoclonality, 1 from group A and 2 from Group B: in the Group A patient, immunodominant clonotypes disappeared, while both patients in Group B retained the oligoclonal repertoire. Lymphocyte subpopulation analysis by flow cytometry was performed in 6 patients of each group. T-LGL proliferations (defined as CD4/CD8 abnormal ratio and CD3+CD8+CD57+ >30%) were found more often in Group B (3/6 cases in Group A versus 6/6 in Group B). They were related to oligoclonal TR gene repertoire in Group A (3/3) but not in Group B patients (3/6 cases). However, true expansions could be considered only in group B patients, since CD8+ lymphocytes >1.0*109/l were seen in all 6 Group B versus only 1/6 group A cases. Self-limiting R-LON was observed in 8 patients (4 in each group), but no association of oligoclonality to R-LON could be found. In conclusion, we report frequent development of oligoclonal T cell populations after Rituximab treatment in two different clinical contexts. The Groups analyzed differed with respect to the extent of oligoclonality, suggesting that the precise clinical setting determines the amplitude of TR repertoire skewing after Ritximab. Sustained oligoclonal cytotoxic expansions were recognised more often among allo-HCT patients, presenting with a highly restricted TR gene repertoire and likely reflecting strong antigenic stimulation by viruses and/or cGVHD aggravated by T cell imbalances induced by Rituximab. DisclosuresStamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Skewing of the T-Cell Receptor Repertoire in Patients Receiving Rituximab after Allogeneic Hematopoietic Cell Transplantation: What Lies Beneath?

The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. DisclosuresNo relevant conflicts of interest to declare.

Skewing of the T Cell Receptor Gene Repertoire and Public Clonotypes in Cytotoxic T Cells of Patients with Chronic Idiopathic Neutropenia: A Role for Antigen Selection in Disease Development

AbstractAbstract 831Chronic idiopathic neutropenia (CIN) is a disorder of neutrophil production usually characterized by benign and asymptomatic course and female predominance. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitor cells, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients. Recently, using flow cytometry and CDR3 spectratyping, we obtained for the first time evidence for expanded T cell populations alluding to repertore skewing in both PB and BM of patients with CIN, but more prominent in the BM CD8+ subset. Prompted by these fndings, here we significantly extended our studies of the cytotoxic T cell responses in CIN in order to obtain a comprehensive view of the role of antigen selection in CIN pathogenesis. The study included 18 patients with CIN, 17 females and 1 male, diagnosed according to the established criterial for CIN. TRBV-TRBD-TRBJ gene rearrangements were amplified on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=6) or BM (n=12) samples. PCR products were subcloned by transformation into E.Coli/TOP10F’ competent bacteria and individual colonies were chosen randomly and subjected to Sanger sequencing. Sequence data were analyzed using the International imMunoGenetics information system and more particularly the IMGT/V-QUEST tool. Overall, 507 TRBV-TRBD-TRBJ gene rearrangements were analyzed (19-30/case) of which 466 were productive since they used functional TRBV genes and also carried in-frame CDR3s. A polyclonal profile was seen in only 3/18 cases (16.7%). The remaining cases were found to carry clusters of identical rearrangements corresponding to distinct immunodominant clonotypes. The frequency of each clonotype was determined by dividing the number of identical sequences by the total number of subcloned sequences analyzed. In 10/18 cases (55.5%), the dominant clonotype accounted for 12.8–22.6% of the total, whereas in 5/18 cases (27.8%) it accounted for 34.2–68.4% of the total. The TRBV28 gene was used by the dominant clonotype of three different CIN cases; the TRBV10-2, TRBV10-3, TRB19, and TRBV7-8 genes were identified in the major clonotypes of two cases each. Importantly, cluster analysis of the CDR3 sequences of all CIN cases of the present study identified 4 different rearrangements that were shared by different patients (public clonotypes). In conclusion, the present study strongly suggests that CIN may result from an autoimmune reaction directed against granulocytic progenitors triggered by a restricted range of, as yet, unidentified antigen(s). The finding of public clonotypes may indicate that public antigenic stimuli and/or shared immune processes underlie CIN development, at least for a proportion of cases. Overall, our results further support the concept that CIN may share common pathophysiologic mechanisms with other disorders characterized by T-cell and cytokine mediated suppression of hematopoiesis, perhaps representing a milder extreme within the spectrum of these acquired immune-mediated BM failure syndromes. Disclosures:No relevant conflicts of interest to declare.

T Cell Receptor Repertoire Landscape Mapping: A Novel Paradigm for T Cell Mediated Autoimmune Disorders Based on Deep Vβ Chain Sequencing

Abstract 1341T cell large granular lymphocytosis (T-LGL), a chronic lymphoproliferation of cytotoxic T cells (CTL) is often associated with lineage-restricted cytopenias. CTL-mediated inhibition of hematopoiesis in T-LGL is, in principle, similar to other T cell-mediated bone marrow failure syndromes. Variation in the CTL target spectrum could explain the lineage-restricted cytopenias in T-LGL and pancytopenia in aplastic anemia (AA). Regardless of effector mechanisms, T-LGL can serve as a simplified, monoclonal model of otherwise polyclonal CTL-mediated AA. The study of TCR Vβ clonotypes has the potential to reveal the nature/derivation of antigenic targets. It is possible that clonal specificity is private, inherently unique, and thus suggestive of the existence of neo-antigens or unique exogenous antigenic drivers. In an alternative scenario, one could expect a high degree of clonal sharing between T-LGL patients who have a similar hematologic presentation and immunogenetic background. Finally, finding of expanded T-LGL clonotypes in controls could point toward autoantigens and breach of tolerance in T-LGL or possibly an inability to maintain viral defense. Consequently, the study of the TCR repertoire may reveal unique patterns allowing for classification of immune-mediated disease.In the past, molecular analysis of the T cell repertoire allowed only a quantitatively limited insight into the clonotypic spectrum. With the advent of next generation sequencing (NGS), the clonotypic repertoire can now be analyzed at an incredible depth. Beginning with a cohort of 143 patients with T-LGL leukemia, we observed a marked increase in clone size as assessed by Vβ flow cytometry in patients with HLA-B7 (p<0.001). Based on this finding, we performed NGS of the TCR CDR3 in 10 patients with CD8 T-LGL leukemia and 3 controls. We selected 7 patients who shared HLA-B7 and 9 with anemia as presentation; 3 had Vβ17 (TRBV19) as the immunodominant clone by flow. Several levels of bioinformatic analysis have been performed; the average depth allowed for characterization of 507574±7818 (338847–666567) sequences. First, we assessed the clonal size, degree of diversity, and determined the frequency of codominant clones. The dominant clone spanned 56.1±34.7 of the TCR repertoire. The diversity, i.e. the number of unique clonotypes per patient, was 3571±3520 (212–11185). Aside from the dominant clonotypes, there were, on average, 107±94 expanded/redundant clonotypes (frequency >5/10K) per patient. In controls, the diversity was 20283±8525 (12428–29349), the most expanded clonotype was 8.3%, and there were an average of 103±36 expanded/redundant sequences.To assess antigenic specificity we first assessed the homology of the CDR3 regions and found that immunodominant clonotype-related clones (>75% homology) were present in other patients, albeit with a lower degree of expansion. In general, accounting for this level of homology, the diversity of patient repertoires was much lower (82% lower) as compared to controls. Interestingly, in 6 patients an additional clone with an amino acid sequence similar to the immunodominant clone (codominant clone) was detected. Similarly, while there was clonotype sharing between the patients, the immunodominant clonotypes were unique among the 10 cases subjected to Vβ NGS. In particular, clustering based on homology revealed cluster sharing with an average of 8 clones structurally related to the immunodominant clone. Most importantly, the immunodominant clones and those with >75% homology to immunodominant clones were generally found at extremely low (<10) copy numbers in controls. However, dominant and codominant clonotypes were not found in a database of over 6K clonotypes associated with various autoimmune and infectious conditions.While these results are preliminary, clonotype sharing at the deepest level of molecular resolution between patients and controls suggests that antigens driving the immune response in T-LGL are unique to patients or may be related to other immunogenetic factors and not related to auto-antigens which would be expected to elicit responses at a lower level. Our study of T-LGL patients demonstrates that analogous analyses, currently ongoing in AA, may reveal distinct and possibly diagnostic landscapes which would allow for sub-classification of disease and identification of patterns predicting response to immunosuppression. Disclosures:No relevant conflicts of interest to declare.

Clonotype Analysis of Cytomegalovirus-Specific Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.

Molecular Analysis of Alloreactive CTL Post-Hemopoietic Stem Cell Transplantation

The development of laboratory tests for the diagnosis and monitoring of graft-vs-host disease (GVHD) is hampered by a lack of knowledge of minor histocompatibility Ags triggering alloresponses. We hypothesized that the unique molecular structure of the TCR could be used as a marker for the unidentified Ags and exploited for molecular monitoring of GVHD posttransplant. To identify alloreactive T cell clones, we performed in vitro allostimulation cultures for a cohort of patients undergoing hemopoietic stem cell transplantation and determined the sequence of the CDR3 of immunodominant alloreactive clones; 10 corresponding clonotypes restricted to activated T cells were identified. As an alternative method for the identification of alloreactive clones, molecular TCR analysis was applied to biopsies of GVHD-affected tissues. Culture- and biopsy-derived clonotypes were used to design sequence-specific quantitative PCR assays to monitor the levels of putative allospecific clonotypes in posttransplant blood samples and subsequent biopsies. Because of the rational design of the methods used to identify immunodominant clonotypes, we were able to follow the behavior of potentially GVHD-specific T cells during the transplant course. Based on our results, we conclude that molecular T cell diagnostics can be a powerful tool for monitoring immune responses posttransplantation.

Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome

AbstractImmune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable β-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.

Molecular Identification of Alloreactive CTL Precursors in Hematopoietic Stem Cell Transplantation.

One of the leading complications in HSCT is GvHD mediated by allospecific cytotoxic T cell (CTL) clones that develop from pre-primed or naïve alloreactive precursors present in the graft. The ability to identify GvHD-specific CTL may facilitate development of monitoring tools for management of GvHD. The sequence (or clonotype) of the complementarity determining region 3 of the variable beta (VB) chain of T cell receptor (TCR) can be used as a molecular marker of individual T cell clones that can be detected in blood or tissues affected by GvHD. We have previously shown that although GvHD is a polyclonal process, immunodominant clonotypes and consequently CTL clones can be identified and quantitated (Exp. Hem. 32: 1010–1022; Br. J. Haematol. 129: 411–419). Although these clonotypes likely correspond to CTL clones participating in the GvHD process their specificity is unknown and may be directed against infectious agents or autoantigens.To more rationally utilize clonotypic diagnostics using truly allospecific CTL clones we developed new methods of selection of immunodominant alloreactive clonotypes. These clonotypes can be isolated from in vitro activated CTL populations following an allostimulation assay or from tissues affected by GvHD, indirectly indicating their target specificity. In 8 HSCT donor/patient pairs we performed allostimulation cocultures using recipient cells as targets and graft lymphocytes as effectors; activated donor CD8+ T cells were sorted based on CD69 expression and immunodominant CTL clonotypes were analyzed in the activated fraction. were studied. Several immunodominant clonotypes were identified and 11 were found to be restricted to activated CTL. The frequency of 5 immunodominant clonotypes from 2 patients was followed in the post-transplant course by direct sequencing of the appropriate VB family-specific PCR. For 4 of the 5 putatively allospecific clones a clonotype-specific quantitative PCR assay was designed and performed, illustrating that levels of the clonotypes in blood can be monitored using this technology. By direct sequencing, allospecific clonotypes were not detected in biopsies of GvHD-affected tissues. By more sensitive clonotype-specific quantitative PCR assay allostimulation-derived clonotype was also detected in skin biopsy obtained for GvHD diagnosis following transplantation. We have also used a reverse strategy for detection of allsopecific clonotypes. GvHD affected tissues were used as a source of clonotype isolation; VB-specific multiplex PCR assay performed on biopsy-derived DNA from 6/8 of the HSCT recipients was performed and a total of 12 immunodominant clonotypes were identified in 5 patients. We showed that identical CTL clones can be detected in various tissues affected by GvHD (e.g. gut and skin). Moreover, we demonstrate that tissue-derived clonotypes can also be used for clonotype-specific quantitative PCR. For example, 1/3 patients studied tissue-derived clonotype was detected also in blood.Our studies demonstrate the feasibility of identifying alloreactive precursors in HSC grafts pre-transplant or during the first bout of GvHD. Once such CTL marker clonotypes are identified, they may be selectively depleted from the graft or simply be used as an individualized marker of GvHD activity.

New Molecular Strategies for Detection and Quantitation of Clonal Cytotoxic T Cell Responses in Myelodysplasia and Aplastic Anemia.

Clinical similarities suggest that a T cell-mediated immune attack is involved not only in the pathophysiology of AA but also certain forms of MDS. Such a mechanism may result from a CTL attack directed against normal hematopoiesis or may reflect an immune surveillance reaction. Unlike in clonal T-LGL, immune responses in bone marrow failure states are polyclonal. We hypothesized that if immunodominant CTL clones are involved in the mechanisms of cytopenia in MDS and AA, the clonal size should correspond to the activity of disease. New molecular technologies based on TCR B-chain CDR3 region analysis allow for identification and monitoring of immunodominant T cell clones. These techniques, initially established in T-LGL (Wlodarski et al, Blood 2005), serve here as a basis for the systematic analysis of polyclonal CTL responses in bone marrow failure. We analyzed blood and paraffin-embedded marrow specimens from 30 AA, 21 PNH, 36 MDS and 60 T-LGL patients. By crude flow cytometric VB utilization analysis, we observed a continuum of VB skewing patterns from extreme monoclonal VB over-representation in T-LGL to multiple smaller expansions identified in AA and MDS. To determine whether the VB expansions are due to oligoclonal CTL proliferations, we have amplified and cloned the VB CDR3 regions of expanded VB families and sequenced large numbers of clonotypes. In both AA and MDS, most of the expanded VB families showed oligoclonality consistent with the presence of immunodominant clones. Pathogenic immunodominant clonotypes were not present in HLA-matched sibling donors or healthy identical twins discordant for the disease. A novel TCR VB multiplex PCR was applied for direct identification of immunodominant clones in the corresponding paraffin-embedded biopsies. Using this technique we retrospectively demonstrated the presence of skewed pattern of CTL clones in marrow biopsies of AA and some MDS patients; we also identified immunodominant clones in most of the biopsies studied.Moreover, biopsy-derived clonotypes were detected in blood; in many instances these clonotypes were also immunodominant as precisely quantitated by a novel clonotypic Taqman PCR utilizing CB- and clonotype-specific primer and JB-specific Taqman probes. Using a modified clonotypic PCR, blood-derived clonotypes were also found in patients' bone marrow biopsies, but not in controls. The frequency of dominant VB CDR3 clonotypes and the overall TCR repertoire were measured before and after administration of immunosuppressive therapy. Notably, the size of immunodominant clones decreased parallel to hematopoietic improvement, while the overall variability of TCR repertoire increased. Conversely, nonpathogenic clones remained unchanged. Globally, our analysis showed that immunodominant CTL clonotypes can be detected at comparable rates in AA and MDS irrespective of the morphologic subtype. Immunodominant clones derived from blood or even historical marrow specimens can be used as markers of the activity of the disease. Beyond the previously reported molecular TCR analysis in AA and MDS, we show that the CDR3 sequences of immunodominant clones can be exploited to design individualized quantitative assays that would facilitate recognition of clonotypic patterns in patients. In the future, the analysis of CDR3 amino acid sequence patterns may be used for structural comparisons of CTL clones in MDS and AA.

Pathologic clonal cytotoxic T-cell responses: nonrandom nature of the T-cell–receptor restriction in large granular lymphocyte leukemia

AbstractT-cell large granular lymphocyte (T-LGL) leukemia is a clonal lymphoproliferation of cytotoxic T cells (CTLs) associated with cytopenias. T-LGL proliferation seems to be triggered/sustained by antigenic drive; it is likely that hematopoietic progenitors are the targets in this process. The antigen-specific portion of the T-cell receptor (TCR), the variable beta (VB)–chain complementarity-determining region 3 (CDR3), can serve as a molecular signature (clonotype) of a T-cell clone. We hypothesized that clonal CTL proliferation develops not randomly but in the context of an autoimmune response. We identified the clonotypic sequence of T-LGL clones in 60 patients, including 56 with known T-LGL and 4 with unspecified neutropenia. Our method also allowed for the measurement of clonal frequencies; a decrease in or loss of the pathogenic clonotype and restoration of the TCR repertoire was found after hematologic remission. We identified 2 patients with identical immunodominant CDR3 sequence. Moreover, we found similarity between multiple immunodominant clonotypes and codominant as well as a nonexpanded, “supporting” clonotypes. The data suggest a nonrandom clonal selection in T-LGL, possibly driven by a common antigen. In contrast, the physiologic clonal CTL repertoire is highly diverse and we were not able to detect any significant clonal sharing in 26 healthy controls.

Antagonism of Antiviral and Allogeneic Activity of a Human Public CTL Clonotype by a Single Altered Peptide Ligand: Implications for Allograft Rejection

Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801(+) individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801(FLRGRAYGL). Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 muM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.

Prospective Molecular Identification of Alloreactive CTL Clones in Allogeneic Hematopoietic Stem Cell Transplantation.

The process of immune recovery after allogeneic HSCT can be characterized by an often profound oligoclonality of the TCR spectrum which may reflect: 1) A decreased diversity within the T cell population or 2) Expansion of individual clones that may be caused by specific antigenic drive exerted by pathogens (e.g., CMV) or alloantigens during the process of GvHD. Novel technologies based on the molecular analysis of the TCR repertoire can be applied to study clonal responses, including multiplex amplification of rearranged TCR VB chains followed by sequencing and quantitation of their contribution to the entire T cell repertoire. We initially studied the T cell repertoire after allogeneic HSCT in sibling (N=20) and matched unrelated (N=9) transplants. VB spectratyping was performed on CD8+ T cells in 22 patients; of the expanded VB families tested, 61.2% (30 of 49) were mono- or oligoclonal by genotyping. The clonal size and structure was determined by sequencing. Immunodominant clones contributed up to 5.4% (avg. 1.4%; range 0.1–5.4%) of all CD8+ T cells, indicating that certain stimuli may drive expansion of immunodominant clones. We originally hypothesized that these expanded clones were allospecific and likely played a role in GvHD; however, we found no correlation between the presence of significant expansions and grade III/IV GvHD. Therefore, in order to identify alloreactive CTL clones and their clonotypic markers, an alternative approach was devised.The proposed technique utilizes an allostimulation step: recipient cells serve as targets to induce activation of allospecific donor cells. Donor alloreactive cells are identified by their expression of activation markers, such as CD25 or CD69. After sorting, allospecific T cells are used as a source of cDNA for identification and quantitation of allospecific clonotypes. In this fashion, we have analyzed patients undergoing allogeneic sibling and matched unrelated donor grafting (N=7). Prior to transplant, allostimulation was performed and alloreactive CD8-derived clonotypes were subjected to molecular analysis. VB families represented within alloresponsive CTL populations that were oligoclonal by genotyping were subcloned and a large number of CDR3 clones were sequenced to identify the immunodominant clonotypes. Sequences have been derived from activated CD8+ donor cells in 6 cases; an average of 4 (range 1–7) VB families per pair have been characterized.. Although the presence of multiple VB families with a diversified CDR3 spectrum suggests the polyclonal nature of alloresponsive clones, immunodominant clones were identified. A total of 13 immunodominant clonotypes have been identified in 5 patients. Five such clones were identified in one donor/recipient pair; in each pair at least one immunodominant clonotype was isolated. Up to 18 clones per VB family were sequenced, and the average expansion contributed 56% to the entire VB family (range 15–100%).Clonotype-specific primers have been designed from two expanded clones and used to detect the allospecific clones in post-transplant blood samples in one patient/donor pair. In sum, molecularly defined marker clonotypes indicative of alloresponsive CTLs in HSCT can be individually and prospectively isolated. Such clonotypes may find application in tissue and blood diagnosis of GvHD.

Pathologic Clonal CTLResponses - Non Random Nature of the TCR Restriction in LGL Leukemia.

T-LGL leukemia, a clonal lymphoproliferation of CTL associated with cytopenias, seems to be triggered/sustained by antigenic drive. It is likely that hematopoietic progenitors are the targets in this process. We hypothesized that CTL clones evolve not randomly but in the context of an autoimmune response. We studied 75 patients with suspected LGL leukemia. The detection strategy for signature clonotypes included VB flow cytometric typing and VB specific PCR or multiplex PCR on cDNA from sorted CD8+ cells, followed by colony sequencing of the VB CDR3 regions (clonotypes). In 60 patients, we were able to isolate and characterize 86 pathologically expanded clonotypes (in 22 patients >1 immunodominant clone was detected); in most of the cases, the over-represented VB families nominally were >20% of all CD8+ cells. Clonal frequency was 65%± 30% of a given VB family or 35.4%± 31% of the total CD8+ population. By comparison, the most expanded clones in controls constitute to only 0.7% of the CD8+ repertoire.Two patients showed an identical immunodominant clonotype also shared with a third patient in whom it was not expanded. We also found sequence identity between an immunodominant and a minor clonotype and between 2 minor clonotypes in 2 LGL patient pairs. In 2 other patient pairs, a major clonotype showed a striking homology to a minor clonotype. Not only were identical clonotypes shared between the patients, a high degree of similarity was found between a large number of minor clonotypes. Likely, these minor clonotypes are the remnants of the initial polyclonal response. When up to 172 clones were sequenced per VB family in 24 healthy donors, only one shared clonotype was found in 2 donors and only a total of 11 sequences were detected to show the level of homology as detected in LGL. Immunodominant clonotypes can be used as tumor markers or to study expansion of LGL clones in correlation with disease activity. Identification of pathologic clonotypes allowed for design of a clonotype-specific real time PCR that allowed for monitoring of the clonal behavior over the time. For 7 patients followed serially either by sequencing or clonotype-specific Taq-Man PCR, a marked decrease in the frequency of the immunodominant clone was observed with successful therapy. This process resulted in increased clonal diversity and decreased redundancy. Some clonotypes that appeared post therapy showed sequence similarity to the original immunodominant clones.Given the normally immense variability of the TCR repertoire and the very low frequency of shared clonotypes identified in healthy donors, our findings suggest a non-random nature of LGL transformation. This possibly occurred in the context of initially polyclonal immune response directed against common antigenic target. Clonotypic sequences may be used to monitor the frequency of malignant T cell clones, but such an approach may also be applied for polyclonal responses in which clonotypes derived from most significantly expanded T cell clones may serve as marker for specific autoimmune processes. Finally, the availability of the clonotypic sequences may allow for measurements of anti-idiotypic responses and study of the applicability of clonotype-based anti-idiotypic vaccination as a potential therapeutic modality for T cell malignancies.

Efficient Identification of T-Cell Clones Associated with Graft-Versus-Host Disease (GvHD) in Target Tissue for Subsequent Detection in Peripheral Blood.

We hypothesized that after allogeneic hematopoietic stem cell transplant (HSCT), GvHD-affected tissues harbor expanded “immunodominant” T-cell clones, and characterization of these clones can be used to develop markers of disease. A multiplex PCR was used to detect T-cell receptor variable beta (VB) chain rearrangements in target tissue. Molecular analysis of the amplified VB CDR3 sequences allowed for identification and quantitation of putative disease-associated “clonotypes” and for the development of clone-specific PCR. We studied 5 HSCT patients for the presence of signature clonotypes in 10 skin biopsies taken during diagnostic GvHD work-up. Size distribution analysis of VB PCR products showed a skewed peak pattern in 9 biopsies; immunodominant clones (per definition frequency ≥30%) were detected in 6/7 biopsies with histologically confirmed GvHD, consistent with the presence of expanded clonotypes and the oligoclonal nature of the tissue-specific alloresponse. Immunodominant clones were also found in 2 of 3 biopsies not diagnostic for GvHD but obtained based on strong clinical suspicion, raising the possibility that they were associated with early evolving GvHD not distinguishable by histology. For example, when serial skin biopsies were analyzed, a GvHD-positive post-transplant d63 biopsy contained an immunodominant clone (frequency 60%), which was also detected in a subsequent biopsy positive for GvHD (frequency 33%). Similar results were seen in another patient, in whom serial biopsies taken on d214 (not diagnostic) and d217 (GvHD-positive) showed an identical immunodominant clone, not present in a d13 GVHD-negative biopsy. This finding suggests that the d214 biopsy might have contained early GvHD that was not detectable morphologically. In a patient who rejected an initial MUD graft (Tx 1) and then received a MUD SCT (Tx 2) from a different, unrelated donor, immunodominant clones were identified in GvHD-positive biopsies following each transplant, that were distinct for each graft. To examine whether immunodominant clonotypes derived from biopsies could be used as markers of disease, clonotypic PCR was developed for an immunodominant biopsy-derived clonotype for each transplant. The Tx 1 clonotype was detected in blood and skin following Tx 1, but not in tissue or blood taken after Tx 2. Specificity and correct size of the clonotypic PCR product were confirmed by both Genescan analysis and sequencing. Clonotypic PCR designed for an immunodominant clonotype from the Tx 2 donor detected the putative allospecific clonotype in serial samples after the second engraftment. Neither clonotype could be found in either donor, indicating that the disease-associated clones expanded to detectable levels following transplant. These results indicate that clonotypic PCR can distinguish distinct GvHD-associated clonotypes from different donors in both blood and tissue following transplant. Monitoring of the relative frequency of disease-associated clones in recipient blood indicated a significant peripheral expansion of disease-associated clones at the time of active GvHD. Our results demonstrate an efficient method for identification of disease-associated clonotypic markers, which can be used to aid diagnosis and monitoring of GvHD.

Shared and individual specificities of immunodominant cytotoxic T-cell clones in paroxysmal nocturnal hemoglobinuria as determined by molecular analysis

Objective Similar immune mechanisms have been suggested to operate in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH), and the presence of PNH clones in AA may indicate that an immune reaction directed against hematopoietic stem cells may be responsible for the immune selection pressure leading to PNH evolution. We previously described expansions of selective cytotoxic T-lymphocyte (CTL) clones in AA patients. Materials and methods We applied a molecular analysis of the T-cell receptor repertoire to study the characteristics of CTL response in patients with various forms of PNH. Immunodominant T-cell clones were detected using combined flow cytometric and molecular analysis of the variable beta (VB) chain and CDR3 representation, followed by determination of the frequency of individual CDR3 clonotypes. Clonotypic polymerase chain reaction (PCR) was performed to establish clonotypic utilization pattern. Results In patients with a past history of AA, and when subgrouped by current blood counts as “hypoproliferative” PNH patients (in contrast to purely hemolytic form of PNH), more pronounced skewing of VB family utilization was found, consistent with T-cell responses involving several immunodominant CTL clones. Sequences of the PNH-derived clonotypes were used to design PCR-based assays for the utilization analysis of individual clones in PNH patients. The clonotypic distribution pattern established by this PCR method indicated that immunodominant T-cell specificities were shared between some patients but also may be found at low frequencies in controls. Conclusion Analysis of the CDR3 sequence pattern as a marker for expanded immunodominant clonotypes may have an application in the study of T-cell responses in PNH.

Application of the Molecular Analysis of the T-Cell Receptor Repertoire in the Study of Immune-Mediated Hematologic Diseases

The basis for the vast recognition spectrum of the T-cell receptor (TCR) can be determined by the rearrangement and recombination of the variable, diversity and joining regions of the variable portions of β (B) and α (A) chains as well as their recombination and modification. Analysis of the TCR rearrangement has been routinely used to detect clonality for the diagnosis of lymphoid malignancies. However, molecular analysis of the TCR repertoire can be a powerful tool in the study of T-cell responses to pathogens and in autoimmune diseases. The concept of the oligoclonality in the context of cellular immune responses is based on the presence of immunodominant T-cell clones within distinct T-cell subpopulations used for analysis. Under normal circumstances, a limited number of clones undergo periodic expansions in reaction to foreign antigens. Under pathologic conditions, though, the derailment of immune regulation allows expansions of specific and potentially pathogenic T-cell clones. For example, large granular lymphocyte (LGL) leukemia illustrates an extreme expansion of a single T-cell clone associated with a distinct autoimmune pathology, which suggests an exaggerated clonal response to a specific antigenic target. In immune-mediated bone marrow failure syndromes, clonal rearrangement of the TCR cannot be detected in unseparated blood or marrow. Nevertheless, individual T-cell clones can significantly expand and may allow for demonstration of oligoclonality in selected T-cell populations. These subpopulations are defined, for example, by a specific β (B)-chain usage or other phenotypic markers. Given the diversity of the TCR recognition spectrum, the task of identifying immunodominant clonotypes derived from unique complementarity determining region-3 (CDR3) sequences is very complex. However, expanded T-cell clones likely represent immunodominant responses which can be detected on the molecular level using analysis of the individual TCR VB-chain representation, CDR3 size fragment skewing, and determination of the frequency of individual clonotypic sequences. In the future, TCR VB clonotypes may be applied as a diagnostic tool, analogous to serologic markers. As an investigative tool in hematology, molecular analysis of the TCR utilization pattern and the detection of immunodominant clonotypes represents a novel approach in the study of immune-mediated hematologic diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS), anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GvHD).

A Structural Basis for the Selection of Dominant αβ T Cell Receptors in Antiviral Immunity

We have examined the basis for immunodominant or “public” TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen, the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1α and CDR2α loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRα constant domain thought to form part of the docking site for CD3ϵ. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.