T-cell Receptor Diversity Analysis In Patients With Wiskott-aldrich Syndrome Gene Mutations And Dock8Gene Mutations

PART ONE AND TWO T-CELL RECEPTOR DIVERSITYANALYSIS IN PATIENTS WITH WISKOTT-ALDRICHSYNDROME GENE MUTATIONSBackground: Wiskott-Aldrich syndrome (WAS) is a severe disordercharacterized by thrombocytopenia, eczema, immunodeficiency, andincreased risk of autoimmune disease and lymphoid malignancies. WASaffects one to ten in every million male newborns. The disease is caused bymutations in the WAS gene on the X chromosome, which encodes the WASprotein (WASp). WASp is a hematopoietic specific regulator of actinnucleation, and is activated in response to signaling arising at the cellmembrane. The average life expectancy of patients who have mutations thatabolish WASP expression is approximately15years. The immunodeficiencycaused by lack of WAS protein (WASP) expression has been mainlyattributed to defective T-cell functions. Whether WASP mutations havedifferential effects on the T-cell receptor (TCR) assembly of selected T-cell subsets is unknown.Objective: We aimed to identify the degree and the pattern of skewingin the variable region of the β-chain (Vβ) TCR repertoire in different T-cellsubsets from patients with WAS.Methods: We collected blood samples from patients aged from2m to22years old, among whom74%(17/23) were younger than three years old.We got PBMC from these samples, and sorted CD4+, CD8+T cells, na veand memory CD4+and CD8+T cells with flow, then extracted RNA andcDNA from total peripheral T cells and sorted T cells.1. Using CDR3spectratyping, we first analyzed the length of TCRCDR3in total peripheral T cells and sorted T cells, and compared to that ofthe age-matched healthy controls. To define each individual profile asnormal or skewed, a previously reported complexity scoring system wasused. The number of TCR Vβ subfamilies with skewed CDR size patterns, asshown by a complexity score of <4, was determined for each subject. Then,the mean complexity score, and the frequency of skewed TCR Vβ of total Tcells and sorted T cells were statistically analyzed.2. Using high-throughput sequencing, we sequenced the CDR3regionof na ve CD4+, na ve CD8+，and memory CD4+T cells from12WASpatients and12age-matched controls. The diversity of TCR repertoire wascalculated based on the Simpson Index of Diversity (Ds) and ShannonWiener index (H’). The private and public sequences of different people were analyzed by bioinformatic methods.Results:1. We first analyzed the length of TCR CDR3in total peripheral T cellsfrom seven of the23patients (P1to P7), compared to seven age-matchedhealthy controls using CDR3spectratyping. Our results show that themajority of Vβ subfamilies in T cells from control subjects exhibited six ormore peaks with a Gaussian distribution, indicating a polyclonal Vβrepertoire. In contrast, a number of Vβ subfamilies in T cells from patientsdisplayed skewed spectratyping profiles, indicating clonal or oligoclonalproliferation of T cells (see Fig1.3, A). Using these data, we furthercalculated the mean complexity score based on the amplification of all23Vβ segments. Consistent with spectratyping profiles, the mean complexityscore of patients (4.76; range,4.08–5.06) was significantly lower than that ofcontrols (5.23; range,4.95–5.48, P=.002; see Fig1.3, B).2. To determine which T-cell subtypes contribute to the alteration ofTCR spectratype profiles in patients, we compared the diversity of TCRrepertoires in sorted CD4+and CD8+T cells of six patients (P2and P7toP11) with that of six controls using Vβ spectratype analysis and complexityscoring. As shown in Fig1.5, A, most TCR Vβ subfamilies in patients’CD4+T cells showed a Gaussian distribution, while the TCR repertoire wasmostly skewed in CD8+T cells. The mean complexity scores of both CD4+(4.81; range,4.56–5.01) and CD8+T cells (3.76; range3.43–4.02) from WAS patients were significantly lower than those in control CD4+(5.22;range,5.07–5.34) and CD8+(4.65; range,4.12–4.99) T cells (CD4, P=.002;CD8, P=.002; see Fig1.5, B).3. Amplification of all23different Vβ segments showed that themajority of Vβ subfamilies in the na ve CD4+, na ve CD8+, and memoryCD4+T-cell subpopulations from healthy controls exhibited a Gaussiandistribution; however, memory CD8+T cells were mostly skewed (Fig1.6,A). In WAS patients, na ve CD8+T cells and memory CD4+and CD8+Tcells showed skewed distributions of Vβ subfamilies, but na ve CD4+T cellsdisplayed a normal distribution similar to that of the controls (Fig1.6, A). Inthe patient group, the mean complexity scores of naive CD8+,memoryCD4+and CD8+T cells, but not na ve CD4+T cells, was significantly lowerthan that of the healthy control group.4. The frequency of unevenly distributed Vβ subfamilies among all23Vβ subfamilies was determined in total peripheral T cells in patients withWAS mutations and in the control group. The median frequency of skewedTCR Vβ subfamilies in patients was17.39(range,0-47.83), which was notsignificantly different from the median frequency in normal controls (4.35;range,0-17.39; Fig3, A). As the degree to which skewing is attributable toeach subset could not be detected due to the overlap of CD4and CD8T cells,we determined the frequency of skewed TCR Vβ subfamilies in sortedCD4+and CD8+T cells. Compared to controls, the frequency of TCR Vβ subfamilies both in patients’ CD4+T cells (P=.03) and CD8+T cells (P=.01) showed significantly higher frequencies of skewed CDR3sizepatterns (Fig1.7, B). To determine whether patients’ na ve or memory Tcells was associated with the high frequency of Vβ skewing, sorted na veand memory CD4+and CD8+T cells were analyzed separately. We foundthat only na ve CD8+and memory CD4+T cells in the patient group showedsignificantly higher frequencies of skewed TCR Vβ subfamilies whencompared with those of the control group (Fig1.7, C). These results furtherdemonstrate that TCR Vβ repertoire diversity in na ve CD8+T and memoryCD4+T cells is significantly skewed in patients with WASP deficiency.5. Using HTS, in total, we generated approximately1.19billioneffective sequence reads corresponding to sequenced cDNA of CDR3, for72distinct T cell populations isolated from na ve CD4+, memory CD4+andna ve CD8+T cell subsets from12controls and12patients. There were nosignificant differences of sequence reads and CDR3for different subsets ofT cells between the patient and control group. However, the productive readsof memory CD4+T cells and the number of unique peptides of na ve CD8+Tcells were significantly lower in patient group than those in control group (P=.03and P=.02, respectively). To statistically describe the overall diversityof patients’ repertoire, we turned to Ds and H’, we found that both Ds and H’of the patient group were lower than those of the control group in all T-cellsubsets (Fig2.1), which indicates that the TCR repertoire of patients were less diverse than those of controls. However, only the H’ of memory CD4and na ve CD8T cells in patients displayed significantly less diversity (P=.003, and P=.022, respectively; Fig2.1, A), when compared to controls.Conclusion: These data provide the first report of the TCRVÎ' diversityin sorted na ve and memory CD4and CD8T in WAS, and have importantimplications for the design of vaccine strategies for the WAS patients. PART THREE T-CELL RECEPTOR DIVERSITYANALYSIS IN PATIENTS WITH DOCK8GENEMUTATIONSBackground: DOCK8immunodeficiency syndrome (DIDS) is acombined immunodeficiency characterized by recurrent viral infections,severe atopy, and early onset malignancy. DIDS are caused by mutations inthe gene encoding the DOCK8protein, which maps to the chromosomallocus9p24.3. DOCK8belongs to the DOCK180superfamily of proteins,which represent novel guanine nucleotide exchange factors for Rho familyGTPases. DOCK180-related guanine nucleotide exchange factors functiondownstream of multiple cell surface receptors to induce actin cytoskeletalrearrangement, lamellipodia formation, cell migration, integrin-mediated adhesion, phagocytosis, cell fusion, cell polarization, and synapseformation.The mortality of DIDS is relatively high, within the reported32cases,7patients had died within their6to21years. As DOCK8is widelyexpressed in human, the DOCK8mutations may affect multiple organs andtissues.DOCK8deficiency exhibites various abnormality in the immunesystem, and the most striking findings are the lymphopenia and antibodyabnormalities. The recurrent infection of DIDS is closely related to abnormalT cell function, and abundant T cell repertoire is critical for the response tomounting infection. However，Whether DOCK8mutations have differentialeffects on the T-cell receptor (TCR) assembly of selected T-cell subsets isunknown.Objective: We aimed to identify the degree and the pattern of skewingin the variable region of the β-chain (Vβ) TCR repertoire in different T-cellsubsets from patients with DOCK8deficiency.Methods: We collected blood samples from4DIDS patients and6healthy controls. We got PBMC from these samples, and sorted CD4+,CD8+T cells, na ve and memory CD4+and CD8+T cells with flow, thenextracted RNA and cDNA from total peripheral T cells and sorted T cells.1. Using CDR3spectratyping, we first analyzed the length of TCRCDR3in total peripheral T cells and sorted T cells, and compared to that ofthe age-matched healthy controls. To define each individual profile as normal or skewed, a previously reported complexity scoring system wasused. The number of TCR Vβ subfamilies with skewed CDR size patterns, asshown by a complexity score of <4, was determined for each subject. Then,the mean complexity score, and the frequency of skewed TCR Vβ of total Tcells and sorted T cells were statistically analyzed.2. Using high-throughput sequencing, we sequenced the CDR3regionof total T cells and sorted T cells from two DIDS patients, and comparedwith that of na ve CD4+, na ve CD8+，and memory CD4+T cells from12WAS patients and12age-matched controls. The diversity of TCR repertoirewas calculated based on the Simpson Index of Diversity (Ds) and ShannonWiener index (H’) and D50.Results:1. We first analyzed the length of TCR CDR3in total peripheral T cellsfrom four patients with DOCK8mutations, compared to six age-matchedhealthy controls using CDR3spectratyping. Our results show that themajority of Vβ subfamilies in T cells from control subjects exhibited six ormore peaks with a Gaussian distribution, indicating a polyclonal Vβrepertoire. In contrast, a number of Vβ subfamilies in T cells from patientsdisplayed skewed spectratyping profiles, indicating clonal or oligoclonalproliferation of T cells (see Fig3.1, A). Using these data, we furthercalculated the mean complexity score based on the amplification of all23Vβ segments. However, no significant difference was found between patients and controls in the mean complexity score (Fig3.1, B). And then,the frequency of unevenly distributed Vβ subfamilies among all23Vβsubfamilies was determined in total peripheral T cells in patients withDOCK8mutations and in the control group. Compared to controls, thefrequency of TCR Vβ subfamilies in DIDS patients showed significantlyhigher frequencies of skewed CDR3size patterns (P=.01, Fig3.1, C).2. To determine which T-cell subtypes contribute to the alteration ofTCR spectratype profiles in patients, we analyzed the diversity of TCRrepertoires in sorted CD4+and CD8+T cells, and sorted na ve and memoryCD4+and CD8+T cells of two patients (P3,D1and P4,D2) using Vβspectratype analysis and complexity scoring. As shown in Fig3.2, A, mostTCR Vβ subfamilies in patients’ CD4+T cells, na ve and memory CD4+Tcells showed a Gaussian distribution, while the TCR repertoire was mostlyskewed in CD8+, na ve and memory CD8+T cells. The mean complexityscores and frequency of skewed TCR VÎ' in both na ve and memory CD8+Tcells from DIDS patients were significantly different from those in na ve andmemory CD4+T cells (see Fig3.2, B). However, when compared with thecontrol and WAS patients, only the mean complexity score and thefrequency of skewed TCR VÎ' in na ve CD8+T cells were significantlydifferent from that in control group and WAS patients group （see Fig3.2, C,D）, Which indicates that the TCR diversity in na ve CD8+T cells in DIDSwas significantly skewed. 3. Using HTS, in total, we generated approximately100millioneffective sequence reads corresponding to sequenced cDNA of CDR3, for18distinct T cell populations isolated from CD4+,CD8+, na ve and memoryCD4+and CD8+T cell subsets from two DIDS patients. There were nosignificant differences of sequence reads and CDR3for different subsets ofT cells between different T cell subsets. However, the productive reads, andthe number of unique peptides and D50of CD8+T, na ve and memoryCD8+T cells were significantly lower than those in CD4+T, na ve andmemory CD4+T cells (Fig3.3).4. To statistically describe the overall diversity of patients’ repertoire,we turned to Ds, H’, and D50, we found that Ds, H’ and D50of the patientgroup were lower than those of the control group in all T-cell subsets (Fig3.4), which indicates that the TCR repertoire of patients were less diversethan those of controls. When compared with the control, only the H’ and D50of na ve CD8T cells in patients with DOCK8mutations displayedsignificantly less diversity (P <.0001， P=.03ï¼›Fig3.4); when comparedWAS patients, only the Ds, H’ and D50in na ve CD8T cells were lower thanthat in WAS patients, the Ds, H’ and D50in na ve and memory CD4T cellswere higher than that in WAS patients, which proves that the TCR diversityin na ve CD8+T cells in DIDS was significantly skewed, even comparedwith WAS patients.5. By HTS, we sequenced the CDR3of the PBMC, CD3, CD4, CD8, na ve and memory CD4and CD8T cells from one DIDS patients (P3, D2).There were no significant differences of sequence reads and CDR3fordifferent subsets of T cells between different T cell subsets. However, theproductive reads, the number of unique peptides and D50of PBMC, CD3+Tcells were significantly lower than those in CD4+T, na ve and memoryCD4+T cells. Which indicates that TCR diversity analysis in detailed sortedT cells may be more fairly reflect the truly situation in patients than that intotal PBMC or CD3+T cells.Conclusion: These data provide the first report of the TCRVÎ' diversityin total T cells, and sorted T cells in DIDS, and have important implicationsfor the design of vaccine strategies for the DIDS patients. Our analysis alsoprovides the necessary of sorting when analyzing TCR diversity.