Vbeta Repertoire Research Articles

Hypomorphic splice site IL2RG variants associated with Cryptosporidium liver disease

BackgroundX-linked severe combined immunodeficiency (X-SCID) is caused by mutations in IL2RG encoding for the common gamma chain (γc). This disease is typically fatal within the first year without immune reconstitution. We present two patients with IL2RG variants leading to a phenotype of Cryptosporidium-related sclerosing cholangitis. MethodsMedical history and laboratories were reviewed for two patients with IL2RG variants at c.270G. For one patient, we examined cDNA, γc expression, TCR repertoire, cytokine signaling, in vitro B-cell plasmablast differentiation, and in vitro T-cell differentiation of peripheral blood CD34+ cells. ResultsTwo unrelated male patients (17 and 21 years old) were identified with liver disease, recurrent childhood pneumonia and resultant bronchiectasis. P1 has persistent warts. Elevated transaminases in P1 led to liver imaging and biopsy consistent with sclerosing cholangitis. Stool PCR was positive for Cryptosporidium. P2 was diagnosed at age 6 with cryptogenic cirrhosis. He underwent orthotopic liver transplant at age 19; diarrhea and cholestatic liver disease developed 4 months post-transplant, and cryptosporidium was found in stool and liver. Cryptococcus meningitis also developed on immunosuppression. Both patients were hemizygous for an IL2RG variant resulting in p.W90C (P1:c.270G>T; P2:c.270G>C). P1 had mildly reduced CD4+ lymphocytes, CD4+ and CD8+ central/ effector memory T-lymphocytes, memory B-lymphocytes, and normal NK counts. P1 cDNA revealed a leaky splicing defect with a mix of exons 2–4 skipping along with full-length transcript encoding p.W90C. Cells from heterozygous mother expressed wild-type sequence suggesting skewed X-inactivation. Surface protein expression and distribution of TCR V-Beta repertoire was largely normal. IL-2-mediated STAT5 phosphorylation was impaired in T- and NK-cells. P1’s B-lymphocytes proliferated in response to in vitro IL-21/CD40L co-stimulation but failed to differentiate into CD27+CD38+ plasmablasts (< 1%). In vitro T-cell differentiation showed impaired survival of developing thymocytes, despite capability of maturing intoTCRaB+ CD3+ T cells. ConclusionsWe present two patients hemizygous for IL2RG variants leading to milder clinical phenotype compared to classic X-SCID. Consistent with other diseases of impaired IL-21 signaling, there was significant Cryptosporidium-related liver disease in addition to clinical manifestations of hypomorphic IL-2 signaling defect. Further work is necessary to address restoring IL-21 signaling to improve liver disease.

Heterozygous Variants in the DNA-binding Domain of c-Myb May Affect Normal B/T Cell Development.

Heterozygous Variants in the DNA-binding Domain of c-Myb May Affect Normal B/T Cell Development.

Clonal Expansion of ∝β-T Lymphocytes With Inverted Jβ1 Bias in Familial Hemophagocytic Lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare but fatal disease in infancy. There are no previous reports on the clonality of T cells in FHL patients. We analyzed here the clonality of alphabeta-T cells in 5 FHL patients using an inverse reverse transcriptase-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region gene (TCR V), a joining region gene of the beta chain (Jbeta)-PCR, a single-strand conformation polymorphism (SSCP), and sequence analysis. A high frequency (15%) of Vbeta and Valpha families was observed in 3 of 5 and 4 of 4 patients examined, respectively. In 19 Vbeta repertoires, including all highly frequent Vbeta, the Jbeta-PCR analysis showed restricted usage of the Jbeta family, indicating a marked bias to Jbeta1 subsets (the mean rate of Jbeta1:Jbeta2 was 87:13 in 65% of the alphabeta-T cells) in widespread alphabeta-T cells (in all patients but 1). In all patients, the clonality of specific Vbeta-Jbeta fragment expanded was confirmed by SSCP and sequence analysis. These results suggest that the existence of clonal expansion and restricted Jbeta1 usage of T cells in FHL is genetically associated with the pathogenesis and the immunodysfunction of the disease. These results help to explain some of the abnormal functional behaviors of T cells in FHL and raise new questions regarding the mechanisms responsible for the restricted clonal diversity.

Bipotent Lymphoid Progenitors Independently Maintain Long-Term Genetically Engineered T and NK Cell Production in Humans

Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental setting has been available to validate such statistical prediction neither in the mouse nor in humans. We here report the first formal evidence, directly in vivo in humans, that a population of bipotent lymphoid progenitors is capable of surviving and maintaining de novo T/NK production for at least 15 years after loss of transplanted HSC. In 5 SCID-X1 patients treated with gammaretroviral vector HSC-GT we observed that genetically engineered myeloid and B cells have stayed constantly below detection limits starting from 6 months after GT. On the contrary T and NK cells remained vector positive for up to 19 years (average vector copies/cell equal 2.1 and 2.3 respectively). We initially thought that such data would be consistent with permanent loss of engineered HSC and survival of vector-marked long-living circulating mature lymphocytes. However, strikingly and unexpectedly, by a comprehensive immunophenotyping of T cells overtime, we detected vector-positive short-living CD45RA+CD62L+CD95- naïve T cells (Tn) in the circulation of these patients. We first obtained functional validation of the identity of such Tn cells by IFN-y productionassays and we confirmed positive thymic activity by measurement of TREC content. High throughput sequencing of 1,193 T-cell receptor (TCR) rearrangements in FACS-sorted T cell subtypes confirmed that these engineered Tn cells had normal TCR diversity and that new rearrangements were detectable overtime. Similarly, Vbeta repertoire measured by spectratyping displayed normal profiles in all patients. These results suggested that a de novo T-cells production is maintained in these patients by a putative LtLP population in absence of supply by gene-corrected HSC. Using Tn data as surrogate markers of LtLP clonal dynamics and composition, we collected and analysed 12,756 unique IS from 5 populations including Tn cells in a window of 10.1 to 14.9 years after the observed loss of transplanted HSC. Clonal diversity was stable in all T subtypes and IS sharing across subpopulations and timepoints was high and consistent with active in vivo output by LtLP and differentiation of Tn into memory/effector T cells. Analyzing nature and recapture probability of IS we observed that IS in LtLP are significantly enriched in genes involved in lymphocyte survival/activation and we could estimate that T cell production is currently sustained by 2,092-6,056 individual engineered LtLP clones. Moreover, by studying 651 IS collected from CD3-CD56+ purified NK cells we observed that up to 41.2% of them were shared with independently analyzed Tn suggesting that the LtLP active in these patients have a dual T/NK restricted potential. Lastly, we detected and tracked overtime 52 clones bearing IS in MECOM, CCND2 and LMO2 including the IS originally associated with vector-induced leukemia in one patient. Our data show absence of any sustained clonal expansions within the engineered LtLP population for up 19 years after GT, the longest follow up available to date in clinical HSC-GT. In conclusion, our data provide the first formal evidence in vivo in human that a de novo production of genetically engineered T and NK cells can be physiologically maintained by a population of LtLP surviving up to 15 years after loss of transplanted HSC (manuscript submitted). Identification and exploitation of such human LtLP population might be crucial for the development of next generation GT and cancer immunotherapy approaches. Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Thrasher:Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Systemic lupus erythematosus favors the generation of IL-17 producing double negative T cells

Mature double negative (DN) T cells are a population of αβ T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.

Histopathologic assessment of cultured human thymus.

The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.

Combination of Triptorelin with Nivolumab in ICI Resistant Advanced Melanoma

Abstract Background It has been shown that androgens are immunosuppressive. Androgen deprivation with GnRH agonists regenerates the thymus and its functions in adults, leading to increase in blood and lymphoid organ lymphocytes, in naive lymphocytes with expansion of TCR Vbeta repertoire, and in Tumour Infiltrating Lymphocytes (TILs) in prostate tumours. This phase I study evaluated the safety of triptorelin in combination with nivolumab, as well as its potential to reverse resistance to PD-1 inhibitors in male melanoma patients. Methods Treatment consisted of triptorelin 3.75 mg i.m. every 4 weeks and nivolumab 3 mg/kg i.v. every 2 weeks. Bicalutamide 50 mg p.o. QD was added for the first 28 days. Evaluation of response was performed after 3 months. Triptorelin PK was assessed and various PDy markers were measured in blood and tumour samples. Planned treatment duration was 48 weeks. Results Fourteen male patients were included, of whom 11 were white and 3 were black, with mean age 65 years (range 45-82). At screening 4 were locally advanced while 10 had distant metastases (2 with M1a, 1 with M1b, 6 with M1c and 1 with M1d). Ten patients had cutaneous melanoma, 2 patients had mucosal melanoma, and 2 had an unknown primary. Safety: No grade 4 AEs were reported. Five grade 3 AEs were reported in 4 patients, of which one (abdominal pain) was attributed to triptorelin and resolved after 39 days (causing no treatment interruption as it started on the day treatment was discontinued due to progression), and one (neutropenia) was considered related to nivolumab and resolved following treatment interruption for 14 days. Efficacy: BOR (RECIST 1.1) was assessed as 2 PR, 5 SD, and 7 PD. The two patients with PR showed reductions from baseline in Target Lesions of 76% (one pancreas metastasis) and32% (two inguinal lymph nodes), following an initial pseudoprogression. Conclusion The association of triptorelin to nivolumab was well tolerated and yielded partial response in two patients. Legal entity responsible for the study Debiopharm International S.A. Funding Debiopharm International S.A. Disclosure C. Robert: Advisory / Consultancy: Novartis, Bristol-Myers Squibb, MSD, Roche, Sanofi, Amgen, Pierre Fabre. F.J. Lejeune: Honoraria (self): Debiopharm International SA. C. Lebbe: Honoraria (self): Roche, Bristol-Myers Squibb, Novartis, MSD, Amgen, Pierre Fabre, Pfizer, Incyte; Advisory / Consultancy: Roche, Bristol-Myers Squibb, Novartis, MSD, Amgen; Travel / Accommodation / Expenses: Bristol-Myers Squibb, MSD; Speaker Bureau / Expert testimony: Roche, Bristol-Myers Squibb, Novartis ,Amgen; Advisory / Consultancy: Aventis. T. Lesimple: Research grant / Funding (self): Roche; Advisory / Consultancy: MSD, Novartis, Pierre Fabre. E. Lundstrom: Full / Part-time employment: Debiopharm International SA. V. Nicolas: Full / Part-time employment: Debiopharm International SA. B. Gavillet: Full / Part-time employment: Debiopharm International SA. V. Gregoire: Full / Part-time employment: Debiopharm International SA. P. Crompton: Full / Part-time employment: Debiopharm International SA.

Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis

The thymus is a primary lymphoid organ that plays a critical role in the development of adaptive T cell immunity and central tolerance. Bone marrow-derived lymphoid progenitor cells migrate into the thymus and interact with thymic epithelial cells (TECs) through sequential positive and negative selection to mature. Thymus-educated mature T cells express a diverse, MHC-restricted and self-tolerant T cell receptor (TCR) repertoire that protects against infection and prevents autoimmunity. Patients born with congenital thymic aplasia, due to 22q11 Deletion Syndrome, or mutations in TBX1, FOXN1 or CHD7, present with complete absence of T cells and a severe combined immunodeficiency (SCID)-like phenotype. Bone marrow transplantation does not cure the thymic defect in these patients and severe infections occur within the first year of life if left untreated. Allogenic thymus transplantation has provided proof of principle that HLA-unmatched pediatric donor thymic tissues can lead to successful immune reconstitution with the emergence of a diverse TCR V-beta repertoire. However, post-transplant organ-specific autoimmunity remains a major concern. Currently allogeneic thymus transplantation is no longer available in the US leaving a deadly therapeutic void for patients born without thymus. Patient-specific or histocompatible thymic tissues derived from pluripotent stem cells could address the critically unmet need, and also a broader range of clinical applications including immune reconstitution post hematopoietic stem cell transplantation (HSCT) and tolerance induction for solid donor organs.The thymus contains two major non-hematological components: the thymic stromal cells and the extracellular matrix (ECM). The thymic stromal layer is composed of thymic epithelial cells and mesenchymal cells. The thymic ECM forms a three-dimensional (3D) network to provide physical support and nutrition to thymic stromal cells.Methods: To address the need for histocompatible regenerative thymic tissues, we aim to differentiate fully functional thymic epithelial progenitor cells (TEPCs) from human pluripotent stem cells (hPSCs) and further generate 3D transplantable organoids using engineered matrix proteins that mimic the native thymic microenvironment.Results: We have developed a novel platform to generate hPSC-derived TEPCs by dissecting the key signaling pathways that govern human thymic ontogeny. These hPSC-derived TEPCs express the defining markers of TEPC-fate, such as FoxN1, Cytokeratin 8, Cytokeratin 5, Delta-like Canonical Notch Ligand 4 (DLL4) and MHC class II. Previous studies have shown FoxN1 to be the master regulator controlling thymic development, however, little is known about its regulatory network. Elucidating and validating the factors that initiate and maintain FoxN1 expression is the key to successfully engineer sustainable thymic tissues. We have identified a combination of morphogens that can maintain the expression of FoxN1, DLL4 and AIRE of primary TECs in culture. To gain insight into the composition of primary thymic ECM proteins and adapt their characteristics beyond the features of commercially available 3D hydrogels, we analyzed a series of human fetal thymic tissues using whole transcriptome analysis. Our current work focuses on adapting our 2D culture protocol to sustain hPSC-TEPCs in 3D matrix-based organoids. Ongoing studies test the capacity of hPSC-TECPs to promote T cell maturation and the development of a diverse TCR repertoire in an athymic xenograft mouse model (NSG-FoxN1null).Conclusions: hPSC can be differentiated in vitro into TEPC-fate and developed into thymic organoids using custom-designed protein matrices. Studies to test sustainability and functionality of the engineered thymic organoids in vivo are currently under way. DisclosuresNo relevant conflicts of interest to declare.

High Number of Activated CD8+ T Cells Targeting HIV Antigens Are Present in Cerebrospinal Fluid in Acute HIV Infection.

Central nervous system (CNS) infiltration by CD8 T cells is associated with neuroinflammation in many neurodegenerative diseases, including HIV-associated dementia. However, the role of CD8 T cells in the CNS during acute HIV infection (AHI) is unknown. We analyzed the phenotype, gene expression, T cell receptor (TCR) repertoire, and HIV specificity of CD8 T cells in cerebrospinal fluid (CSF) of a unique cohort captured during the earliest stages of AHI (n = 26), chronic (n = 23), and uninfected (n = 8). CSF CD8 T cells were elevated in AHI compared with uninfected controls. The frequency of activated CSF CD8 T cells positively correlated to CSF HIV RNA and to markers of CNS inflammation. In contrast, activated CSF CD8 T cells during chronic HIV infection were associated with markers of neurological injury and microglial activation. CSF CD8 T cells in AHI exhibited increased functional gene expression profiles associated with CD8 T cells effector function, proliferation, and TCR signaling, a unique restricted TCR Vbeta repertoire and contained HIV-specific CD8 T cells directed to unique HIV epitopes compared with the periphery. These results suggest that CSF CD8 T cells in AHI expanding in the CNS are functional and directed against HIV antigens. These cells could thus play a beneficial role protective of injury seen in chronic HIV infection if combination antiretroviral therapy is initiated early.

Deciphering the Different Types of Refractory Celiac Disease

Universite Paris Descartes-Sorbonne Paris Centre Gastroenterology department, Hopital Europeen Georges Pompidou APHP, Paris Inserm U989, Hopital Europeen Georges Pompidou APHP Paris, France Fifteen years ago has emerged the concept of refractory celiac disease (RCD). [1,2] RCD is defined by persisting malabsorption and villous atrophy after one year of strict GFD ascertained by a dietician. RCD has been subdivided into two subgroups according to the normal (type I RCD (RCDI)) or abnormal phenotype of intraepithelial lymphocytes (IEL) (type II RCD (RCDII). RCDI is indistinguishable from active CD and is characterized by a polyclonal repertoire. Survival of patients with RCDI is slightly inferior to that of those with celiac disease. [3,4] In contrast, RCDII is a severe enteropathy with frequent intractable ulcerative duodeno-jejunitis. RCDII is characterized by a clonal repertoire and is considered as a low-grade intraepithelial lymphoma. [1,2] The five year survival rate of patients with RCDII is around 50%. This poor prognosis is due to severe malnutrition and increased risk of overt lymphoma. One of the current challenges in RCD is to ascertain the type of RCD. Indeed, diagnosis of RCD has become highly specialized. It requires small bowel endoscopic specialized investigations such as capsule endoscopy and double balloon enteroscopy. [5,6] Intestinal biopsy needs to be studied by different complementary techniques such as immunohistochemistry, flow cytometry, and Multiplex PCR, all necessary to ascertain the precise diagnosis. Recent studies have reported case series of chronic intestinal diseases mimicking the both types of RCD. RCDI needs to be distinguished from other causes of non clonal villous atrophy such as enteropathy associated with common variable immunodeficiency (CVID). [7] Demonstration of HLA haplotypes encoding HLA-DQ2 or DQ8 can be useful as their absence excludes CD or RCD as a cause of villous atrophy. [8] Nevertheless, genotypes HLA-DQ2/DQ8 are also found in 77% of CVID patients with enteropathy mimicking CD defined by intestinal intra-epithelial lymphocytosis. [7] So evidence of serum hypogammaglobulinemia and histopathological features such as intestinal plasmocytic rarefaction or nodular lymphoid hyperplasia appear essential to distinct CVID enteropathy from RCDI. [7] RCDII needs to be distinguished from other cases of clonal enteropathy with villous atrophy. Flow cytometry allows to tell the difference between RCDII and other intestinal clonal malignancies with villous atrophy. Indeed, demonstration of excess of CD4+IEL with a specific Vbeta repertoire is essential for diagnosis of intestinal CD4 lymphoproliferations. [9] Diagnosis may be particularly tricky when CD4 lymphoproliferation [9] or large granular lymphocytic leukemia (LGL) [10] complicate an authentic CD. Consequently, spectrum of RCD has been recently substantially extended. Advances in accuracy of diagnostic tools could reduce the number of undiagnosed RCD and increase the frequency of RCD currently estimated to 1 to 2% of CD patients. [11,12] Besides epidemiological interest, improvement of diagnostic tools allowed precise diagnosis of RCD type and appropriate treatment. By example, distinction between RCDII and CD complicated by LGL is crucial as cyclosporin, inefficient in RCDII, is in contrast very useful for treating LGL. Indeed, treatment of RCD remains another challenge particularly in case of RCDII patients who have poor prognosis and for whom referral treatment does not yet exist. Immunosuppressors are poorly efficient and may possibly trigger overt lymphoma. [13] Purine analogues such pentostatine or cladribine (2 CDA) have been largely used in the past with slight therapeutic effect. [4,14,15] Autologous haematopoietic stem cells transplantation represents an interesting alternative but when combined to chemotherapy for hopping sustained reduction of abnormal IEL. [16,17] We are currently evaluating this strategy in a prospective phase II trial. Many advances in the understanding of the pathogenesis of RCDII were made by deciphering the anti-apoptotic signaling pathway of the cytokine IL-15 which prevents the elimination of IEL in CD and RCDII. [18] The IL-15 induced antiapoptotic signaling pathway includes activation of IL15Rβγ, Jak3, STAT5 and Bcl-xL [19] which represent so many therapeutic targets. Blocking the effect of IL-15 appears of a particular interest in RCDII but may probably be useful in other types of RCD with possible involvement of IL-15 such as RCDI or LGL-complicated CD. [20] Continuing the characterization of the different forms of RCD will probably guide next pathogenic studies and permit to increase our therapeutic efficacy in the future.

CD4 Treg and CD4 Tcon Utilize Distinct TCR Vbeta Repertoires in Response to Alloantigen

Background: Regulatory CD4 T cells (Treg) are potent to suppress the responses of conventional CD4+ (Tcon) and CD8+ T cells to alloantigens and prevent graft-vs.-host diseases (GVHD). Since the mechanisms for thymic selection of Treg and Tcon are distinct, we hypothesized that the two cells types use distinct TCR repertoires in the response to same alloantigen. We have used high throughput deep sequencing to study the T cell receptor (TCR) Vbeta CDR3 repertoire of CD4 Treg and CD4 Tcon at baseline and again after expansion with alloantigen.Methods: CD4+CD25+CD127- Treg and CD4+CD25-CD127- Tcon were FACS-sorted from healthy donor PBMCs or umblical cord blood mononuclear cells. Aliquots of sorted Treg and Tcon were expanded separately by DC from the same Major Histocompatibility (MHC)-mismatched donor. Treg were cultured with DCs, IL-2, IL-15 and rapamycin, while Tcon were cultured with DCs and IL-2. Genomic DNA from baseline or expanded purified Treg and Tcon was sequenced, at least 10^6 deep, by Adaptive Biotechnologies multiplex kit to detect unique T cell receptor (TCR) Vbeta CDR3 sequences. Data was analyzed by the algorithm established for the Adaptive Biotechnologies software and characterized according to the IMGT (International ImmunoGeneTics information system) nomenclature.Results: The TCR Vbeta CDR3 sequences of baseline natural Treg and Tcon T cell have minimal overlap, indicating that the thymus shapes distinct TCR repertoires in these two cell types. However, the CDR3 length and the frequency of nucleotide deletion or insertion at the Vbeta-Dbeta and Dbeta-Jbeta junction were similar. By employing the "robust regression model", we identified expanded TCR Vbeta CDR3 sequences among both Treg and Tcon after in vitro culture with the same alloantigens. These expanded Treg and Tcon used unique TCR Vbeta CDR3 sequences that are not shared by the other cell type. Expanded Treg and Tcon displayed fewer nucleotide deletions and insertions at the Vbeta-Dbeta and Dbeta-Jbeta junction than at baseline. The frequency of the expanded sequences, insertions and deletions were of the same magnitude in Treg and Tcon suggesting that both undergo similar processes of antigen-driven TCR selection and magnitude of cell expansion in vitro.Conclusion: CD4 Treg and Tcon utilize largely distinct TCR Vbeta CDR3 repertoires at baseline and after expansion against the same alloantigens. Questions remain whether Treg and Tcon recognize the same or distinct peptides from the same antigen, and whether they bind peptide with different avidity. TCR Vbeta CDR3 deep sequencing ought to be used to track single Treg and Tcon after adoptive cell therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alemtuzumab Achieved Durable Hematologic Response In Heavily Treated T-Large Granular Lymphocytosis Irrespective To STAT3 Mutation Or V-Beta Clone Size

▪ BackgroundT-large granular lymphocytosis (T-LGL) is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes, presenting with immune mediated cytopenias and associated with autoimmune syndromes. Immunosuppressive therapy (IST) with methotrexate, cyclosporine, or cyclophosphamide can improve the cytopenias in about half the patients but can lead to significant toxicity in older patients. The anti CD52 antibody alemtuzumab is a potent immunosuppressive agent with a good safety profile. We therefore initiated a pilot phase II study to evaluate alemtuzumab as a treatment for T-LGL. Methods20 consecutive patients with T-LGL were enrolled from October 2006 to August 2012 at National Institutes of Health (www.clinicaltrials.gov - NCT00345345). After a 1 mg test dose, alemtuzumab was administered at 10 mg/dose/day intravenously for 10 days. Peripheral blood, bone marrow, and plasma samples were collected from patients before and at 3 or 6 months after treatment. Blood was analyzed for 1) lymphocytes subsets, T-cell receptor V-beta repertoire and CD57 and CD52 expression by flow cytometry (LSR II, BD, San Jose, California), 2) plasma cytokines using a a magnetic bead based Luminex assay (Affymetrix, CA, USA), 3) STAT3 mutation by direct Sanger sequencing and 4) expression level of 84 genes of the JAK-STAT signaling pathway quantified by PCR array 384 well from SABiosciences (Frederick, MD, USA). ResultsWe report here the results of treatment with alemtuzumab in 20 T-LGL patients enrolled in the first stage of the protocol. Three had associated myelodysplasia (MDS) and two had T-LGL following hematopoietic stem cell transplantation (HSCT). The median age was 61 years (range, 26-82). The median number of prior therapeutic interventions for T-LGL leukemia was 3 (range, 0-7) and the median time from diagnosis to alemtuzumab therapy was 1096 days (range, 18-6054). The median follow-up for all patients is 508 days (range, 99-1481) and for surviving patients 650 days (range, 120-1481). One patient was lost to follow-up 4 months after alemtuzumab therapy. Alemtuzumab was generally well tolerated. Labeled infusion related reactions were common and managed symptomatically. Prolonged and subclinical EBV and CMV reactivations were common but there were no cases of EBV or CMV disease without instituting prophylactic or pre-emptive therapy. Hematological response as defined by protocol was observed in 11 of 20 patients by 3 months after treatment. No patient with MDS or post HSCT responded to alemtuzumab. Four patients relapsed and received a second round of immunosuppression. One patient achieved stable blood counts on cyclosporine, three received alemtuzumab with one patient responding but relapsing 1 year later. STAT3 mutations in the SH2 domain identified in 10 of 20 patients did not correlate with response to alemtuzumab (5 responders and 5 non-responders).Treatment with alemtuzumab reduced significantly the absolute clonal population of T-cytotoxic lymphocytes, as identified by their V-beta receptor phenotype, but they tended to persist in frequency in the peripheral blood of responders. The expanded V-beta clone expressed both CD52 positive and negative cells and both compartments reduced in size after the treatment. When compared with healthy volunteers T-LGL patients had a distinct plasma cytokine signature (IL-12p40, TRAIL, IL22, IP10, MCP1, M-CSF, PDGF-AA, LIF, SCF) as well as JAK-STAT pathway activation prior to treatment but neither was correlated to clinical responses to alemtuzumab, likely due to the various prior IST regimens. ConclusionThis is the largest cohort of T-LGL patients treated with alemtuzumab yet reported. Treatment was well tolerated and at this dose minimal side effects were observed. Alemtuzumab treatment in previously heavily treated T-LGL patients results in over 50% response rate and represents a good treatment option for this condition. Disclosures:Off Label Use: Alemtuzumab for T-LGL.

SP0126 NKT and Related Cells: Key Roles in Spondyloarthritis?

Natural killer T (NKT) cells are a unique T cell subset with features of both innate and adaptive immunity. Several subsets of NKT cells exist, the most known is characterized...

Expression of functional T-cell markers and T-cell receptor Vbeta repertoire in endomyocardial biopsies from patients presenting with acute myocarditis and dilated cardiomyopathy

To quantify and functionally characterize the intramyocardial T-cells in endomyocardial biopsies (EMBs) from patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM). Expression of genes characterizing Th1 [interferon (IFN)γ, Tbet-1, Eomesodermin, interleukin (IL)-27], Th2 (IL-4, IL-5, GATA3), Th17 (IL-17), regulatory [regulatory T-cells (Treg); FoxP3, TGFβ, IL-10], anergic (GRAIL), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A), as well as of functional T-cell receptor Vbeta (TRBV) families were investigated in EMBs from AMC patients (n= 58) and DCM patients (n= 34) by pre-amplified real-time reverse transcription-polymerase chain reaction. These data were compared with EMBs from n= 19 controls. Expression of CD3d, CD3z, and TRBC (T-cell receptor beta constant region) were associated with the immunohistological diagnosis of inflammatory cardiomyopathy (DCMi). In EMBs from DCM patients with increased CD3d expression, significantly increased markers of Th1 (IFNγ, T-bet, Eomesodermin), regulatory T-cells (Treg; FoxP3, TGFβ), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A) were present, while no differential polarization of T-cells was found in EMBs form AMC patients. A differential dominance of distinct functional TRBV families was associated with different cardiotropic viruses: TRBV 11 and 24 with Parvovirus B19; TRBV4, 10 and 28 with human herpes virus type 6; and TRBV14 for Coxsackie virus, respectively. The T-cell infiltrates in human DCMi are characterized by differential expression of functional T-cell markers indicating Th1, Treg, and CTLs, while no major role could be confirmed for Th17. The virus-associated differential TRBV dominance suggests an antiviral specificity of virus-induced T-cell responses in human DCMi.

Change in expression pattern of TCR–CD3 complex in patients with multiple myeloma

In haematological malignancy, cell-mediated immunity has been shown to be suppressed in advanced disease. This immune dysfunction may be due, in part, to altered expression of the T cell receptor (TCR)–CD3 complex. The distribution and clonality of the TCR Vbeta repertoire and the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in T cells from patients with multiple myeloma (MM) were investigated. Specific Vbeta subfamily primers, reverse transcription polymerase chain reaction, and the GeneScan® technique were used to analyse the expression of the TCR Vbeta subfamily and the clonality of Vbeta T cells in 11 patients with MM. Real-time reverse transcription polymerase chain reaction was used to detect the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in peripheral blood mononuclear cells from 19 patients with MM. The beta2-microglobulin gene was used as an endogenous reference. A total of 5–22 Vbeta subfamily T cells were detected in different patients (mean value of expressed Vbeta subfamilies was 12·55±6·11), whereas all 24 Vbeta genes were identified in all control samples. The most frequently expressed Vbeta subfamilies were Vbeta1 (100%), Vbeta2, Vbeta3, Vbeta9, Vbeta13, and Vbeta16 (81·8%), while the expression of Vbeta20 was undetectable in all MM samples. Oligoclonal expansion of one or more Vbeta subfamily of T cells was detected in all patients. Such expansions involved different MM stages, and the numbers of expanded clonal Vbeta subfamilies seemed higher in stage I/II groups than in stage III; however, there was no significant difference. Among MM samples, of the Vbeta subfamily members, Vbeta13, Vbeta1, and Vbeta21, were expanded most frequently. A significant decrease in the expression level of the CD3gamma gene was observed in MM samples; in contrast, a higher expression of CD3epsilon was found in the MM group than in the healthy group. The expression pattern of the four CD3 chains was epsilon>zeta>delta>gamma in peripheral blood mononuclear cells from MM, while a gamma>epsilon>zeta>delta expression pattern was found in healthy controls. In conclusions, the present study presents precise data on changes in the variability of Vbeta patterns and expression of TCR signal transduction molecules in MM patients compared to controls, which may be associated with immune dysfunction. This study contributes to a better understanding of the cellular immune features in MM patients.

Highly skewed T-cell receptor V-beta chain repertoire in the bone marrow is associated with response to immunosuppressive drug therapy in children with very severe aplastic anemia

One of the major obstacles of immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA) comes from the often months-long unpredictability of bone-marrow (BM) recovery. In this prospective study in children with newly diagnosed very severe AA (n=10), who were enrolled in the therapy study SAA-BFM 94, we found a dramatically reduced diversity of both CD4+ and CD8+ BM cells, as scored by comprehensive V-beta chain T-cell receptor (TCR) analysis. Strongly skewed TCR V-beta pattern was highly predictive for good or at least partial treatment response (n=6, CD8+ complexity scoring median 35.5, range 24–73). In contrast, IST in patients with rather moderate reduction of TCR V-beta diversity (n=4, CD8+ complexity scoring median 109.5, range 82–124) always failed (P=0.0095). If confirmed in a larger series of patients, TCR V-beta repertoire in BM may help to assign children with SAA up-front either to IST or to allogeneic stem-cell transplantation.

Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation

AbstractAbstract 831After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

An early commitment to expression of a particular TCRVβ chain on CD8+ T cells responding to attenuated Plasmodium berghei sporozoites is maintained following challenge with infectious sporozoites

Protection induced by irradiated Plasmodium berghei sporozoites (Pbgamma-spz) in mice is linked to CD8(+) T cells specific for exo-erythrocytic-stage Ags, and intrahepatic memory CD8(+) T cells are associated with protracted protection. However, the Ag specificity of the protective CD8(+) T cells remains largely unknown. In this study, we characterized the TCR Vbeta usage by intrahepatic CD8(+) T cells during gamma-spz immunization and after the challenge with infectious Pb sporozoites. The repertoire of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells was diverse and conserved between individual mice, and did not change with immunization. In contrast, preferential usage of one or more TCR Vbeta subset was observed in effector memory (T(EM)) CD8(+) T cells after immunization. The expanded TCR Vbeta varied between individual mice but Vbeta4, 6, 7, 8.3, 9 and 11 were the most frequently expressed. In addition, there was a correlation in the TCR Vbeta usage by gamma-spz-induced CD8(+) T(EM) in the liver and blood of individual mice. The expansion pattern of blood CD8(+) T(EM) did not change with challenge and remained the same for 8 weeks thereafter. These results demonstrate that immunization with gamma-spz skews the TCR Vbeta repertoire of CD8(+) T(EM), and commitment to a particular TCR Vbeta expression is maintained long-term.

Recurrent respiratory papillomatosis: a complex defect in immune responsiveness to human papillomavirus‐6 and ‐11

Recurrent respiratory papillomatosis (RRP) is a rare disease of the larynx caused by infection with human papillomaviruses (HPV) -6 or -11, associated with significant morbidity and on occasion mortality. Here we summarize our current understanding of the permissive adaptive and innate responses made by patients with RRP that support chronic HPV infection and prevent immune clearance of these viruses. Furthermore, we provide new evidence of T(H)2-like polarization in papillomas and blood of patients with RRP, restricted CD4 and CD8 Vbeta repertoires, the effect of HPV-11 early protein E6 on T-cell alloreactivity, enriched Langerhans cell presence in papillomas, and evidence that natural killer cells are dysfunctional in RRP. We review the immunogenetic mechanisms that regulate the dysfunctional responses made by patients with RRP in response to HPV infection of the upper airway. In addition, we are identifying T-cell epitopes on HPV-11 early proteins, in the context of human leukocyte antigen (HLA) class II alleles enriched in RRP that should help generate a therapeutic vaccine. Taken together, RRP is a complex, multigene disease manifesting as a tissue and HPV-specific, immune deficiency that prevents effective clearance and/or control of HPV-6 and -11 infection.

T cells with a specific motif in the CDR3-beta region protect mice from CT26 tumor growth (131.46)

Abstract Vaccination with peptide mimics of tumor antigens, or mimotopes, is a potential strategy for antigen-specific immunotherapy of cancer. To determine how to elicit the most effective antigen-specific cytotoxic response against tumors, we have assembled a set of mimotopes of AH1, the immunodominant antigen from the mouse CT26 tumor. Although the AH1 antigen is an ineffective immunogen, vaccination with some of the mimotopes is very successful in protecting mice from tumor growth. The majority of AH1-specific T cells responding to the vaccine or tumor express V-beta 8 gene segments. However, tumor protection by the mimotope vaccines correlates with stimulation of T cells with a more restricted TCR V-beta repertoire. These T cells express receptors that encode a relatively short CDR3-beta motif. This motif consists of a small amino acid, a non-hydrophobic amino acid, a germline-encoded tyrosine, and the J-beta 2.6 gene segment. These results suggest that protective mimotopes enhance antitumor immunity, in part, by increasing the expansion of a subset of functional tumor-specific T cells that naturally respond to the tumor, not a new repertoire of T cells only elicited by the mimotope. We will test the hypothesis that specific T cells with this motif impart increased protection because they have higher affinity for the antigen complex than specific T cells without the motif.