Broad T-cell Receptor Repertoire Research Articles

IPSC-Derived Thymic Epithelial Progenitor Cells As Cell-Based Therapy to Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients

Hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for many malignant and non-malignant diseases to date. The thymus plays a key role in post-transplant immune reconstitution. The outcome of a HSCT and the recipient thymic function are intimately intertwined. In fact, the production of mature T cells occurs in the thymus and depends on the interaction of hematopoietic progenitor cells with non-hematopoietic stromal elements, notably thymic epithelial cells (TECs). Through positive and negative selection, a healthy thymus generates a broad and self-tolerant T cell receptor (TCR) repertoire that protects from infection and prevents autoimmunity. In addition, the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance. At odds with its importance for successful transplant outcomes, the thymus is exquisitely sensitive to a wide range of insults encountered during the peri-transplant period, including cytotoxic agents, steroids and irradiation. In the allogeneic transplant setting, acute graft-versus-host disease (GVHD) can cause further thymic injury. Thymic injury in turn, leads to susceptibility to infections and autoimmune complications. Moreover, it is now widely recognized that thymic injury leading to defective deletion of self-reactive T-cells along with misguided T-helper cell polarization and B-cell activation constitute major drivers of chronic GVHD. Together these represent the leading causes of non-relapse-related transplant mortality and morbidity. As such, HSCT can precipitate a vicious cycle, in which the success of the HSCT procedure hinges on intact thymic function, but the HSCT process itself often causes irrecoverable thymic injury (Figure1). Thymic function naturally declines with age with little to no endogenous potential to regenerate. Current treatment options for thymic injury during HSCT are limited to symptomatic therapy. Therefore, novel approaches to cure irreversible thymic injury are critically needed.To address this need, our goal is to generate fully functional thymic epithelial progenitor cells (TEPCs) from hPSCs as a stem cell-based therapy for thymic regeneration and restoration of immune competence in HSCT recipients. Building on the efficient induction of definitive endoderm from human pluripotent stem cells (hPSCs) by directing signals controlling lineage bifurcations, we have developed a novel platform that drives TEPC development through sequential induction of the intermediaries anterior foregut (AFG) and pharyngeal pouch endoderm (PPE). Here we show that TEPCs derived in vitro from hPSCs or hESCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs, respectively. In addition, in vitro derived TEPCs express MHC class II to varying degrees (Figure 2). To optimize our in vitro platform, modifications of key-signaling pathways in the differentiation protocol are validated by comparing the transcriptome of hPSC-derived TEPCs to that of primary human medullary and cortical thymic epithelial cells using next generation sequencing. Our ongoing work is testing the capacity of hPSC-derived TEPCs to promote T-cell maturation, positive and negative selection resulting in the development of a broad and self-tolerant TCR repertoire from adoptively transferred human HSPCs in a humanized mouse model.Through sequential validation of fidelity, functionality, transplantability and sustainability of hPSC-derived TEPCs, we strive to develop an experimental model that can be translated by a clinical Good Manufacturing Practice (cGMP) facility for use in bone marrow transplant recipients.Ultimately, the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting. TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ. Leveraging the body's own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro-generated MART-1-specific CD8 T cells display a broader T-cell receptor repertoire than exvivo naïve and tumor-infiltrating lymphocytes.

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved invitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated invitro. To test this notion, we compared the TCR-Vα/Vβ (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated invitro to that from exvivo naïve T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naïve T cells and TILs, which showed the expected narrow TRAV repertoire, invitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated invitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

AbstractHematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report

BackgroundCancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing.MethodsVaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization.ResultsA peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now.ConclusionsTherapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial.

Treatment of Pediatric Acute Graft-versus-Host Disease—Lessons from Primary Immunodeficiency?

Allogeneic hematopoietic stem cell transplant (HSCT) is used to treat increasing numbers of malignant and non-malignant disorders. Despite significant advances in improved human leukocyte antigens-typing techniques, less toxic conditioning regimens and better supportive care, resulting in improved clinical outcomes, acute graft-versus-host disease (aGvHD) continues to be a major obstacle and, although it principally involves the skin, gastrointestinal tract, and liver, the thymus is also a primary target. An important aim following HSCT is to achieve complete and durable immunoreconstitution with a diverse T-cell receptor (TCR) repertoire to recognize a broad range of pathogens providing adequate long-term adaptive T-lymphocyte immunity, essential to reduce the risk of infection, disease relapse, and secondary malignancies. Reconstitution of adaptive T-lymphocyte immunity is a lengthy and complex process which requires a functioning and structurally intact thymus responsible for the production of new naïve T-lymphocytes with a broad TCR repertoire. Damage to the thymic microenvironment, secondary to aGvHD and the effect of corticosteroid treatment, disturbs normal signaling required for thymocyte development, resulting in impaired T-lymphopoiesis and reduced thymic export. Primary immunodeficiencies, in which failure of central or peripheral tolerance is a major feature, because of intrinsic defects in hematopoietic stem cells leading to abnormal T-lymphocyte development, or defects in thymic stroma, can give insights into critical processes important for recovery from aGvHD. Extracorporeal photopheresis is a potential alternative therapy for aGvHD, which acts in an immunomodulatory fashion, through the generation of regulatory T-lymphocytes (Tregs), alteration of cytokine patterns and modulation of dendritic cells. Promoting normal central and peripheral immune tolerance, with selective downregulation of immune stimulation, could reduce aGvHD, and enable a reduction in other immunosuppression, facilitating thymic recovery, restoration of normal T-lymphocyte ontogeny, and complete immunoreconstitution with improved clinical outcome as the ability to fight infections improves and risk of secondary malignancy or relapse diminishes.

Broad T-cell receptor repertoire in T-lymphocytes derived from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.

Transplantation of TcRαβ/CD19 Depleted Stem Cells From Haploidentical Donors: Robust Engraftment and Rapid Immune Reconstitution In Children with High Risk Leukemia

Abstract 1005In haploidentical transplantation with mobilized peripheral blood stem cells (PBSC’s), in vitro T-cell depletion of the graft is an effective method to prevent or completely avoid Graft-versus-Host Disease (GvHD). In order to increase the T-cell depletion efficacy of PBSC’s while maintaining the anti-tumor and anti-infectious properties of the graft, we have investigated a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, Natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The αβ+ T-cells and CD19+ B cells were then removed using the CliniMACS system. So far, 23 patients have been treated in two centers, namely Tübingen and Rome. Graft manipulation resulted in a consistent efficiency of αβ+ T-lymphocyte removal at the two centers. The overall depletion of αβ+ T-cells was 4.5 log (range 3.8–5.0) and 4.3 log (range 3.7–5.0) in Tübingen and Rome, respectively, with a median number of transplanted αβ+ T-cells of 14 × 103/kg. The recovery of CD34+ stem cells was 72% and 89% in the two centers, and the median number of infused CD34+ stem cells was 11.9 ×106/kg (range 7.5 –30 × 106/kg) and 13.3 ×106/kg (range 8.3 –19.8 × 106/kg), respectively. Patients were given 107 × 106/kg (range 35 –186 ×106/kg) and 123 × 106/kg (range 51–202 ×106/kg) CD56+ NK cells in Tübingen and Rome, respectively. The median number of infused γδ+ T-lymphocytes was 11.9 ×106/kg (range 7.5–30.2 × 106/kg) and 10.3 ×106/kg (range 6.5–25.1 × 106/kg) respectively.The 10 patients transplanted in Tübingen had advanced/refractory leukemias (ALL, n=5; AML, n=5; active disease, n=6; 2nd transplantation, n=2; CR2, n=2). For this poor-prognosis patients, a reduced-intensity conditioning regimen (melphalan, thiotepa, fludarabin or clofarabin and OKT-3 or ATG) was used. No further post-transplant GvHD prophylaxis was given. All 10 patients engrafted. The median time to reach neutrophil (PMN) and platelet (PLT) recovery was 9 (range 8– 12) and 15 days (range 6 –28) respectively. All patients reached complete donor chimerism and showed a very rapid immune reconstitution with 350 (range 21–824) CD3+ T cells/μl, 66 (12–177) CD3+4+/μl and 599 (227–1390) CD56+ NK cells/μl at day +28 posttransplant. Three patients had no signs of acute GvHD, 5 patients had GvHD grade I and 2 patients had skin GvHD grade II. Only 1 patient experienced a transient grade 3 GvHD of the skin which required only topical treatment. No patient experienced chronic GvHD. Three patients relapsed after transplantation, 7 patients are in remission for 5 months (range 3 –12). There was no transplant-related death sofar.The second cohort given transplantation in Rome comprised 13 patients with ALL (9), AML (3) and NHL (1). All children but 1 had relapsed/refractory disease. In particular, 11 patients were transplanted in CR and 2 with active disease. Conditioning regimen was myeloablative and consisted of fractionated TBI, Thiotepa, fludarabine and ATG (8 mg/Kg). No further post-transplant GvHD prophylaxis was given. All patients engrafted, the median time to reach PMN and PLT recovery being 11 (range 7–13) and 12 (range 10–16) days, respectively. Only 2 patients had skin grade I acute GvHD. No patient experienced chronic GvHD. With a median follow-up of 4 months (range 1–9) 10 patients are alive and disease-free; 2 patients relapsed (1 died) and 1 had fatal lung aspergillosis. In both cohorts, γδ+ T cells started to expand faster than αβ+ T cells in the early post-transplant period, whereas at day +100, αβ+ T-cells were predominant. In addition to a rapid reconstitution of αβ+ T-lymphocytes, Vbeta spectratyping revealed a broad T-cell receptor repertoire early after transplantation.Altogether, these data indicate that transplantation of TcR αβ+/CD19 depleted cells from a haploidentical donor results in sustained engraftment, rapid immune reconstitution and low incidence of both acute and chronic GvHD. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up. Disclosures:No relevant conflicts of interest to declare.

Interleukin-7 Permits Th1/Tc1 Maturation and PromotesEx vivoExpansion of Cord Blood T Cells: A Critical Step toward Adoptive Immunotherapy after Cord Blood Transplantation

Donor leukocyte infusions (DLI) in the allogeneic hematopoietic transplant setting can provide a clinically relevant boost of immunity to reduce opportunistic infections and to increase graft-versus-leukemia activity. Despite significant advances in applicability, DLI has not been available for single-unit recipients of unrelated cord blood transplant. Ex vivo expansion of cord blood T cells can be achieved with interleukin (IL)-2 and CD3/CD28 costimulatory beads. However, significant apoptosis occurs in proliferating T cells, diminishing the yield and skewing the CD4/CD8 ratio in the T-cell population, jeopardizing the potential efficacy of DLI. In this study, we show that interleukin (IL)-7 not only reduces apoptosis of activated T lymphocytes and enhances their proliferation but also promotes functional maturation, leading to secretion of IFN-gamma and other key cytokines. Recognizing that infused T lymphocytes will need to meet microbial antigens in secondary lymphoid organs to generate effectors, we also show that expansion with IL-7 promotes the preservation of a polyclonal broad T-cell receptor repertoire and a surface phenotype that favors lymph node homing. Expanded lymphocytes lack alloreactivity against recipient and other allogeneic cells, indicating a favorable safety profile from graft-versus-host disease. Nevertheless, expanded T cells can be primed subsequently against lymphoid and myeloid leukemia cells to generate tumor-specific cytotoxic T cells. Taken together, our findings offer a major step in fulfilling critical numerical and biological requirements to quickly generate a DLI product ex vivo using a negligible fraction of a cord blood graft that provides a flexible adoptive immunotherapy platform for both children and adults.

Natural killer T cells are not the predominant T cell in asthma and likely modulate, not cause, asthma

Asthma is a multifactorial disease of the airways characterized by airway inflammation, mucus hypersecretion, and airway hyperresponsiveness. Conventional MHC class II-restricted CD4(+) T cells are considered a key cell in asthma pathogenesis because they have a broad T-cell receptor repertoire, providing specificity and reactivity to diverse protein allergens. This notion was challenged when a study found that invariant Natural Killer (NK) T cells were the predominant T cells in the lung and bronchoalveolar lavage fluid of all asthmatic subjects studied. This finding was provocative because invariant NKT cells have a very limited T-cell receptor repertoire and are specific for a restricted set of lipid antigens that bind to CD1d, a nonpolymorphic MHC-like molecule. However, multiple subsequent studies failed to replicate the initial study and instead found that invariant NKT cells are present as a small fraction of the total T cells in the asthmatic lung. Thus, we believe that although CD1d-restricted NKT cells might play a role in modulating the asthmatic phenotype, they are not the critical drivers of the asthmatic response, a role we believe is still held by conventional MHC class II-restricted CD4(+) T cells.

Characterization of MHC Class-I Restricted TCRαβ+CD4−CD8− Double-Negative T Cells Recognizing the gp100 Antigen from a Melanoma Patient after gp100 Vaccination

The immune attack against malignant tumors requires the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR)αβ+ CD4− CD8− double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.

Characterization of MHC class-I restricted TCRalphabeta+ CD4- CD8- double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination.

The immune attack against malignant tumors require the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR) alphabeta+ CD4- CD8- double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-gamma and TNF. Although lacking the CD8 molecule the gp100-specific DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.

Characteristics of T-cell receptor repertoire and myelin-reactive T cells reconstituted from autologous haematopoietic stem-cell grafts in multiple sclerosis.

Multiple sclerosis is thought to involve aberrant immune responses to myelin autoantigens. Haematopoietic stem-cell transplantation (HSCT) is in clinical trials for progressive multiple sclerosis based on the rationale that it destroys aberrant immune system, while recapitulation of lymphocyte ontogeny might alter the immune system and slow down disease progression. This study was undertaken to analyse characteristics of the T-cell receptor (TCR) repertoire, serum cytokine profile and the T-cell responses to myelin basic protein (MBP) in the reconstituted immune system in progressive multiple sclerosis. The study revealed that, following autologous HSCT, the T-cell immunity recovered in two distinctive phases. The first phase was characterized by limited T-cell immunity as a result of selective expansion of pre-existing T cells commonly expressing the TCR beta chain variable region (TCR BV) 20 and increased serum cytokine production during the first several months. The second phase of T-cell reconstitution coincided with increased thymic T-cell output 9-12 months after HSCT. T cells reconstituted from stem-cell grafts had the distinctive properties of comprehensive T-cell immunity and a broad TCR repertoire. T cells recognizing MBP were initially depleted by immunoablation and rapidly expanded from the reconstituted T-cell repertoire in 12 months. The reconstituted MBP-reactive T cells exhibited a broader epitope recognition repertoire while maintaining the same skewed reactivity pattern compared with that seen at baseline. The findings have important implications in the understanding of the role of HSCT as a potential treatment for multiple sclerosis.

Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.