Eliana Ruggiero

Thymus-autonomous T cell development in the absence of progenitor import

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High-resolution analysis of the human T-cell receptor repertoire

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires.

Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory

Antigen exposure and differentiation phenotype influence long-term persistence of memory T cells after hematopoietic stem cell transplant. Committing T cells to memory Adoptive cell transfer is an increasingly successful therapy for a variety of diseases; however, little is known about what regulates the survival of these cells in humans. Now, Oliveira et al. leverage patients who have received genetically modified hematopoietic stem cells to track T cells over time. They found labeled effector memory, central memory, and stem memory T cells 2 to 14 years after infusion in all patients. Antigen recognition was critical in driving persistence and expansion. The clones that survived long-term appeared to initiate preferentially from central and stem cell memory T cell populations. These data suggest that the original phenotype of infused cells may influence long-term persistence of adoptively transferred cells. Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK+ cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)– and Flu-specific TK+ cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRβ clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

Lentivirus-induced ‘Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Identity, potency, in vivo viability, and scaling up production of lentiviral vector-induced dendritic cells for melanoma immunotherapy

SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (“SmyleDCpp65”). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1−/−.IL2γc−/− mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

TCR sequences and tissue distribution discriminate the subsets of naïve and activated/memory Treg cells in mice

Analyses of the regulatory T (Treg) cell TCR repertoire should help elucidate the nature and diversity of their cognate antigens and thus how Treg cells protect us from autoimmune diseases. We earlier identified CD44hiCD62Llow activated/memory (am) Treg cells as a Treg‐cell subset with a high turnover and possible self‐specificity. We now report that amTreg cells are predominantly distributed in lymph nodes (LNs) draining deep tissues. Multivariate analyses of CDR3 spectratyping first revealed that amTreg TCR repertoire is different from that of naïve Treg cells (nTreg cells) and effector T (Teff) cells. Furthermore, in deep‐ versus superficial LNs, TCR‐β deep sequencing further revealed diversified nTreg‐cell and amTreg‐cell repertoires, although twofold less diverse than that of Teff cells, and with repertoire richness significantly lower in deep‐LN versus superficial‐LN Treg cells. Importantly, expanded clonotypes were mostly detected in deep‐LN amTreg cells, some accounting for 20% of the repertoire. Strikingly, these clonotypes were absent from nTreg cells, but found at low frequency in Teff cells. Our results, obtained in nonmanipulated mice, indicate different antigenic targets for naïve and amTreg cells and that amTreg cells are self‐specific. The data we present are consistent with an instructive component in Treg‐cell differentiation.

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

BackgroundReactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCpp65”) that accelerated antigen-specific T cell development.MethodsHere we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.ResultsUpscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.ConclusionThese results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.ZusammenfassungHintergrundDie Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCpp65”) vorstellen, die zu einer beschleunigten Entwicklung antigen-spezifischer T-Zellen führt.VerfahrenIn der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert.ErgebnisseDie standardisierte Produktion des IDLVs und die Herstellung von SmyleDCpp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCpp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCpp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCpp65 validiert.FazitDie weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen.

The Contained Self-Reactive Peripheral T Cell Repertoire: Size, Diversity, and Cellular Composition

Individual self-reactive T cells have been discovered in both humans and mice. It is difficult to assess the entire contained self-reactive peripheral T cell repertoire in healthy individuals because regulatory T cells (Tregs) can render these cells anergic and, therefore, functionally indistinguishable. We addressed this issue by removing regulatory T cells, thereby allowing us to characterize the exposed self-reactive T cells. This resulted in activation of approximately 4% of both CD4+ and CD8+ T cells. Activation and division of these cells was not a bystander product of Ag-independent signals but required TCR stimulation. Analysis of TCR sequences showed that these responding cells were polyclonal and encompassed a broad range of structural TCR diversity. Adoptive transfer of naive and effector/memory T cell populations showed that even the naive T cell pool contained self-reactive T cell precursors. In addition, transfer of mature thymocytes showed that this response was an intrinsic T cell property rather than a peripheral adaptation. Finally, we found that the unexpectedly strong contribution of the naive CD5low T cell pool showed that the overall self-reactive response has not only a diverse polyclonal TCR repertoire, but also comprises a broad range of affinities for self.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms

Oncogenic JAK mutation sensitizes myeloproliferative neoplasms to immune checkpoint inhibition. Cancers JAK up an immune checkpoint Myeloproliferative neoplasms, a group of hematologic cancers, are associated with mutations activating the JAK2 oncogene. JAK2 is located on chromosome 9, in the vicinity of the immunosuppressive PD-L1 gene, and Prestipino et al. discovered that myeloproliferative cancers with overactive JAK2 generally have increased PD-L1 as well. Although PD-L1 helps cancers evade the immune system, immune checkpoint inhibitors developed in recent years provide a way to block its function and turn PD-L1 expression into a therapeutic vulnerability for the tumors, as the authors demonstrate in this study. Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.