An Alternate Pathway for T Cell Development Supported by the Bone Marrow Microenvironment: Recapitulation of Thymic Maturation
In the principal pathway of α/β T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.
Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures: No relevant conflicts of interest to declare.
Abstract There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.
Abstract B cell receptor signaling (BCR) in chronic lymphocytic leukemia (CLL) drives tumor cell proliferation and survival. Inhibition of Bruton's tyrosine kinase (BTK), a key enzyme in the BCR pathway, has proved to be efficacious even in poor-risk and chemo-refractory patients. However resistance to the BTK inhibitor ibrutinib has been shown to emerge in a subset of CLL patients. Of importance, the C481S BTK mutation conferred resistance by preventing the covalent binding of ibrutinib to its target cysteine 481 in BTK. Vecabrutinib (formerly known as SNS-062, a succinate salt) is a novel, highly potent, next generation noncovalent BTK inhibitor which demonstrated biochemical and cellular activity against C481S BTK mutant in vitro. However, the efficacy of vecabrutinib and its impact on the T-cell microenvironment has not been studied in in vivo preclinical CLL models. In the present study, the efficacy of vecabrutinib was investigated using the Eµ-TCL1 adoptive transfer model. Mice were randomized to treatment with either 40mg/kg vecabrutinib succinate, twice daily by oral gavage (n=6) or vehicle control (n=6). The mice were sacrificed after 2 weeks of treatment and changes in tumor burden as well as alterations in T-cell microenvironment were analysed in detail. Treatment with vecabrutinib decreased tumor burden as observed by a significant decrease in WBC count (36.5 vs. 17.1 giga/L; P=0.002), spleen weight (median 0.56g vs. 0.31g; P=0.005) and liver weight (median 1.5g vs. 1.2g; P=0.005) compared to vehicle treatment. Correspondingly, the CD5+ CD19+ tumor cells were significantly decreased in blood (P=0.002) and spleen (P=0.002) while no significant difference was observed in bone marrow (P=0.818) upon treatment with vecabrutinib. Since BTK inhibition is known to reshape the tumor microenvironment, we studied the impact of vecabrutinib specifically on T-cell subsets. Firstly, no difference in the proportions of CD4 or CD8 expressing T-cells was observed in mice treated with vehicle or vecabrutinib. However, of interest, the percentage of CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) were significantly decreased upon treatment with vecabrutinib in peripheral blood (P=0.026) and spleen (P=0.009). The decrease in Tregs was due to reduced proliferation of these cells upon exposure to the drug as measured by Ki-67 staining. Also, the Tregs expressing the maturation and activation markers such as CD103 and GITR were significantly decreased in blood and spleen upon drug treatment. Further, we analysed the changes in CD8 T-cell subsets following treatment with vecabrutinib. Treatment with the drug resulted in expansion of the CD127+ CD44- naïve CD8 T-cells in blood, bone marrow and spleen (all P values 0.002) while the CD127+ CD44+ memory CD8 T-cells were significantly decreased in bone marrow and spleen (all P values 0.009). Also, the CD127low CD44int-hi effector CD8 T-cells were decreased in blood (P=0.004), bone marrow (P=0.004) and spleen (P=0.002) upon vecabrutinib treatment. Therefore, vecabrutinib treatment did not alter the percentage of CD4+ and CD8+ T cells in mice however, significant changes in the subset composition of the CD4 and CD8 T cells were observed. Lastly, to analyse the impact of vecabrutinib on survival, a cohort of mice (n=12) were transplanted with 5 million splenic tumor cells isolated from Eµ-TCL1 transgenic mice. After allowing for engraftment, the mice were randomized to treatment with the drug (n=6) or vehicle (n=6). Of note, the mice treated with the drug showed a significant increase in survival (median 35 days from transplant; P<0.001) compared to treatment with vehicle (median 28 days). In summary, vecabrutinib was efficacious in vivo in a preclinical CLL adoptive transfer model, decreasing tumor burden in different organs and significantly improving survival. Treatment with the drug altered the T-cell architecture in vivo. Of interest, the immunosuppressive Tregs, which protect the tumor from immune surveillance were decreased in various tissue compartments; however, a decrease in the effector CD8 T cells might impact anti-tumor immunity if there is a consistent effect upon drug treatment. Vecabrutinib antitumor activity and effects on T-cell populations in vivo in this preclinical CLL model are intriguing, merits further investigation and supports the ongoing phase 1b/2 study in patients with previously treated B-lymphoid malignancies (NCT03037645). Disclosures Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Taverna:Sunesis Pharmaceuticals: Employment. Stilgenbauer:Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Aplastic anemia (AA) is a hematopoietic disorder resulted from immune-related hypocellular hematopoiesis in bone marrow (BM). It has been clearly addressed that the activated T cells contribute to the exhaustion of hematopoietic progenitors and hypo-hematopoiesis. The adipogenic BM is one of the characteristics to make AA diagnosis. However, little is known about the relationship of intra-BM immune imbalance and hematopoietic microenvironment abnormity in this disease entity. Functional hematopoiesis relies on not only abundant hematopoietic stem cells (HSCs) but also the balanced supportive hematopoietic niche. Intra-BM immune balance, at either cellular or cytokine level, is one of the key footstones to maintain hematopoietic microenvironment. Various intra-BM immune cellular components play both sides of one coin. Among them, myeloid-derived suppressive cells (MDSCs) are heterogeneous myeloid progenitor cells characterized by the negative immune response in cancers and other inflammatory diseases. In BM aspiration and biopsy samples from the patients who were diagnosed as AA in our study, massive activated lymphocytes infiltration and adipocytes accumulation were observed. Interestingly, the absolute numbers of immune modulatory MDSCs either in AA patients' PB or in BM of immune-related AA mice were reduced, indicating a potential link between polarized BM adipo-osteogenic microenvironment and immune disorder under AA circumstance. We thus adopted AA mice model to look into the embedded details both in vivo and in vitro. We clarified that BM components were more vulnerable to the attack of CD8+ T cells than that of CD4+ T cells. Taking into the fact that BM adipocytes are more abundant either in AA patients or in AA mice models, we differentiated mesenchymal stromal cells (MSCs), the major BM stroma cells, into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation in BM microenvironment. Interestingly, CD8+ T cells and interferon-γ(IFN-γ) exerted dramatically adipocytic stimulation on BM-MSCs either in vitro or in vivo, by determination of increasing expression of adipogenetic genes including Ap2, Perilipin, Pparg and Cebpα, as well as staining of Oil Red O and perilipin. To dissect intra-BM cellular immune balance, MDSCs were isolated as representative immune regulating population to investigate their function on osteo-adipogenic balance. Interestingly, not CD11b+Ly6G+Ly6C-granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+monocytic-MDSCs (mMDSCs) inhibited both T cell proliferation and IFN-γ production. Addition of L-NMMA, the antagonist of iNOS pathway in mMDSCs-containing system restored T cell proliferative curve and cell numbers, whereas Nor-NOHA, the antagonist of Arg-1 pathway didn't abrogate mMDSCs' immune-regulation properties, indicating that mMDSCs inhibited T cell proliferation via iNOS pathway. We then performed single dose or multi-dose injection of mMDSCs in AA mice to see whether mMDSCs are able to reconstitute the impacted hematopoiesis. Single injection of mMDSCs was able to prevent from CTL infiltration in a very short term. However, multi-injection of mMDSCs showed significant benefit in overall survival rate compared to AA mice. We further detected the function of mMDSCs on polarized BM-MSCs adipo-osteogenic differentiation potential. To detect sequential BM adipogenetic progression in AA microenvironment, we performed in vivo fluorescent microscopy on AP2 (Fabp4)-Cre×mT/mG reporting mice at different transfusion time points of T cells and mMDSCs. GFP-expressing AP2+ adipocytes accumulated adjacently to perivascular niches whose boarders were labelled by Dextran-CY5 in a time-dependent manner after T cell infusion. Monocytic MDSCs transfused AA mice showed decreased GFP+ adipocytes which was coincident with our in vitro findings. In conclusion, intra-BM immune balance is one of the environmental factors seesawing by activating and suppressive ends to support functional hematopoiesis. Adoptive transfusion of mMDSCs, the immune-suppressive population might be a novel immune-regulating strategy to treat AA, relying on not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment. Figure Disclosures No relevant conflicts of interest to declare.
Abstract Abstract 4221 Background & Purpose: Recent findings regarding leukemia stem cell (LSC) have emphasized the importance of suppression of LSC for the achievement of durable remission, the first requisite to establish cure of leukemia. For this subject, successful graft-vs.-leukemia (GvL) effect in allogeneic hematopoietic stem cell transplantation (allo-HSCT) against human leukemias has strongly illustrated the importance of anti-leukemia immunity. Additionally, WT1, one of well-known leukemia-associated antigens, has been obviously demonstrated to be expressed by LSCs in bone marrow niche (Saito Y et al, Sci Transl Med.2010). On the other hand, cell-cycle quiescence of LSCs in bone marrow niche is importantly implicated in their chemoresistance. Taking all above, in this study, we set out to answer questions whether therapeutically adopted T-cell immunity towards WT1 enabled to suppress LSC in vivo, and whether cell-cycle status of leukemia cells affected the sensitivity to cyotocidal activity mediated by WT1-specific cytotoxic T cells (CTLs). Methods: Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. Written informed consent was given by all patients, healthy volunteers in accordance with the Declaration of Helsinki. Peripheral CD8+ T cells obtained from AML or ALL patients in complete remission (CR) or healthy individuals were gene-modified to express HLA-A*2402-restricted and WT1235–243 nonamer -specific T-cell receptor (TCR) using our unique TCR-a/b gene expression vector carrying silencers for endogenous TCRs (WT1-siTCR vector) were generated as effector cells. Bone marrow CD34+ leukemia (L-BMCD34+) cells isolated using immunomagnetic beads from HLA-A*2402 positive or negative patients with AML or ALL were serially transplanted into NOD/scid/γcnull (NOG) mice as previously reported (Ochi T et al. Blood, 2011). 12 weeks later, engrafted leukemia cells in murine bone marrow were examined using a flowcytometry. In some experiments, after engrafted, first transplanted mice were treated with intraperitoneal administration of high dose (150mg/kg) of cytosine arabinoside (Ara-C). A week later, those mice received intravenous administration of gene-modified autologous CD8+ T cells to express WT1-specific TCR or non-gene-modified (NGM) ones in combination with intraperioneal administration of 500u of IL-2 every 2 days. A week after therapeutic T-cell infusion, bone marrow cells were harvested, and transplantation into second mice. 12 weeks later, engrafted human leukemia cells in murine bone marrow were assessed. Next, using a time-lapse photo assay and fluorescent ubiqutination-based cell-cycle indicator (Fucci)-labeled K562-A24 cells which are known to produce high amounts of WT1 mRNA and are positive for HLA-A*2402, we directly assessed the impact of cell-cycle status of leukemia cells on their sensitivity to redirected CTL towards WT1 in vitro. Results: Using isolated L-BMCD34+ cells, LSCs were detectable as leukemia initiating cell in serially transplanted NOG mice. High dose of Ara-C treatment alone was unable to eradicate LSCs. An experiment using samples from a patient with HLA-A*2402+ ALL revealed that intravenously infused gene-modified autologous peripheral CD8+ T cells in CR successfully reduced leukemia burden in bone marrow which were refractory to high dose of Ara-C. In serial transplantation experiments using samples from AML patients, therapeutic infusion of redirected CD8+ T cells to express WT1-specific TCR, but not NGM ones in nadir state successfully eradicated LSCs out of murine bone marrow. In vitro time-lapse photo assay directly illustrated that retargeted CD8+T cells towards WT1 killed fucci-labeled K562-A24 cells irrelevantly to cell-cycle status of target leukemia cells. Summary: In this study, when leukemia mass burden was reduced, therapeutically infused gene-modified CD8+ T cells targeting WT1 successfully enabled to inhibit LSCs in vivo. Furthermore cell-cycle status of leukemia cells which is importantly implicated in their chemoresistance in bone marrow niche, did not affect WT1-specific cytocidal activity mediated by genetically redirected CTLs at all. Although it is preliminary, our observation encourages us to actively introduce redirected T cell-based antileukemia adoptive immunotherapy, aiming at a cure of leukemias. Disclosures: No relevant conflicts of interest to declare.
Abstract Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of a malignant disease of peripheral CD4+ T cells called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although major target of HTLV-1 is CD4+ T cells, other hematopoietic cells such as CD8+ T cells and monocytes are also infected with HTLV-1. Since the receptors of HTLV-1 are glucose transporter 1 and neuropilin 1, which are found on various cell surfaces, it is possible that HTLV-1 infects various hematopoietic cells and hematopoietic stem cells (HSCs). However, the previous studies could not detect HTLV-1 in HSCs. To assess the distribution of infected cells and expression of viral genes in various tissues, a nonhuman primate model, Japanese macaques (JMs) infected with simian T-cell leukemia virus type 1 (STLV-1) was utilized in this study. STLV-1 is a close relative of HTLV-1, and the dynamics of viral replication and proliferation of infected cells are very similar to each other. Indeed, STLV-1 caused malignant lymphomas in STLV-1 infected monkeys. Therefore, STLV-1 infected JMs are good models of HTLV-1 carriers. Using this model, we first analyzed transcription level of two viral genes, tax and STLV-1 bZIP factor, in multiple tissues, and found that tax was highly expressed in bone marrow compared to other tissues. Since Tax is a potent activator of viral transcription, this result suggested that viral replication occurred in bone marrow. To evaluate which cells express Tax in bone marrow, we performed flow cytometric analysis of bone marrow mononuclear cells from STLV-1 infected monkeys, and found that not only CD4+ T cells but also non T cells (CD3 negative cells) expressed Tax. To determine whether hematopoietic stem cells are infected with HTLV-1, we next performed highthroughput sequencing of HTLV-1 integration sites in multiple cell lineages using a next generation sequencer. Since HTLV-1 is randomly integrated into genome of infected cells, and each infected cell can proliferate clonally, we can interpret that cells sharing same integration sites are derived from the same precursor cell. Blood samples from patients with HAM/TSP were separated into five cell types: CD4+ T cells, CD8+ T cells, B cells, monocytes, and neutrophils. Integration sites of HTLV-1 provirus were analyzed by next generation sequencing using different chips for each cell type in order to avoid cross contamination between the samples. The results showed that some of these cells have same integration sites between different cell types. More than 40 percent of HTLV-1 infected B cells, monocytes and neutrophils had shared the same integration sites, suggesting that HSCs in the HAM/TSP patients were infected with HTLV-1. Importantly, most HTLV-1 infected CD4+ T cells and CD8+ T cells had independent integration sites, while some of them shared the same integration sites with non T cell subsets. These results implied that most T-cell clones were generated by de novo infection in the periphery, but a part of infected T cells were derived from infected HSCs. To assess whether HTLV-1 infected HSCs persist in vivo, we again analyzed integration sites in neutrophils from the same HAM/TSP patients after 1 year. About 50 percent of integration sites in HTLV-1 infected neutrophils were detected after 1 year. Considering short lifespan of neutrophils, this result suggests that HTLV-1 infected HSCs could be maintained for at least 1 year in vivo. To visualize HTLV-1 infection in myeloid cells, we performed immunofluorescence staining of neutrophils from HAM/TSP patients. Tax and myeloperoxidase were detected in neutrophils. Finally, we evaluated the significance of infected monocytes in viral transmission. We isolated monocytes from HAM/TSP patients and co-cultured with JET WT35 cell, which is a subline of Jurkat containing a fluorescent reporter of viral infection. The result demonstrated that monocyte from HAM/TSP patients could be a source of infective HTLV-1. In conclusion, our findings suggest that HSCs infected with HTLV-1 survive for long time in vivo, and could be reservoirs of the virus. Since ATL is a difficult disease to cure, further studies are required to understand the nature of HTLV-1 infection. Disclosures No relevant conflicts of interest to declare.
Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.
Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.
Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.
BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.
