In Vivo Evidence That Peptide Vaccination Can Induce HLA-DR-Restricted CD4+ T Cells Reactive to a Class I Tumor Peptide

Abstract Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

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Co-Administration Of Gene-Modified CD4+ T Cells Targeting HLA Class I-Restricted WT1 Epitope Diversely Enhances The Antitumor Effect Mediated By Redirected T-Cell Based Adoptive Immunotherapy Against Human Leukemia

Blood ◽

10.1182/blood.v122.21.144.144 ◽

2013 ◽

Vol 122 (21) ◽

pp. 144-144

Author(s):

Hiroshi Fujiwara ◽

Fumihiro Ochi ◽

Toshiki Ochi ◽

Hiroaki Asai ◽

Yukihiro Miyazaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Cd8 T Cells ◽

Adoptive Immunotherapy ◽

Cd4 T Cells ◽

Hla Class I ◽

Class I ◽

Leukemia Cells

Abstract Purpose In the context of redirected T-cell based antitumor adoptive immunotherapy, the therapeutic roles played by co-infused CD4+ T cells genetically redirected to the predefined HLA class I-restricted epitope which had been originally recognized by effector CD8+ T cells has not yet been fully discussed. In this study, using an HLA class I-restricted WT1 -specific T-cell receptor (TCR) gene transfer, we in detail examined antileukemia functionality mediated by these gene-modified CD4+ T cells co-infused with similarly gene-modified effector CD8+ T cells as the redirected T cell-based adoptive immunotherapy. Methods Using our unique retroviral vector expressing HLA-A*2402-restricted and WT1235-243-specific TCR a/b genes and shRNAs for endogenous TCRs (WT1-siTCR vector), we genetically modified both CD4+ and CD8+ T cells from the same healthy donor or leukemia patients (termed WT1-siTCR/CD4 and WT1-siTCR/CD8, respectively). First, target-responsive cellular outputs mediated by WT1-siTCR/CD4 was thoroughly examined using flowcytometry, ELISA, 51Cr-release assay, CFSE dilution assay and bioluminescence assay. Next we similarly assessed impacts of WT1-siTCR/CD4 on the antileukemia functionality mediated by concurrentWT1-siTCR/CD8 both in vitro and in vivo. Eventually, we assessed the in vivo therapeutic efficacy of combined administration of WT1-siTCR/CD8 with WT1-siTCR/CD4 using a xenografted mouse model. Results The transcription factor profile demonstrated that WT1-siTCR/CD4 turned a terminal effector, but not regulatory phenotype. Activated WT1-siTCR/CD4 expressed cell-surface CD40L. Target-responsive cytokine production profile of WT1-siTCR/CD4 represented the Th1 helper function in the context of HLA-A*2402. HLA class II molecules expressed by leukemia cells facilitated the recognition of leukemia cells by WT1-siTCR/CD4 in the context of HLA-A*2402. WT1-siTCR/CD4 displayed the delayed cytocidal activity determined by 51Cr release assay. WT1-siTCR/CD4 could produce IFN-g in response to freshly isolated leukemia cells. WT1-siTCR/CD4 displayed the leukemia trafficking activity in vivo. WT1-siTCR/CD4 represented the potential to migrate into bone marrow via CXCR4/CXCL12 axis both in vitro and in vivo. Concurrent WT1-siTCR/CD4 augmented IFN-g production and cytotoxic degranulation mediated by WT1-siTCR/CD8 in response to the cognate epitope via humoral factors. Consequently, the cytocidal activity against autologous leukemia cells mediated by WT1-siTCR/CD8 was augmented in the presence of WT1-siTCR/CD4, both of them generated from normal lymphocytes of the same patient with leukemia in a complete remission. Upon the target recognition, activated WT1-siTCR/CD4 recruited WT1-siTCR/CD8 via CCL3/4-CCR5 axis. Proliferative response and differentiation into central memory T-cell subset mediated by WT1-siTCR/CD8 in response to the cognate epitope and leukemia cells were enhanced in the presence of autologousWT1-siTCR/CD4, but not gene-modified CD4+ T cells (NGM-CD4). CD127 expression on activated WT1-siTCR/CD8 also increased in parallel to this differentiation. Co-infused WT1-siTCR/CD4 augmented the tumor trafficking and persistence of WT1-siTCR/CD8 in vivo, resulting in the greater suppression of leukemia cells in a xenografted mouse model. Finally, in the therapeutic mouse model, co-infusion of WT1-siTCR/CD8 with of WT1-siTCR/CD4 significantly suppressed the growth of inoculated leukemia cells compared to that in mice received co-infusion of WT1-siTCR/CD8 with NGM-CD4 (Fig.1). Correlation between the therapeutic efficacy and survival of infused gene-modified T cells was also observed. Conclusion In results, the combined infusion of WT1-siTCR/CD8 with WT1-siTCR/CD4, but not NGM-CD4 obviously demonstrates the enhanced antileukemia efficacy via diverse mechanisms. Now we have just started a clinical trial using gene-modified T cells with WT1-siTCR vector for the treatment of patients with refractory acute myeloid leukemia and myeloid dysplastic syndrome. Because redirected T cells employed in this trial encompassed both WT1-siTCR/CD4 and WT1-siTCR/CD8, we are planning to clinically verify the significance of WT1-siTCR/CD4 in the redirected T cell-based antileukemia adoptive immunotherapy. (Fig.1) Disclosures: No relevant conflicts of interest to declare.

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MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

Cancer Immunology Immunotherapy ◽

10.1007/s00262-012-1336-z ◽

2012 ◽

Vol 62 (2) ◽

pp. 359-369 ◽

Cited By ~ 16

Author(s):

Carolina M. Soto ◽

Jennifer D. Stone ◽

Adam S. Chervin ◽

Boris Engels ◽

Hans Schreiber ◽

...

Keyword(s):

T Cells ◽

Mhc Class I ◽

Cd4 T Cells ◽

Class I ◽

Wild Type

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Enhancing the Efficacy of T Cell Receptor (TCR) Gene Therapy by Co-Transfer of TCR and Additional CD3 Molecules Into CD4+ T Cells

Blood ◽

10.1182/blood.v120.21.2044.2044 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2044-2044

Author(s):

Emma K Nicholson ◽

Maryam Ahmadi ◽

Angelika Holler ◽

Rebecca Pike ◽

Ben Carpenter ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Cd4 T Cells ◽

Specific Antigen ◽

Surface Expression ◽

Class I ◽

Tumour Regression

Abstract Abstract 2044 Introduction: The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method: The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results: CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion: Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures: Stauss: Cell Medica: Scientific Advisor Other.

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Endogenous T Cell Receptors Drive Non-Specific Suppression by Foxp3 MHC Class I-Restricted T Cell Receptor-Transduced Polyclonal CD4+ T Cells in Models of Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v120.21.3152.3152 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3152-3152

Author(s):

Benjamin J Uttenthal ◽

Emma Nicholson ◽

Ben Carpenter ◽

Sara Ghorashian ◽

Graham P Wright ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class I ◽

Cd4 T Cells ◽

Class I ◽

Graft Versus Host ◽

Specific Suppression ◽

Mhc Class I Molecule

Abstract Abstract 3152 Alloreactive immune responses directed against malignant cells in recipients of allogeneic hematopoietic stem cell transplants are able to cure patients with hematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD). Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and possibly reducing the potential for non-specific immune suppression. We have generated ‘converted’ Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into polyclonal conventional CD4+ T cells. We used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH-TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI. In vitro, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells were hyporesponsive to stimulation and suppressed the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells significantly reduced early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, polyclonal CD4+ T cells transduced with Foxp3 and the MH-TCR caused marked suppression of allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH-TCR conferred specificity to their cognate antigens in vitro, the potent suppression in these in vivo models was independent of the cognate antigen for the transduced TCRs. This non-specific suppression was markedly reduced when class I-restricted TCRs were transduced into OT-II Rag1-/- CD4+ T cells that are transgenic for a single endogenous TCR. These findings demonstrate an important role for the endogenous TCRs in driving non-specific suppression by polyclonal CD4+ T cells transduced with Foxp3 and class I-restricted TCRs, and suggest that strategies to downregulate endogenous TCRs will be required to achieve antigen-specific suppression in TCR gene-modified regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

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Myeloid Neoplasm-Related Gene Abnormalities Differentially Affect FLT3-Ligand Mediated Dendritic Cell Differentiation From Murine Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v116.21.3899.3899 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3899-3899

Author(s):

Jiro Fujita ◽

Masao Mizuki ◽

Masayasu Otsuka ◽

Sachiko Ezoe ◽

Hirokazu Tanaka ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Class Ii ◽

Class I ◽

Related Gene ◽

Hematopoietic Stem ◽

Myeloid Neoplasm ◽

Mild Impairment ◽

And Function

Abstract Abstract 3899 DCs play important roles in tumor immunology. In patients with myeloid neoplasm such as AML, CML, and MDS, there are spontaneously-differentiated DCs in vivo from leukemic cells (in vivo leukemic DCs), which are thought to retain leukemia-associated antigens (LAAs). Therefore it is postulated that in vivo leukemic DCs affect host immune responses in a LAA-specific manner. However, there have been only a few concise examinations about their subsets, maturation state, or function. DC differentiation is crucially regulated by STAT3/5 and in part associated with myeloid differentiation. Myeloid neoplasm develops from hematopoietic stem/progenitors cells (HSCs/HPCs) bearing various gene abnormalities, named class I and class II mutations, which contribute to growth augmentation and myeloid differentiation block, respectively. Therefore, this study tested the hypothesis that myeloid neoplasm-related gene abnormalities may affect steady-state DC differentiation from HSCs/HPCs. We first established an efficient and reproducible in vitro FLT3-ligand (FL)-mediated DC (FL-DC) differentiation system from murine HSCs/HPCs-rich population, lineage-, Sca-I+, c-Kithigh cells (LSKs). After 9 days of culture, the proportion of whole FL-DCs (CD11c+ cells), pDCs (CD11c+B220+ cells) and cDCs (CD11c+B220- cells) were reproducibly constant (whole FL-DCs; 87.5 ± 0.9 %, pDCs; 30.4 ± 6.4 %, cDCs; 57.1 ± 6.4 %, pDCs/cDCs ratio; 0.55 ± 0.19). FL-DCs from LSKs efficiently stimulated allogeneic CD4+ T cells. FL-DCs from LSKs yielded high amount of type I interferon by CpG-stimulation. These results indicated that our culture method efficiently and reproducibly induced functionally competent FL-DCs from LSKs. Next, we transduced various myeloid neoplasm-related gene abnormalities, named class I and II mutations, into LSKs, and then analyzed their effects on FL-DC differentiation from LSKs. We selected FLT3-ITD, FLT3-TKD, constitutive active (CA)-N-Ras, c-Kit-TKD, TEL/PDGFRβ, and FIP1L1/PDGFRα as representative of class I mutations, and AML1/ETO, PML/RARα, CBFβ/MYH11, and AML1dC as representative of class II mutations, respectively (Table). All class II mutations consistently showed mild impairment of FL-DCs keeping comparable pDCs/cDCs ratio with control. In contrast, class I mutations induced the heterogeneous impairment of FL-DCs. Both FLT3-ITD and FLT3-TKD showed a mild decrease in whole FL-DCs retaining comparable pDCs/cDCs ratio with control. CA-N-Ras showed the mild impairment of whole FL-DCs with a severe decrease in pDCs/cDCs ratio. c-Kit-TKD, TEL/PDGFRβ, and FIP1L1/PDGFRα displayed a severe decrease in both whole FL-DCs and their pDCs/cDCs ratio. whole FL-DCs (%) pDCs/cDCs ratio mock 85.4 ± 3.0 0.50 ± 0.21 FLT3-WT 46.1 ± 5.7 0.42 ± 0.19 FLT3-ITD 30.6 ± 14.5 0.60 ± 0.23 FLT3-TKD 29.4 ± 10.0 0.82 ± 0.48 CA-N-Ras 55.2 ± 6.3 0.02 ± 0.01 c-Kit-TKD 9.4 ± 5.7 0.19 ± 0.10 TEL/PDGFRβ 13.3 ± 2.9 0.05 ± 0.04 FIP1L1/PDGFRα 14.8 ± 4.7 0.03 ± 0.04 AML1/ETO 51.6 ± 8.4 0.63 ± 0.11 PML/RARα 35.6 ± 3.0 0.60 ± 0.30 CBFβ/MYH11 40.9 ± 17.3 0.48 ± 0.27 AML1dC 55.8 ± 7.9 0.29 ± 0.15 Time course study showed that each differentiation pattern of CA-N-Ras, c-Kit-TKD, TEL/PDGFRβ, or FIP1L1/PDGFRα was quite different from that of control. The analysis of the effects of signal transduction molecules revealed that CA-STAT5 and CA-MEK1, but not CA-STAT3 and CA-PI3 kinase, severely inhibited pDC differentiation. These data suggested that class I mutations differentially regulated FL-DC differentiation, possibly via their individual sets and magnitude of each constitutive active signal transduction pathway. We next investigated whether this DC differentiation heterogeneity seen in class I mutations influence on DC maturation. Overall, surface expressions of MHC II, CD80, and CD86 on whole FL-DCs induced by class I mutations were higher than those induced by control. FLT3-ITD-expressing FL-DCs, showing relatively immature phenotype among class I mutations, stimulated allogeneic CD4+ T cells comparably with control. CA-N-Ras-expressing FL-DCs, showing relatively mature phenotype among class I mutations, more efficiently stimulated allogeneic CD4+ T cells. These data suggested that class I mutations caused heterogeneous maturation and function of FL-DC. In conclusion, FL-DC differentiation from LSKs, its maturation, and function were affected in a myeloid neoplasm-related gene abnormality-specific manner. Disclosures: No relevant conflicts of interest to declare.

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