IL-15 Augments in-Vitro Expansion and Functional Activity of Antigen-Specific Effector Memory T-Cells (TEM) While Co-Expression of IL-15 and IL-15 Rα on Antigen Presenting Cells Also Promotes Expansion of Central Memory T-Cells (TCM)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3541-3541
Author(s):  
Aisha Hasan ◽  
Annamalai Selvakumar ◽  
Bo Dupont ◽  
Michel Sadelain ◽  
Isabelle Riviere ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic Activity ◽  
Memory T Cells ◽  
Antigen Presenting Cells ◽  
Effector Memory ◽  
Central Memory T Cells ◽  
Receptor Alpha ◽  
Antigen Presenting

Abstract Adoptive immunotherapy with in-vitro expanded antigen-specific T cells (TC) is often hampered by the extended culture times required to generate sufficient numbers of antigen- specific TC and their limited persistence in-vivo. IL-2 predominantly supports the generation of short-lived effector memory (TEM) and effector (TE) CD8+ TC without expanding the CD62L+ and CCR7+ central memory T cells (TCM), which may persist for long periods in vivo. We examined the potential of IL-15 to foster growth of CMVpp65- specific TC as well as expansion of both TEM and TCM CD8+ TC. Accordingly, TC from 3 seropositive donors bearing HLA A0201 were sensitized in-vitro using NIH 3T3 based artificial antigen presenting cells transduced to express B7.1, ICAM-1, LFA-3, β2-M and HLA A0201 heavy chain as well as CMVpp65 (A2-AAPC) in presence of exogenous IL-2, IL-15 or IL-15 plus IL-2. To assess if trans-presentation of IL-15 by the receptor alpha on AAPCs leads to enhancement of IL-15 activity, we sequentially transduced A2-AAPCs with the IL-15 and IL-15 receptor alpha cDNA (A2- AAPC IL-15/IL-15Rα). TC were then cultured using A2-AAPC with either IL-2 (20U/ml) IL-15 (10ng/ml) IL-2 plus IL-15 or with A2-AAPC IL-15/IL-15Rα or A2-AAPC IL-15Rα + IL-2 (20U/ml). TC sensitized using either A2-AAPC IL-15/IL-15Rα or A2-AAPC + IL-15 demonstrated ~ 1500–2000 fold expansion of CMVpp65 A2-NLV tetramer (+) CD8+ TC, compared to a 300–600 fold expansion using A2-AAPC + IL-2 after 21–28 days in culture. Cultures containing IL-15 generated 25–70 ×106 A2-NLV tetramer (+) CD8+ TC in comparison to 10–18 ×106 in cultures with IL-2. At 7–10 days, ~20% of the tetramer (+) TC demonstrated a TCM phenotype (CD62L+ and CCR7+) in all cultures, with 75% TEM and 5% TE. By 21- 28 days, no TCM were detected among tetramer (+) TC in cultures containing exogenous IL-2, IL-15 or IL-2 plus IL-15. However, TC sensitized with A2-AAPC IL-15/IL-15Rα still contained 10–12% (~7×106) tetramer (+) CD8+ TCM which further increased through day 35. In functional assays, TC sensitized in the presence of IL-15 or AAPCs expressing IL-15 and IL-15Rα elicited superior CMV-specific responses with 7–20% CD8+ TC demonstrating CMVpp65 epitope specific interferon gamma production compared to 2–3% in cultures with IL-2. Cytotoxic activity against CMV pp65 peptide loaded autologous EBV transformed B-cell lines (E:T= 10:1) was higher for TC sensitized with IL-15 (80%) compared to cultures with IL-2 (60%). The cytotoxic activity against HLA mismatched targets or K562 was <5% in all cultures. In conclusion, our data demonstrate higher yields and augmented function in antigen specific T cells cultured with IL-15. TC sensitized with A2-AAPC-CMVpp65 together with IL-15 +/− IL-2 only supported sustained expansion of TEM and TE. In contrast, TC sensitized with A2-AAPC IL-15/IL-15Rα also supported sustained expansion of TCM.

scholarly journals Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Manoj Patidar ◽  
Naveen Yadav ◽  
Sarat K. Dalai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Half Life ◽  
Therapeutic Potential ◽  
Chimeric Protein ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Antigen Presenting

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

scholarly journals Nanoparticles Engineered as Artificial Antigen-Presenting Cells Induce Human CD4+ and CD8+ Tregs That Are Functional in Humanized Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Sophia Giang ◽  
David A. Horwitz ◽  
Sean Bickerton ◽  
Antonio La Cava
Keyword(s):  
T Cells ◽  
Lupus Erythematosus ◽  
T Regulatory Cells ◽  
Antigen Presenting Cells ◽  
Plga Nanoparticles ◽  
Nsg Mice ◽  
Artificial Antigen ◽  
Antigen Presenting

Artificial antigen-presenting cells (aAPCs) are synthetic versions of naturally occurring antigen-presenting cells (APCs) that, similar to natural APCs, promote efficient T effector cell responses in vitro. This report describes a method to produce acellular tolerogenic aAPCs made of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and encapsulating IL-2 and TGF-β for a paracrine release to T cells. We document that these aAPCs can induce both human CD4+ and CD8+ T cells to become FoxP3+ T regulatory cells (Tregs). The aAPC NP-expanded human Tregs are functional in vitro and can modulate systemic autoimmunity in vivo in humanized NSG mice. These findings establish a proof-of-concept to use PLGA NPs as aAPCs for the induction of human Tregs in vitro and in vivo, highlighting the immunotherapeutic potential of this targeted approach to repair IL-2 and/or TGF-β defects documented in certain autoimmune diseases such as systemic lupus erythematosus.

Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2766-2766
Author(s):  
Seema Rawal ◽  
Nathan Fowler ◽  
Min Zhang ◽  
Zhiqiang Wang ◽  
Tariq Muzzafar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Memory T Cells ◽  
T Cell Subsets ◽  
Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

scholarly journals Stimulation of mature unprimed CD8+ T cells by semiprofessional antigen-presenting cells in vivo.

1992 ◽  
Vol 176 (5) ◽  
pp. 1291-1302 ◽  
Author(s):  
H Kosaka ◽  
C D Surh ◽  
J Sprent
Keyword(s):  
T Cells ◽  
Parental Strain ◽  
Antigen Presenting Cells ◽  
Class I ◽  
Small Subset ◽  
Effector Cells ◽  
Cd8 Cells ◽  
Antigen Presenting

To test whether unprimed CD8+ cells can recognize class I alloantigens presented selectively on non-bone marrow (BM)-derived cells, unprimed parental strain CD8+ cells were transferred to long-term parent--&gt;F1 BM chimeras prepared with supralethal irradiation. Host class I expression in the chimeras was undetectable on BM-derived cells and, in spleen, was limited to low-level staining of vascular endothelium and moderate staining of follicular dendritic cells (a population of nonhemopoietic cells in germinal centers). Despite this restricted expression of antigen, acute blood-to-lymph recirculation of parental strain T cells through the chimeras led to selective trapping of 95% of CD8+ cells reactive to normal F1 spleen antigen presenting cells (APC) in vitro. Subsequently, a small proportion of the trapped cells entered cell division and gave rise to effector cells expressing strong host-specific CTL activity. The activation of host-specific CD8+ cells was also prominent in double-irradiated chimeras, and cell separation studies showed that the effector cells were generated from resting precursor cells rather than from memory-phenotype cells. It is suggested that the non-BM-derived cells in the chimeras acted as semiprofessional APC. These cells were nonimmunogenic for most host-reactive CD8+ cells but were capable of stimulating a small subset of high-affinity T cells. The possible relevance of the data to the prolonged immunogenicity of vascularized allografts in humans is discussed.

scholarly journals 751 Neo-X-Prime bispecific antibodies targeting CD40 and tumor antigens promote cross-presentation of tumor exosome-derived neoantigen and induce superior anti-tumor responses compared to CD40 mAb

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A785-A785
Author(s):  
Karin Hagerbrand ◽  
Mattias Levin ◽  
Laura Von Schantz ◽  
Laura Varas ◽  
Anna Säll ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Antigens ◽  
Antigen Presenting Cells ◽  
Immunological Memory ◽  
Cell Repertoire ◽  
Cross Presentation ◽  
Antigen Presenting

BackgroundAlligator's Neo-X-Prime platform aims to enable antigen presenting cells to efficiently enhance priming of tumor neoantigen-specific T cells with the goal of overcoming PD-1 resistance in certain tumor types. We hypothesize that binding of a CD40 x TAA bispecific antibody (bsAb) to CD40 on dendritic cells (DCs) and a tumor-associated antigen (TAA) on tumor exosomes or tumor debris leads to (i) activation of the DC, (ii) uptake of the tumor material, (iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and, iv) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool.MethodsFunctionality was evaluated in vitro using CD40 reporter cells and monocyte-derived DCs, co-cultured with cells expressing TAA. Further, co-localization of TAA-expressing cellular debris with a CD40-expressing human B cell line in the presence of bsAbs was assessed using live cell imaging. In vivo, anti-tumor efficacy and immunological memory were assessed in human CD40 transgenic (hCD40tg) mice bearing MB49 bladder carcinoma tumors transfected with human TAA or controls. T cells isolated from OVA-specific TCR-transgenic mice were used to evaluate the effect of Neo-X-Prime bsAbs on antigen-specific T cell expansion in the presence of hCD40tg DCs and exosomes from MB49 tumors transfected with both human TAA and OVA using flow cytometry.ResultsUsing CEA as a highly expressed TAA, we have developed lead Neo-X-Prime CD40-CEA bsAbs engineered to achieve an optimal profile. Further, using Neo-X-Prime concept molecules targeting EpCAM, we have demonstrated the ability to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells that is dependent on the receptor density of the TAA. We have further shown that addition of Neo-X-Prime bsAbs to a co-culture of murine DCs, T cells and tumor-derived exosomes induces increased expansion of model neoantigen-specific T cells. In vivo, Neo-X-Prime bsAbs display a potent, TAA-dependent anti-tumor effect that is superior to CD40 mAbs. Cured mice develop a broad immunological memory that is not dependent on expression of the TAA. The tumor-localizing property of Neo-X-Prime bsAbs also shows potential for improved safety compared to CD40 monospecific antibodies.ConclusionsNeo-X-Prime bsAbs have the potential to tumor-selectively target CD40-expressing antigen-presenting cells to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.Ethics ApprovalAll experiments were performed after approval from the Malmö/Lund Animal Ethics Committee.

scholarly journals Treg-expressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on antigen-presenting cells

2021 ◽  
Vol 118 (30) ◽  
pp. e2023739118
Author(s):  
Murat Tekguc ◽  
James Badger Wing ◽  
Motonao Osaki ◽  
Jia Long ◽  
Shimon Sakaguchi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Synapse Formation ◽  
Cytotoxic T Lymphocyte ◽  
Antigen Presenting Cells ◽  
Programmed Death ◽  
Antigen Presenting

Foxp3-expressing CD4+CD25+ regulatory T cells (Tregs) constitutively and highly express the immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, Treg-expressed CTLA-4 down-regulates the expression of CD80/CD86 costimulatory molecules on antigen-presenting cells (APCs). Here, we show that Treg-expressed CTLA-4 facilitated Treg-APC conjugation and immune synapse formation. The immune synapses thus formed provided a stable platform whereby Tregs were able to deplete CD80/CD86 molecules on APCs by extracting them via CTLA-4–dependent trogocytosis. The depletion occurred even with Tregs solely expressing a mutant CTLA-4 form lacking the cytoplasmic portion required for its endocytosis. The CTLA-4–dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 down-regulation or blockade by Treg-expressed membrane CTLA-4 or soluble CTLA-4-immunoglobulin (CTLA-4-Ig), respectively, disrupted cis-CD80/programmed death ligand-1 (PD-L1) heterodimers and increased free PD-L1 on dendritic cells (DCs), expanding a phenotypically distinct population of CD80lo free PD-L1hi DCs. Thus, Tregs are able to inhibit the T cell stimulatory activity of APCs by reducing their CD80/CD86 expression via CTLA-4–dependent trogocytosis. This CD80/CD86 reduction on APCs is able to exert dual suppressive effects on T cell immune responses by limiting CD80/CD86 costimulation to naïve T cells and by increasing free PD-L1 available for the inhibition of programmed death-1 (PD-1)–expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in combination may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing immune responses, including tumor immunity.

Human CMVpp65-Specific Stem Cell-like Memory T-Cells (TSCM) Are a Principal Source for Rapid Repopulation of Immunodominant Central Memory (TCM) and Effector Memory (TEM) in the Circulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5728-5728
Author(s):  
Tzu-Yun Kuo ◽  
Aisha Hasan ◽  
Manuel Guerreiro ◽  
Richard J. O'Reilly
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Amino Acid Sequences ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Tnf Α ◽  
Artificial Antigen ◽  
Ifn Γ ◽  
Antigen Presenting

Abstract Latent CMV infection is controlled by a limited repertoire of immunodominant T-cells specific for viral peptides, particularly CMVpp65 and CMV IE-1. The antigen-specific T-cell subsets responsible for maintaining memory T-cells in this repertoire and repopulating them in response to periodic viral reactivations remain unclear. In this study, we generated T-cells specific for CMVpp65 from naïve (TN), TSCM, TCM and TEM subsets isolated from the blood of HLA-A*0201 normal seropositive donors and then comparatively characterized NLV-HLA-A*0201 Tetramer+ T-cells from each of these subsets. Following in vitro sensitization with artificial antigen-presenting cells transduced to express HLA-A*0201 and CMVpp65 and expansion with IL-7, IL-15 and IL-2, Tet+ T-cells were regularly generated from CD62L+CD45RO-CD95- TN and from CD62L+CD45RO-CD95+ TSCM, as well as TCM and TEM. Isolated Tet+ TN, TSCM, TCM and TEM were each able to generate IFN-γ, TNF-α and granzyme B. Each Tet+ subset also expressed similar level of PD-1 and KLRG-1. However, Tet+ TN and TSCM expressed higher levels of CD27 and lower levels of CD57 than TCM or TEM. Tet+ TSCM were distinguished from Tet+ TN, TCM and TEM by a significantly greater level of proliferation and by their rapid and selective expansion of NLV-specific T-cells bearing TCRs identical by amino acid sequences to those expressed by TEM and TCM in the blood. Thus, Tet+ TSCM rather than Tet+ TN constitute the principal repertoire for repopulation of immunodominant memory T-cells sustained in the circulation. Disclosures Hasan: Atara: Patents & Royalties.

scholarly journals Polarizing CD8+ Central Memory T Cells and Th1 Cells By Lenalidomide Contributes to the Antitumor Function of CD19 CAR-T Cells in Killing Diffused Large B Cell Lymphoma in Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4190-4190
Author(s):  
Zhen Jin ◽  
Han Liu ◽  
Molly Allen ◽  
Xiaoyang Li ◽  
Rufang Xiang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
Car T Cells ◽  
Car T

Abstract Background CD19-CAR T cells with costimulatory ligand of CD28 or 4-1BB have acquired well response in ALL and CLL, whereas it shows less effective in B-cell NHL. The microenvironment of lymphomas is much more complicated than that of leukemia, which containing physical barriers and higher immunosuppression levels preventing lymphoma cells from T cell attack. To overcome such T cell toleration, one can optimize T cell fitness by adding co-stimulatory domain or polarizing T cell differentiation. Some pre-clinical studies have reported the 3rd generation of CD19-CAR T cells with CD28 and 4-1BB domain in treating ALL, but the results were in controversy. Lenalidomide has been proved to have direct anti-tumor effects in killing DLBCL cell lines except its immunomodulatory functions. Therefore, we did preliminary investigation in vitro to seek whether the combination of lenalidomide and CD19 CAR-T cells with both CD28 and 4-1BB costimulatory domain could acquire better effects Method We first verified the proliferation inhibition of lenalidomide in treating both ABC-DLBCL cell lines (Su-DHL2 and OCI-Ly3) and GCB-DLBCL cell line OCI-Ly1. CY cell was primary cells isolated from GCB-DLBCL patients in Rui-jin Hospital. Under the maximum observed plasma concentration of lenalidmomide (2.2¦ÌM), the growth inhibition in both GCB-CY and OCI-Ly1 cell line was minimal, whereas the impact on ABC-DLBCL cell lines was more obvious. We further examined the efficiency of lenalidomide in vivo using a patient-derived mouse model. The primary lymphoma cells were obtained from a ABC-DLBCL patient and subcutaneously transplanted into NOD/SCID mouses. However, daily treated with lenalidomide could not delay the tumor growth (p>0.05) (Fig A, B, C). We next isolated CD3+ T cells from healthy donors, expanded with CD3/CD28 beads. The pLenti-EF1¦Á-CD19-28-BB-¦Æ-mcherry lentiviral vectors was generated and transduced in the expanded T cells to generate CD19 CAR-T cells. T cells transduced with pLenti-EFI¦Á-Actin-mcherry lentiviral vector were used as control. CD19-CAR T cells and T cells transdued with Actin-mcherry were pretreated with 2¦ÌM lenalidomide for 72 hours. LDH assay was then performed to identify the cytotoxicity of CD19-CAR T cells against CY in 7 hours. We found that lenalidomide substantially enhanced the anti-tumor function of CD19 CAR T cells and it also promoted the CD19-CAR T cells proliferation to some extent (Fig D, E). We therefore used three DLBCL patients CAR-T cells to identify the cytokine secretion. It was found that lenalidomide promoted Th1-biased cytokines secretion (IL-2, IFN-¦Ã, TNF-¦Á) and decreased Th2-biased cytokines (IL-6, IL-10). Interestingly, CAR-T cells secreted less IFN-¦Ã and TNF-¦Á but more IL-6 and IL-10 in killing OCI-Ly3 compared with OCI-Ly1 and CY (Fig F). The results leaded us to next determine the CD19-CAR T cell differentiation. A comparable increase of CD8+CD45RA-CD62L+ CD19 CAR T cells was observed as well as the CD4+CCR6-CCR4-CXCR3+ subset, indicating lenalidomide could induce CD19 CAR T cells differentiate to CD8+ central memory T cells and Th1 cells (Fig G). As the central memory T cells are more likely to home to the lymph nodes, we found that lenalidomide considerably increased the CD19-CAR T cell migration toward CCL21 and CCL19 in transwell system (Fig H). Conclusion In conclusion, our results indicate that lenalidomide could polarize CD19-CAR T cells to CD8 TCM and Th1 subset, which might contribute to the enhanced antitumor function of CD19 CAR-T cells. Meanwhile, by overexpressed CD62L, lenalidomide could promote the migrating capability of CD19 CAR-T cells. More in-vivo work shall be done to determine the combination therapy in the future. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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