Induction, Selection and Expansion of Acute Myeloid Leukemia Reactive Autologous T Cells for Adoptive Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1061-1061
Author(s):  
Rui-kun Zhong ◽  
Edward D. Ball
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor ◽  
Adoptive Immunotherapy ◽  
Cell Number ◽  
Cell Killing ◽  
Specific Lysis ◽  
Gm Csf ◽  
Highly Active ◽  
Ifn Γ

Abstract To generate highly AML reactive autologous CTL, the optimal protocol of growth factor combination and sequence of administration were screened and compared in an AML-DC culture system aimed to induce AML cell DC differentiation, activate and expand naïve T cells against AML; and simultaneously activate and expand possible rare existing AML reactive memory T cells. Primary AML peripheral blood or marrow cells containing more than 90% AML blasts were cultured in 96 well plates. Every single well was regarded and treated as an independent culture. Various growth factor combinations of GM-CSF, IL2, IL-3, IL-4, IL7, IL12, CD40L, TNF-α, anti-CTLA-4 mAb were compared for efficiency of AML cell DC differentiation induction and autologous T cell activation in 7 day culture. Significantly enhanced CD80/CD86 expression and total cell number (include both AML-DC and T cells, see attached figure) in the culture was observed when GM-CSF/IL-4 combined with IL-12. The T cells in each well were than expanded with high dose IL-2 (6000 u/ml) and CTL activity against autologous AML were examined by both 51Cr release assay and culture supernatant IFN-γ concentration assay. Multiple wells of AML cell culture from the same patient with the same cell number and culture condition exhibited substantial functional variation demonstrated by MHC-restricted autologous AML cell killing (3 - 70% specific lysis) and IFN-γ secretion (25 - 2994 pg/ml), indicating that the T cells randomly put in the independent wells vary greatly in efficiency of AML antigen recognition and activation. IL-12 alone exerted significant enhanced IFN-γ secretion in 4 of 5 patients. IL-12 combined with GM-CSF/IL-2/IL-4/IL-7 enhanced IFN-γ secretion compared with IL-12 alone in 3 of 5 patients. CTLA-4 blockade further significantly enhanced T cell activity in 5 of 5 patients. Highly active CTL lines were selected from high IFN-γ secretion wells and rapidly expanded using OKT-3/CD28 coated, mitomycin-c treated autologous AML cells. After expansion, the highly active lines still maintained high autologous AML cell killing compared to the low active lines. A therapeutic quantity of AML reactive autologous T cells (20.2±30.1)x109, (n=5) can be obtained after 6 weeks of culture from (6.4±2.9)x107 AML PBMC. This study demonstrated AML cells cultured in IL-12 or IL-12 combined with GM-CSF/IL-2/IL-4/IL-7/anti-CTLA4 mAb activated the potential of the patient immune system and may maximize the possibility of AML reactive CTL generation for adoptive immunotherapy. Figure Figure

The Response to Repeated Vaccination with PR1 and WT1 Peptides Admixed in Montanide Adjuvant Is Transient and Fails to Induce Sustained Anti-Leukemia Responses in Patients with Myeloid Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4096-4096
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Stephan Mielke ◽  
Behnam Jafarpour ◽  
Bipin N. Savani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Gm Csf ◽  
Cell Responses ◽  
Ifn Γ

Abstract Abstract 4096 Poster Board III-1031 We previously demonstrated the immunogenicity of a combined vaccine approach employing two leukemia-associated antigenic peptides, PR1 and WT1 (Rezvani Blood 2008). Eight patients with myeloid malignancies received one subcutaneous 0.3 mg and 0.5 mg dose each of PR1 and WT1 vaccines in Montanide adjuvant, with 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF). CD8+ T-cell responses against PR1 or WT1 were detected in all patients as early as 1 week post-vaccination. However, responses were only sustained for 3-4 weeks. The emergence of PR1 or WT1-specific CD8+ T-cells was associated with a significant but transient reduction in minimal residual disease (MRD) as assessed by WT1 expression, suggesting a vaccine-induced anti-leukemia response. Conversely, loss of response was associated with reappearance of WT1 transcripts. We hypothesized that maintenance of sustained or at least repetitive responses may require frequent boost injections. We therefore initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies. Five patients with acute myeloid leukemia (AML) and 2 patients with myelodysplastic syndrome (MDS) were recruited to receive 6 injections at 2 week intervals of PR1 and WT1 in Montanide adjuvant, with GM-CSF as previously described. Six of 7 patients completed 6 courses of vaccination and follow-up as per protocol, to monitor toxicity and immunological responses. Responses to PR1 or WT1 vaccine were detected in all patients after only 1 dose of vaccine. However, additional boosting did not further increase the frequency of PR1 or WT1-specific CD8+ T-cell response. In 4/6 patients the vaccine-induced T-cell response was lost after the fourth dose and in all patients after the sixth dose of vaccine. To determine the functional avidity of the vaccine-induced CD8+ T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of PR1 and WT1 peptides (0.1 and 10 μM) was measured by IC-IFN-γ staining. Vaccination led to preferential expansion of low avidity PR1 and WT1 specific CD8+ T-cell responses. Three patients (patients 4, 6 and 7) returned 3 months following the 6th dose of PR1 and WT1 peptide injections to receive a booster vaccine. Prior to vaccination we could not detect the presence of PR1 and WT1 specific CD8+ T-cells by direct ex-vivo tetramer and IC-IFN-γ assay or with 1-week cultured IFN-γ ELISPOT assay, suggesting that vaccination with PR1 and WT1 peptides in Montanide adjuvant does not induce memory CD8+ T-cell responses. This observation is in keeping with recent work in a murine model where the injection of minimal MHC class I binding peptides derived from self-antigens mixed with IFA adjuvant resulted in a transient effector CD8+ T cell response with subsequent deletion of these T cells and failure to induce CD8+ T cell memory (Bijker J Immunol 2007). This observation can be partly explained by the slow release of vaccine peptides from the IFA depot without systemic danger signals, leading to presentation of antigen in non-inflammatory lymph nodes by non-professional antigen presenting cells (APCs). An alternative explanation for the transient vaccine-induced immune response may be the lack of CD4+ T cell help. In summary these data support the immunogenicity of PR1 and WT1 peptide vaccines. However new approaches will be needed to induce long-term memory responses against leukemia antigens. To avoid tolerance induction we plan to eliminate Montanide adjuvant and use GM-CSF alone. Supported by observations that the in vivo survival of CD8+ T-effector cells against viral antigens are improved by CD4+ helper cells, we are currently attempting to induce long-lasting CD8+ T-cell responses to antigen by inducing CD8+ and CD4+ T-cell responses against class I and II epitopes of WT1 and PR1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunogenicity of personalized dendritic-cell therapy in HIV-1 infected individuals under suppressive antiretroviral treatment: interim analysis from a phase II clinical trial

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Marcella Vassão de Almeida Baptista ◽  
Laís Teodoro da Silva ◽  
Sadia Samer ◽  
Telma Miyuki Oshiro ◽  
Iart Luca Shytaj ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Cell Therapy ◽  
Antiretroviral Treatment ◽  
Gm Csf ◽  
Ifn Γ ◽  
Dendritic Cell Therapy ◽  
Hiv 1

Abstract Background We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. Methods PBMCs were obtained from 10 HIV+ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient’s HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients’ cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4+ and CD8+ T-cells. Results The protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4+ and CD8+ T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4+ T-cells. The number of candidates that increased in vitro the cytokine levels in CD4+ and CD8+ T cells upon stimulation with Gag peptides from baseline to day 15 and from baseline to day 30 and day 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. Conclusions MDDC had a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment. Trial registration NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829, posted November 11th, 2016)

Latent-Membrane 2a Specific CTL’s but Not Latent-Mebrane 1 Specific CTL’s Lysis Efficiently Hodgkin Lymphoma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4991-4991
Author(s):  
Max S. Topp ◽  
Jan Diekamm ◽  
Olaf Beck ◽  
Georg Rauser ◽  
Kai Hauschulz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Cytokine Production ◽  
Cd8 T Cell ◽  
Rapid Expansion ◽  
Target Cells ◽  
Specific Lysis ◽  
Healthy Donors

Abstract Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.

NK Cell KIR Reconstitution after Allogeneic Hematopoietic Cell Transplant (HCT) Is Affected by T Cell Number and Function.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1406-1406
Author(s):  
Sarah Cooley ◽  
Valarie McCullar ◽  
Rosanna Wangen ◽  
Tracy L. Bergemann ◽  
John E. Wagner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor ◽  
Nk Cells ◽  
Clinical Outcomes ◽  
Nk Cell ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Naive T Cells ◽  
Naïve T Cells

Abstract NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P < .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P < .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

Generation of CMV Specific T Cells From CMV Seronegative Donors by T Cell Receptor RNA Transfer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3577-3577
Author(s):  
Simone Thomas ◽  
Sebastian Klobuch ◽  
Mirjam H.M. Heemskerk ◽  
Diana Stolle ◽  
Katrin Besold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Human Fibroblasts ◽  
Expression Level ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Peptide Epitope ◽  
Retroviral Transduction ◽  
Ifn Γ

Abstract Abstract 3577 Poster Board III-514 Reactivation of latent human cytomegalovirus (CMV) infection is a frequent complication in CMV seropositive patients after allogeneic hematopoietic stem cell transplantation (HSCT). Although antiviral drug therapy is successfully used to reduce the risk of CMV disease, long-term virus control requires the re-establishment of protective antiviral T cell immunity in the host. The latter is challenging, particularly if the donor is CMV seronegative and thus, no CMV specific T cells are being transferred from donor to recipient during HSCT. Grafting nonreactive T cells of CMV seronegative donors by virus-antigen specific T cell receptors (TCR) may be an efficient means to transfer CMV specific T cell function into allogeneic HSCT recipients. In this study, we intended to reprogram T cells of CMV seronegative donors with human TCR recognizing the immunodominant HLA-A*0201 binding peptide epitope NLVPMVATV (495-503) derived from the CMV pp65 protein. A common approach for TCR gene transfer into T cells is retroviral transduction bearing the risk of insertional mutagenesis which hampers clinical translation. In addition, heterologous recombination between introduced and naturally expressed TCR chains might lead to the induction of harmful autoimmunity. Herein we used in vitro transcribed RNA encoding the CMV pp65/HLA-A*0201-specific TCR for electroporation of anti-CD3 stimulated T cells in peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. This procedure resulted in transient surface expression of the introduced TCR chains up to 5 days as shown by flow cytometry. Maximum expression level was observed at 4 to 24 h after electroporation, with up to 70% of total CD8+ and CD4+ T cells staining positive for the vβ13.1 subfamily domain of the TCRβ chain. After introduction of TCR RNA, the intensity of CMV pp65/HLA-A*0201 tetramer staining was 60% and 50% of total CD8+ and CD4+ cells, respectively. In IFN-γ ELISPOT and 51Chromium-release assays, TCR RNA transfected T cells recognized HLA-A*0201 expressing T2 cells pulsed with titrated amounts of CMV pp65 (495-503) peptide. Minimal peptide concentration triggering specific lysis was 0.1 nM to 1 nM at a CD8+ to target (CD8+:T) ratio of 2:1. The EC50 value (0.2 nM) was in the same range of avidity compared to that of a retrovirally transduced counterpart construct of this TCR. Most importantly, TCR recipient CD8+ T cells gained the ability to lyse HLA-A*0201 positive human fibroblasts upon infection with CMV. Specific lysis between 20% and 100% was observed at a CD8+:T ratio of 1:1 or higher. We next sorted CD8+ T cells from PBMC of CMV seronegative donors into naïve and memory cells according to expression of the differentiation markers CD45RA and CD45RO. Although 90% of naïve CD8+ T cells stained positive for the CMV pp65/HLA-A*0201 tetramer after electroporation of TCR RNA, they mediated only marginal lysis toward CMV infected fibroblasts. In contrast, TCR RNA transfected memory CD8+ T cells showed strong lysis against CMV infected fibroblasts at a CD8+:T ratio of 0.7:1 or higher. Specific lysis was detected for at least 3 days after electroporation. In summary, our data demonstrate that nonreactive human T cells can be successfully redirected with CMV pp65 TCR RNA. The expression level of the introduced TCR is sufficient to trigger IFN-γ production and cytolytic activity toward CMV infected human fibroblasts. Electroporation of TCR RNA is comparably easy and eliminates the risk of retroviral transduction. We therefore believe that CMV pp65 TCR RNA has the potential to be further developed as a therapeutic “off-the-shelf” reagent for patients who undergo drug-resistant CMV reactivation after HSCT. Disclosures: No relevant conflicts of interest to declare.

T-Cell Immunity to Two HLA-A2-Restricted Self-Determinants of Cyclin E May Contribute to Remission After Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 686-686
Author(s):  
Hong He ◽  
Ken Ishiyama ◽  
Gheath Alatrash ◽  
Yukio Kondo ◽  
Sijie Lu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Specific Lysis ◽  
Cyclin E1 ◽  
Specific Ctls ◽  
Ifn Γ ◽  
Change In Mean ◽  
Constitutively Active

Abstract Abstract 686 Cyclin E1 (CCNE1) and cyclin E2 (CCNE2) are tightly regulated cell cycle genes in normal cells but are over-expressed and constitutively active in breast cancer and in the majority of hematological malignances. To validate CCNE as a potential target antigen for T-cells in leukemia, we first confirmed aberrant CCNE1 and CCNE2 protein in PBMC from 26 (93%) of 28 patients (CML = 16; AML = 7; ALL =2; NHL = 3) by Western Blot compared to 4 (33%) of 12 healthy controls (p < 0.0005). Next, we screened the sequences of CCNE1 and CCNE2 for HLA-A*0201 binding motifs and identified a pair of homologous nonameric peptides with highest predicted binding to HLA-A*0201 using an NCBI algorithm. The peptides, denoted CCNE1M (144ILLDWLMEV152) and CCNE2L (144ILLDWLLEV152), differed at P7 (M or L), and both differed from mouse sequence at P1 (V). Synthetic mouse and human peptides were used to confirm high affinity HLA-A2 binding on T2 cells by FACS analysis and peptide-pulsed T2 were used to elicit peptide-specific CTLs from healthy HLA-A2+ PBMC in vitro. CCNE1M-CTL lines specifically lysed both CCNE1M-loaded and CCNE2L-loaded T2 cells, while no CTL could be elicited with mouse peptide. Similarly, CCNE2L-stimulated CTL lines killed CCNE1M-loaded and CCNE2L-loaded T2 cells but not non-loaded T2 cells. Using CCNE1M and CCNE2L HLA-A2 tetramers, we found that either tetramer could bind equally to either the CCNE1M- or CCE2L-derived CTL lines, suggesting that both peptides could be cross-recognized by CTL lines elicited with either peptide. To further study the cross-recognition and potential immune dominance of both peptides and to determine their potential anti-leukemia activity, CCNE1M- and CCNE2L-CTL clones were derived by limiting dilution assay. Two peptide-specific CTL clones from each of the lines showed 25% and 26% specific lysis, respectively, of leukemia cells at E:T 10:1. Neither CCNE-specific CTLs showed lysis of BM cells that were obtained from the same patient during remission, nor HLA-A2+ BM cells from a healthy donor. Next, we compared the T-cell antigen receptor (TCR) avidity of these CCNE1M- and the CCNE2L-CTL clones by measuring tetramer dissociation half-times (t1/2) at 25°C using CCNE1M/HLA-A2 and CCNE2L/HLA-A2 (and control pp65/HLA-A2) tetramers analyzed by flow cytometry. The decay of normalized (to time = 0) tetramer-bound fluorescence versus time was linear for each clone with either tetramer (R2 = 0.85 to 0.91), showing that tetramer binding avidity could be used to proportionally determine TCR affinity. Furthermore, first order kinetics could be used to determine the t1/2 of each of the clones. The t1/2 of CCNE1M/HLA-A2 tetramer was 85 min and 25 min, respectively, while the t1/2of CCNE1L/HLA-A2 was 30 min and 11 min, respectively, for the CCNE1M-CTL and the CCNE2L-CTL. This suggests that while both peptides were cross recognized by unique T-cell clones (with unique TCR, determined by TCR-Vβ sequence comparisons), CCNE1M appeared to be immunodominant. To determine whether immune response (IR) to either peptide occurred in leukemia patients, we studied PBMC from 18 patients (10 CML; 8 ALL) before and 3–6 mo after SCT with CCNE1M/HLA-A2- and CCNE2L/HLA-A2-tetramer assay. The mean number of CCNE1M-CTL and CCNE2L-CTL cells increased after SCT (p< 0.002 in CCNE1M-CTL and CCNE2L-CTL) compared to no change in mean number of pp65-CTL before/after SCT. IR (defined as ≥ 20% increase of specific CTL after SCT) to either CCNE1M or CCNE2L did not correlate with type of leukemia, donor-recipient HLA disparity (matched or mismatched), or disease status prior to SCT by Fisher's exact test. However, in 8 CML patients not in remission prior to SCT, IR to either CCNE1 or CCNE2 occurred more frequently in patients who achieved CR compared to those that did not achieve CR after SCT (100% vs. 33%, respectively; p < 0.04). These findings were confirmed in an additional 25 AML patients with active disease at SCT. To study whether the peptide-specific CTL were functional, we measured IFN-γ and TNF-αa production after peptide stimulation by Luminex bead assay and by intracellular cytokine flow cytometry (CFC). The assays showed production of IFN-γ and TNF-αa cytokines by T-cells after stimulation with CCNE1M or CCNE2Lpeptides. Taken together, these results show that CCNE1M and CCNE2Lself-peptides from constitutively active cell cycle proteins are novel leukemia-associated antigens that could be studied in immunotherapy strategies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunogenicity of Personalized Dendritic-cell Therapy in HIV-1 Infected Individuals Under Suppressive Antiretroviral Treatment: Interim Analysis From a Phase II Clinical Trial.

2021 ◽  
Author(s):  
Marcela Vassão de Almeida Batista ◽  
Laís Teodoro Silva ◽  
Sadia Samer ◽  
Telma Miyuki Oshiro ◽  
Iart Luca Shytaj ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Treatment ◽  
Ex Vivo ◽  
Gm Csf ◽  
T Cell Immune Responses ◽  
Ifn Γ ◽  
Dendritic Cell Therapy ◽  
Hiv 1

Abstract BackgroundWe developed a personalized Monocyte-Derived Dendritic Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses.MethodsPBMCs were obtained from 10 HIV+ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient’s HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients’ cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4+ and CD8+ T-cells. ResultsThe protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4+ and CD8+ T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4+ T-cells. The number of candidates that increased in vitro the cytokine levels in CD4+ and CD8+ T cells upon stimulation with Gag peptides from baseline to days 15 and from baseline to days 30 and days 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. ConclusionsMDDC has a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment.NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829, posted November 11th, 2016).

scholarly journals Idiotype Immunization Combined With Granulocyte-Macrophage Colony-Stimulating Factor in Myeloma Patients Induced Type I, Major Histocompatibility Complex–Restricted, CD8- and CD4-Specific T-Cell Responses

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2459-2466 ◽  
Author(s):  
Anders Österborg ◽  
Qing Yi ◽  
Lotta Henriksson ◽  
Jan Fagerberg ◽  
Susanne Bergenbrant ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Response ◽  
Type I ◽  
Gm Csf ◽  
Histocompatibility Complex ◽  
Macrophage Colony Stimulating Factor ◽  
Ifn Γ ◽  
Macrophage Colony ◽  
Stimulating Factor

Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-γ/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I–restricted type I T-cell response.

scholarly journals Single cell immune profiling reveals a developmentally distinct CD4+ GM-CSF+ T cell lineage that induces GI tract GVHD

2022 ◽  
Author(s):  
Clint Piper ◽  
Emma Hainstock ◽  
Cheng Yin-Yuan ◽  
Yao Chen ◽  
Achia Khatun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cell Population ◽  
Cell Populations ◽  
Sequencing Analysis ◽  
Gi Tract ◽  
Gm Csf ◽  
Mediators Of Inflammation ◽  
Ifn Γ

Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft versus host disease (GVHD). CD4+ T cells that produce GM-CSF have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if there exists a unique CD4+ GM-CSF+ subset that differs from other defined T helper (TH) subtypes. Using single cell RNA sequencing analysis, we identified two CD4+ GM-CSF+ T cell populations that arose during GVHD and were distinguishable by the presence or absence of IFN-γ co-expression. CD4+ GM-CSF+ IFN-γ- T cells which emerged preferentially in the colon had a distinct transcriptional profile, employed unique gene regulatory networks, and possessed a non-overlapping TCR repertoire when compared to CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T cell populations in the colon. Functionally, this CD4+ GM-CSF+ T cell population contributed to pathological damage in the GI tract which was critically dependent upon signaling through the IL-7 receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic TH GM-CSF+ subset that mediates immunopathology.

scholarly journals Intergraft Variability in Nonhematopoietic Immunoregulatory Cell Number and Expression of Immune Checkpoint Inhibitor Receptors and Ligands in Both Allo- and Autografts: Potential Target for Intervention

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3382-3382
Author(s):  
Qingdong Guan ◽  
Scott Gilpin ◽  
James Doerksen ◽  
Lauren Bath ◽  
Tracey Lam ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitor ◽  
Checkpoint Inhibitor ◽  
Cell Number ◽  
Cell Dose ◽  
Cd34 Cells ◽  
Ifn Γ

Abstract Intergraft variability in nonhematopoietic immunoregulatory cell number and expression of immune checkpoint inhibitor receptors and ligands in both allo- and autografts: potential target for intervention Qingdong Guan,1-3 Scott Gilpin,3 James Doerksen,3 Lauren Bath,3 Tracey Lam,3 Kristjan Paulson,4 Pascal Lambert,4 Yun Li,1,3 Donna A.Wall1-4 1, Department of Pediatrics and Child Health, 2, Immunology, University of Manitoba; 3, Manitoba Center for Advanced Cell and Tissue Therapy; 4, CancerCare Manitoba The number of CD34+ hematopoietic stem/progenitor cells (HSC) in HSC products is the main and often sole characterization of the graft used in HSCT. However CD34+ cells make up only 0.3-5% of the graft with the rest of the cells being lymphocytes and immature myeloid and granulocytic cells, including myeloid-derived suppressor cells (MDSC). We examined a cohort of HSC products collected from 2010-2014. Filgrastim and chemotherapy was used to mobilize 60 multiple myeloma and 34 lymphoma patients. Filgrastim-mobilized healthy donor products used in allografts (N=68) was a comparator. Aliquots stored in liquid nitrogen were analyzed for cell phenotype with a focus on immunoregulatory populations. We found CD33+CD15-CD14+HLA-DR-/low monocytic (M-MDSC) ranged from 0-59% in the infused graft. Similarly CD3+T lymphocyte ranged from 2-80% in the graft. There were 10-50 fold more M-MDSC than CD34+ cells with the infused M-MDSC cell dose ranging from 0-600×106/kg (Fig 1). Similarly CD3+T cell dose ranged from 4-670×106/kg (Fig1). M-MDSC were functional as they could suppress T cell proliferation and IFN-γ secretion, but promote regulatory T cell development in vitro. We examined receptor-ligand relations between M-MDSC and T cells and markers of T exhaustion. M-MDSC expressed variable PD-L1 (19.3±13.9% for MM, 10.4±4.4% for lymphoma and 7.0±4.8% for allografts), and CD86 (48.3±17.1% for MM, 59.9±15.4% for lymphoma and 57.8±17.0% for allografts), the ligands for PD-1 and CTLA-4, respectively. Blocking PD-L1-PD-1 signaling pathway using anti-PD-L1 or anti-PD1 partially reversed the suppressive functions of M-MDSC. Compared to allografts, CD4+T and CD8+T cells in the autografts showed poor proliferation, decreased the secretion of IFN-γ and/or granzyme B, and increased inhibitory receptors PD-1 and CTLA-4 on their surface - markers of T cell exhaustion. Levels of PD-L1 and CD86 on M-MDSC were correlated with expression of inhibitory receptors PD-1 and CTLA-4 on T cells, respectively. Taken together, our pilot data showed variable numbers of M-MDSC are infused with HSC grafts. These cells have strong immune regulatory function in vitro. T cells in autografts have high levels of T cell exhaustion markers and are less functional. It indicated immune function may be enhanced by interfering with PD1/PDL1 or CTLA-4. The numbers of M-MDSC and T cells are in the range of a cellular therapy product and may be targeted for enhance/inactivation pre- or peri-transplant immune function. Figure 1. The infusion cell dose of CD34+ stem cells, M-MDSC and CD3+T. Disclosures No relevant conflicts of interest to declare.

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