Adoptive Transfer of Treg-Depleted Donor Th1 and Th2 Cells Safely Accelerates Alloengraftment After Low-Intensity Chemotherapy

Abstract Abstract 521 Ex-vivo culture of murine donor CD4+ T cells using rapamycin, co-stimulation, and IL-4 yielded a defined T cell population (T-rapa cells) that favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate these findings, we conducted a multi-center clinical trial (NCT0074490) to evaluate T-rapa cell therapy after allogeneic HCT. T-rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation in media containing IL-4, IL-2, and rapamycin. T-rapa cells had a mixed Th2/Th1 phenotype with minimal Treg content (intra-cellular flow, n=48 products; median transcription factor expression: 11.5% [GATA-3], 5.1% [T-bet], and 0.1% [FoxP3]). Median T-rapa cell cytokine secretion (pg/ml; re-stimulation at harvest) was 1.3 [IL-4], 20.6 [IL-5], 9.7 [IL-10], 23.7 [IL-13], 34.7 [IFN-g], and 17.1 [IL-2]. Patients received an HLA-matched sibling, T cell-replete, G-CSF mobilized allograft, and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT). Two protocol arms evaluated T-rapa cell therapy after induction chemotherapy and outpatient, low-intensity preparative chemotherapy (Table I). First, patients (n=25) were accrued to arm A to evaluate T-rapa infusion at d +14 post-HCT; subsequently, accrual was initiated to arm B (n=25) to evaluate T-rapa infusion on d0 of HCT. Arm A was then expanded to n=40 patients. Patients accrued to arms A and B were similar for recipient age, high-risk malignancy diagnosis, chemotherapy refractoriness, and prior regimen number (Table I). Most recipients were not in remission at the time of HCT. High-risk NHL was the most frequent diagnosis (25/65 patients), followed by non-high-risk NHL (11/65), AML/MDS (8/65), myeloma (7/65), CLL (6/65), Hodgkin's disease (5/65), and CML (3/65). Arm A and B recipients had similar mean donor myeloid cell chimerism at d +14, +28, and +100 (arm A, 43%, 74%, and 89%; arm B, 50%, 62%, and 84%). At d +14, arm A and B recipients also had mixed donor T cell chimerism (mean values, 60% in each arm; Table I). At d +28 and +100, T cell chimerism increased in arm A to 80% and 89%; in arm B, these values increased to only 67% and 69%. Four recipients on arm B had < 10% donor T cell chimerism at d +100; in contrast, the lowest donor T cell chimerism value observed at d +100 on arm A was 36%. T-rapa therapy on arm A was relatively safe as there was: no engraftment syndrome, a 10% rate of acute grade II to IV GVHD, a 67% incidence of chronic GVHD, and no transplant-related mortality (Table I). On arm A, 37.5% (15/40) of recipients are in sustained complete remission, with a median survival probability of 63.6% at 24 months post-HCT. Therefore, pre-emptive donor lymphocyte infusion with ex-vivo manufactured T-rapa cells that express a balanced Th2/Th1 effector phenotype represents a novel approach to safely accelerate alloengraftment and harness allogeneic GVT effects after low-intensity conditioning.Table IArm AArm BLow-Intensity Regimen Induction Chemotherapy1EPOCH-FREPOCH-FR 2Terminal Chemotherapy3Flu (120 mg/m2)EPOCH-FRCy (1200 mg/m2)T-Rapa Cell TimingD +14 post-HCTD 0 of HCTPatient Characteristics & of Patients Accrued4025 Age (median, range)55 (25–67)51 (32–66) & of Prior Regimens3 (1–6)3 (1–8) High-Risk Malignancy65% (26/40)52% (13/25) Chemotherapy Refractory50% (20/40)48% (12/25) CR (at time of HCT)25% (10/40)8% (2/25)% Donor T Cell ChimerismMean Median (Range)Mean Median (Range)Day 14 post-HCT6061(8–97)6060(4–100)Day 28 post-HCT8089(27–100)6773(10–100)Day 100 post-HCT8993(36–100)6982(0–100)Clinical Results Engraftment Syndrome0% (0/40)0% (0/25) Acute GVHD10% (4/40)23% (5/22) Chronic GVHD67% (22/33)75% (15/20) Complete Remission38% (15/40)28% (7/25) Transplant-related Mortality0% (0/40)0% (0/25) Percent Survival65% (26/40)40% (10/25) Median Survival27.5 mo11.2 mo Survival Prob. at 24 mo63.6%44.0%1EPOCH-FR, EPOCH with fludarabine (Flu) and rituximab.2Terminal (preparative) chemotherapy administered one week prior to HCT.3Flu/Cy [cyclophosphamide] doses are total doses, given over 4 days (Cy dose is 75% lower than 4800 mg/m2 “reduced-intensity” Cy dose). Disclosures: No relevant conflicts of interest to declare.

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Selective Allodepletion by TH9402-Mediated Photosensitization Results in Early Full Donor T Cell Reconstitution in the Absence of High-Grade, Acute GvHD and Is Associated with Favorable Outcome after HLA Matched Sibling SCT for Hematologic Malignancies

Blood ◽

10.1182/blood.v112.11.1168.1168 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1168-1168

Author(s):

Stephan Mielke ◽

Aarthi Shenoy ◽

Vicki S. Fellowes ◽

Katayoun Rezvani ◽

Bipin N. Savani ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

High Risk ◽

Ex Vivo ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Infectious Complications ◽

High Risk Population ◽

Donor T Cells

Abstract Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from allografts to prevent graft versus host disease (GvHD) while conserving useful donor immunity. We developed a semi-closed, GMP-quality, clinical scale SD process where donor-derived lymphocytes are stimulated with patient-derived T-cell antigen presenting cells in an ex vivo mixed lymphocyte reaction (MLR). Alloactivated donor T cells preferentially retain the photosensitizer 4,5-dibromorhodamine 123 (TH9402), rendering them susceptible to elimination by exposure to visible light in a photodepletion device (Kiadis Pharma Inc, The Netherlands). After Food and Drug Administration and Institutional Review Board approval we initiated a clinical trial where HLA-identical sibling recipients with hematological (non T-cell) malignancies received a CD34-cell selected transplant (Miltenyi, Germany) containing less then 1 × 104 T cells/kg together with 5 × 106/kg viable SD donor T cells on day 0, using an age-adapted, radiation-based preparative regimen (FluCyTBI). Low-dose cyclosporine was used as sole immunosuppression in the absence of GvHD. Eleven patients (median age 43 (28–68) years with ALL, MDS, CML, mantle cell lymphoma (MCL), or AML) were transplanted with a median follow-up of 240 (43–400) days. Nine patients were considered high risk. Patients received a stem cell product containing a median of 6.0 (3.9–9.5) ×106/kg CD34+ stem cells in addition to 5×106/kg SD T cells. Absolute lymphocyte recovery was rapid (median 834 (384–2486) cells/μL day 30 post transplant) [Fig A]. Early T cell chimerism was donor-dominated (median 66% (6–95) on day 14, and 97% (82–100) on day 30, and 100% (92–100) on day 45 [Fig B]. One patient received an unmanipulated DLI to treat a delayed fall in T cell chimerism. Three patients developed steroid-sensitive grade II aGvHD of skin (N=2) and gut (N=1) but no grade III–IV aGvHD occurred after transfusion of the photodepleted lymphocytes [Fig C]. Two patients developed limited chronic GvHD. Only one patient, transplanted for refractory MCL, relapsed 340 days after transplant. One patient died of infectious complications and GvHD 330 days after transplant after receiving an unmanipulated DLI in her home country for suspected, but subsequently unconfirmed relapse. Eight patients reactivated CMV but were successfully treated. These results demonstrate for the first time clinical feasibility of photodepletion-based SD stem cell allotransplants in matched siblings. Robust lymphocyte recovery and early donor chimerism with a low relapse incidence in a high-risk population suggest functionality of SD T cells in the absence of severe GvHD, which should allow further reduction of immunosuppression to optimize disease control in future studies.

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Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells Among the 3 Subtypes of Type 1 Diabetes

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa447 ◽

2020 ◽

Vol 105 (10) ◽

pp. 3141-3151 ◽

Cited By ~ 1

Author(s):

Daisuke Chujo ◽

Akitsu Kawabe ◽

Maya Matsushita ◽

Nobuyuki Takahashi ◽

Chiharu Tsutsumi ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Cd4 T Cells ◽

Ex Vivo ◽

Th2 Cells ◽

Cell Phenotype ◽

Islet Antigen ◽

Ex Vivo Assay

Abstract Context Type 1 diabetes (T1D) is classified into 3 subtypes: acute-onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). The differences in the type of cellular autoimmunity within each subtype remain largely undetermined. Objective To determine the type and frequency of islet antigen-specific CD4+ T cells in each subtype of T1D. Participants Twenty patients with AT1D, 17 with SP1D, 18 with FT1D, and 17 persons without diabetes (ND). Methods We performed an integrated assay to determine cellular immune responses and T-cell repertoires specific for islet antigens. This assay included an ex vivo assay involving a 48-hour stimulation of peripheral blood mononuclear cells with antigen peptides and an expansion assay involving intracytoplasmic cytokine analysis. Results The results of the ex vivo assay indicated that glutamic acid decarboxylase 65 (GAD65)-specific interleukin-6 and interferon-inducible protein-10 (IP-10) responses and preproinsulin (PPI)-specific IP-10 responses were significantly upregulated in AT1D compared with those of ND. Furthermore, GAD65- and PPI-specific granulocyte colony-stimulating factor responses were significantly upregulated in FT1D. Expansion assay revealed that GAD65- and PPI-specific CD4+ T cells were skewed toward a type 1 helper T (Th1)- cell phenotype in AT1D, whereas GAD65-specific Th2 cells were prevalent in SP1D. GAD65-specific Th1 cells were more abundant in SP1D with human leukocyte antigen-DR9 than in SP1D without DR9. FT1D displayed significantly less type 1 regulatory T (Tr1) cells specific for all 4 antigens than ND. Conclusions The phenotypes of islet antigen-specific CD4+ T cells differed among the three T1D subtypes. These distinct T-cell phenotypes may be associated with the manner of progressive β-cell destruction.

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Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

Blood ◽

10.1182/blood-2008-03-143636 ◽

2008 ◽

Vol 112 (6) ◽

pp. 2232-2241 ◽

Cited By ~ 48

Author(s):

Jeff K. Davies ◽

John G. Gribben ◽

Lisa L. Brennan ◽

Dongin Yuk ◽

Lee M. Nadler ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Viral Infections ◽

Ex Vivo ◽

Chronic Gvhd ◽

Costimulatory Blockade ◽

Donor T Cells

AbstractWe report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 ×106/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

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Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

10.21203/rs.3.rs-35386/v1 ◽

2020 ◽

Author(s):

Thomas Vollbrecht ◽

Aaron O. Angerstein ◽

Bryson Menke ◽

Nikesh M. Kumar ◽

Michelli Faria Oliveira ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Ex Vivo ◽

Latent Reservoir ◽

Antigen Specificity ◽

Pcr Assay ◽

Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

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Human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential

10.22541/au.161654362.26820666/v1 ◽

2021 ◽

Author(s):

Johan Verhagen ◽

Edith Van der Meijden ◽

Vanessa Lang ◽

Andreas Kremer ◽

Simon Völkl ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cd4 T Cells ◽

Cellular Therapy ◽

Therapeutic Potential ◽

Cell Receptor ◽

Haematopoietic Stem Cell ◽

T Cell Therapy ◽

Immunological Protection

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of 9 immunodominant epitopes and characterise T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, by using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.

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Advances in Adoptive Cell Therapy Using Induced Pluripotent Stem Cell-Derived T Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.759558 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ratchapong Netsrithong ◽

Methichit Wattanapanitch

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cell Biology ◽

Ex Vivo ◽

Adoptive Cell Therapy ◽

Basic Knowledge ◽

Car T Cells ◽

Car T ◽

Induced Pluripotent

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells holds impressive clinical outcomes especially in patients who are refractory to other kinds of therapy. However, many challenges hinder its clinical applications. For example, patients who undergo chemotherapy usually have an insufficient number of autologous T cells due to lymphopenia. Long-term ex vivo expansion can result in T cell exhaustion, which reduces the effector function. There is also a batch-to-batch variation during the manufacturing process, making it difficult to standardize and validate the cell products. In addition, the process is labor-intensive and costly. Generation of universal off-the-shelf CAR T cells, which can be broadly given to any patient, prepared in advance and ready to use, would be ideal and more cost-effective. Human induced pluripotent stem cells (iPSCs) provide a renewable source of cells that can be genetically engineered and differentiated into immune cells with enhanced anti-tumor cytotoxicity. This review describes basic knowledge of T cell biology, applications in ACT, the use of iPSCs as a new source of T cells and current differentiation strategies used to generate T cells as well as recent advances in genome engineering to produce next-generation off-the-shelf T cells with improved effector functions. We also discuss challenges in the field and future perspectives toward the final universal off-the-shelf immunotherapeutic products.

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Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

Journal of Experimental Medicine ◽

10.1084/jem.20160576 ◽

2016 ◽

Vol 213 (11) ◽

pp. 2413-2435 ◽

Cited By ~ 53

Author(s):

Yi Wang ◽

Cindy S. Ma ◽

Yun Ling ◽

Aziz Bousfiha ◽

Yildiz Camcioglu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Ex Vivo ◽

Cell Phenotype ◽

Loss Of Function ◽

Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

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Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽

10.1161/hyp.64.suppl_1.001 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Kristine M Wadosky ◽

Sri N Batchu ◽

Angie Hughson ◽

Kathy Donlon ◽

Craig N Morrell ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cd4 T Cells ◽

White Blood Cells ◽

Interleukin 4 ◽

Th2 Cells ◽

Salt Hypertension ◽

Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

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