In vivo Inhibition of Human CD19-Targeted Effector T Cells by Natural T Regulatory Cells in a Xenotransplant Murine Model of B Cell Malignancy

Wild-type (WT) NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) when given 0.05% NaI in their drinking water, whereas B cell–deficient NOD.H-2h4 mice are SAT resistant. To test the hypothesis that resistance of B cell–deficient mice to SAT was due to the activity of regulatory CD4+CD25+ T (T reg) cells activated if autoantigen was initially presented on non–B cells, CD25+ T reg cells were transiently depleted in vivo using anti-CD25. B cell–deficient NOD.H-2h4 mice given three weekly injections of anti-CD25 developed SAT 8 wk after NaI water. Thyroid lesions were similar to those in WT mice except there were no B cells in thyroid infiltrates. WT and B cell–deficient mice had similar numbers of CD4+CD25+Foxp3+ cells. Mice with transgenic nitrophenyl-specific B cells unable to secrete immunoglobulin were also resistant to SAT, and transient depletion of T reg cells resulted in severe SAT with both T and B cells in thyroid infiltrates. T reg cells that inhibit SAT were eliminated by day 3 thymectomy, indicating they belong to the subset of naturally occurring T reg cells. However, T reg cell depletion did not increase SAT severity in WT mice, suggesting that T reg cells may be nonfunctional when effector T cells are activated; i.e., by autoantigen-presenting B cells.

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Abstract 44: Failure of Protective Autoimmunity in Mouse and Human Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.44 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Dennis Wolf ◽

Teresa Gerhardt ◽

Nathaly Anto Michel ◽

Bjarke Hansen ◽

Alessandro Sette ◽

...

Keyword(s):

T Cells ◽

Lymph Nodes ◽

Mhc Class Ii ◽

T Regulatory Cells ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

T Cell Response ◽

Regulatory Cells ◽

Protective Autoimmunity

Background: In atherosclerosis, CD4 + T helper cells recognize auto-antigens including ApoB, the main protein in low-density lipoprotein (LDL). However, atherosclerosis-specific, auto-reactive CD4 + T cells have not been detected in vivo , and their function is unknown. Methods and Results: We have previously identified peptides derived from mouse ApoB that bind with high affinity to the MHC class II molecule of C57BL/6 mice (I-A b ). We designed and validated a new multimer of a recombinant MHC-II molecule fused to one ApoB auto-epitopes, P6 (TGAYSNASSTESASY, P6:I-A b ), that enabled detection of low-affinity, P6-reactive CD4 + T cells. Using this P6:I-A b multimer, we identified ApoB-reactive CD4 + T cells in healthy, young C57BL/6 mice that were predominately differentiated T-regulatory cells (T regs ) and expressed IL-10, a known atheroprotective cytokine. This population was detectable in lymph nodes and already showed a memory phenotype in young animals without atherosclerosis. In Apoe -/- mice, adoptively transferred ApoB P6-specific T regs accumulated in the aorta and draining lymph nodes and gave rise to pathogenic T H 1 and T H 17 cells. This phenotypic switch was caused by enhanced plasticity of antigen-specific T regs as evidenced by multiple clusters of intermediate T reg -T eff phenotypes in single cell RNA sequencing of 4485 antigen-specific CD4 + T cells. In the plaque, many T cells were ex-T regs as identified by a FoxP3 lineage tracker mouse, suggesting that atherosclerosis-specific CD4 + T cells lost their regulatory capacity. Vaccination with P6 maintained a protective phenotype in antigen-specific T regs and protected from atherosclerosis. In humans, ApoB-specific CD4 + T cells from atherosclerotic patients showed the same cytokine patterns found in mouse CD4 + T cells, suggesting that autoimmunity to ApoB is protective first, but later gives rise to a pathogenic CD4 + T cell response that aggravates atherosclerosis. Conclusion: Protective T-regulatory cells recognizing peptide antigens of ApoB exist in naïve mice, protect against atherosclerosis, but convert into pathogenic T H 1 and -17 cells during the natural course of disease in mice and humans. These results call for immunomodulatory therapies to maintain protective autoimmunity.

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Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells

Blood ◽

10.1182/blood.v81.12.3343.3343 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3343-3349 ◽

Cited By ~ 10

Author(s):

BK Link ◽

GJ Weiner

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Hybridoma Cells ◽

Activated T Cells ◽

Human B Cells ◽

B Cell Malignancy ◽

Cell Malignancy

Abstract Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

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Tumor-Targeted Human CD4+ CD25hi Regulatory T Cells Effectively Inhibit T Cell –mediated Rejection of CD19+ Tumors in An in Vivo xenotransplant Tumor Model

Blood ◽

10.1182/blood.v112.11.3901.3901 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3901-3901

Author(s):

James Lee ◽

Michel Sadelain ◽

Renier J. Brentjens

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

B Cell ◽

Clinical Setting ◽

Effector T Cells ◽

Effector T Cell ◽

Human T Cells ◽

Anti Tumor Activity

Abstract The genetic targeting of human T cells to selected tumor antigens offers a novel means to investigate human immunobiology and treat cancer. T cells may be genetically modified to target specific antigens through the introduction of genes encoding chimeric antigen receptors (CARs). We have previously demonstrated that human T cells targeted in this manner to the CD19 antigen, expressed on normal B cells as well as most B cell tumors, eradicate systemic human CD19+ B cell malignancies in SCID-Beige mice. However, in the clinical setting, the anti-tumor efficacy of these T cells may be impaired by endogenous suppressive elements of the host immune system, including CD4+ CD25hi Foxp3+ regulatory T cells (Tregs). Significantly, Tregs are often increased in the blood and infiltrate the tumor of cancer patients which has been correlated with poor patient outcome and ineffective anti-tumor immunity. In order to study the in vivo impact of Tregs on adoptive therapy with CD19 targeted effector T cells, we developed a murine model wherein human Tregs, similarly targeted to the tumor, are infused prior to adoptive transfer of targeted cytotoxic T cells. To do so, we initially isolated natural Tregs from healthy donor peripheral blood mononuclear cells. Isolated Tregs were subsequently modified to express CARs through retroviral gene transfer. Subsequently, CAR+ Tregs were rapidly expanded either by activation on NIH-3T3 fibroblasts modified to express CD19 and the CD80 costimulatory ligand (3T3(CD19/CD80)), or non-specifically using CD3/CD28 antibodycoated magnetic beads. Expanded CAR+ Tregs exhibited potent suppressive function in vitro inhibiting both effector T cell proliferation as well as cytotoxicity. In vivo, CAR+ Tregs specifically traffic to established tumor in SCID-Beige mice. Significantly, injection of CD19-targeted Tregs into SCID-Beige mice bearing established human CD19+ tumors at 24 hours prior to infusion with CD19-targeted effector T cells, completely abrogated effector T cell function even at Treg:Teff ratios as low as 1:8. We further found that full suppression was dependant both on Treg localization to the tumor site as well as in vivo activation through the CAR. Finally, we show that a pre-conditioning regimen with low-dose cyclophosphamide, which failed to eradicate tumor, was able to reverse the CAR+ Treg mediated inhibition and restore the anti-tumor activity by the targeted effector T cells. In conclusion, we have developed a robust model ideally suited to the study of in vivo Treg-Teff interactions. Furthermore, the data generated from this model to date have significant implications with respect to the application of adoptive T cell therapies in the clinical setting. Namely, the presence of endogenous Tregs at the site of tumor is likely to significantly compromise the anti-tumor activity of adoptively transferred tumor targeted T cells. This inhibition may be reversed by preconditioning regimens designed to eradicate endogenous Tregs. The findings presented here should be considered in the design of future clinical trials utilizing T cell-based adoptive therapies of cancer.

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A Subpopulation of Follicular Lymphoma Tumor Infiltrating T Cells Shows Suppressed Common Gamma Chain Cytokine Signaling.

Blood ◽

10.1182/blood.v114.22.759.759 ◽

2009 ◽

Vol 114 (22) ◽

pp. 759-759

Author(s):

June H Myklebust ◽

Jonathan M Irish ◽

Roch Houot ◽

Joshua Brody ◽

Debra K Czerwinski ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Single Cell ◽

T Regulatory Cells ◽

Cell Lymphoma ◽

Cell Subset ◽

Regulatory Cells ◽

Gamma Chain ◽

The Common

Abstract Abstract 759 Introduction: Tumor infiltrating T cells present within biopsy specimens of human B cell non-Hodgkin's lymphomas (NHL) provide a valuable opportunity to examine immune system function in the presence of cancer. We recently used flow cytometry to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a novel lymphoma cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome. Here, we turned our attention to signaling differences in subsets of the tumor-infiltrating T cells from FL and two other NHLs, diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Signaling differences that distinguish the tumor infiltrating T cells from each malignancy might be expected to be a reflection of the specific disease microenvironment, whereas T cell signaling differences distinguishing cases of the same malignancy might be related to the biology of each patient's tumor. Methods: Single cell flow cytometry measurements of signaling were acquired for samples of DLBCL (N=13), MCL (N=20), and FL (N=14). Phosphorylation of 14 signaling proteins was measured under 12 stimulation conditions in every cell, including lymphoma B cells and tumor-infiltrating T cells within the same specimen. Stimulation conditions included those that were B cell specific (BCR crosslinking, CD40 ligand), T cell specific (IL-7), and those that stimulated both B and T cells (IL-4, IL-10, IL-21, PMA + ionomycin, and IFN-γ). Results: Striking differences were observed in the signaling responses of tumor infiltrating T cells. T cells infiltrating FL patient samples showed significantly lower responses to cytokines where signal transduction is mediated by the common γ chain receptor. Specifically, we observed significant lower induction of p-STAT6 after IL-4 stimulation, p-STAT5 after IL-7 stimulation, and p-STAT3 after IL-21 stimulation (p < 0.001 for FL vs. MCL in all cases). In contrast, receptor-independent signaling was not significantly different as FL tumor infiltrating T cells responded at a level comparable to MCL and DLBCL tumor infiltrating T cells when stimulated with PMA and ionomycin. The lower response to common γ chain family cytokines could be the result of a partial suppression of all tumor infiltrating T cells or a complete suppression of a distinct subset. To distinguish between these possibilities, we analyzed signaling in tumor infiltrating T cell subsets. This single cell approach showed that tumor infiltrating T cells were a heterogeneous mixture of non-responsive cells and highly responsive T cells in response to cytokines. Specifically, the mean percentage of T cells that did not induce p-STAT3 after IL-21 stimulation was 50.3% in FL samples in contrast to only 26.2% in MCL samples. Phenotypic analysis showed that the vast majority of T cells infiltrating FL patient samples were CD4+CD45RO memory cells, and the single cell signaling approach revealed that the FL nonresponsive T cell subset had this phenotype. Furthermore, FL T cells were composed of a significantly larger fraction of T regulatory cells than MCL T cells, on average 17% FoxP3+CD25+ cells compared to only 9% in MCL (p<0.0002). Experiments are ongoing to test whether the prevalence of T regulatory cells influence the signaling capacity of the remaining CD4 conventional T cells. Conclusions: A subpopulation of tumor infiltrating T cells within FL patient samples has reduced responsiveness to the common gamma chain family members IL-4, IL-7 and IL-21, and distinguishes FL from DLBCL and MCL. These results may reflect a more suppressive microenvironment in FL. Disclosures: No relevant conflicts of interest to declare.

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A Transgenic Mouse Model of Aggressive B-Cell Malignancy for Evaluating Anti-Human CD37 Therapeutics

Blood ◽

10.1182/blood.v120.21.188.188 ◽

2012 ◽

Vol 120 (21) ◽

pp. 188-188

Author(s):

Kyle A Beckwith ◽

Frank W Frissora ◽

Matthew R Stefanovski ◽

Jutta Deckert ◽

Carlo M Croce ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Mouse Model ◽

B Cell ◽

Transgenic Mouse ◽

In Vivo Studies ◽

B Cell Malignancy ◽

Cell Malignancy

Abstract Abstract 188 BACKGROUND: Introduction of the anti-CD20 antibody rituximab has led to remarkable progress in the development of targeted therapies for CLL and other B-cell malignancies. Despite prolonging patient survival, therapies targeting CD20 have not been curative. In recent years, alternative targets for therapeutic antibodies have emerged. One of the most promising targets has been CD37, which is highly expressed on malignant B-cells in chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. The recent interest in this target has led to the generation of novel anti-CD37 therapeutics that could benefit from more extensive preclinical evaluation. However, preclinical development of these agents has been limited by the absence of appropriate leukemia animal models that provide targets expressing human CD37 (hCD37). Here we describe the development and characterization of a transgenic mouse where CLL-like leukemic B-cells express hCD37 and aggressively transplant into syngenic hosts. We demonstrate the utility of this unique mouse model by evaluating the in vivo efficacy of IMGN529, a novel antibody-drug conjugate targeting hCD37 that consists of the CD37-targeting K7153A antibody linked to the maytansinoid DM1 via the thioether SMCC linker. METHODS: The hCD37 transgenic mouse (hCD37-Tg) founder lines were generated by conventional methodology at the OSU Transgenic Facility. B-cell specific expression of hCD37 is driven by immunoglobulin heavy chain promoter and Ig-μ enhancer elements. Founder lines were evaluated by RT-PCR and flow cytometry to confirm RNA and protein expression, respectively. These lines were then crossed with the EμTCL1 mouse model of CLL to generate hCD37xTCL1 mice that develop CD5+CD19+hCD37+ leukemia. For in vivo studies, splenocytes from a leukemic hCD37xTCL1 donor were injected i.v. into healthy hCD37-Tg mice. Mice were randomly assigned to the following treatment groups (n=8–10 per group): IMGN529 conjugate, its K7153A antibody component, or negative controls (isotype antibody-DM1 conjugate or trastuzumab). Upon diagnosis of leukemia, a 10 mg/kg dose was administered i.p. and repeat doses were given 2 times per week for 3 weeks (70 mg/kg total). Peripheral blood disease was monitored by flow cytometry, using counting beads to obtain the absolute number of leukemic CD5+CD19+ B-cells. CD37 expression levels were determined by quantitative flow cytometry. In vitro cytotoxicity was evaluated after 24 hour incubation by flow cytometry with Annexin V and propidium iodide staining. RESULTS: IMGN529 and its K7153A antibody component demonstrated comparable in vitro activity against freshly isolated human CLL cells even in the absence of cross-linking agents (mean IMGN529 cytotoxicity=50.04% vs. 48.85% for K7153A; p=0.175; n=9). Both compounds also demonstrated cytotoxicity against hCD37 Tg B-cells ex vivo in a cross-linking dependent manner, and while expression of hCD37 in hCD37-Tg animals was B-cell specific, the expression levels were substantially lower than those observed in human CLL cells. In vivo studies with transferred hCD37xTCL1 splenocytes demonstrated rapid and complete depletion of CD5+CD19+ leukemic B-cells in response to IMGN529 conjugate, but not K7153A antibody treatment. After 1 week of IMGN529 treatment, peripheral blood leukemia was nearly undetectable and previously detected massive splenomegaly was no longer palpable. In contrast, leukemic counts and spleen sizes continued to increase in control cohorts. CONCLUSIONS: In summary, our group has generated a mouse model that develops a transplantable CD5+CD19+ leukemia expressing hCD37. We demonstrate the utility of this model for both in vitro and in vivo testing of therapeutics targeting hCD37. In addition, preclinical mouse studies expose the robust anti-leukemic effects of IMGN529 in this in vivo model of aggressive B-cell malignancy, despite the relatively low expression of hCD37 on the leukemic B-cells. Our engraftment model shows that IMGN529 is capable of eliminating widespread and highly proliferative mouse leukemia by a mechanism that is both CD37 antigen and conjugate dependent. Therefore, we propose that this novel therapeutic may also exhibit substantial efficacy in a wide range of human B-cell malignancies, even those with relatively low CD37 expression. [This work was supported by NIH (NM, JCB), LLS (NM, JCB) and Pelotonia (KAB)]. Disclosures: Deckert: ImmunoGen Inc.: Employment.

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Clinical Immunotherapy of B-Cell Malignancy Using CD19-Targeted CAR T-Cells

Current Gene Therapy ◽

10.2174/1566523213666131223130554 ◽

2014 ◽

Vol 14 (1) ◽

pp. 35-43 ◽

Cited By ~ 22

Author(s):

John Maher

Keyword(s):

T Cells ◽

B Cell ◽

Car T Cells ◽

B Cell Malignancy ◽

Car T ◽

Cell Malignancy

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Development of CAR T-cell lymphoma in two of ten patients effectively treated with piggyBac modified CD19 CAR T-cells

Blood ◽

10.1182/blood.2021010813 ◽

2021 ◽

Author(s):

David C Bishop ◽

Leighton E Clancy ◽

Renee Simms ◽

Jane Burgess ◽

Geetha Mathew ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Complete Remission ◽

B Cell ◽

Viral Vectors ◽

Cell Transplant ◽

Car T Cells ◽

B Cell Malignancy ◽

Car T ◽

Cell Malignancy

CD19-specific chimeric antigen receptor (CAR19) T-cells effectively induce remission of B-cell malignancy, but the cost and complexity of production using viral vectors is a factor limiting widespread application. Furthermore, the small cargo capacity of viral vectors may hamper future development of more heavily engineered CAR T-cells. We demonstrated the feasibility of generating CAR19 T-cells from HLA-matched donors of sibling allogeneic hematopoietic stem cell transplant (HSCT) patients via a simple and inexpensive method using the high-capacity piggyBac transposon. A cohort of 10 patients with relapsed or refractory B-cell acute lymphoblastic leukemia or aggressive lymphoma following HSCT were the first human subjects to receive piggyBac-generated CAR19 T-cells. Treatment with intra-patient escalating doses of CAR19 T-cells was effective, with all 9 evaluable patients achieving complete remission. At a median follow-up of 18.0 months, 5 patients remained in complete remission of B-cell malignancy. One patient died of viral sepsis. Four patients developed cytokine release syndrome of maximum grade 2, and no neurotoxicity or new graft-versus-host disease occurred. However, two patients developed malignant CAR19 T-cell tumors, one of whom was successfully treated; one patient died of the secondary tumor. The piggyBac system represents a feasible alternative to viral vectors for the generation of effective CAR19 T-cells, but its oncogenic potential in the context of the described production process will need to be addressed before any further clinical use is possible. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

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CD4+CD25+ T Cells Can Inhibit CD8 T Cell Mediated GVHD: Requirement for In Vivo Recognition of Allogeneic Host MHC Class II Antigens.

Blood ◽

10.1182/blood.v106.11.1307.1307 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1307-1307

Author(s):

Robert B. Levy ◽

Angela Jones

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

T Regulatory Cells ◽

Cd8 T Cell ◽

Class Ii ◽

Regulatory Cells

Abstract CD4 regulatory T (Treg) cells have shown promise in the transplantation mileu including the ability to inhibit the development of graft vs host disease (GVHD) following allogeneic hematopoietic stem cell transplants (HCT). The antigen specificity of the Treg population(s) involved is not yet clear nor is the role of their activation following transplant. We are interested in determining the requirement for recognition of host MHC antigens following infusion of CD4+CD25+ T cells in an experimental model of GVHD. To clearly distinguish the requirements of regulatory vs GVH reactive cells, a model of CD8 T cell mediated GVHD was developed using highly purified BALB/c (H2d) donor CD8+ T cells (Miltenyi column, 95-98%). CD8 T cells were transplanted together with T cell depleted (TCD) BALB/c BMC into 12.0 GY (6.0 Gy split dose) TBI conditioned C57BL/6 (B6, H2b) recipients. To support development of GVHD by these cells, resistance was inhibited by treatment of recipients with anti-NK1.1mab (PK136) at Days -1, 0 and +7. BALB/c CD8+ T cells at doses of 5.0x106 but not 2.5x106 induced weight loss and some lethality in B6 recipients. 5x106 CD8+ T cells were then transplanted into B6-MHC class II−/ − recipients. GVHD symptoms including weight loss and lethality were readily apparent in these mice post-transplant. Interestingly, GVHD was consistently more severe with respect to the induction of weight loss and lethality in MHC Class II−/ − vs B6-wt recipients. Highly enriched BALB/c CD4+CD25+ T cells (> 95%) were produced from spleen and lymph node cells following negative (B-cells, CD8 and NK) and positive (CD25) selection using Miltenyi magnetic bead columns. Co-transplant of 1x106 CD4+CD25+ T cells together with BALB/c CD8+ T cells into B6 recipients inhibited GVHD as assessed by the absence of weight loss and lethality compared to B6 recipients of CD8+ T cells alone. In contrast, BALB/c CD4+CD25+ T cells failed to protect B6-MHC class II−/ − recipients from severe CD8+ T cell mediated GVHD. These findings demonstrate that donor CD4+ T regulatory cells can suppress GVHD inducing CD8+ T cells after the former recognize host class II alloantigen following transplant. We hypothesize that activated CD4+CD25+ T regulatory cells inhibit GVH reactive T cells at the host APC interface. Future studies in this model can be designed to examine ex-vivo activated and expanded CD4+CD25+ T regulatory populations. Transplant of such cells will enable us to address questions regarding the importance of in vivo recognition of host class II in the regulation of GVHD by these cells.

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