scholarly journals 794 Long-term anti-tumor preclinical efficacy of an optimized anti PD-1/IL-7 bifunctional antibody sustaining activation of progenitor stem-like CD8 TILs and disarming Treg suppressive activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A829-A829
Author(s):  
Aurore morello ◽  
Margaux Seite ◽  
Justine Durand ◽  
Geraldine Teppaz ◽  
Virginie Thepenier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Drug Exposure ◽  
Cell Subset ◽  
Therapy Response ◽  
Tumor Model ◽  
Mouse Tumor ◽  
Suppressive Activity ◽  
Immunodeficient Mice

BackgroundDespite the PD-(L)1 therapy success, a majority of patients remain resistant. PD-1+IL7R+ progenitors CD8 TILs is a key T-cell subset associated with durable PD-(L)1 therapy response. However, this subset may rapidly undergo apoptosis and/or being fully exhausted after PD-(L)1 blockade. Some cytokines have the potential to strengthen PD-(L)1 therapy by promoting T cell survival, however, their clinical developments are limited by a shortened half-life and systemic toxicity. To redirect immunotherapy to tumor-specific T cells, expressing PD1, we propose to selectively deliver the pro-survival IL-7 to PD-1+ T cells using a bifunctional anti-PD1/IL-7 mutein antibody. We previously described that the anti-PD1/IL-7v abrogated suppressive activity of human Treg. Here we evaluated its preclinical anti-tumor efficacy and how it promotes the response of PD1+IL7R+ tumor-specific T cells.MethodsProliferation, IFN-γ, IL-7R signaling, and NFAT assays were tested to determine the mechanism of this antibody. For the suppressive assay, CD4 Treg and autologous CD8 Teff were co-cultured. In vivo experiments were performed in hPD-1 KI immunocompetent or humanized immunodeficient mice.ResultsThe anti-PD1/IL-7v antibody design has been optimized with a monovalent approach to enhance its biological activity: (1) preserved PD-1 antagonist activity, (2) improved pSTAT5 IL7R signaling, and (3) enhanced in vivo drug exposure and antitumor efficacy. An IL7 mutein has been designed to improve activity on PD1+ T cells while sparing PD1neg T cells.Using a chronic antigen stimulation model, anti-PD1/IL-7v restores the proliferation & survival of both early and fully exhausted CD8+ or CD4+ T cells. Similarly, anti-PD1/IL-7v, but not anti-PD1 alone, reactivates exhausted TILs isolated from human resected tumors. Gene expression analysis by Nanostring showed increase cytotoxicity, antigen presentation, and chemokines signatures. In vivo, anti-PD1/IL-7v demonstrated high monotherapy efficacy (90%) in a PD-1 sensitive orthotopic immunocompetent mouse tumor model as well as in a PD-1 refractory tumor model with 70% of CR vs 15% for anti-PD-1 alone. A selective higher expansion of stem-like/progenitors CD8 TILs was observed after therapy with anti-PD1/IL-7v compared to anti-PD1. Memory immune response was demonstrated in 100% of cured mice after tumor rechallenge in the absence of new treatment in 3 different tumor models. Finally, using two different humanized mouse models implanted with human tumors (A549 or MDA-MB231), we confirmed significant preclinical monotherapy efficacy of the anti-PD-1/IL7v.ConclusionsThese data highlight the potential of anti-PD1/IL-7 bifunctional drug to overcome immunotherapy resistance and to promote durable anti-tumor efficacy by preferentially reinvigorating PD-1+IL7R+ stem-like progenitors CD8 T cells.

scholarly journals Anti–4-1bb Monoclonal Antibodies Abrogate T Cell–Dependent Humoral Immune Responses in Vivo through the Induction of Helper T Cell Anergy

1999 ◽  
Vol 190 (10) ◽  
pp. 1535-1540 ◽  
Author(s):  
Robert S. Mittler ◽  
Tina S. Bailey ◽  
Kerry Klussman ◽  
Mark D. Trailsmith ◽  
Michael K. Hoffmann
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
B Cells ◽  
Humoral Immunity ◽  
Cd8 T Cells ◽  
Immunodeficient Mice ◽  
Humoral Immune

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti–mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti–4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell–independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti–4-1BB mAb was given within 1 wk after immunization. Anti–4-1BB inhibition was observed in mice lacking functional CD8+ T cells, indicating that CD8+ T cells were not required for the induction of anergy. Analysis of the requirements for the anti–4-1BB–mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti–4-1BB–treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti–4-1BB–treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti–4-1BB–treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti–4-1BB mAbs.

scholarly journals Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

2020 ◽  
Vol 8 (2) ◽  
pp. e001250
Author(s):  
Benson Chellakkan Selvanesan ◽  
Kiran Meena ◽  
Amanda Beck ◽  
Lydie Meheus ◽  
Olaya Lara ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
Mouse Tumor ◽  
Epitope Spreading ◽  
Stromal Compartment ◽  
Positive Effects

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

Modeling Human T Cell Acute Lymphoblastic Leukemia In Vitro and In Vivo with LMO2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3648-3648
Author(s):  
James A Kennedy ◽  
Sara Berthiaume ◽  
Frederic Barabe
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cell ◽  
Leukemic Cell ◽  
Human Cells ◽  
Immunodeficient Mice ◽  
Human T Cell

Abstract Abstract 3648 The studies identifying gene translocations and mutations in T-ALL cell lines and/or in patients have contributed significantly to the understanding of the genetic abnormalities involved in T-ALL. However, studies on the biology of these genes, the targeted cells, the sequence and the number of hits required to convert a primary human hematopoietic stem cell (HSC)/progenitor cell into a fully transformed leukemic cell require good experimental models of human T cell development both in vivo and in vitro. The only in vivo model of human T cell leukemogenesis came unexpectedly from the gene therapy trial on patients with X-linked severe combined immunodeficiency (SCID-X1). Three to five years after gene therapy, 4 out of 10 patients in the trial developed clonal T-ALL. In these patients, retroviral integrations were found in proximity to the LMO2 promoter in the malignant clones, leading to aberrant expression of the oncogene. However, little is known on the effect of LMO2 overexpression in human cells and how it facilitates the development of T-ALL. We have developed in vivo and in vitro models to study the role of T cell oncogenes in human cells. Using the OP9-DL1 co-culture system to differentiate human HSC into mature T cells in vitro, we culture human HSC transduced with lentiviruses expressing LMO2. LMO2 overexpressing cells are blocked at the double negative stage (CD4-CD8-) of differentiation when co-cultured on OP9-Delta-Like1 stroma and proliferate 50 to 100 times more than control cells. However, these cells are not immortalized and cultures lasted approximately 80 days. LMO2 overexpression have no effect on myeloid differentiation in vitro. In vivo, LMO2 transduced human HSC/progenitor cells engraft the bone marrow of immunodeficient mice to levels comparable to control cells, while normal myeloid and B cell populations 20–24 weeks post-transplantation. LMO2 transduced cells have an increased capacity to generate T cells in the thymus in comparison to control cells (42% engraftment vs 8%, p<0.0001). Surprisingly, thymic and peripheral LMO2 cells are not blocked in their differentiation. LMO2 cells did not engraft secondary mice, confirming that LMO2 doesn't induce self-renewal of human HSC. However, the increase in thymic repopulation by LMO2 cells and the lack of differentiation block in vivo suggest that LMO2 overexpression generates an abnormal T cell population with an increase repopulation advantage (increase proliferation or decrease apoptosis) in the thymus which becomes the substrate for additional genetic/epigenetic events. To test this hypothesis, we tried to immortalize LMO2 cells in vitro with secondary hits. Our preliminary results show that insertional mutagenesis can immortalized LMO2 cells in vitro. However these cells are not able to engraft immunodeficient mice or generate leukemia in vivo. The addition of intracellular NOTCH to one immortalized LMO2 cell line allows these cells to engraft and generate human T-ALL in vivo. Globally, these results show that T cell oncogenes can be studied in primary human hematopoietic cells both in vitro and in vivo. Also, at least three hits are required to transform a human primary HSC/progenitor cell into a leukemic cell able to engraft and generate leukemia in vivo. It also suggests that a non-engrafting cell can be turned into a leukemic cell generating leukemia in vivo, implying that a cell can regain self-renewing properties. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Elimination of syngeneic sarcomas in rats by a subset of T lymphocytes.

1980 ◽  
Vol 152 (4) ◽  
pp. 823-841 ◽  
Author(s):  
E Fernandez-Cruz ◽  
B A Woda ◽  
J D Feldman
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Subset ◽  
Suppressor Cells ◽  
Effector Cells ◽  
Long Duration ◽  
Ia Antigens

Established subcutaneous Moloney sarcomas (MST-1) of large size and long duration were eliminated from syngeneic rats by intravenous infusion of varying numbers of specific syngeneic effector T lymphocytes. Spleen cells from BN rats in which tumor had regressed were cultured in an in vitro mixed lymphocyte tumor cell culture (MLTC) to augment cytotoxicity of effector cells. In the MLTC a T cell subset was expanded in response to MST-1 antigens and transformed into blast elements. With these changes, there was an increase in the W3/25 antigen on the T cell surface, a decrease of W3/13 antigen, and an increase in the number of T cells with Ia antigens. The subset associated with elimination of established tumors was a blast T cell W3/25+, W3/13+, as detected by monoclonal antibodies to rat T antigens. The W3/25+ subset was poorly cytotoxic in vitro for MST-1 and apparently functioned in vivo as an amplifier or helper cell in the tumor-bearing host. The W3/25- population was a melange of cells that included (W3/13+, W3/25-) T cells, null cells, Ig+ cells, and macrophages, and was associated with enhancement of tumor in vivo, suggesting the presence of suppressor cells.

scholarly journals T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.

1996 ◽  
Vol 183 (5) ◽  
pp. 2361-2366 ◽  
Author(s):  
J C Becker ◽  
J D Pancook ◽  
S D Gillies ◽  
K Furukawa ◽  
R A Reisfeld
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Tumor Model ◽  
Melanoma Metastases ◽  
Tumor Eradication

Induction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8 + T cells. Such CD8 + T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell-dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs.

scholarly journals CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells in mice

2020 ◽  
Author(s):  
Takayoshi Yamauchi ◽  
Toshifumi Hoki ◽  
Takaaki Oba ◽  
Kristopher Attwood ◽  
Xuefang Cao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Critical Role ◽  
Cell Subset ◽  
Antitumor Efficacy ◽  
Preclinical Model ◽  
Therapeutic Vaccines ◽  
T Cell Subset

AbstractThe use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early-phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 vaccination and the generation of antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination, suggesting that the expanded CX3CR1+ CD8+ T cell subset represents the post-differentiated in vivo effective CX3CR1-negative CD8+ T cell subset. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

173 An in vivo CRISPR/Cas9 screening platform to identify T cell enhancing edits in distinct solid tumor microenvironments

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A187-A187
Author(s):  
Amy Becker ◽  
Troy Luster ◽  
Ishina Balwani ◽  
Nachiket Shevale ◽  
Jingwei Sun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Model Systems ◽  
Mouse Tumor ◽  
Target Discovery ◽  
Knock Out ◽  
Cell Therapies ◽  
Screening Platform

BackgroundChimeric antigen receptor (CAR)-based T cell therapy and other forms of adoptive cell therapies (ACTs) have shown remarkable success in the treatment of hematologic malignancies; however, reports of clinical activity in solid tumors are limited to date. One key therapeutic challenge presented by solid tumors is the immunosuppressive tumor microenvironment (TME). Adding to the complexity, it is becoming increasingly clear that TMEs are heterogeneous (broadly classified as ‘inflamed,’ ‘immune excluded’ and ‘immune dessert’), utilizing different mechanisms of immunosuppression. Instrumental to overcoming the barriers presented by solid tumors will be the development of T cells with immune- enhancing edits that improve penetration, potency and persistence, while also preventing exhaustion in hostile TMEs. T cells with these properties may help in the development of ACTs in solid tumors.MethodsCRISPR/Cas9-based functional genetic screens in T cells can enable prioritization of known targets and uncover novel targets to improve the design of genetically reprogrammed cell therapies, in an unbiased fashion. Most CRISPR screens to date have been performed in vitro with tumor cells due to the complexity of setting up CRISPR screens in primary T cells, particularly for in vivo target discovery. Here, we describe the development and careful optimization of an in vivo mouse CRISPR-screening platform to identify knock-out targets in primary T cells, with the goal of increasing T cell abundance and persistence in tumors with different TMEs. Using a mouse retroviral system to express single-guide RNA (sgRNA) libraries in T cells from Cas9 transgenic mice, we performed in vivo screens in syngeneic, fully immune-competent mouse tumor models.ResultsWe identified both known and potential novel regulators of T cell activation and persistence. Importantly, we have discovered knock-out targets that accumulate in multiple, distinct TMEs and other targets that are TME-specific. The use of sub-genomic- focused libraries allowed us to rapidly screen in multiple tumor model systems and reproducibly identify hits across individual mice.ConclusionsWe have developed a fully optimized an in vivo genetic screen, which could be a rich source for target discovery, and can enable identification of functional regulators of T cells for rapid incorporation into CRISPR-engineered T cell therapies for different solid TMEs.

Chimeric Immunoreceptor (T-Body) Targeting of Acute B-Cell Lymphoblastic Leukemia (B-ALL) through Lentivirus Engineering of Primary Human T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3267-3267
Author(s):  
Michael C. Milone ◽  
Jonathan Fish ◽  
Carmine Carpenito ◽  
Mehdi Lakal ◽  
Ella Ofori ◽  
...  
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Subset ◽  
Transduction Efficiency ◽  
Single Chain ◽  
Immunodeficient Mice ◽  
T Cell Subset ◽  
Engineered T Cells

Abstract T-bodies that combine the antigen recognition capability of single chain antibody molecules (scFv) with the signal transduction domains of activating receptors can be used to generate T cells with novel, engineered specificities independent of their endogenous TCR. We have developed a series of T-bodies with specificity towards CD19, a molecule with expression that appears restricted to normal and malignant B cells. These T-bodies contain the activating signals from the costimulatory T cell receptors, CD28 and/or 4-1BB, in tandem with the TCR-ζ signal transduction domain. Using lentiviral gene transfer and T cells expansion via bead-immobilized anti-CD3 and anti-CD28 antibodies, we can generate T cells 95% of which express the T-bodies. Lentiviral transduction combined with this efficient T cell expansion system has the potential to produce >1011 antigen-specific T cells without the need for a cell selection step. Interestingly, we have noted significant constitutive signaling effects of T-body overexpression on transduced T cells that may been overlooked previously due to lower transduction efficiency. Despite the observed constitutive effects, we show that lentiviral-engineered T cells expressing anti-CD19 T bodies efficiently kill CD19 expressing cell lines and primary B-ALL cells in an antigen-specific manner. T-body engineered T cells also produce cytokines in response to antigen triggering with different patterns of cytokine production by T cells engineered with different T-bodies. In particular, a T-body receptor containing the 4-1BB signal transduction domain combined with the TCR-ζ domain stimulates a >6-fold increase in IL-6 production compared with other receptor combinations in CD4+ T cells. In contrast, a CD28 and TCR-ζ domain containing T body uniquely enhances IL-2 (>3-fold) and TNF-α (> 7-fold) secretion by CD8+ T cells. These results indicate that the functional characteristics of our tested T-body signaling constructs are distinct and depend upon the particular receptor and T cell subset combination. We are also comparing the anti-tumor response generated by adoptively transferred T-body engineered T cells in leukemia bearing immunodeficient mice as a pre-clinical, therapeutic model. In addition to elucidating important functional differences of our lentiviral constructs, these studies indicate that an important variable in clinical trials will be optimizing the cytosolic signaling domain for the desired T cell subset. The present results suggest that distinct combinatorial signaling domains may be required for optimal function in CD4 and CD8 engineered T cells.

Apoptotic Pathways of Human Regulatory T Cells in Chronic Graft Versus Host Disease (cGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 225-225
Author(s):  
Kazuyuki Murase ◽  
Yutaka Kawano ◽  
Jeremy Ryan ◽  
Ken-ichi Matsuoka ◽  
Gregory Bascug ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Cell Subset ◽  
Mitochondrial Pathway ◽  
T Cell Subsets ◽  
T Cell Subset ◽  
Mitochondrial Membrane Depolarization

Abstract Abstract 225 CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play a central role in the maintenance of self-tolerance and immune homeostasis. After allogeneic stem cell transplantation, impaired recovery of Treg is associated with the development of cGVHD. Interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in Treg deficiency. In patients with cGVHD, we previously demonstrated that Treg proliferate at high levels but this subset is also highly susceptible to apoptosis leading to inadequate Treg numbers (Matsuoka et al. JCI 2010). We also reported that low-dose IL-2 administration resulted in selective expansion of Treg in vivo and clinical improvement of cGVHD (Koreth et al. NEJM 2011). To identify mechanisms responsible for increased Treg susceptibility to apoptosis in cGVHD we used a new flow cytometry-based assay to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assessment allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets, including CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. Expression of Bcl-2, CD95 and Ki67 were also studied in each T cell subset. We studied peripheral blood samples from 36 patients with hematologic malignancies (median age 59 yr) who are > 2 years post HSCT (27 patients with cGVHD and 9 patients without cGVHD) and 15 patients who received daily subcutaneous IL-2 for 8 weeks for treatment of steroid-refractory cGvHD. Severity of cGVHD was classified according to NIH criteria. In patients without cGVHD, BH3 priming was similar in all 3 T cell subsets (CD4 Treg, CD4 Tcon and CD8). In patients with cGVHD, CD4 Treg were more primed than CD4 Tcon when challenged with BIM, BAD, PUMA, BMF and the combination of BAD + NOXA peptides (p<0.01 – 0.0001). Treg were more primed than CD8 T cells when challenged with PUMA peptide (p<0.0001), but priming in Treg and CD8 T cells was similar for other BH3 peptides in patients with cGVHD. We also compared BH3 priming of each T cell subset in patients with different grades of cGVHD. When challenged with BH3 peptides, Treg, Tcon and CD8 T cells were less primed in patients with severe cGVHD. In patients with cGVHD, Treg expressed higher levels of Ki-67, higher levels of CD95 and lower levels of Bcl-2 than Tcon. Expression of CD95 did not vary with severity of GVHD in any T cell subset, but expression of Bcl-2 was significantly increased in all subsets in patients with severe cGVHD. Increased BH3 priming and high expression of CD95 indicate that Treg are more susceptible to apoptosis than Tcon in cGVHD. However, both Treg and Tcon become less primed and Bcl-2 levels increase in severe cGVHD suggesting that these cells are less susceptible to mitochondrial pathway apoptosis. Since the total number of Treg and Tcon are significantly reduced in patients with cGVHD, these findings suggest that the remaining circulating cells are relatively resistant to mitochondrial pathway apoptosis. CD95 expression in Treg remains high indicating no change in death receptor pathway apoptosis. Daily treatment with low-dose IL-2 for 8 weeks selectively expands Treg in vivo in patients with severe cGVHD. As the number of Treg increase, BH3 profiling shows that these cells gradually become more primed and therefore more susceptible to mitochondrial pathway apoptosis. Taken together, these studies help define the complex and distinct pathways that regulate survival in different T cell subsets and changes in these pathways that occur in patients with chronic GVHD. These pathways play important roles in the maintenance of T cell homeostasis and targeting these complex pathways can provide new opportunities to promote immune tolerance after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document