A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

2019 ◽  
Vol 15 (11) ◽  
pp. 2229-2239 ◽  
Author(s):  
Zhuoran Tang ◽  
Fengzhen Mo ◽  
Aiqun Liu ◽  
Siliang Duan ◽  
Xiaomei Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Tumor Cell Apoptosis ◽  
Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

Sirolimus Inhibits Proliferation but Enhances Suppressive Activity of Rhesus Macaque Natural CD4 Regulatory T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1008-1008
Author(s):  
Karnail Singh ◽  
Natalia Kozyr ◽  
Linda Stempora ◽  
Allan D Kirk ◽  
Christian P Larsen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
T Cell Proliferation ◽  
Suppressive Activity ◽  
Suppressive Capacity

Abstract Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p<0.01) A mechanistic evaluation of the increase potency observed with sirolimus pulsed Tregs (SPTs) has revealed several key differences that distinguish these cells from the less-potent unpulsed Tregs: SPTs were found to undergo fewer rounds of proliferation in an MLR when compared with unpulsed Tregs (14% proliferation in SPTs versus 37% proliferation in un-pulsed Tregs, p= 0.015), suggesting that the suppressive capability of Tregs may be inversely related to their proliferative capacity. SPTs were also shown to have significantly increased expression of CD25 (p=0.04) and total CTLA4 (p= 0.009) compared to unpulsed Tregs, implicating signaling through both of these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Christian Binder ◽  
Felix Sellberg ◽  
Filip Cvetkovski ◽  
Erik Berglund ◽  
David Berglund
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mixed Lymphocyte Reaction ◽  
T Cell Proliferation ◽  
Allogeneic Mixed Lymphocyte Reaction ◽  
Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

scholarly journals Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 300 ◽  
Author(s):  
Konstantina Antoniou ◽  
Fanny Ender ◽  
Tillman Vollbrandt ◽  
Yves Laumonnier ◽  
Franziska Rathmann ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
C5a Receptor ◽  
The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

Donor Requirements For CD4+CD25+FoxP3+ Regulatory T Cells Capable Of Suppressing CD4+ and CD8+ Conventional T Cell Proliferation and Graft Versus Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4484-4484 ◽  
Author(s):  
Antonio Pierini ◽  
Lucrezia Colonna ◽  
Maite Alvarez ◽  
Dominik Schneidawind ◽  
Byung-Su Kim ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Animal Models ◽  
Graft Versus Host Disease ◽  
Third Party ◽  
T Cell Proliferation ◽  
Graft Versus Host

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1427-1427
Author(s):  
Suzanne L Tomchuck ◽  
Jin He ◽  
Ross W. Perko ◽  
Scarlett Evans ◽  
Amy McKenna ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
T Cell Proliferation ◽  
Gamma Delta T Cells ◽  
Memory Cells ◽  
Gamma Delta ◽  
Gamma Delta T Cell

Abstract Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

scholarly journals The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types

2021 ◽  
Vol 12 ◽  
Author(s):  
Danielle Minns ◽  
Katie J. Smith ◽  
Gareth Hardisty ◽  
Adriano G. Rossi ◽  
Emily Gwyer Findlay
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Full Range ◽  
T Cell Proliferation ◽  
Cell Contact ◽  
Activation Process ◽  
Naive T Cells ◽  
Naïve T Cells

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions in vitro do not reflect the full range of interactions found in vivo; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil-released intracellular contents enhance proliferation. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain the contradictory results observed previously.

Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1032-1032
Author(s):  
Saisha Abhay Nalawade ◽  
Paul Shafer ◽  
Pradip Bajgain ◽  
Katie McKenna ◽  
Arushana Ali ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Nuclear Translocation ◽  
T Cell Proliferation ◽  
Tumor Site ◽  
Car T Cells ◽  
Car T

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Cord Blood Nucleated Cells Induce Delayed Proliferative and Cytotoxic T Cell Alloreactivity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5138-5138
Author(s):  
Dolores Mahmud ◽  
Sandeep Chunduri ◽  
Javaneh Abbasian ◽  
John Maciejewski ◽  
Ronak Iqbal ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
T Cell Proliferation ◽  
Cd34 Cells ◽  
Ctl Activity

Abstract Transplantation of HLA-mismatched nucleated cells from cord blood (CB) has reduced risks of graft rejection and severe acute graft-versus-host disease. In this study we analyzed the in-vitro alloantigen presenting capacity of cord blood nucleated cells. CB mononuclear cells (MNCs) or immunomagnetically-selected CD34+ cells, or CD14+ monocytes, were irradiated and tested as stimulators of allogeneic blood T cells in primary (stimulator:responder ratio = 1:1) or secondary (stimulator:responder ratio = 1:2) mixed leukocyte culture (MLC), or in cytotoxic T-lymphocytes (CTL) assays. CB-MNCs failed to induce allogeneic T cell proliferation in 6-days primary MLC, whereas CD34+ or CD14+ cells stimulated brisk T cell responses. A suppressive effect of CB-MNCs was ruled out since CD3+ cell-depletion of CB-MNCs did not restore CB immunogenicity and the addition of increasing doses of CB-MNCs did not inhibit T cell alloreactivity to CD34+ cells. Despite allogeneic T cells were unresponsive to CB-MNCs after primary and secondary MLC, T cell anergy was ruled out since T cells that were unresponsive after primary MLC proliferated potently in secondary MLC stimulated with CB CD34+ cells, and even more with CB monocyte-derived dendritic cells (Mo-DC) generated in-vitro with GM-CSF and IL-4. Interestingly, after co-culture with irradiated allogeneic T cells for 6 days, CB-MNCs showed a greater proportion of CD86+ cells and elicited allo- T cell proliferation. In addition, allo-CTL activity was induced by CB-MNCs only after restimulating effector cells for 3–4 weeks (26±7% lysis of antigen-specific PHA-blast at 50:1 E:T ratio), and was comparable to CTL activity induced after 1 week by Mo-DC generated from the same CB. When T cell effectors were stimulated by combining two incompatible cord blood MNCs mixed together, CTL activity was then detected after 4 weeks against both of them regardless of the CB:CB cell ratio. These results show an impaired allo-APC activity of CB-MNCs, without suppressive or tolerogenic activity. These findings might partially explain the initial engraftment of combined HLA mismatched CB grafts in vivo, however they also suggest that a delayed T cell response may occur due to CB-derived APCs activating CTLs.

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