Depletion of CD25+ T cells from hematopoietic stem cell grafts increases posttransplantation vaccine-induced immunity to neuroblastoma

Abstract A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4+ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4+ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25+ Treg cells was similar to that achieved by in vivo depletion of all CD4+ T cells. Depletion of CD25+ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4+ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8+ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

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Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽

10.1182/blood.v108.11.866.866 ◽

2006 ◽

Vol 108 (11) ◽

pp. 866-866

Author(s):

Carolina Berger ◽

Michael C. Jensen ◽

Stanley R. Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Annexin V ◽

T Cell Memory ◽

T Cell Clones ◽

Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for >30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

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Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.1182.1182 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1182-1182

Author(s):

Eva M Wagner ◽

Aline N Lay ◽

Sina Wenzel ◽

Timo Schmitt ◽

Julia Hemmerling ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Cell Depletion ◽

Down Regulation ◽

T Cell Depletion ◽

Hematopoietic Stem

Abstract The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

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Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells

Journal of Experimental Medicine ◽

10.1084/jem.20131459 ◽

2014 ◽

Vol 211 (3) ◽

pp. 529-543 ◽

Cited By ~ 95

Author(s):

Julio Gomez-Rodriguez ◽

Elizabeth A. Wohlfert ◽

Robin Handon ◽

Françoise Meylan ◽

Julie Z. Wu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Signaling Pathways ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Mammalian Target Of Rapamycin ◽

Cell Receptor ◽

Treg Cells ◽

Cytokine Signaling

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk−/− CD4+ T cells develop higher percentages of functional FoxP3+ cells, associated with increased sensitivity to IL-2. Itk−/− CD4+ T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk−/− cells also exhibit reduced IL-2–induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4+ T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4+ T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.

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Alloantigen-activated (AAA) CD4+ T cells reinvigorate host endogenous T cell immunity to eliminate pre-established tumors in mice

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02102-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Kazuhiro Mochizuki ◽

Shogo Kobayashi ◽

Nobuhisa Takahashi ◽

Kotaro Sugimoto ◽

Hideki Sano ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Antitumor Immunity ◽

Unmet Need ◽

T Cell Response ◽

Hematopoietic Stem ◽

Cell Immunity ◽

Allogeneic Lymphocytes

Abstract Background Cancer vaccines that induce endogenous antitumor immunity represent an ideal strategy to overcome intractable cancers. However, doing this against a pre-established cancer using autologous immune cells has proven to be challenging. “Allogeneic effects” refers to the induction of an endogenous immune response upon adoptive transfer of allogeneic lymphocytes without utilizing hematopoietic stem cell transplantation. While allogeneic lymphocytes have a potent ability to activate host immunity as a cell adjuvant, novel strategies that can activate endogenous antitumor activity in cancer patients remain an unmet need. In this study, we established a new method to destroy pre-developed tumors and confer potent antitumor immunity in mice using alloantigen-activated CD4+ (named AAA-CD4+) T cells. Methods AAA-CD4+ T cells were generated from CD4+ T cells isolated from BALB/c mice in cultures with dendritic cells (DCs) induced from C57BL/6 (B6) mice. In this culture, allogeneic CD4+ T cells that recognize and react to B6 mouse-derived alloantigens are preferentially activated. These AAA-CD4+ T cells were directly injected into the pre-established melanoma in B6 mice to assess their ability to elicit antitumor immunity in vivo. Results Upon intratumoral injection, these AAA-CD4+ T cells underwent a dramatic expansion in the tumor and secreted high levels of IFN-γ and IL-2. This was accompanied by markedly increased infiltration of host-derived CD8+ T cells, CD4+ T cells, natural killer (NK) cells, DCs, and type-1 like macrophages. Selective depletion of host CD8+ T cells, rather than NK cells, abrogated this therapeutic effect. Thus, intratumoral administration of AAA-CD4+ T cells results in a robust endogenous CD8+ T cell response that destroys pre-established melanoma. This locally induced antitumor immunity elicited systemic protection to eliminate tumors at distal sites, persisted over 6 months in vivo, and protected the animals from tumor re-challenge. Notably, the injected AAA-CD4+ T cells disappeared within 7 days and caused no adverse reactions. Conclusions Our findings indicate that AAA-CD4+ T cells reinvigorate endogenous cytotoxic T cells to eradicate pre-established melanoma and induce long-term protective antitumor immunity. This approach can be immediately applied to patients with advanced melanoma and may have broad implications in the treatment of other types of solid tumors.

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The Absence of CCR7 on Donor T Cells Leads to Attenuated Graft-Versus- Host Disease (GVHD) with a Preserved Graft-Versus-Leukemia (GVL) Effect

Blood ◽

10.1182/blood.v112.11.810.810 ◽

2008 ◽

Vol 112 (11) ◽

pp. 810-810

Author(s):

James M. Coghill ◽

Karen N. Hogan ◽

Jonathan S. Serody

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Model ◽

Rank Test ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Mastocytoma Cells

Abstract The development of GVHD depends on the trafficking of donor effector T cells (Teffs) into recipient secondary lymphoid tissue early after hematopoietic stem cell transplantation (HSCT). Chemokine receptor 7 (CCR7) has been shown to be critical for the movement of naïve T cells into lymph nodes and for the organization of T cell and B cell regions in the spleen. Using a murine transplant model, we set out to study the contribution of CCR7 to both GVHD induction and the immunomodulatory properties of regulatory T cells (Tregs) in the HSCT setting. Methods: C57BL/6 (H-2b; termed B6) mice served as bone marrow (BM) donors, and B6xDBA/2 F1 (H-2bxd; termed B6D2) mice functioned as recipients. Teffs and Tregs were obtained from wild-type (WT) B6 or CCR7 knockout (CCR7−/−) mice extensively backcrossed on a B6 background. For Teff studies, recipient animals were lethally irradiated to 950 rads on day –1 and administered 3 ×106 T-cell depleted (TCD) BM cells +/− 4×106 splenic Teffs from WT or CCR7−/− donors on day 0. For GVL studies, 25,000 P815 murine mastocytoma cells were coadministered with the BM/Teff inoculum on day 0. For Treg studies, mice received TCD BM +/− 1×106 WT or CCR7−/− Tregs on transplant day 0, with 4×106 WT Teffs dosed on transplant day +2. Results: WT and CCR7−/− Teffs generated GVHD responses that were nearly identical during the first 7–10 post transplant. Thereafter, those mice receiving CCR7−/− Teffs demonstrated a significant attenuation of their GVHD, with 83% surviving long term. In contrast, those animals receiving WT Teffs exhibited relentlessly progressive disease and a 92% mortality rate by day +70 (see figure; P=0.0006 for comparison between WT and CCR7−/− Teff groups by the log-rank test). In spite of their relatively impaired capacity to generate lethal GVHD, CCR7−/− Teffs were found to produce complete donor reconstitution of the CD4+ and CD8+ T cell compartments by transplant day +40, albeit at a slower rate than that observed with WT Teffs. CCR7−/− Teffs also demonstrated significant anti-tumor activity in-vivo. B6D2 mice challenged with P815 tumor cells and TCD BM all died with massive splenomegaly and diffuse tumor infiltration of the liver by transplant day +20. Animals receiving WT Teffs at the time of P815 challenge all died of GVHD, with no tumor noted at autopsy. In contrast, 75% of the mice administered P815 cells and CCR7−/− Teffs survived long term with no signs of malignancy and only mimimal evidence of GVHD clinically. These findings did not appear to be the result of an impaired ability of CCR7−/− Teffs to respond to alloantigens in-vitro, as proliferative responses of sort-purified CD25- CD4+ and CD8+ T cells from WT and CCR7−/− mice were similar in one-way mixed lymphocyte reactions. Surprisingly, CCR7−/− Tregs appeared to be capable of protecting against lethal GVHD when administered two days prior to WT Teffs, with 100% of recipients surviving to day +70. Their GVHD scores, however, were somewhat higher than in those mice receiving WT Tregs. Conclusions: CCR7 is not required for the initial phase of GVHD induction, but appears important for the perpetuation and augmentation of disease 10–14 days post transplantation. The attenuated in-vivo allo-immune responses observed with CCR7−/− Teffs appear to be independent of any intrinsic deficiency in the proliferative capacity of the T cells themselves. CCR7−/− Teffs are capable of generating a potent GVL effect in our P815 tumor model. CCR7 does not appear to be required for Tregs to protect against lethal GVHD when they are administered in advance of WT Teffs. At the time of this writing we have successfully generated CCR7−/− eGFP+ mice, and in-vivo trafficking studies are currently planned to elucidate a mechanism for the above findings. Figure Figure

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Multiple developmental pathways lead to the generation of CD4 T-cell memory

International Immunology ◽

10.1093/intimm/dxaa051 ◽

2020 ◽

Vol 32 (9) ◽

pp. 589-595 ◽

Cited By ~ 1

Author(s):

Shintaro Hojyo ◽

Damon Tumes ◽

Akihiko Murata ◽

Koji Tokoyoda

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Memory Cell ◽

Cd4 T Cell ◽

Developmental Pathways ◽

T Cell Memory ◽

Cell Memory ◽

Memory Cd4 T Cells

Abstract Long-term immunological memory mediated by CD4 T cells provides a rapid protection against previously encountered pathogens or antigens. However, it is still controversial how memory CD4 T cells are generated and maintained. Unclear definitions of T-cell memory may be partially responsible for this controversy. It is becoming clear that diverse pathways are responsible for the differentiation and long-term persistence of memory T cells. We herein discuss the diversity of memory cell generation, describing a novel population of resting memory CD4 T cells and their precursors.

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Hematopoietic Stem Cell Infected with HTLV-1 Functions As a Viral Reservoir In Vivo

Blood ◽

10.1182/blood.v128.22.1343.1343 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1343-1343 ◽

Cited By ~ 4

Author(s):

Jun-ichirou Yasunaga ◽

Rie Furuta ◽

Michi Miura ◽

Kenji Sugata ◽

Akatsuki Saito ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Leukemia ◽

Hematopoietic Stem ◽

Integration Sites ◽

Infected Cells

Abstract Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of a malignant disease of peripheral CD4+ T cells called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although major target of HTLV-1 is CD4+ T cells, other hematopoietic cells such as CD8+ T cells and monocytes are also infected with HTLV-1. Since the receptors of HTLV-1 are glucose transporter 1 and neuropilin 1, which are found on various cell surfaces, it is possible that HTLV-1 infects various hematopoietic cells and hematopoietic stem cells (HSCs). However, the previous studies could not detect HTLV-1 in HSCs. To assess the distribution of infected cells and expression of viral genes in various tissues, a nonhuman primate model, Japanese macaques (JMs) infected with simian T-cell leukemia virus type 1 (STLV-1) was utilized in this study. STLV-1 is a close relative of HTLV-1, and the dynamics of viral replication and proliferation of infected cells are very similar to each other. Indeed, STLV-1 caused malignant lymphomas in STLV-1 infected monkeys. Therefore, STLV-1 infected JMs are good models of HTLV-1 carriers. Using this model, we first analyzed transcription level of two viral genes, tax and STLV-1 bZIP factor, in multiple tissues, and found that tax was highly expressed in bone marrow compared to other tissues. Since Tax is a potent activator of viral transcription, this result suggested that viral replication occurred in bone marrow. To evaluate which cells express Tax in bone marrow, we performed flow cytometric analysis of bone marrow mononuclear cells from STLV-1 infected monkeys, and found that not only CD4+ T cells but also non T cells (CD3 negative cells) expressed Tax. To determine whether hematopoietic stem cells are infected with HTLV-1, we next performed highthroughput sequencing of HTLV-1 integration sites in multiple cell lineages using a next generation sequencer. Since HTLV-1 is randomly integrated into genome of infected cells, and each infected cell can proliferate clonally, we can interpret that cells sharing same integration sites are derived from the same precursor cell. Blood samples from patients with HAM/TSP were separated into five cell types: CD4+ T cells, CD8+ T cells, B cells, monocytes, and neutrophils. Integration sites of HTLV-1 provirus were analyzed by next generation sequencing using different chips for each cell type in order to avoid cross contamination between the samples. The results showed that some of these cells have same integration sites between different cell types. More than 40 percent of HTLV-1 infected B cells, monocytes and neutrophils had shared the same integration sites, suggesting that HSCs in the HAM/TSP patients were infected with HTLV-1. Importantly, most HTLV-1 infected CD4+ T cells and CD8+ T cells had independent integration sites, while some of them shared the same integration sites with non T cell subsets. These results implied that most T-cell clones were generated by de novo infection in the periphery, but a part of infected T cells were derived from infected HSCs. To assess whether HTLV-1 infected HSCs persist in vivo, we again analyzed integration sites in neutrophils from the same HAM/TSP patients after 1 year. About 50 percent of integration sites in HTLV-1 infected neutrophils were detected after 1 year. Considering short lifespan of neutrophils, this result suggests that HTLV-1 infected HSCs could be maintained for at least 1 year in vivo. To visualize HTLV-1 infection in myeloid cells, we performed immunofluorescence staining of neutrophils from HAM/TSP patients. Tax and myeloperoxidase were detected in neutrophils. Finally, we evaluated the significance of infected monocytes in viral transmission. We isolated monocytes from HAM/TSP patients and co-cultured with JET WT35 cell, which is a subline of Jurkat containing a fluorescent reporter of viral infection. The result demonstrated that monocyte from HAM/TSP patients could be a source of infective HTLV-1. In conclusion, our findings suggest that HSCs infected with HTLV-1 survive for long time in vivo, and could be reservoirs of the virus. Since ATL is a difficult disease to cure, further studies are required to understand the nature of HTLV-1 infection. Disclosures No relevant conflicts of interest to declare.

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In Vivo Administration of Interleukin 15 (IL-15) Safely Promotes the Survival and Proliferation of Adoptively Transferred Antigen-Specific T Cell Clones

Blood ◽

10.1182/blood.v112.11.679.679 ◽

2008 ◽

Vol 112 (11) ◽

pp. 679-679

Author(s):

Carolina Berger ◽

Michael Berger ◽

Michael C Jensen ◽

Stanley R Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

T Cell Memory ◽

Ki 67

Abstract The adoptive transfer of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) that have been expanded in vitro is a promising treatment for human malignancies and infections. Interleukin (IL)-2 is frequently administered to support the in vivo survival of transferred T cells, but causes systemic toxicity when given in high doses and promotes the expansion of CD4+ regulatory T cells, which can inhibit antitumor immunity. IL-15, like IL-2, belongs to the four α-helix bundle family of cytokines and shares functional activities with IL-2, including binding to the IL-2 receptor (R) β and γc signaling components and promoting the proliferation of activated T cells in vitro. Despite the similar structure and in vitro function of IL-2 and IL-15, mice deficient in IL-15 or IL-15Rα have a marked reduction in natural killer (NK) cells, NKT cells, and CD8+ memory cells, whereas mice deficient in IL-2 or IL-2Rα have lymphoid hyperplasia and autoimmunity. Because of its critical role in the maintenance of T cell memory, IL-15 is an attractive alternative to IL-2 for augmenting adoptively transferred T cell immunity in humans. We administered IL-15 subcutaneously to nonhuman primates and evaluated toxicity, immunological effects, and peak and trough plasma levels. After establishing a safe regimen of IL-15 dosing, we evaluated the ability of IL-15 to support the survival of adoptively transferred CD8+ effector T cell (TE) clones in vivo. Results: IL-15 was administered subcutaneously to five macaques at doses ranging from 2.5 – 15 μg/kg, given either daily or every 3 days, respectively. The animals were monitored for clinical toxicity and plasma levels. Peripheral blood T cell subsets were enumerated at intervals and evaluated for phenotype and expression of Ki-67, a nuclear antigen expressed by cells undergoing proliferation. Daily administration of high-dose IL-15 resulted in a pronounced increase in the absolute numbers and Ki-67-expression of CD8+ T cells and NK cells, respectively, and preferentially expanded CD8+CD95+CCR7− effector memory (TEM) and CD8+CD95+CCR7+ central memory T cells (TCM). However, daily IL-15 in doses of 5 – 15 μg/kg was associated with accumulation of IL-15 in serum, and caused toxicities that were reversible when IL-15 was discontinued. By contrast, intermittent IL-15 treatment every 3 days was safe and induced only a moderate increase in NK cells, CD8+ TEM and TCM, and enhanced expression of Ki-67 in these cell subsets. This coincided with an increase of the absolute number of cytomegalovirus (CMV)-specific CD8+ T cells in the peripheral blood, but total numbers of CD4+ FoxP3+ T cells were not increased with IL-15. We then examined the ability of IL-15 administered every 3 – 4 days for 3 weeks to support the in vivo persistence of TCM-derived CMV-specific CD8+ TE clones that were marked to express a truncated macaque CD19 surface molecule and transferred to the animals without prior lymphodepletion. As previously reported, CD8+ TE clones derived from TCM precursors survive in vitro in low-doses of IL-15 in the absence of T cell receptor stimulation, persist long term in vivo after transfer and revert to the memory pool (Berger et al, JCI2008, 118:294). In comparison with animals that received CD8+ T cells alone in which transferred T cells persisted at a stable level of 0.2 – 0.8% of circulating CD8+ cells, the administration of IL-15 after T cell transfer resulted in the establishment of a high-level T cell response (10 – 15% of CD8+ T cells; >100 cells/μL) that persisted for >6 months after IL-15 was discontinued. The CD19+CD8+ TE clones re-acquired a memory T cell phenotype in vivo and expressed bcl-2, bcl-xL and Ki-67 comparable to endogenous CD8+ T cells. The transferred cells were present in large numbers in bone marrow and lymph node samples obtained on day 14 and day 56 after infusion suggesting that they efficiently occupied niches of T cell memory. This data in a large animal model predictive of clinical translation demonstrates that IL-15 can be safely administered, exerts a profound immunologic effect, and dramatically augments the long-term survival of ex vivo expanded antigen-specific CD8+ CTL clones after adoptive transfer without promoting in vivo expansion of CD4+ Foxp3+ regulatory cells. Thus, IL-15 may be a safer and more effective alternative to IL-2 and/or lymphodepletion to support the in vivo persistence of adoptively transferred tumor or virus-specific T cells in human immunotherapy.

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A Critical Role of MyD88 Signaling in Donor T Cells for Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia Effect (GVL) After Experimental Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

Blood ◽

10.1182/blood.v120.21.1893.1893 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1893-1893

Author(s):

Ji-Young Lim ◽

Dae-Chul Jeong ◽

Hyewon Youn ◽

Eun-Young Choi ◽

Chang-Ki Min

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Critical Role ◽

Hematopoietic Stem ◽

Donor T Cells ◽

Myd88 Signaling

Abstract Abstract 1893 The therapeutic potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT) relies on the graft-versus-leukemia effect (GVL) to eradicate residual tumor cells by immunologic mechanisms. However, graft-versus-host disease (GVHD) remains the major toxicity of allo-HSCT. Alloreactive donor T cells are important effector cells in the development of GVHD, and proinflammatory cytokines enhance the generation of donor antihost cytotoxic function. Myeloid differentiation factor (MyD88) is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the Toll-like receptor (TLR) family. TLR engagement on professional antigen-presenting cells induces their maturation, resulting in optimal T-cell activation. However, recent advances indicate that the adjuvant effects of certain TLR agonists may also be attributed to the activation of TLRs and MyD88 directly in T cells. Both CD4 and CD8 T cells express functional TLRs. It remains to be defined whether direct TLR signaling on donor T cells is critical for GVHD or GVL activity. We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) experimental allo-HSCT model, which differs at major and minor histocompatibility loci, to address the role of donor T cell MyD88 signaling on GVHD and GVL. Lethally irradiated recipient mice were transplanted TCD-BM (5 × 106) together with either wild-type (WT) or MyD88 knock out (KO) mice spleen T cells (1 × 106) on day 0 and then host-type P815 mastocytoma or L1210 leukemia (H-2d) cells were injected either intravenously (3 × 103) or subcutaneously (1 × 106) on day 1 to generate a GVHD/GVL model. First of all, clinical GVHD scores were comparable between recipients of WT T cells and MyD88 KO T cells. At 70 days post-allo-HSCT, 50 % of allogeneic recipients of WT T cells died due to severe GVHD, but necropsy showed no evidence of tumor. In contrast, 83.5% of those of MyD88 KO T cells died with gross evidence of tumors (P<.05). Moreover, subcutaneous tumors in the allogeneic recipients receiving MyD88 KO T cells exhibited markedly increased growth in vivo compared to those receiving WT T cells (tumor volume on day 41, 15205.6 vs. 373.9 mm3, P<.01). GVHD mortality is critically dependent on donor CD4 T cells in this donor/recipient strain combination (B6 → B6D2F1) and CD8 T cells that mediate cytotoxicity are more potent effectors of GVL. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two T cell types was examined; apoptosis of CD8 T cells in recipients of MyD88 KO T cells was significantly enhanced compared to those of WT T cell recipients (P<.01) whereas apoptosis of CD4 T cells was comparable between two groups. Resultingly, the percentages of CD8 T cells in recipients of MyD88 KO T cells were significantly lower (P<.01). We next examined the effects of MyD88 signaling in donor T cells on cytolytic activity to host antigens. Splenocytes harvested from WT mice showed stronger cytolytic activity against P815 targets compared to those from MyD88 KO mice (P<.01). After allogeneic mixed leukocyte reaction, responder T cells from MyD88 KO mice showed markedly reduced IFN-γ, MCP-1 and IL-17A production with a significant augmentation in IL-10 secretion. We further evaluated the effect of T-cell MyD88 deficiency on GVL mediated by the intensity of total body irradiation (TBI) conditioning (1300 vs. 900 cGy, Exp Hematol 2011; 39: 1018–29). Enhanced GVL in the allogeneic recipients receiving 1300 cGy TBI was not shown in the recipients of MyD88 KO T cells. In summary, these results highlight a critical role for MyD88 signaling in T-cell activation and cytotoxicity, offering the opportunity for improving GVL activity by targeting TLR-MyD88 signaling within donor T cells. Furthermore, these data demonstrated that MyD88 deficiency in T cells can impair cytolytic function or subsequent GVL activity of CD8 T cells without significant change in the severity of CD4-dependent GVHD. This difference is attributed to the fact that MyD88 deficiency in T cells causes an enhanced apoptosis of donor CD8 T cells but not donor CD4 T cells in vivo after HSCT. Disclosures: No relevant conflicts of interest to declare.

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