NKG2D, An NK Cell Activating Receptor on CD8+ T Cells, Plays An Essential Role In Killing Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2087-2087 ◽  
Author(s):  
Laleh Talebian ◽  
Kenneth Meehan ◽  
Megan E Patey ◽  
Dawn A Fisher ◽  
Zbigniew Szczepiorkowski ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Mhc I ◽  
Myeloma Cells ◽  
Post Transplant ◽  
Autologous Transplant ◽  
Activating Receptor

Abstract Abstract 2087 Autologous stem cell transplant for myeloma improves response rates, but the immunologic mechanisms contributing to this improvement are unknown. Our results indicate that this beneficial effect may be due to a subpopulation of CD8+T cells that express an NK cell activating receptor, called NKG2D. NKG2D is present on some CD8+ T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. Three NKG2D ligands (MICA, ULBP1, and ULBP3) are expressed on patients’ myeloma cells. Our data indicate that NKG2D expression can be up-regulated on some CD8+T cells making these cells highly effective at killing myeloma cells. We previously developed an ex vivo expansion method that enriches for NKG2D+CD8+T cells using mobilized blood progenitor cells (Cytotherapy 2008). These ex vivo expanded NKG2D+CD8+ T cells aggressively lysed myeloma cells and blocking the NKG2D receptor significantly inhibited this killing (p<0.0009). Due to these intriguing laboratory results, we conducted a phase II trial using adoptive cellular immunotherapy following autologous transplant, using ex vivo expanded cells enriched for NKG2D+CD8+ T cells. Myeloma patients received high-dose melphalan (200mg/m2) followed by an autologous transplant. Low dose IL-2 (6×105 IU/m2/d × 20 days) and GM-CSF (250 μg/m2/d) were administered following transplant. The ex vivo expanded cells (1 × 109 CD3+ T cells per infusion) were administered at weeks 1, 2, 3, and 8 following transplant. Nineteen of twenty-three patients are evaluable. Median engraftment of neutrophils was 13 days (range: 12–16 days) and 16 days for platelets (range: 12–26 days). All nineteen patients completed the full course of post-transplant IL-2. There were no treatment-related deaths. Due to the required CD3+T cell number in each of the ex vivo expanded cell infusions, fifteen patients received the ex vivo expanded cells (4 infusions n = 9; 3 infusions n = 3; 2 infusions n = 2; 1 infusion n = 1). Transplant-related adverse effects (> Grade 3) included nausea/vomiting (n=2), fever (n=7), elevated AST/ALT (n=2), anorexia (n=2), pneumonia (n=2), enteritis (n=1), diarrhea (n=2), pulmonary embolism (n=1), or typhlitis (n=2).There was an increased number and function of NKG2D+CD8+T cells circulating in vivo post-transplant. At 1 month post-transplant, there was an increase in the number of NKG2D+CD8+T cells (p < 0.0004), CD3+CD8+ T cells (p < 0.027), CD8+CD56+T cells (p < 0.0086), and NKG2D+CD56+ T cells (p < 0.0036). The circulating NKG2D+CD8+T cells recognized and lysed autologous myeloma cells (p<0.005) and lysis was significantly inhibited by blocking NKG2D (p<0.0014). NKG2D ligand expression on patients’ myeloma cells strongly correlated with % cell lysis. These results suggest that NKG2D+CD8+ T cells recognize and kill autologous myeloma cells in an NKG2D-dependent manner. Since myeloma cells down regulate MHC-I, NKG2D+CD8+T cells’ MHC-I unrestricted killing of myeloma cells may improve outcomes in transplanted myeloma patients. Ongoing experiments will identify the kinetics of NKG2D+CD8+ T cells following transplant and the molecular mechanisms of cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2076-2082 ◽  
Author(s):  
Anja Fuchs ◽  
Marina Cella ◽  
Takayuki Kondo ◽  
Marco Colonna
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Close Contact ◽  
Killer Cell ◽  
Adaptor Protein ◽  
Interferon Α ◽  
Memory Cd8 T Cells ◽  
Activating Receptor

Abstract Natural killer (NK) cell-mediated cytotoxicity is triggered by multiple activating receptors associated with the signaling adaptor protein DNAX activation protein 12/killer cell-activating receptor-associated protein (DAP12/KARAP). Here, we show that one of these receptors, NKp44, is present on a subset of natural interferon-producing cells (IPCs) in tonsils. NKp44 expression can also be induced on blood IPCs after in vitro culture with interleukin 3 (IL-3). Crosslinking of NKp44 does not trigger IPC-mediated cytotoxicity but, paradoxically, inhibits interferon α (IFN-α) production by IPCs in response to cytosine-phosphate-guanosine (CpG) oligonucleotides. We find that IPCs in tonsils are in close contact with CD8+ T cells and demonstrate that a subset of memory CD8+ T cells produces IL-3. Therefore, IL-3-mediated induction of NKp44 on IPCs may be an important component of the ongoing crosstalk between the innate and adaptive immune response that allows memory CD8+ T cells to control the IPC response to virus. (Blood. 2005;106: 2076-2082)

Platelet MHC Class I Mediates CD8+ T Cell Suppression During Sepsis

Blood ◽  
2021 ◽  
Author(s):  
Li Guo ◽  
Sikui Shen ◽  
Jesse W Rowley ◽  
Neal D. Tolley ◽  
Wenwen Jia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
Class I ◽  
Functional Responses ◽  
Class I Mhc ◽  
Mhc I

Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased, and have been associated with adverse clinical events, including increased platelet-T cell interactions. Sepsis is associated with reduced CD8+ T cell numbers and functional responses, but whether platelets regulate CD8+ T cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (e.g., IFN-g and LPS). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage specific MHC-I deficient mouse strain (B2mf/f--Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo during sepsis. Loss of platelet MHC-I reduced sepsis-associated mortality in mice in an antigen specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen specific CD8+ T cells, and regulate CD8+ T cell number, functional responses, and outcomes during sepsis.

Paradoxical Immunological Activity of Flagellin: A Novel Method to Reduce GvHD In Allogeneic HSCT Recipients.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1465-1465
Author(s):  
Mohammad S Hossain ◽  
Andrew T Gewirtz ◽  
John Roback ◽  
Edmund Waller
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Transplant ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Abstract 1465 Background: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Using flagellin, a bacterial protein that agonistically binds with TLR5, we previously reported that two doses of flagellin (before irradiation and after allogeneic HSCT with 5×106 splenocytes) in H-2K à H-2b model significantly reduced GvHD and had 67% long-term survival by 132 days post-transplant whereas control recipients had severe acute GvHD and 100% early mortality within 15 days post transplant. Here, we report the mechanism by which flagellin reduces GvHD using the same H-2K à H-2b HSCT murine model. Methods: 3×106 splenocytes and 5 ×106 T cell depleted bone marrow (BM) cells harvested from the congeneic H-2K donors were transplanted into lethally irradiated (11Gy) C57BL/6 (H-2b) recipients. 50-μg HPLC purified LPS-free flagellin diluted in ice-cold PBS were administered i.p 3 hours before irradiation and 24 hours after transplant. Control recipients were treated with PBS. Recipients (6/group) were sacrificed on day 4 and 10 after post transplant. Serum was collected to determine cytokines by ELISA and splenocytes were analyzed by FACS to determine immune cells phenotypes. Results: Although both Fla and PBS-treated recipients showed identical weight-loss within day 10 post transplant, surprisingly significantly lower numbers of cells/spleen were determined in the spleens of Fla recipients compared to control recipients [Fla 0.9 ± 0.1 (x106) vs PBS 1.7 ± 0.4(x106), p=0.002] on day 4 post transplant but not on day 10 [Fla 186.1 ± 35.1 (x106) vs PBS 151.2 ± 40.5(x106), p=0.43] post-transplant. We investigated the paradoxical immune response of flagellin on donor T cells on day 4-post transplant. First, we determined the numbers of donor spleen-derived Thy1.2+ T cells per spleen. The numbers of donor spleen-derived T cells per spleen were significantly lower in Fla recipients compared to PBS-treated recipients [Fla 0.005 ± 0.002 (x103) vs PBS 0.04 ± 0.03(x103), p=0.02]. Accordingly, donor spleen-derived both CD4 and CD8 T cells per spleen of Fla recipients were also found significantly lower compared to PBS-treated recipients (CD4, p=0.04; CD8, p= 0.003). The CD62L, a naïve and also markers for allo-reactive T cells that cause GvHD were found significantly lower in both CD4 and CD8 T cells (CD4, p= 0.03; CD8, p=0.003) and the inducible co-stimulatory molecule 1 (ICOS-1), another prominent T cells activation marker were also found significantly lower (CD4, p= 0.04; CD8, p=0.007) in Fla recipients compared to PBS-treated recipients. These lower immune phenotypes of donor T cells in Fla recipients may reduce the initiation of GvHD at the early time points of transplant. However, flagellin-induced reduction of donor T cells activity were not suppressed considerably as the numbers of donor spleen-derived CD4 T cells expressing activation markers such as CD25 {Fla 0.5 ± 0.2 (x103) vs PBS 4.5 ± 0.5 (x103), p=0.07} and CD69 {Fla 0.5 ± 0.2 (x103) vs PBS 6.5 ± 5.0 (x103), p=0.08} per spleen were not found significantly different. On the other hand, the numbers of CD8 T cells those expressed CD25 {Fla 0.2 ± 0.04 (x103) vs PBS 1.3 ± 0.7 (x103), p=0.01} and CD69 {Fla 0.4 ± 0.1 (x103) vs PBS 2.4 ± 1.7 (x103), p=0.03} per spleen were found significantly lower in Fla recipients compared to PBS-treated recipients. Although over 90% of donor spleen CD4 T cells of both Fla and PBS-treated recipients expressed PD-1, 24% and 32% respectively, expressed IFN-γ after vitro PMA stimulation. Similar results were found in case of CD8 T cells. The over expression of PD-1 in HSCT recipients, thus did not make the donor T cells exhausted as they expanded over 100 times within day 10 post transplant with the expression of PD-1. Although similar level of serum IFN-γ was determined between the Fla and PBS-treated recipients on day 10 post-transplant, IFN-γ level was found below detection limit on day 4 post-transplant. The serum levels of IL-1β and IL-10 were undetectable on both days 4 and 10 post-transplant. Serum LPS were found identical in both Fla and PBS-treated groups determined on day 4 and 10 days post transplant. Conclusion: Flagellin protected allogeneic HSCT recipients from lethal GvHD by reducing donor CD62L+ and ICOS-1+ T cells expansion and activation within 4 days post-transplant without compromising their normal immune responses. Disclosures: No relevant conflicts of interest to declare.

The natural killer-activating receptor, NKG2D, on CD3+CD8+ T cells plays a critical role in identifying and killing autologous myeloma cells

Transfusion ◽  
2014 ◽  
Vol 54 (6) ◽  
pp. 1515-1521 ◽  
Author(s):  
Laleh Talebian ◽  
Dawn A. Fischer ◽  
Jillian Wu ◽  
Jacqueline Y. Channon ◽  
Charles L. Sentman ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Cd8 T Cells ◽  
Critical Role ◽  
Myeloma Cells ◽  
Activating Receptor

P16.06 Immunophenotyping of tumor-infiltrating T cells in primary CNS lymphoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
S Schliffke ◽  
C Maire ◽  
M Holz ◽  
C Bokemeyer ◽  
M Westphal ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Primary Cns Lymphoma ◽  
Great Promise ◽  
Cns Lymphoma ◽  
High Dose ◽  
Dismal Prognosis

Abstract BACKGROUND Primary CNS lymphoma represents a malignant disease with dismal prognosis. Standard of care is high dose chemotherapy and radiation. However, this combination cannot be applied to the elderly and fragile population. Immunotherapy holds great promise to be effective in these patients. This study therefore aims to explore the phenotype of tumor-infiltrating lymphocytes (TIL) in order to analyze the potential for immune checkpoint inhibition. MATERIAL AND METHODS We performed ex vivo multicolor flow-cytometry on surgical specimens of nine patients with intracerebral lymphoma, including seven with primary CNS lymphoma after isolation of TILs following standard protocols. Data was analyzed using a Fortessa LSR flow cytometer and Diva software. The study was approved by the local ethics committee (PV4904). RESULTS Our ex vivo phenotyping demonstrated a predominant infiltration of CD8+ T cells, which outnumber CD4+ T cells by a ratio of 2:1 (p&lt;0.01). Regulatory T cells (Tregs) were not increased in the tumor microenvironment and the NK cell frequency was reduced compared to the peripheral blood. While CD4+ T helper cells displayed significantly increased surface expression of multiple activation and checkpoint markers, including TIGIT, PD-1, Tim3 and CD57, cytotoxic CD8+ T cells predominantly expressed only TIGIT and PD-1. On average 70% and 80% of CD8+ T cells expressed PD-1 and TIGIT, respectively, compared to 35% and 60% of PD-1 and TIGIT on CD4+ T cells (p&lt;0.05). CD8+ T cells furthermore showed an increased expression of CD39 and a simultaneous downregulation of CD73, both ectoenzyms involved in the modulation of intratumoral ATP, thereby indicating a metabolic immune modulation by the tumor. CONCLUSION Taken together, our study demonstrates a strong infiltration of cytotoxic CD8+ T cells into cerebral lymphoma, which potentially can be disinhibited using checkpoint immunotherapy. Our profiling suggests that PD-1 and TIGIT present appealing targets for such kind of immune disinhibition.

Donor’s NK Cells May Influence the Engraftment in Pediatrics Patients after T-Cell Depleted Haploidentical Stem Cell Transplant for Thalassemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4595-4595
Author(s):  
Antonella Isgro ◽  
Marco Marziali ◽  
Pietro Sodani ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Mixed Chimerism ◽  
Effector Cells ◽  
Post Transplant

Abstract In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cells ablates recipient T cells which reject the graft, and ablates recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD (Ruggeri et al., Science 2002). NK cell alloreactivity also boosts very rapid rebuilding of donor adaptive immunity to infections. In this study we analysed the potential role of NK cells after haploidentical transplant in b-thalassemia patients. T and B cell depletion was carried out with CD34+ coated magnetic microbeads and the CliniMACS device (Miltenyi Biotec©) from peripheral blood and bone marrow of donors (the mothers) and resulted in grafts consisting of stem cells and effector cells (NK cells, monocytes) with the addition of bone marrow mononuclear cells (BMMNCs 3 × 105/kg of the recipient). A total of 11 pediatric patients with b-thalassemia received T and B cell depleted transplants from their haploidentical mothers with a median number of 15 ×106 CD34 stem cells. To analyse the mechanisms involved in immunological reconstitution post transplant, we analysed T cell subsets by flow cytometry, particularly NK sets (CD3- CD56+, CD3− CD16+ and CD56+CD16+ NK cells) at day + 20 and + 60 post transplant. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD3-CD56+bright cells. Interestingly, a direct correlation has been observed between the percentages of CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, 27 ± 28% in the three patients with a stable mixed chimerism after BM transplant (70–80% of donor cells) and 1.4 ± 1% in the four patients with rejection). In all the patients the origin of the NK subsets was from the mothers. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%), in parallel with the increase of the CD3-CD16+ NK cells (potent cytotoxic effector cells) especially in the patients with full engraftment (in mean 47 ± 20% vs. 28 ± 31% in mixed chimerism) NK CD56+bright cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factor (IL-2) in the bone marrow. Interestingly we observed higher percentages of NK subsets just twenty days post transplant in the patients with full engraftment respect the mixed chimerism and the rejection, suggesting a role of donor NK cells on improved engraftment and on prevention of the rejection with the attack of the host lympho-hematopoietic cells. These observations may suggest the importance of NK subsets analyses at the first time of the transplant as an useful parameter for the outcome of the transplant and/or the use of donor’s alloreactive NK cells especially in haploidentical recipients.

CMV Specific T Cells for Adoptive Transfer Exhibit Multiple Effector Functions Associated with Protective Immunity Including the Concurrent Production of Cytokines and Cytolytic Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3481-3481
Author(s):  
Leighton E Clancy ◽  
Kenneth P Micklethwaite ◽  
Emily Blyth ◽  
Upinder Sandher ◽  
David J Gottlieb
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Protective Immunity ◽  
Stem Cell Transplant ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Haemopoietic Stem Cell ◽  
Ifn Γ ◽  
Cmv Reactivation

Abstract Background: Cytomegalovirus (CMV) reactivation post allogeneic haemopoietic stem cell transplant (HSCT) causes significant morbidity. Adoptive transfer of ex-vivo generated CMV specific T cells has the potential to restore immunity, prevent CMV reactivation and circumvent the need for pharmacotherapy. Donor derived CMV specific T cells were generated for prophylactic infusion into haemopoietic stem cell transplant recipients as part of a phase I/II clinical trial aiming to reduce the incidence of CMV reactivation. T cells were expanded by co-culturing with dendritic cells transfected with Ad5F35pp65, a recombinant adenovirus promoting the presentation of epitopes derived from the immunodominant CMV antigen pp65. The aim of this study was to determine if ex vivo expanded CMV specific T cells have the capacity to produce different cytokines and chemokines associated with protective immunity. Results: Ex vivo expanded Ad5F35pp65 stimulated cultures were primarily CD3+ T cells (median 92%) with a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. An assay measuring antigen specific production of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor (TNF) and macrophage inflammatory protein 1β (MIP-1β) by intracellular cytokine flow cytometry was established to quantify the frequency of T cells with specificity towards CMV or adenovirus and to assess the quality of responses reflected by the simultaneous production of multiple cytokines. Ad5F35pp65 stimulated cultures were greatly enriched for CMV specific T cells producing cytokines in response to pp65 (mean 59%, 19.1–90%) compared to the starting PBMC population (0.5–1.5%). Responses directed towards the adenovirus hexon protein were also detected accounting for 0.65 to 9% of T cells. CMV specific CD8 T cells predominantly produced IFN-γ and MIP-1β followed by TNF with means of 61.3%, 59% and 44% respectively. The majority of CMV specific CD8+ T cells produced both IFN-γ and MIP-1β (80–96%) with a substantial proportion of these also producing TNF (72%, 44–88.5%). IL-2 producing CD8+ T cells were less frequent, ranging from 0.5–40%. However, IL-2 producers consistently exhibited the highest level of functionality with the production of all four cytokines. Furthermore, IFN-γ producing CD8+ T cells mobilized CD107, a marker of degranulation and cytotoxic activity. In the majority of cases, fewer CD4+ T cells exhibited specificity towards CMV pp65 (30%, 4–49.5%) however greater than 80% co-produced IFN-γ, MIP-1β and TNF. Furthermore, a high proportion of CD4+ T cells also produced IL-2 (53.4%). Adenovirus specific T cell responses were detected in all cultures but were mainly confined to the CD4+ population. Finally, we examined CMV specific responses in the starting donor PBMC population. Cytokine production profiles of CMV specific CD4 and CD8 T cells closely resembled those of ex vivo generated cultures suggesting the uniform expansion of CMV specific functional subsets. In conclusion, these results demonstrate that ex vivo expanded CMV specific T cells intended for adoptive transfer perform several functions associated with protective immunity in vivo, including the capacity to kill infected cells and the simultaneous production of multiple cytokines.

Recombinant Human Interleukin-7 (CYT107) Enhances CD4 and CD8 T Cell Recovery Following T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplant In Patients with Myeloid Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 674-674
Author(s):  
Miguel-Angel Perales ◽  
Jenna D. Goldberg ◽  
Leuren Lechner ◽  
Jianda Yuan ◽  
Esperanza Papadopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Transplant ◽  
Immune Recovery ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Interleukin 7 ◽  
T Cell Reconstitution

Abstract Abstract 674 Immune recovery is an important determinant in multiple outcomes following allogeneic hematopoietic stem cell transplant (allo-HSCT). Delays in B and T cell reconstitution are associated with an increased risk of infection, relapse and secondary malignancy. Strategies to enhance post-transplant T-cell reconstitution could therefore improve morbidity and mortality after allo-HSCT. The cytokine Interleukin-7 (IL-7) is a unique therapeutic candidate to promote immune reconstitution because it has a central role in T cell development and survival. Murine models of allo-HSCT have demonstrated that IL-7 can enhance thymopoiesis as well as promote peripheral T cell survival and expansion. Initial clinical trials performed with recombinant human IL-7 (rhIL-7) have demonstrated a dose-dependent expansion of CD4+ and CD8+ T cells with an acceptable toxicity profile in patients with solid tumors or HIV infection. Hence we are conducting a phase I trial of post-transplant administration of rhIL-7 (CYT107, Cytheris Inc) in recipients of a T cell depleted (TCD) allo-HSCT to determine the safety, toxicity and biological activity on T cell reconstitution. To date, 9 patients (AML=7, MDS=2), with a median age of 59.3 years (range 27–67 years) have been treated with escalating doses of rhIL-7 (3 at 10 mcg/kg, 6 at 20 mcg/kg) administered subcutaneously weekly for 3 weeks following TCD allo-HSCT from an HLA compatible donor. Accrual is ongoing in the final cohort (30 mcg/kg). Recombinant hIL-7 was started at a median of 96 days post allo-HSCT (range 61–244 days). Most patients experienced transient minor injection site reactions. One patient (20 mcg/kg) developed a biopsy proven hypersensitivity drug rash a week after the first injection and was removed from the study (evaluable for toxicity but not immune recovery endpoints). No other significant injection-related toxicities have occurred, and no patients have developed GVHD. No anti-IL-7 antibodies or neutralizing antibodies have developed following rhIL-7 injection. Two of 9 patients with high-risk AML have relapsed (4 and 9 months post rhIL-7), an incidence consistent with published data in patients undergoing allo-HSCT for AML in CR, irrespective of T-cell depletion. Eight patients remain alive with a median follow-up of 14.5 months post rhIL-7 administration. At baseline, the median T cell counts were 91/mm3 (range 5 – 219 /mm3), 43/mm3 (range 9 – 299 /mm3) and 0 (range 0 – 17 /mm3) for CD4+, CD8+ and CD45RA+ T cells, respectively. Preliminary assessment of the immunological effects of rhIL-7 in 8 evaluable patients has demonstrated an increase in CD4+ T cells exhibiting a naïve or central memory phenotype (69% median increase over baseline at day 21 – range 8% to 35-fold increase), and CD8+ T cells exhibiting a naïve or effector memory phenotype (94% median increase over baseline at day 28 – range 0 to 11-fold increase). There was no observed effect on the frequency of CD4+CD25+FoxP3+ T cells or CD19+ B cells. TCR excision circles (TREC) analysis performed on CD4+ and CD8+ subsets in the first 6 patients, using absolute quantification real-time PCR, demonstrated increases in TRECs in 5/6 patients indicating enhanced T cell production. Finally, all 3 CMV-seropositive patients developed CD8+ T cell CMV-specific responses detected by intracellular IFNγ production to overlapping CMV-pp65 pentadecapeptides peptide pools after administration of rhIL-7. In one patient, we also analyzed CMV-specific T-cell frequency using HLA-A*0201 restricted MHC-tetramers. The highest CMV-specific response levels were noted in this patient with a history of CMV viremia and low-level CMV-specific CD8+ T cells prior to rhIL-7 (5.3-fold increase to the A0201-restricted immunodominant NLV peptide by tetramer assay after rhIL-7). Our pre-clinical data and early clinical results suggest that administration of rhIL-7 in recipients of a TCD allo-HSCT has minimal toxicity and can enhance post-transplant immune recovery without causing GVHD. Disclosures: Perales: Cytheris: Research Funding. Croughs:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morre:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van den Brink:Cytheris: Research Funding.

The Critical Requirement of the NKG2D Receptor On CD8+ T Cells in Killing Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4733-4733
Author(s):  
Laleh X. Talebian ◽  
Dawn A Fischer ◽  
Zbigniew M Szczepiorkowski ◽  
Charles L Sentman ◽  
Kenneth R Meehan
Keyword(s):  
T Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Class I ◽  
Myeloma Cells ◽  
Nkg2d Receptor ◽  
Tumor Cell Killing

Abstract Abstract 4733 Autologous stem cell transplant for myeloma improves survival, but the immunologic mechanisms accounting for this improvement are unknown. Our data indicate that NKG2D+CD8+T cells may be one reason for this benefit. NKG2D, one of four NK cell activating receptors, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. We identified methods for ex vivo expansion of cyclophosphamide – mobilized blood progenitor cells (BPCs) from myeloma patients, as part of a clinical trial (Cytotherapy 2008). Mobilized BPC are cultured in serum-free media with IL-2 (50 IU/ml) and OKT3 (50ng/ml). After 7 days, the CD8+ T cells are isolated, evaluated and tested. Myeloma cells from patients' marrow aspirates are used as targets in cytotoxicity assays. Phenotypic analysis of the expanded cells and the patients' primary myeloma cells was performed using flow cytometry. Ex vivo expansion of cyclophosphamide-mobilized BPCs induces CD8+ T cells that acquire the NKG2D receptor during ex vivo expansion (P < 0.03). Three of the 6 known NKG2D ligands are strongly expressed on patients' myeloma cells, including MICA, ULBP1 and ULBP3. Using cytotoxicity assays, autologous effector cells recognize and kill the patient's autologous tumor cells (E:T 50:1, P< 0.001). When using K562 leukemia cells as targets (lack MHC class I), the NKG2D+CD8+ T cells kill K562, supporting an MHC class I-independent mechanism of tumor cell killing. Blocking the NKG2D receptor on CD8+ T cells prevents killing of autologous myeloma cells (P < 0.009). NKG2D-mediated cytotoxicity correlates with the amount of ligand expression on the target. The NKG2D receptor on CD8+T cells recognizes ligands expressed on autologous myeloma cells. The NKG2D+CD8+T cells aggressively kill myeloma cells in a non–MHC restricted and TCR-independent manner. Blocking NKG2D on CD8+ T cells prevents killing of autologous myeloma cells. Since tumor cells often down regulate MHC Class I expression, the ability of NKG2D+CD8+ T cells to recognize and kill autologous myeloma cells in a non-MHC restricted manner may contribute to the beneficial effects in the treatment of myeloma. Ongoing experiments are testing these methods in mouse models and defining the molecular mechanisms involved. Disclosures: Szczepiorkowski: Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding. Meehan:Berlex Pharmaceutical: Research Funding.

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