NK cells negatively regulate CD8 T cells to promote immune exhaustion and chronic Toxoplasma gondii infection

AbstractNK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii (T. gondii) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and reduces parasite reactivation. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell behavior. This splenic CD49a-CD49b+NKp46+ NK cell population develops during the early chronic phase of infection and increases through the late chronic phase of infection. They are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. They are also absent from brain. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced. Blockade of NKp46 also rescued the chronically infected mice from death. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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Immune Cell Dysfunction in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL)

Blood ◽

10.1182/blood.v120.21.3875.3875 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3875-3875 ◽

Cited By ~ 2

Author(s):

Marion E Cole ◽

Alexander MacFarlane ◽

Mowafaq Jillab ◽

Mitchell R Smith ◽

Adam D Cohen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Nk Cell ◽

Granzyme B ◽

Immune Dysfunction ◽

T Cell Subsets ◽

Cell Compartment

Abstract Abstract 3875 Introduction: Immunologic environment influences progression of lymphoid malignancies. Specifically, shifts in subsets of natural killer (NK) and T cells as well as tumor expression of inhibitory ligands may contribute to ability to evade host detection. Immune dysfunction may be particularly important in CLL/SLL, as prevalent circulating tumor cells engage in persistent, widespread interactions with immune cells; commonly-used mAb therapies (e.g. rituximab, alemtuzumab) rely upon ADCC mediated by NK cells and other innate effectors; and disease course is highly variable and not fully accounted for by tumor-intrinsic prognostic factors. Therefore, to better characterize the immune system in CLL/SLL, we prospectively assessed NK and T cell frequency, phenotype, and function in a series of CLL/SLL patients. Methods: Serial blood samples (up to 3 samples each, 3–6 months apart) were collected from 31 untreated CLL/SLL patients (median age 66) and 15 healthy age-matched controls (HC), and peripheral blood lymphocytes (PBL) analyzed directly ex vivo by multiparameter flow cytometry (160 distinct parameters evaluated, primarily on T and NK cells). NK cell-mediated natural and antibody-dependent cytotoxicity were also assessed by CD107a degranulation assay following PBL co-culture with rituximab, 721.221 EBV-transformed lymphoma cells, or both. Differences in parameters between patients and controls, or between progressors and non-progressors [categorized based on updated NCI-WG criteria (Blood 2008;111:5446)] were analyzed by Wilcoxon rank-sum test. All subjects signed IRB approved informed consent forms. Results: CLL/SLL VS. HC: CLL/SLL samples displayed a marked decrease in the ability of the cytolytic CD56dim NK cells to degranulate in response to tumor, both with or without rituximab (Table 1). CD56dim NK cells from CLL/SLL patients also displayed a more immature phenotype (↓CD57, ↓NKG2D, ↑CD27, ↓KIR) than those from HC, suggesting either a block in differentiation or elimination of the most-differentiated cells. NK cell expression of NKp44, CD69, CD62L, CD137, granzyme B, perforin, or PD-1, as well as tumor-induced NK cell production of IFNγ, did not differ. CLL/SLL patients had increased total T cells with a decreased CD4:CD8 ratio, associated with increased total number of CD8 T cells, greater activation of naive CD4 T cells and transition to a memory phenotype. Treg (CD4+CD25+FoxP3+) frequency was significantly higher in CLL/SLL patients (4.5% vs. 1.8% of CD4 T cells, p=0.005), as was PD-1 expression on both CD4 and CD8 T cells, while CD137 and ICOS expression was similar in both groups. PROGRESSORS VS. NON-PROGRESSORS: With median follow-up of 16.5 months (range 1–37), 7 of 31 patients have met criteria for progression. Compared to non-progressors, progressors showed changes in the CD56bright NK cell compartment suggestive of increased activation and accelerated differentiation, with increased expression of CD69, granzyme B, perforin, CD16, and KIR. However, no significant functional differences in NK cells, or consistent differences in T cell subsets, have been observed to date. Conclusions: CLL/SLL patients have a shift toward less mature NK cells, associated with deficits in NK cell degranulation against tumor targets, compared with healthy donors. Those CLL/SLL patients who progressed had greater CD56 bright NK cell phenotypic aberrancies than non-progressors, though these findings require confirmation with a larger cohort. Taken together, our findings support the hypothesis that immune dysfunction in CLL/SLL may be due in part to a block in NK cell differentiation or loss of more mature cells, and current studies are exploring these possibilities and potential mechanisms. Given these findings, along with the immunosuppressive changes observed in the T cell compartment (↑Tregs, ↑PD-1), these data support therapeutic strategies in CLL/SLL aimed at augmenting NK and/or T cell function. Disclosures: No relevant conflicts of interest to declare.

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451 Combining Bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) pairs local innate activation with systemic CD8+ T cell expansion to enhance anti-tumor immunity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0451 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A478-A478

Author(s):

Annah Rolig ◽

Daniel Rose ◽

Saul Kivimae ◽

Werner Rubas ◽

William Redmond

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cell Expansion ◽

Nk Cell ◽

Tumor Regression ◽

Cell Activity ◽

Cd8 T Cell ◽

Post Treatment

BackgroundPreviously, we demonstrated that radiation therapy (RT) combined with Bempegaldesleukin (BEMPEG;NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, led to enhanced anti-tumor efficacy through a T cell-dependent mechanism. However, we observed only modest systemic responses to BEMPEG/RT across several murine tumor models. Therefore, we explored alternative approaches to improve systemic tumor-specific immunity. We evaluated whether intratumoral NKTR-262, a polymer-modified toll-like receptor (TLR) 7/8 agonist, combined with systemic BEMPEG treatment resulted in improved tumor-specific immunity and survival compared to BEMPEG combined with RT. We hypothesized that BEMPEG/NKTR-262 immunotherapy would promote synergistic activation of local immunostimulatory innate immune responses followed by systemic adaptive immunity to significantly improve tumor regression and overall survival.MethodsTumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; iv), RT (12 Gy x 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell activation status in the blood and/or tumor (7 days post-treatment) and NK cell activity in the tumor (1, 3 days post-treatment). The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell activity was determined in vitro by tracking apoptosis in an Incucyte assay. Data are representative of 1–2 independent experiments (n=5–14/group) and statistical significance was determined by 1-way ANOVA (p-value cut-off of 0.05).ResultsBEMPEG/NKTR-262 resulted in significantly improved survival compared to BEMPEG/RT. BEMPEG/NKTR-262 efficacy was NK and CD8+ T cell-dependent, while BEMPEG/RT primarily relied on CD8+ T cells. Response to BEMPEG/NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p<0.05). In the tumor, NKTR-262/BEMPEG induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+, TIM-3+), compared to BEMPEG/RT. Indeed, CD8+ T cells isolated from BEMPEG/NKTR-262-treated tumors had greater cytolytic capacity than those from BEMPEG/RT-treated mice. CD8+ T cell expansion (blood) and activity (tumor) depended upon the initial NK response, as neither occurred in the absence of NK cells. BEMPEG/NKTR-262 uniquely induced the expansion of early and high effector NK cells.ConclusionsCombining BEMPEG with NKTR-262 lead to an early and robust NK cell expansion not observed in the BEMPEG/RT combination. The improved tumor regression and survival was dependent on the NKTR-262 driven expansion of NK cells. A clinical trial of BEMPEG/NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640).

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Role of Recipient CD8+ T Cell Exhaustion in the Rejection of Adoptively Transferred Haploidentical NK Cells

Blood ◽

10.1182/blood.v128.22.503.503 ◽

2016 ◽

Vol 128 (22) ◽

pp. 503-503

Author(s):

Robin Williams ◽

Sarah Cooley ◽

Veronika Bachanova ◽

Thomas A Waldmann ◽

Bruce R. Blazar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cell Expansion ◽

Nk Cell ◽

Cd8 T Cell ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Refractory Aml

Natural killer (NK) cells are cytotoxic innate lymphoid cells, which play a major role in tumor surveillance. We have tested the safety and efficacy of allogeneic NK cell adoptive transfer from heathy haploidentical donors and demonstrate that in vivo expansion and persistence of the adoptively transferred NK cells at Day 14 after infusion correlates with 30-50% remission in patients with refractory AML. However, the factors that influence successful persistence of donor-derived NK cells are unclear. We hypothesized that recipient T cells play a role in the rejection of allogeneic NK cells and a correlation could exist between persistence of donor-derived NK cells and exhaustion in recipient T cells. T cell exhaustion, a well-established state of T-cell dysfunction occurring in response to chronic and continuous antigen stimulation, is well-documented in human cancer, and characterized by progressive and hierarchical loss of effector functions including sustained up-regulation and co-expression of multiple inhibitory receptors such as PD-1 and Tim-3 and altered expression of key transcription factors including the gain of Eomes and T-bet. We used samples from a phase I/II trial of CD3/CD19 depleted, IL-15-activated, haploidentical donor NK cells delivered following conditioning with cyclophosphamide (50mg/kg) and fludarabine (35 mg/m2 x 3days) in adults with chemotherapy refractory AML. Patients received donor NK cells on Day 0 followed by 10 doses of recombinant human (rh) IL-15 (2 mcg/kg/day) manufactured by the NCI and delivered SQ on Days 1-5 and 8-12. A significant proportion of patients experienced donor NK cell expansion at Day 14 (expanders), but there were some that did not (non-expanders). Therapeutic benefit has only been noted among the expanders. We examined samples from a total of 10 patients with refractory AML, 5 expanders and 5 non-expanders, along with their 10 respective donors. Cryopreserved patient PBMCs were thawed and rested overnight in RPMI-1640 with 2% FBS. The cells were stained for viability, for surface markers using antibodies against CD3, CD8, CD56, PD-1, and Tim-3, intracellularly stained for Eomes and T-bet. We evaluated CD8+ T cell expression of PD-1 and Tim-3, in expanders and non-expanders, prior to chemotherapy and at Day 14. Paired donor T cells from the non-mobilized apheresis products served as controls. Prior to chemotherapy, both patient groups had equivalently elevated expression of both PD1 and Tim-3 on CD8+ T cells. However, at Day 14, the expanders had persistence of PD-1 and Tim-3 while expression on non-expander CD8+ T cells fell to donor level (Figure 1A). Furthermore, expanders had a significantly higher proportion of CD8+ T cells that either co-expressed PD-1 and Tim-3 (p=0.017) or had a PD-1high phenotype (p=0.032) at Day 14, both of which are suggestive of an exhausted state, as opposed to an activated one (Figure 1B,C). Next, we examined Eomes and T-bet expression in recipient T cells. While generally low among healthy T cell populations, as T cells become exhausted, they gain expression of these transcription factors. We looked specifically at the expression of these transcription factors in the recipient CD8+ T cell populations with the highest likelihood of being exhausted, i.e. those co-expressing PD-1 and Tim-3 or those with the PD-1high phenotype. Eomes expression in recipient PD-1high CD8+ T cells and in PD-1+Tim-3+ CD8+ T cells at Day 14 was significantly higher (p=0.01 and p=0.04, respectively) among expanders compared to non-expanders (Figure 2A,B). Likewise, T-bet expression was greater (p=0.004) among expanders in the PD-1high population (Figure 2A). There was no difference in the T-bet expression in PD-1+Tim-3+ CD8+ T cells between groups (Figure 2B). While all patients with refractory AML receiving NK cell adoptive transfer had an elevated percentage of CD8+ T cells with an exhausted phenotype prior to therapy, only patients with donor-derived NK cell expansion had persistence of the exhausted T cell phenotype at Day 14. Thus, T cell mediated rejection is a major obstacle to overcome for successful adoptive NK cell transfer which could in part be aided by a link between recipient T cell exhaustion and expansion of NK cells. This might further suggest that IL-15 reverses T cell exhaustion among those who failed to achieve donor-derived NK cell expansion. Disclosures Miller: Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

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Transient and persistent effects of IL-15 on lymphocyte homeostasis in nonhuman primates

Blood ◽

10.1182/blood-2010-03-275438 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3238-3248 ◽

Cited By ~ 72

Author(s):

Enrico Lugli ◽

Carolyn K. Goldman ◽

Liyanage P. Perera ◽

Jeremy Smedley ◽

Rhonda Pung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

Nk Cell ◽

The Body ◽

Effector Memory ◽

Memory Cd8 T Cells

Abstract Interleukin-15 (IL-15) is a cytokine with potential therapeutic application in individuals with cancer or immunodeficiency to promote natural killer (NK)– and T-cell activation and proliferation or in vaccination protocols to generate long-lived memory T cells. Here we report that 10-50 μg/kg IL-15 administered intravenously daily for 12 days to rhesus macaques has both short- and long-lasting effects on T-cell homeostasis. Peripheral blood lymphopenia preceded a dramatic expansion of NK cells and memory CD8 T cells in the circulation, particularly a 4-fold expansion of central memory CD8 T cells and a 6-fold expansion of effector memory CD8 T cells. This expansion is a consequence of their activation in multiple tissues. A concomitant inverted CD4/CD8 T-cell ratio was observed throughout the body at day 13, a result of preferential CD8 expansion. Expanded T- and NK-cell populations declined in the blood soon after IL-15 was stopped, suggesting migration to extralymphoid sites. By day 48, homeostasis appears restored throughout the body, with the exception of the maintenance of an inverted CD4/CD8 ratio in lymph nodes. Thus, IL-15 generates a dramatic expansion of short-lived memory CD8 T cells and NK cells in immunocompetent macaques and has long-term effects on the balance of CD4+ and CD8+ T cells.

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AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽

10.1182/blood.v108.11.455.455 ◽

2006 ◽

Vol 108 (11) ◽

pp. 455-455 ◽

Cited By ~ 1

Author(s):

Federico Mingozzi ◽

Marcela V. Maus ◽

Denise E. Sabatino ◽

Daniel J. Hui ◽

John E.J. Rasko ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Cell Population ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Transgene Expression ◽

Cd8 T Cell ◽

Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

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Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.3300.3300 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3300-3300

Author(s):

Don Benson ◽

Leslie Andritsos ◽

Mehdi Hamadani ◽

Thomas Lin ◽

Joseph Flynn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Nk Cell ◽

Disease Status ◽

Lymphocytic Leukemia ◽

T Cell Subsets ◽

Cell Recovery

Abstract Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL. Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry. Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets. Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

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Myeloid Chimerism Reflects Engraftment of Donor Hematopoiesis, Whereas T Cell Chimerism Reflects Survival and Expansion of Donor and Recipient Residual Mature T Cells Early After T Cell Depleted Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.4475.4475 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4475-4475

Author(s):

Jessica C. Harskamp ◽

Esther H.M. van Egmond ◽

Hans L. Vos ◽

Stijn J.M. Halkes ◽

Roel Willemze ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cd8 T Cells ◽

Nk Cell ◽

Median Frequency ◽

Cell Compartment ◽

Hematopoietic Lineage ◽

Chimerism Analysis

Abstract Abstract 4475 Allogeneic stem cell transplantation (alloSCT) is frequently complicated by life-threatening graft versus host disease (GVHD). Previous studies demonstrated that T cell depletion (TCD) of the graft significantly decreases the incidence and severity of GVHD, and is associated with a higher percentage of patients with mixed chimerism (MC). In most studies chimerism analysis is performed on the total bone marrow (BM) leukocyte fraction, and changes in chimerism are related to engraftment. In this study we investigated whether MC in the total BM leukocyte fraction truly reflects engraftment or if it is influenced by survival and expansion of donor and recipient residual mature T cells, and whether hematopoietic lineage specific chimerism analysis is therefore a better method to determine engraftment. It is likely that chimerism analysis of the stem cell compartment is best reflected in peripheral blood (PB) in those cells that are continuously produced and short lived, such as monocytes and granulocytes, and therefore PB myeloid chimerism primarily reflects engraftment. In contrast, previous studies have shown by T cell receptor excision circle analysis that T cell neogenesis is virtually absent in the first 6 months after alloSCT, and that predominantly memory T cells are present in PB and BM. Therefore, we hypothesize that MC of these long lived T cells merely reflects survival and expansion of recipient and donor residual T cells. Since the life span of B and NK cells is longer than myeloid cells, but shorter than T cells, we anticipate that in the first 6 months after alloSCT, B and NK cell chimerism reflects a combination of survival and neogenesis. To analyze these hypotheses we performed hematopoietic lineage specific chimerism analysis on PB cells of 22 patients (median age 52 years, range 23-73, 11 males) receiving a TCD alloSCT between June and November 2008 after a myeloablative (n=11) or non myeloablative conditioning regimen (n=11) for AML, ALL, high risk MDS, multiple myeloma, CML, CLL or NHL. At intervals of 6 weeks PB was collected, and monocytes, granulocytes, B and NK cells, CD4+ and CD8+ T cells were sorted. The total leukocyte fraction was obtained by erythrocyte lysis of BM. DNA was isolated to perform chimerism analysis using short tandem repeats - PCR. Our results show that in the BM leukocyte fraction 47% of the patients were MC at 3 months after alloSCT, with a median frequency of patient cells of 4%. However, of the patients with MC in the total leukocyte fraction, 67% was complete chimeric in the myeloid subsets and MC in the T cell compartment. In the PB myeloid subsets (monocytes and granulocytes) less than 28% of the patients were MC during the first 6 months after alloSCT with a median frequency of patient cells less than 5%. In the B and NK cell subsets, at most time points more patients were MC (7-43%) with higher frequencies of patient cells (2-14%) compared to the myeloid subsets. The CD4 and CD8 T cell subsets showed the highest frequencies of MC in numbers of patients (31-61%) as well as the highest MC frequencies of patient cells (13-80%). Phenotypic analysis of the T cell compartment showed that 98% of the CD4 and CD8 T cells were memory cells during the first 6 months after alloSCT. Preliminary data indicate that the median percentage of donor derived T cells increased during the first 6 months after alloSCT, correlating with development of mild GVHD, suggesting that T cell chimerism is influenced by immunogenic triggers. In conclusion, these results illustrate that for engraftment and neogenesis of donor hematopoiesis, myeloid chimerism analysis provides more accurate information than total BM leukocyte chimerism analysis, since the results are greatly influenced by T cell chimerism. Since almost all T cells were memory cells within the first 6 months after alloSCT, T cell chimerism analysis reflects survival and expansion of mature donor as well as recipient T cells, and can therefore not be used to measure engraftment. Disclosures: No relevant conflicts of interest to declare.

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The tumor suppressor TSLC1/NECL-2 triggers NK-cell and CD8+ T-cell responses through the cell-surface receptor CRTAM

Blood ◽

10.1182/blood-2005-02-0817 ◽

2005 ◽

Vol 106 (3) ◽

pp. 779-786 ◽

Cited By ~ 114

Author(s):

Kent S. Boles ◽

Winfried Barchet ◽

Tom Diacovo ◽

Marina Cella ◽

Marco Colonna

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Suppressor ◽

Cell Surface ◽

Nk Cells ◽

Cd8 T Cells ◽

Nk Cell ◽

Cell Junctions ◽

Cell Surface Receptor ◽

Cytotoxic Lymphocytes

AbstractThe tumor suppressor in lung cancer-1 (TSLC1) gene is frequently silenced in human lung carcinomas, and its expression suppresses tumorigenesis in nude mice. TSLC1 encodes a cell-surface protein called Necl-2 that belongs to the Nectin and Nectin-like (Necl) family of molecules. Necl-2 mediates epithelial cell junctions by homotypic contacts and/or heterotypic interactions with other Nectins and Necls. Thus, it inhibits tumorigenesis by ensuring that epithelial cells grow in organized layers. Here, we demonstrate that natural killer (NK) cells and CD8+ T cells recognize Necl-2 through a receptor known as class I-restricted T-cell–associated molecule (CRTAM), which is expressed only on activated cells. CRTAM–Necl-2 interactions promote cytotoxicity of NK cells and interferon γ (IFN-γ) secretion of CD8+ T cells in vitro as well as NK cell–mediated rejection of tumors expressing Necl-2 in vivo. These results provide evidence for an additional mechanism of tumor suppression mediated by TSLC1 that involves cytotoxic lymphocytes. Furthermore, they reveal Necl-2 as one of the molecular targets that allows the immunosurveillance network to distinguish tumor cells from normal cells.

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Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions

Open Biology ◽

10.1098/rsob.160293 ◽

2016 ◽

Vol 6 (11) ◽

pp. 160293 ◽

Cited By ~ 5

Author(s):

Lee Kim Swee ◽

Zhen Wei Tan ◽

Anna Sanecka ◽

Nagisa Yoshida ◽

Harshil Patel ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Toxoplasma Gondii ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Effector Function ◽

Effector Functions ◽

Physiological Conditions ◽

Cell Clones

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii . We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro . Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.

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