γδ T Cells in Normal Murine Spleen Assist Immunized αβ T Cells in the Adoptive Cell Transfer of Contact Sensitivity: Effect of <i>Bordetella pertussis</i>. Cyclophosphamide, and Antisuppressor T Cell Monoclonal Antibodies

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.

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Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000311 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000311 ◽

Cited By ~ 6

Author(s):

Lucine Marotte ◽

Sylvain Simon ◽

Virginie Vignard ◽

Emilie Dupre ◽

Malika Gantier ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Adoptive Transfer ◽

Adoptive Cell Transfer ◽

Effector Memory ◽

High Avidity ◽

Cell Transfer ◽

T Cell Clones ◽

Cell Clones

BackgroundGenome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far,PDCD1editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.MethodsHere we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to editPDCD1gene in human effector memory CD8+T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validatedPDCD1editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain’s sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR.ResultsHere we demonstrated the feasibility to editPDCD1gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent onPDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model.ConclusionThe use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

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An innate-like Vδ1+ γδ T cell compartment in the human breast is associated with remission in triple-negative breast cancer

Science Translational Medicine ◽

10.1126/scitranslmed.aax9364 ◽

2019 ◽

Vol 11 (513) ◽

pp. eaax9364 ◽

Cited By ~ 23

Author(s):

Yin Wu ◽

Fernanda Kyle-Cezar ◽

Richard T. Woolf ◽

Cristina Naceur-Lombardelli ◽

Julie Owen ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

T Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Γδ T Cells ◽

Γδ T Cell ◽

Human Breast ◽

Cell Compartment ◽

Αβ T Cells

Innate-like tissue-resident γδ T cell compartments capable of protecting against carcinogenesis are well established in mice. Conversely, the degree to which they exist in humans, their potential properties, and their contributions to host benefit are mostly unresolved. Here, we demonstrate that healthy human breast harbors a distinct γδ T cell compartment, primarily expressing T cell receptor (TCR) Vδ1 chains, by comparison to Vδ2 chains that predominate in peripheral blood. Breast-resident Vδ1+ cells were functionally skewed toward cytolysis and IFN-γ production, but not IL-17, which has been linked with inflammatory pathologies. Breast-resident Vδ1+ cells could be activated innately via the NKG2D receptor, whereas neighboring CD8+ αβ T cells required TCR signaling. A comparable population of Vδ1+ cells was found in human breast tumors, and when paired tumor and nonmalignant samples from 11 patients with triple-negative breast cancer were analyzed, progression-free and overall survival correlated with Vδ1+ cell representation, but not with either total γδ T cells or Vδ2+ T cells. As expected, progression-free survival also correlated with αβ TCRs. However, whereas in most cases TCRαβ repertoires focused, typical of antigen-specific responses, this was not observed for Vδ1+ cells, consistent with their innate-like responsiveness. Thus, maximal patient benefit may accrue from the collaboration of innate-like responses mounted by tissue-resident Vδ1+ compartments and adaptive responses mounted by αβ T cells.

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Mycobacterium-Specific γ9δ2T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity

Infection and Immunity ◽

10.1128/iai.01262-15 ◽

2015 ◽

Vol 84 (2) ◽

pp. 580-589 ◽

Cited By ~ 10

Author(s):

Getahun Abate ◽

Charles T. Spencer ◽

Fahreta Hamzabegovic ◽

Azra Blazevic ◽

Mei Xia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Γδ T Cells ◽

Cd40 Ligand ◽

Cell Contact ◽

Protective Effects ◽

Inhibitory Effects ◽

Content Type ◽

T Cell Lines ◽

Αβ T Cells

Numerous pathogens, includingMycobacterium tuberculosis, can activate human γ9δ2T cells to proliferate and express effector mechanisms. γ9δ2T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We preparedM. tuberculosis-specific γ9δ2T cell lines to study their direct protective effects and APC functions forM. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ9δ2T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ9δ2T cells enhance the expansion ofM. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ9δ2T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ9δ2T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4+αβ T cells and γ9δ2T cells provide similar immune enhancing/APC functions forM. tuberculosis-specific T cells. These effector and helper effects of γ9δ2T cells further indicate that these T cells should be considered important new targets for new TB vaccines.

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Recovery Of Gamma/Delta+ T Cells After Transplantation With Alpha-Beta+/CD19+ Lymphocyte Depleted Hematopoietic Stem Cells From HLA-Haploidentical Donors

Blood ◽

10.1182/blood.v122.21.3245.3245 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3245-3245

Author(s):

Irma Airoldi ◽

Ignazia Prigione ◽

Alice Bertaina ◽

Claudia Cocco ◽

Daria Pagliara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Zoledronic Acid ◽

Γδ T Cells ◽

Leukemia Cells ◽

Γδ T Cell ◽

Hematopoietic Stem ◽

Functional Studies ◽

Αβ T Cells ◽

Over Time

Abstract HLA-haploidentical hematopoietic stem cell transplantation (HSCT) using CD34+ selected cells is a widely used procedure, which, however, is complicated by delayed immune reconstitution. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature natural killer (NK) cells and γδ+ T cells in the graft. These cells can exert a graft-versus-leukemia (GvL) effect and reduce the risk of infection. In particular, unconventional γδ T cells play a critical role in both innate and adaptive immunity and exert HLA-unrestricted cytotoxicity against both solid and hematological tumors, thus potentially acting as beneficial effector cells in transplanted patients. Moreover, such grafts may limit the risk of graft-versus-host disease and prevent EBV-related lymphoproliferative disease. We performed phenotypic and functional studies on γδ T cells collected from 20 pediatric patients (pts, 13 males, 7 females, median age 10 years, range 6 months to 16 years) that received this type of allograft. Eighteen pts had acute leukemia and 2 non-malignant disorders. Ex vivo assays of peripheral blood γδ T cell phenotype and function were performed weekly until Hospital discharge and monthly until 6 months after HSCT. Phenotype of γδ T cells was analysed by flow cytometry. Analyses were performed on mononuclear cells labelled with mAb panels (CD3, CD45, pan-γδ, anti-Vδ1, -Vδ2, -Vγ9, CD45RO, CD45RA, CD27, CD16, CD56) allowing the identification of the main γδ+ T cell subsets, including Vδ1+ and Vδ2+ cells, naïve, central memory (CM), effector memory (EM) and terminally differentiated (TD) γδ T cells. Functional studies were performed using γδ T cells shortly after collection from pts, as well as after in vitro expansion with zoledronic acid and IL-2 for 10 days. Cytotoxic activity of γδ T cells was tested against primary leukemia cells, through CD107a degranulation assay and/or standard 51Cr-release assay. In the first 4 weeks after HSCT, T cells were consistently of the γδ subset (>90% of CD45+CD3+ cells); by contrast, αβ+ T cells gradually increased over time. In approximately half of the pts, the percentage of αβ T cells exceeded that of γδ T cells already starting from 30 days after HSCT. γδ T cells consisted of Vδ2+Vγ9+ and Vδ1+Vγ9+/- cells, and marginally of the Vδ1-Vδ2-Vγ9- population. Detailed phenotypic characterization of Vδ1+ and Vδ2+ γδ T cells revealed that, at day +20 after HSCT, 44% of Vδ1+ cells were CM (identified as CD45RO+CD27+ cells), 26% naïve (CD45RO-CD27+), 21.4% TD (CD45RO-CD27-) and 6.1% EM (CD45RO+CD27-). Similarly, 55.4% of Vδ2+ γδ T lymphocytes were CM, 9.8% naïve, 11.4% TD and 23.1% EM. The proportion of the different Vδ2+ γδ T cell subset did not change significantly over time, especially when comparing that present at day +20 after HSCT (time point, TP1) with that measured 30 days after the attainment of a 1:1 ratio of αβ-to- γδ T cells (TP2) (Figure 1, left panel). By contrast, by comparing TP1 and TP2, we found that Vδ1+ CM γδ T cells decreased and EM cells increased over time, while naïve or TD Vδ1+ γδ T cells did not change (Figure 1, right panel). In transplanted pts experiencing cytomegalovirus (CMV) reactivation, γδ T cells mostly consisted of Vδ1+ cells (mean 59.8% of γδ T cells), among which 49% were TD, 22.7% EM, 18.9% CM and 10.1% naïve. Noteworthy, in transplanted pts who did not have CMV reactivation, the main γδ T cells showed a Vδ2+ phenotype. Functional studies revealed that pt-derived γδ T cells consistently expanded in vitro after exposure to zoledronic acid and IL-2, the resulting Vγ9Vδ2 population expressing mainly an EM phenotype. These Vγ9Vδ2 cells exerted cytotoxic activities against primary allogeneic leukemia cells, especially when leukemia cells were pre-treated with zoledronic acid (Figure 2). More importantly, both Vδ1+ and Vδ2+ γδ T cells obtained from transplanted pts showed cytotoxic activity against primary leukemia cells, as assessed by CD107a degranulation assay. In conclusion, we provide the first phenotypic and functional characterization of γδ T cells, analyzed over time in children transplanted with grafts depleted of αβ+ T cells and of B lymphocytes. Our results support the concept that γδ T cells are important effector cells, which can be expanded and activated after exposure to bisphosphonates and IL-2 with the aim of improving their killing capacity against leukemia cells. Disclosures: No relevant conflicts of interest to declare.

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Anti-Tumor Cytotoxicity of γδ T Cells Expanded from Blood Cells of Myeloma and Leukemia Patients Against Self Tumor Cells — Enhancement of the Anti-Tumor Cytotoxicity by Type I IFN, Dendritic Cells, and Activated αβ T Cells.

Blood ◽

10.1182/blood.v110.11.4762.4762 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4762-4762

Author(s):

Anri Saito ◽

Miwako Narita ◽

Norihiro Watanabe ◽

Nozomi Tochiki ◽

Noriyuki Satoh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Hematological Malignancies ◽

Γδ T Cells ◽

Type I ◽

Type I Ifn ◽

Cd69 Expression ◽

Tumor Cytotoxicity ◽

Αβ T Cells

Abstract In order to establish an efficient gd T cell-mediated immunotherapy for hematological malignancies, we tried to clarify whether γδ T cells could be expanded from blood cells of patients with myeloma, lymphoma and acute leukemia by culture with zoledronate and a low dose of IL-2 and whether the expanded patients’ γδ T cells could kill tumor cells including self tumor cells with sparing normal clone cells. In addition, we explored the methods to enhance the anti-tumor cytotoxicity of the expanded γδ T cells by activating them with type I IFN, monocyte-derived dendritic cells (mo-DCs), or ab T cells. Although γδ T cells could be expanded in patients with myeloma, lymphoma and leukemia as well as normal persons, the amplification rates of gd T cells before and after the culture were varied from patient to patient in the patients with hematological malignancies. γδ T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines (RPMI8226, Daudi) as shown in γδ T cells generated in normal persons. In addition, γδ T cells generated in a patient with myeloma and acute leukemia showed a cytotoxic ability against self myeloma or leukemia cells freshly prepared from bone marrow. However, the same γδ T cells were not cytotoxic to normal lymphocytes of the patients. Then the expanded γδ T cells were stimulated with type I IFN, mo-DCs, or αβ T cells and the activation (CD69 expression) and cytotoxicity against tumor cells were examined. By the stimulation with type I IFN, the expression of CD69 and Trail of γδ T cells was increased and the cytotoxic ability of γδ T cells was enhanced at dose-dependent manner of type I IFN. CD69 expression on γδ T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after the addition of mo-DCs of either autologous or allogeneic origin. Activation of γδ T cells with mo-DCs enhanced anti-tumor cytotoxicity of γδ T cells against RPMI8226 and CML blastic crisis cell line (C2F8) in an effector-to-target ratio-dependent manner. Although CD69 expression of γδ T cells was enhanced by the co-culture with allogeneic ab T cells, autologous ab T cells couldn’t activate γδ T cells. However, autologous ab T cells stimulated with IL-2 or PHA could induce the activation of γδ T cells. The activation of γδ T cells with stimulated αβ T cells required cell-to-cell interaction. These findings suggested that αβ T cells stimulated by allogeneic γδ T cells could activate the same allogeneic γδ T cells. The present data demonstrated that γδ T cells, which could be expanded in vitro from blood cells of the patients with myeloma, lymphoma and leukemia by culture with zoledronate and IL-2, possess an enough cytotoxic ability against tumor cells including self tumor cells with sparing normal cells. These findings suggested that in vitro generated patients’ γδ T cells could be applied to γδ T cell-mediated immunotherapy for hematological malignancies. Besides, potent γδ T cells activated by type I IFN, mo-DCs or activated αβ T cells were considered to be applicable for γδ T cell-mediated immunotherapy.

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Donor γδ T Lymphocytes Promote Allogeneic Engraftment Across the Major Histocompatibility Barrier in Mice

Blood ◽

10.1182/blood.v89.3.1100 ◽

1997 ◽

Vol 89 (3) ◽

pp. 1100-1109 ◽

Cited By ~ 34

Author(s):

William R. Drobyski ◽

David Majewski

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Γδ T Cells ◽

Cell Receptor ◽

Marrow Graft ◽

Hematopoietic Reconstitution ◽

Donor T Cells ◽

Αβ T Cells

Abstract T cells that express the αβ T-cell receptor are thought to be the T-cell population primarily responsible for facilitating alloengraftment. The role of γδ+ T cells that comprise only a minority of mature T cells in promoting allogeneic engraftment, however, has not been extensively studied. The purpose of this study was to determine whether γδ T cells were capable of facilitating alloengraftment in murine recipients of major histocompatibility complex-mismatched marrow grafts. We developed a model where engraftment of C57BL/6 × 129/F2 (H-2b) marrow in sublethally irradiated (800 cGy) recipients (AKR/J, H-2k) is dependent on the presence of mature donor T cells in the marrow graft. In this model, donor T-cell engraftment was significantly augmented by as few as 1 × 105 αβ T cells. The role of γδ T cells was then investigated using transgenic donors (C57BL/6 × 129 background) in which a portion of the T-cell receptor–β chain gene was deleted by gene targeting so that these mice lack αβ T cells. Addition of 10 × 106 naive γδ T cells to T-cell depleted marrow grafts was required to significantly increase alloengraftment, although donor T cells averaged <50% of total splenic T cells. To determine whether higher doses of γδ T cells would improve donor engraftment and eradicate residual host T cells, γδ T cells were ex vivo expanded with a γδ T-cell–specific monoclonal antibody and interleukin-2 and then transplanted into irradiated recipients. Transplantation of ≥ 160 × 106 activated γδ T cells was necessary to consistently and significantly augment donor cell chimerism and enhance hematopoietic reconstitution when compared to control mice, but host T cells persisted in these chimeras. Addition of 2.5 × 104 mature αβ T cells, which alone were incapable of facilitating engraftment, to T-cell depleted marrow grafts containing 160 × 106 activated γδ T cells resulted in long-term (<100 day) complete donor engraftment, indicating that limiting numbers of αβ T cells were required in the marrow graft for the eradication of residual host T cells. Using serial weight curves and B-cell reconstitution as end points, clinically significant graft-versus-host disease was not observed in these chimeras under these experimental conditions. These data show that, whereas less potent than αβ T cells, γδ T cells are able to promote engraftment and enhance hematopoietic reconstitution in allogeneic marrow transplant recipients.

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Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

Blood ◽

10.1182/blood-2004-06-2482 ◽

2005 ◽

Vol 105 (1) ◽

pp. 241-250 ◽

Cited By ~ 203

Author(s):

Daniel J. Powell ◽

Mark E. Dudley ◽

Paul F. Robbins ◽

Steven A. Rosenberg

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Late Stage ◽

Adoptive Cell Transfer ◽

Effector Memory ◽

Cell Transfer ◽

Effector Cells

Abstract In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA- CD62 ligand- (CD62L-) CC chemokine receptor 7- (CCR7-) interleukin-7 receptor αLo (IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L- CCR7- profile, which may explain in part their ability to mediate tumor destruction. (Blood. 2005;105:241-250)

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Immunophenotypic Attributes of Benign Peripheral Blood γδ T Cells and Conditions Associated With Their Increase

Archives of Pathology & Laboratory Medicine ◽

10.5858/132.11.1774 ◽

2008 ◽

Vol 132 (11) ◽

pp. 1774-1780

Author(s):

Anja C. Roden ◽

William G. Morice ◽

Curtis A. Hanson

Keyword(s):

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Laboratory Finding ◽

Cell Subset ◽

Γδ T Cells ◽

Lymphocytic Leukemia ◽

Design Flow ◽

Immune System Activation ◽

Αβ T Cells

Abstract Context.—In comparison to αβ T cells, little is known about the immunophenotype of healthy peripheral blood γδ T cells or about conditions associated with expansion of this usually minor T-cell subset. Objective.—To study the immunophenotype of increased nonneoplastic peripheral blood γδ T cells and to determine clinical conditions associated with this laboratory finding. Design.—Flow cytometric T-cell phenotyping studies performed on 352 consecutive peripheral blood specimens were reviewed, and 62 cases (18%) in which γδ T cells comprised either more than 5% of the total lymphocytes or had an absolute count of more than 200 cells per μL or both, were studied further. Clinical data were available from 36 cases. Results.—The γδ T cells often had an immunophenotype distinct from the αβ T cells, with differences in CD5 expression as the most common (n = 17), followed by differences in CD3 (n = 6) and CD7 (n = 3). CD16 coexpression by the γδ T cells was also frequent (n = 20). In 28 (78%) of 36 cases, there were one or more associated conditions: infection/inflammatory disease (n = 18), autoimmune disease (n = 9), lymphoproliferative disorder (n = 6), and splenectomy (n = 3). Conclusions.—Circulating γδ T cells are immunophenotypically distinct from αβ T cells, and mild increases in these cells are not uncommon and may be associated with immune system activation and splenectomy. Recognition of this phenomenon is important because reactive γδ T cells can exhibit distinctive immunophenotypic features that are also encountered in neoplastic conditions, such as T-cell large granular lymphocytic leukemia.

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