Control of chronic lymphocytic leukemia development by clonally-expanded CD8+ T-cells that undergo functional exhaustion in secondary lymphoid tissues

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

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Phenotypic alteration of CD8+ T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming

Oncotarget ◽

10.18632/oncotarget.9941 ◽

2016 ◽

Vol 7 (26) ◽

pp. 40558-40570 ◽

Cited By ~ 18

Author(s):

Jiazhu Wu ◽

Xiaojing Xu ◽

Eun-Joon Lee ◽

Austin Y. Shull ◽

Lirong Pei ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Epigenetic Reprogramming ◽

Lymphocytic Leukemia ◽

Phenotypic Alteration

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Blockade of PD-1 and TIM-3 immune checkpoints fails to restore the function of exhausted CD8+ T cells in early clinical stages of chronic lymphocytic leukemia

Immunologic Research ◽

10.1007/s12026-020-09146-4 ◽

2020 ◽

Vol 68 (5) ◽

pp. 269-279 ◽

Cited By ~ 1

Author(s):

Hadiseh Rezazadeh ◽

Mojgan Astaneh ◽

Mohsen Tehrani ◽

Hadi Hossein-Nataj ◽

Ehsan Zaboli ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Lymphocytic Leukemia ◽

Immune Checkpoints ◽

Clinical Stages

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RHAMM/CD168 Is a Novel Leukemia Associated Antigen with Prognostic Value for Patients with B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v108.11.2773.2773 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2773-2773

Author(s):

Krzysztof Giannopoulos ◽

Alexander Krober ◽

Anna Dmoszynska ◽

Jacek Rolinski ◽

Hartmut Dohner ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Surrogate Marker ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Free Survival ◽

Cell Chronic Lymphocytic Leukemia

Abstract Background and Aims: Differential expression of molecules in patients with B-cell chronic lymphocytic leukemia (B-CLL) might define suitable targets for T cell based vaccines and/or antibody approaches. Methods: We assessed the mRNA expression of the tumor associated antigen (TAA) RHAMM/CD168 defined earlier by serological analysis of cDNA expression libraries (SEREX) from leukemic cells. Results: Peripheral blood mononuclear cells from 40 B-CLL patients and 20 healthy volunteers (HVs) were examined by quantitative RT-PCR. A leukemia-restricted expression of the antigen RHAMM/CD168 was observed in 39/40 B-CLL patients in contrast to the absence of its expression in HVs. To evaluate the immunogenicity of this novel LAA, mixed lymphocyte peptide cultures (MLPCs), followed by enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry assays were performed to detect antigen-specific CD8+ T cells. RHAMM/CD168 specific responses by CD8+ HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. As these CD8+ T cells contribute to the elimination of RHAMM+ CLL cells, we questioned whether expression of the antigen would be associated with a better survival. RHAMM/CD168 expression revealed to be higher in patients with unmutated IgVH status. RHAMM normalized against the housekeeping gene TATA binding protein (TBP), i.e. the RHAMM/TBP ratio was defined as a prognostic surrogate marker for B-CLL. B-CLL patients with a RHAMM/TBP ratio > 1.67 showed a significantly shorter treatment free survival (TFS) (Fig. 1). A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, an antigen correlating with the clinical course of the disease. Therefore, we consider RHAMM/CD168 an interesting target for immunotherapy in early stage B-CLL patients, especially with worse prognosis (IgVH unmutated). Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio

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Defective T Cell Migration in Chronic Lymphocytic Leukemia Is Repaired by Lenalidomide

Blood ◽

10.1182/blood.v112.11.3117.3117 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3117-3117

Author(s):

Alan G. Ramsay ◽

Lena Svensson ◽

Nancy Hogg ◽

John G. Gribben

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Direct Contact ◽

Immune Surveillance ◽

Lymphocytic Leukemia ◽

T Cell Migration

Abstract We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p<0.01). We further postulated that direct contact of CLL tumor cells with healthy T cells would induce this migratory defect. Healthy CD4 or CD8 T cells were cocultured with either allogeneic CLL B cells or allogeneic healthy B cells and subsequently used in migration assays. Co-culture with CLL cells resulted in significantly reduced T cell migration compared with co-culture with healthy B cells (~ 44% reduction in migration, n=6, p<0.01). Evidence that direct contact was required to induce this migratory defect was shown when no effect was observed when cell-cell adhesion was prevented by pretreatment of CLL cells with anti-ICAM-1 blocking antibody prior to primary co-culture with healthy T cells. This cancer-induced migratory defect was repaired when CLL T cells were pretreated with the immunomodulatory drug Lenalidomide (1μM for 1hr). Treatment with this agent enhanced the migratory potential of CLL T cells to a speed comparable to untreated and treated healthy T cells. The finding that lenalidomide can restore rapid migration in patient T cells provides evidence that this agent may increase immune surveillance in CLL patients.

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Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2002-03-0746 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1063-1070 ◽

Cited By ~ 50

Author(s):

Mohammad-Reza Rezvany ◽

Mahmood Jeddi-Tehrani ◽

Hans Wigzell ◽

Anders Österborg ◽

Håkan Mellstedt

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

Abstract T-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.

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Expansion of CMV-specific CD8+CD45RA+CD27- T cells in B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2003-01-0182 ◽

2003 ◽

Vol 102 (3) ◽

pp. 1057-1063 ◽

Cited By ~ 68

Author(s):

Wendelina J. M. Mackus ◽

Florine N. J. Frakking ◽

Annette Grummels ◽

Laila E. Gamadia ◽

Godelieve J. de Bree ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Immune Responses ◽

B Cell ◽

Cd8 T Cells ◽

Cytotoxic T Cells ◽

Lymphocytic Leukemia ◽

The Absolute ◽

Cell Chronic Lymphocytic Leukemia

Abstract In patients with B-cell chronic lymphocytic leukemia (B-CLL), the absolute number of T cells is increased. Although it has been suggested that these T cells might be tumor specific, concrete evidence for this hypothesis is lacking. We performed a detailed immunophenotypic analysis of the T-cell compartment in the peripheral blood of 28 patients with B-CLL (Rai 0, n = 12; Rai I-II, n = 10; Rai III-IV, n = 6) and 12 healthy age-matched controls and measured the ability of these patients to mount specific immune responses. In all Rai stages a significant increase in the absolute numbers of CD3+ cells was observed. Whereas the number of CD4+ cells was not different from controls, patients with B-CLL showed significantly increased relative and absolute numbers of CD8+ cells, which exhibited a CD45RA+CD27- cytotoxic phenotype. Analysis of specific immune responses with tetrameric cytomegalovirus (CMV)–peptide complexes showed that patients with B-CLL had significantly increased numbers of tetramer-binding CMV-specific CD8+ T cells. The rise in the total number of CD8+ cytotoxic T cells was evident only in CMV-seropositive B-CLL patients. Thus, our data suggest that in patients with B-CLL the composition of T cells is shifted toward a CD8+ cytotoxic cell type in an effort to control infections with persistent viruses such as CMV. Moreover, they offer an explanation for the high incidence of CMV reactivation in CLL patients treated with T cell–depleting agents, such as the monoclonal antibody (mAb) alemtuzumab (Campath; α-CD52 mAb). Furthermore, because in CMV-seronegative patients no increase in cytotoxic CD8+ T cells is found, our studies do not support the hypothesis that tumor-specific T cells account for T-cell expansion in B-CLL.

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A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

Frontiers in Immunology ◽

10.3389/fimmu.2021.627020 ◽

2021 ◽

Vol 12 ◽

Author(s):

Piers E. M. Patten ◽

Gerardo Ferrer ◽

Shih-Shih Chen ◽

Jonathan E. Kolitz ◽

Kanti R. Rai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Intraperitoneal Administration ◽

Human Condition ◽

Lymphocytic Leukemia ◽

Lymphoid Tissues ◽

Quiescent State

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

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CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

Blood Advances ◽

10.1182/bloodadvances.2020003795 ◽

2021 ◽

Vol 5 (14) ◽

pp. 2817-2828

Author(s):

Matteo Grioni ◽

Arianna Brevi ◽

Elena Cattaneo ◽

Alessandra Rovida ◽

Jessica Bordini ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Antigen Specificity

Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

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Increased Expression of CD152 by Normal T Lymphocytes in Untreated Patients with B Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.963.963 ◽

2004 ◽

Vol 104 (11) ◽

pp. 963-963

Author(s):

Marina Motta ◽

Bobby Shelvin ◽

Susan Lerner ◽

Michael Keating ◽

William G. Wierda

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Lymphocytic Leukemia ◽

Peripheral Tolerance ◽

Regulatory Function ◽

Cell Phenotype ◽

Costimulatory Receptor

Abstract Patient with chronic lymphocytic leukemia (CLL) have defects in both cellular and humoral immunity. Despite reported increases in absolute T cell counts in untreated patients with CLL, abnormalities of T cell phenotype and function have been described as well as progressive hypogammaglobulinemia. Furthermore, defects are compounded by current treatments for the disease. Expansion and differentiation of normal antigen-specific T cells depends upon two signals: binding of the T cell receptor to antigen presented in the context of self MHC molecules and ligation of a costimulatory receptor. CD28 is the primary T cell surface costimulatory receptor and is constitutively expressed on almost all CD4+ and about 50% of CD8+ T cells. The ligands CD80 and CD86 bind CD28, thereby transducing the second enhancing signal for T cell proliferation and cytokine secretion. CD152 (CTLA-4) has homology to CD28 and binds to CD80 and CD86 with much higher affinity, but plays a critical role in the down regulating T cell responses and maintenance of peripheral tolerance. Surface CD152 is not normally expressed on resting T cells, but is induced upon activation. We hypothesized that in previously untreated patients with CLL, T cell anergy is the result of increased expression of CD152. Therefore, we studied the expression of surface and cytoplasmic CD152 (sCD152 and cCD152, respectively) in freshly isolated T cells from blood (N=40) and bone marrow (N=14) of previously untreated patients with CLL. Also, the activation status of these T cells was evaluated by evaluating IL-2 receptor subunit expression. CD4+ and CD8+ T cells from patients with CLL demonstrated significant increase in sCD152 and cCD152 compared to T cells from normal donors (Table 1). Table 1 Expression of CD152 by T Cells Mean % Positive T Cell Population Normal CLL P-value sCD152 N=13 N=40 CD4+ 0.8 5.0 <.01 CD4+/CD25+ 1.8 11.5 <.05 CD8+ 1.8 5.0 <.05 cCD152 N=13 N=19 CD4+ 6.9 40.4 <.01 CD4+/CD25+ 26.6 48.0 <.01 CD8+ 1.3 16.9 <.05 Furthermore, patients with CLL had an increased proportion of CD4+/CD25+/CD152+ cells. This subpopulation of T cells is known to have a regulatory function. T cells from patients with CLL (N=25) also showed an activated immunophenotype with significantly increased proportion of CD4+ and CD8+ T cells co-expressing the CD122/CD25 subunits of the IL-2 receptor compared to normal donors (N=10). No significant differences were seen in proportion or pattern of expression of these antigens between peripheral blood and bone marrow cells. These findings suggest that the T cells have been activated, however, may be primed for hyporesponsiveness and peripheral tolerance by expression of CD152. Correlations between CD152 expression and relevant clinical and biological variables were made in these previously untreated patients. The number of CD4+/CD152+ and CD4+/CD25+/CD152+ cells from patients with CLL inversely correlated with serum IgG and IgA levels. These findings suggest a further possible involvement of CD152 in the possible suppression of normal B cells in patients with CLL. The proportion of CD4+/CD25+/CD152+ cells also correlated with advanced Rai stage. In summary, T cells from patients with CLL are potentially primed for anergy by expression of CD152. Functional studies to investigate the role of CD152 and CD4+/CD25+/CD152+ cells in patients with CLL are ongoing, with the goal to develop immunotherapeutic strategies.

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