HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL)

The breakthrough development of clinically effective immune checkpoint inhibitors illustrates the potential of T-cell–based immunotherapy to effectively treat malignancies. A remaining challenge is to increase and guide the specificities of anticancer immune responses, e.g., by therapeutic vaccination or by adoptive T-cell transfer. By analyzing the landscape of naturally presented HLA class I and II ligands of primary chronic lymphocytic leukemia (CLL), we delineated a novel category of tumor-associated T-cell antigens based on their exclusive and frequent representation in the HLA ligandome of leukemic cells. These antigens were validated across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. We demonstrate specific immune recognition of these antigens exclusively in CLL patients, with the frequencies of representation in CLL ligandomes correlating with the frequencies of immune recognition by patient T cells. Moreover, retrospective survival analysis revealed survival benefits for patients displaying immune responses to these antigens. These results directly imply these nonmutant self-peptides as pathophysiologically relevant tumor antigens and encourages their implementation for cancer immunotherapy.

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Immune Response Dysfunction in Chronic Lymphocytic Leukemia: Dissecting Molecular Mechanisms and Microenvironmental Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms21051825 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1825 ◽

Cited By ~ 8

Author(s):

Francesca Arruga ◽

Benjamin Baffour Gyau ◽

Andrea Iannello ◽

Nicoletta Vitale ◽

Tiziana Vaisitti ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Immune Responses ◽

T Lymphocyte ◽

Molecular Mechanisms ◽

Cell Activity ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Adaptive Immune

Representing the major cause of morbidity and mortality for chronic lymphocytic leukemia (CLL) patients, immunosuppression is a common feature of the disease. Effectors of the innate and the adaptive immune response show marked dysfunction and skewing towards the generation of a tolerant environment that favors disease expansion. Major deregulations are found in the T lymphocyte compartment, with inhibition of CD8+ cytotoxic and CD4+ activated effector T cells, replaced by exhausted and more tolerogenic subsets. Likewise, differentiation of monocytes towards a suppressive M2-like phenotype is induced at the expense of pro-inflammatory sub-populations. Thanks to their B-regulatory phenotype, leukemic cells play a central role in driving immunosuppression, progressively inhibiting immune responses. A number of signaling cascades triggered by soluble mediators and cell–cell contacts contribute to immunomodulation in CLL, fostered also by local environmental conditions, such as hypoxia and derived metabolic acidosis. Specifically, molecular pathways modulating T-cell activity in CLL, spanning from the best known cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) to the emerging T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)/CD155 axes, are attracting increasing research interest and therapeutic relevance also in the CLL field. On the other hand, in the microenvironment, the B cell receptor (BCR), which is undoubtedly the master regulator of leukemic cell behavior, plays an important role in orchestrating immune responses, as well. Lastly, local conditions of hypoxia, typical of the lymphoid niche, have major effects both on CLL cells and on non-leukemic immune cells, partly mediated through adenosine signaling, for which novel specific inhibitors are currently under development. In summary, this review will provide an overview of the molecular and microenvironmental mechanisms that modify innate and adaptive immune responses of CLL patients, focusing attention on those that may have therapeutic implications.

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A case of chronic lymphocytic leukemia with properties characteristic of natural killer cells

Blood ◽

10.1182/blood.v61.5.940.940 ◽

1983 ◽

Vol 61 (5) ◽

pp. 940-948 ◽

Cited By ~ 39

Author(s):

K Itoh ◽

K Tsuchikawa ◽

T Awataguchi ◽

K Shiiba ◽

K Kumagai

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemia Cells ◽

Surface Markers ◽

T Antigens ◽

Homogeneous Population

Abstract A case of chronic lymphocytic leukemia that consisted of a homogeneous population of cells that had properties similar to those described for natural killer (NK) cells is presented. These leukemic cells had a morphology of large granular lymphocytes (LGL) and receptors for sheep erythrocytes (ER) and for the Fc portion of IgG (Fc gamma-R). They expressed pan-T antigens OKT3 and Leu-4, but neither helper/inducer T- cell differentiation antigens OKT4 and Leu-3a nor cytotoxic/suppressor T-antigens OKT8 and Leu-2a. HNK 1 antigen, which can be expressed on human NK cells, could be detected on almost all leukemic cells (LGL), whereas a myeloid differentiation antigen, OKM1, which can be expressed on macrophages, granulocytes, and NK cells, was not detected. Thus, it was concluded that the leukemia cells had a characteristic profile of the surface markers: ER+, Fc gamma-R+, HNK-1+, OKT3+, Leu-4+, OKT4-, OKT8-, Leu-3a, Leu-2a, and OKM1-. Although freshly isolated leukemic cells showed no cytotoxicity on NK targets, after incubation at 37 degrees C, the cells did show a potent cytotoxicity on targets of erythroleukemic cell, T cell, and monocyte (but not B cell) origins. When the cells were incubated at 37 degrees C, interferon (IFN gamma) was spontaneously produced in the culture fluids. Treatment with anti- HNK-1 and complement completely abrogated expression of NK activity and interferon production of the patient's lymphocytes in culture. These characteristic features of surface markers and functions strongly suggest the possibility that the leukemia cells of this case are of NK cell origin. The relationship between this case and chronic lymphocytic leukemia of T-cell origin is discussed.

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CD40-Activated B-Cell Chronic Lymphocytic Leukemia Cells for Tumor Immunotherapy: Stimulation of Allogeneic Versus Autologous T Cells Generates Different Types of Effector Cells

Blood ◽

10.1182/blood.v93.6.1992.406k23_1992_2002 ◽

1999 ◽

Vol 93 (6) ◽

pp. 1992-2002 ◽

Cited By ~ 113

Author(s):

Raymund Buhmann ◽

Annette Nolte ◽

Doreen Westhaus ◽

Bertold Emmerich ◽

Michael Hallek

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Tumor Antigens ◽

Lymphocytic Leukemia ◽

T Cell Anergy ◽

Cell Anergy ◽

Cell Chronic Lymphocytic Leukemia ◽

Stimulation Of

Although spontaneous remissions may rarely occur in B-cell chronic lymphocytic leukemia (B-CLL), T cells do generally not develop a clinically significant response against B-CLL cells. Because this T-cell anergy against B-CLL cells may be caused by the inability of B-CLL cells to present tumor-antigens efficiently, we examined the possibility of upregulating critical costimulatory (B7-1 and B7-2) and adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen presentation. The stimulation of B-CLL cells via CD40 by culture on CD40L expressing feeder cells induced a strong upregulation of costimulatory and adhesion molecules and turned the B-CLL cells into efficient antigen-presenting cells (APCs). CD40-activated B-CLL (CD40-CLL) cells stimulated the proliferation of both CD4+ and CD8+ T cells. Interestingly, stimulation of allogeneic versus autologous T cells resulted in the expansion of different effector populations. Allogeneic CD40-CLL cells allowed for the expansion of specific CD8+cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells did not induce a relevant CTL response, but rather stimulated a CD4+, Th1-like T-cell population that expressed high levels of CD40L and released interferon-γ in response to stimulation by CD40-CLL cells. Together, these results support the view that CD40 activation of B-CLL cells might reverse T-cell anergy against the neoplastic cell clone, although the character of the immune response depends on the major histocompatibility complex (MHC) background on which the CLL or tumor antigens are presented. These findings may have important implications for the design of cellular immunotherapies for B-CLL.

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Superagonistic Anti-CD28 Antibody TGN1412 as a Potential Immunotherapeutic for the Treatment of B Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2519.2519 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2519-2519 ◽

Cited By ~ 1

Author(s):

Chia-Huey Lin ◽

Thomas Kerkau ◽

Christine Guntermann ◽

Martin Trischler ◽

Niklas Beyersdorf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mhc Class Ii ◽

T Cell Activation ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Activation Markers ◽

Cell Chronic Lymphocytic Leukemia

Abstract B cell chronic lymphocytic leukemia (B-CLL) is characterised by an accumulation of malignant B cells, and impaired humoral and cellular immune responses. Evasion strategies of leukemic cells appear to involve down-regulation of co-stimulatory molecules as well as increased resistance to apoptosis. Here we provide data supporting a novel concept to treat B-CLL with a humanized, superagonistic monoclonal antibody specific for CD28 (TGN1412). Superagonistic anti-CD28 antibodies have been shown to activate human T cells in vitro without requirement for engagement of the T cell antigen receptor (Luhder et al., J. Exp. Med. 2003. 197(8):955–66). Indicative of their activation, TGN1412-triggered T cells from healthy donors upregulate, among other activation markers, CD40L, that has been reported to promote anti-leukemic effects when ectopically expressed on B-CLL cells (Wierda et al., Blood. 2000. 96 (9): 2917–2924). In this report, the responses of PBMCs from B-CLL patients to soluble TGN1412 were examined. We show that in a dose-dependent fashion, polyclonal T cell activation was induced by TGN1412 including proliferation, cytokine production and induction of activation markers such as CD25, CD71, CD134 (Ox40), CTLA-4 (CD152) and CD154 (CD40L). Significantly, modulation of malignant B-CLL cells was also observed. MHC class II molecules (HLA-DR), CD95 and the co-stimulatory molecules CD80 and CD86, but not the proliferation marker Ki-67, were strongly up-regulated upon TGN1412 stimulation. These data suggested that improved antigen-presenting functions of B-CLL cells were induced by TGN1412. Accordingly, preliminary data indicate that B-CLL cells isolated from TGN1412 stimulated cultures induced enhanced proliferation of both allogeneic and autologous T cells, and importantly, TGN1412 activated T cells exhibited enhanced CTL-activity against B-CLL cells. In conclusion, our data suggest that TGN1412 induces polyclonal T cell expansion and activation as well as increased APC function of B-CLL cells. They imply that TGN1412 may have future therapeutic benefit for B-CLL patients.

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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 case of chronic lymphocytic leukemia with properties characteristic of natural killer cells

Blood ◽

10.1182/blood.v61.5.940.bloodjournal615940 ◽

1983 ◽

Vol 61 (5) ◽

pp. 940-948

Author(s):

K Itoh ◽

K Tsuchikawa ◽

T Awataguchi ◽

K Shiiba ◽

K Kumagai

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemia Cells ◽

Surface Markers ◽

T Antigens ◽

Homogeneous Population

A case of chronic lymphocytic leukemia that consisted of a homogeneous population of cells that had properties similar to those described for natural killer (NK) cells is presented. These leukemic cells had a morphology of large granular lymphocytes (LGL) and receptors for sheep erythrocytes (ER) and for the Fc portion of IgG (Fc gamma-R). They expressed pan-T antigens OKT3 and Leu-4, but neither helper/inducer T- cell differentiation antigens OKT4 and Leu-3a nor cytotoxic/suppressor T-antigens OKT8 and Leu-2a. HNK 1 antigen, which can be expressed on human NK cells, could be detected on almost all leukemic cells (LGL), whereas a myeloid differentiation antigen, OKM1, which can be expressed on macrophages, granulocytes, and NK cells, was not detected. Thus, it was concluded that the leukemia cells had a characteristic profile of the surface markers: ER+, Fc gamma-R+, HNK-1+, OKT3+, Leu-4+, OKT4-, OKT8-, Leu-3a, Leu-2a, and OKM1-. Although freshly isolated leukemic cells showed no cytotoxicity on NK targets, after incubation at 37 degrees C, the cells did show a potent cytotoxicity on targets of erythroleukemic cell, T cell, and monocyte (but not B cell) origins. When the cells were incubated at 37 degrees C, interferon (IFN gamma) was spontaneously produced in the culture fluids. Treatment with anti- HNK-1 and complement completely abrogated expression of NK activity and interferon production of the patient's lymphocytes in culture. These characteristic features of surface markers and functions strongly suggest the possibility that the leukemia cells of this case are of NK cell origin. The relationship between this case and chronic lymphocytic leukemia of T-cell origin is discussed.

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HLA-G Antigen Expression Is Associated with a More Aggressive, Unfavorable Outcome and Immunosuppression in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2811.2811 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2811-2811

Author(s):

Holger Nueckel ◽

Vera Rebmann ◽

Duerig Jan ◽

Duehrsen Ulrich ◽

Grosse-Wilde Hans

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Immune Responses ◽

Tumor Cells ◽

Human Leukocyte ◽

Progression Free Survival ◽

Antigen Expression ◽

Hazard Ratio ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

A Prospective Study

Abstract Background: The human leukocyte antigen (HLA)-G molecule exhibits limited tissue distribution and exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells which may favor their escape from antitumor immune responses. The role of HLA-G in B cell chronic lymphocytic leukemia (B-CLL) has not been defined. Experimental design: HLA-G expression was studied retrospectively in circulating B-CLL cells from 47 patients by flow cytometry using the MEM/G9 monoclonal antibody. Results: The proportion of leukemic cells expressing HLA-G varied from 1% to 54%. Patients with < 23% HLA-G positive cells (according to ROC analysis; designated as the HLA-G negative group) had a significantly longer progression-free survival (PFS) time than patients with > 23% of positive cells (median PFS 120 versus 23 months, p=0.0001). Multivariate analysis revealed that HLA-G expression (hazard ratio 4.81; p=0.002) was next to the initial staging according to Binet (hazard ratio 8.6; p=0.0001) the best independent prognostic factor compared to other known prognostic factors like ZAP-70 status (hazard ratio 3.6; p=0.029) or CD38 status (hazard ratio 1.83; p=0.37). Humoral and cellular immunosuppression was significantly more prominent in HLA-G positive as compared to HLA-G negative patients group (median immunglobuline G (g/l) 4.3 versus 7.3; median total T-cells (per μl) 824 versus 2540). Conclusions: In B-CLL the level of HLA-G expression is correlated with the degree of immunosuppression and prognosis. To our knowledge this is the first report that describes an association of HLA-G antigen expression and the course of the disease in B-CLL patients. Thus, HLA-G may contribute to the impairment of immune responses against tumor cells and infections. These findings need to be confirmed in a prospective study.

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HLA Ligandome Profiling Identifies a Novel Category of Pathophysiologically Relevant CLL Associated Antigens

Blood ◽

10.1182/blood.v124.21.715.715 ◽

2014 ◽

Vol 124 (21) ◽

pp. 715-715

Author(s):

Daniel J. Kowalewski ◽

Heiko Schuster ◽

Claudia Berlin ◽

Stefan Kahn ◽

Linus Backert ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Conflicts Of Interest ◽

Peptide Vaccine ◽

Rational Approach ◽

Tumor Control ◽

Immune Recognition ◽

Hla Ligands ◽

Hla Ligandome

Abstract The breakthrough clinical success of immune checkpoint inhibition with PD-1 and CTLA-4 antibodies illustrates the potential of T cell based immunotherapy to effectively treat malignancies. However, a remaining major challenge is to increase the specificity and frequency of anti-cancer immune responses. A rational approach to achieve this goal is therapeutic vaccination, as highlighted by a recent phase II vaccination study using naturally presented HLA ligands, which induced vaccine-specific immune responses that were associated with improved clinical outcome (Walter et al., Nat. Med. 2012). This underscores the potential of identifying patho-physiologically relevant targets by direct differential analysis of the entire landscape of HLA-presented peptides, termed the HLA ligandome. Here we applied this approach to chronic lymphocytic leukemia (CLL) with the aim of developing a CLL-specific multi-peptide vaccine. Using cohorts of 30 patients and 30 healthy volunteers, we comparatively and extensively mapped the HLA ligandome landscape of CLL and identified more than 20,000 different HLA class I and II ligands representing 7,377 source proteins, attaining >95% of the maximum attainable coverage (as extrapolated from saturation analysis). A set of 49 HLA ligand source proteins (225 different HLA ligands) showed CLL-exclusive representation in >20% of CLL patients, thereby constituting the novel category of ligandome-derived tumor associated antigens (LiTAAs). These LiTAAs were validated to be broadly and frequently represented across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. Functional characterization by IFNγ-ELISPOT revealed peptide-specific immune recognition of 14/15 (93.3%) corresponding HLA ligands (LiTAPs) exclusively in CLL patients. These immune responses were strictly CLL (LiTAP-) directed and mediated by functional patient-derived CD8+ T cells. Notably, for immunoreactive LITAPs, a linear correlation between frequencies of representation in the CLL ligandomes and immune recognition by CLL patient PBMC was observed (R²=0.59), suggesting that LiTAP presentation on cancer cells is a direct prerequisite for the presence of anti-LiTAP immune responses. Moreover, we investigated the prognostic relevance of LiTAP-specific immune responses: Retrospective survival analysis of 45 CLL patients analyzed by IFNγ-ELISPOT assays comparing cases with T cell responses specific for 0-1 LiTAPs (n=32) versus >1 LiTAPs (n=13) suggests a prolonged overall survival in the multiple-response cohort as compared to the single/non-responders (P=0.0695, log-rank test, Fig. 1). Taken together these data provide clear evidence for tumor-dependent priming of LiTAP-specific T cells in CLL patients and indicate their involvement in tumor control in vivo. This directly implies these non-mutant self-peptides as pathophysiologically relevant tumor antigens in CLL thereby validating our target identification approach and encouraging future evaluation in clinical vaccination trials. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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