CD38 In Chronic Lymphocytic Leukemia Research Articles

The Value of Neutrophil Gelatinase-Associated Lipocalin Receptor as a Novel Partner of CD38 in Chronic Lymphocytic Leukemia: From an Adverse Prognostic Factor to a Potential Pharmacological Target?

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes that escape death, and correlates with the expression of negative prognostic markers such as the CD38 antigen. Although certain new drugs approved by the US Food and Drug Administration improve the clinical outcome of CLL patients, drug resistance and disease relapse still occur. Like CD38, neutrophil gelatinase-associated lipocalin receptor (NGAL-R) is frequently overexpressed in CLL cells. Here, we evaluated the concomitant surface expression of NGAL-R and CD38 in leukemic blood cells from 52 CLL patients (37 untreated, 8 in clinical remission, and 7 relapsed). We provide evidence of a positive correlation between NGAL-R and CD38 levels both in the interpatient cohorts (p < 0.0001) and in individual patients, indicating a constitutive association of NGAL-R and CD38 at the cell level. Patients with progressing CLL showed a time-dependent increase in NGAL-R/CD38 levels. In treated CLL patients who achieved clinical remission, NGAL-R/CD38 levels were decreased, and were significantly lower than in the untreated and relapsed groups (p < 0.02). As NGAL-R and CD38 participate in CLL cell survival, envisioning their simultaneous inhibition with bispecific NGAL-R/CD38 antibodies capable of inducing leukemic cell death might provide therapeutic benefit for CLL patients.

Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

AbstractPatients 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.

Significance of Interleukin-22 and CD38 in Chronic Lymphocytic Leukemia

Significance of Interleukin-22 and CD38 in Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Cells Impair Mitochondrial Fitness in CD8+ T Cells and Impede CAR T Cell Efficacy

Chronic Lymphocytic Leukemia Cells Impair Mitochondrial Fitness in CD8+ T Cells and Impede CAR T Cell Efficacy

Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that &amp;gt;50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

Mass Cytometry Reveals Heterogeneity in the Exhaustion Profile of T-Cells in Chronic Lymphocytic Leukemia and the CD4:CD8 Ratio May be a Reliable Predictor of CD8+ T Cell Compartment "Fitness"

T cell exhaustion is characterized by coordinated expression of a series of negative checkpoint receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, resulting in T cell dysfunction and immune evasion. Under physiological states, these inhibitory molecules maintain self-tolerance and prevent autoimmunity by applying a break on cytotoxic T cells. In cancer, T-cells exhibit features of T-cell exhaustion including increased expression of PD-1, 2B4 and CD160, coupled with reduced T cell proliferation, altered synapse formation and impaired cytotoxicity. Although the role of the PD-1/PD-L1 axis in mediating T cell defects in chronic lymphocytic leukemia (CLL) is well-studied, the contribution of other checkpoint molecules such as 2B4, CD160 and TIGIT in mediating tumor-induced immune dysfunction remains to be determined.Checkpoint inhibitors have provided a paradigm-shifting approach to cancer treatment. We hypothesized that the expression levels of checkpoint receptors on T-cells, as well as the "fitness" of the T cell compartment may provide a prognostic stratification system to predict response to checkpoint inhibitors in CLL. To determine if the number of inhibitory receptors per cell and their expression level may identify patient-to-patient differences that may not be easily deciphered using conventional research tools, we performed a detailed single-cell analysis of the T-cell repertoire, using 40-parameter mass cytometry (CyTOF) in 12 untreated CLL and 12 healthy controls.Consistent with previous reports, we found that expression of 2B4 (43.7% vs 30.8%), PD1 (28.8% vs 21%) and CD160 (17% vs. 9.7%) was significantly higher on CLL CD8+ T cells compared to healthy controls. In addition, CD8+T cells in CLL expressed higher levels of TIGIT (48.2% vs 25.2%), CD57 (43.9% vs 17.9%) and KLRG1 (49.5% vs. 29.7%). We clearly distinguished 2 patterns of exhaustion marker distribution in CLL. In one group of patients, the expression of checkpoint receptors was similar to that seen in healthy controls, whereas in the second group, CD8+ T-cells expressed higher levels of PD1, 2B4, TIGIT, CD160 as well as markers of terminal differentiation such as CD57 and KLRG1.Compared to healthy donors, CLL was characterized by an inversion in the CD4:CD8 ratio. Interestingly, CD8+ T cells in patients with a low CD4:CD8 ratio (defined as <2.5) expressed significantly higher levels of 2B4 (56.6% vs 31.25%), TIGIT (62.9% vs 37%), CD160 (22.8% vs 12.6%), CD57 (57% vs 28.7), PD-1 (34.6% vs 24.5%) and KLRG1 (62.3% vs 36.3%). In contrast, the expression levels of PD-1, 2B4 and CD160 in CLL patients with a CD4:CD8 ratio of ≥2.5 were similar to that seen in healthy controls, suggesting that the CD4:CD8 ratio may be a valuable marker of T cell exhaustion in CLL.Next, we compared the number of checkpoint molecules expressed per CD8+ T-cells in CLL patients versus healthy donors. Whereas a similar proportion of CD8+ T-cells in CLL (mean 19.56%, range 18.34-31.73%) and healthy donors (mean 22.13%, 14.17-41.19%) expressed one inhibitory receptor, a significantly higher proportion of CLL patients expressed 2 and more inhibitory receptors (mean 28.4, range 10.52-48.78%) compared to healthy controls (mean 15.38%, range 9.67-21.94%). PD-1 was mostly co-expressed with TIGIT, although TIGIT+PD-1+CD4+ and CD8+ T-cells were higher in CLL compared to healthy controls (12.9% vs 7.1%). Interestingly the predominant population of PD-1+CD8+ T cells in CLL was also positive for 2B4 and TIGIT, whereas expression of TIGIT was more diverse and was seen in association with PD-1, 2B4, KLRG1 or CD57.Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. While CLL patients with a normal CD4:CD8 ratio expressed comparable levels of inhibitory molecules to that seen in healthy controls, a low CD4:CD8 ratio was indicative of higher expression of checkpoint molecules. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. A more detailed data and analysis, including transcription and functional profile of exhausted CLL T cells, will be presented in the meeting. DisclosuresWierda:Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding. Jain:Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; Infinity: Research Funding; BMS: Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria.

The diagnostic and prognostic value of CD38 and CD49d expressions in chronic lymphocytic leukemia

Background Little is known about the prognostic importance of coexpression of CD49d and CD38 in chronic lymphocytic leukemia (CLL) patients. Aim This study aimed to investigate the coexpression of both CD38 and CD49d as prognostic and survival markers in CLL patients. Patients and methods Fifty-two patients with newly diagnosed B-cell CLL were included in the study. Twenty age-matched and sex-matched healthy control participants were also included in the study. Patients were subjected to a clinical examination and abdominal ultrasonography and chest radiography. Laboratory investigations including complete blood count, β2 microglobulin, cytogenetic analysis, and immunophenotyping by flow cytometer (B-lymphocyte markers and the expression of CD38 and CD49d) were performed. Results There was a significant decrease in hemoglobin concentration and platelet counts in patients who coexpressed CD49d+/CD38+ compared with patients who expressed CD49+ alone, whereas white blood cell and lymphocyte counts, lactate dehydrogenase, and β2 microglobulin were significantly higher. In addition, CD49d+/CD38+ coexpression was significantly high in advanced stages of CLL. A positive correlation was detected between CD49d expression and poor prognostic parameters in CLL. The median treatment-free time was shorter in CD49d+ patients (32 months) compared with CD49d- patients (98 months). The median treatment-free duration was shorter in CD38+ patients (28 months) compared with CD38- patients (102 months). In the concordant cases of CD49d+/CD38+, the median treatment-free survival was shorter (24 months) in patients with CD49+/CD38+ patients compared with disconcordant cases of CD49d+/CD38- patients (62 months). Conclusion CD38 and Cd49d expressions are considered prognostic markers for CLL patients and they should be assessed to decided on the patient's therapy and to determine disease prognosis. These molecules should also be tested in a large-scale study to determine their potential in preventing frequent relapses and development of resistance to chemotherapy in CLL.

CD38 in Chronic Lymphocytic Leukemia (CLL) – From a Diagnostic Tool to a Therapeutic Target?

CD38 in Chronic Lymphocytic Leukemia (CLL) – From a Diagnostic Tool to a Therapeutic Target?

T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production

AbstractT-cell exhaustion, originally described in chronic viral infections, was recently reported in solid and hematologic cancers. It is not defined whether exhaustion contributes to T-cell dysfunction observed in chronic lymphocytic leukemia (CLL). We investigated the phenotype and function of T cells from CLL patients and age-matched controls. CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD244, CD160, and PD1, with expansion of a PD1+BLIMP1HI subset. These molecules were most highly expressed in the expanded population of effector T cells in CLL. CLL CD8+ T cells showed functional defects in proliferation and cytotoxicity, with the cytolytic defect caused by impaired granzyme packaging into vesicles and nonpolarized degranulation. In contrast to virally induced exhaustion, CLL T cells showed increased production of interferon-γ and TNFα and increased expression of TBET, and normal IL2 production. These defects were not restricted to expanded populations of cytomegalovirus (CMV)–specific cells, although CMV seropositivity modulated the distribution of lymphocyte subsets, the functional defects were present irrespective of CMV serostatus. Therefore, although CLL CD8+ T cells exhibit features of T-cell exhaustion, they retain the ability to produce cytokines. These findings also exclude CMV as the sole cause of T-cell defects in CLL.

Characterizing Immunophenotypic and Functional Pseudo-Exhaustion in T Cells From CLL Patients: The Impact of Lenalidomide

AbstractAbstract 564The ability to evade immune destruction is increasingly being recognised as a crucial feature of cancer cells. Chronic lymphocytic leukemia (CLL) is associated with profound defects in T-cell function, resulting in failure of anti-tumor immunity and increased susceptibility to infections. T cells from CLL patients exhibit functional defects and alterations in gene expression, that show similarities to exhausted T cells in chronic viral infections. However, it is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific T cells. We investigated the phenotype and function of CD8+ T cells from CLL patients and controls matched for age and CMV-serostatus. We demonstrate an increased proportion of CCR7- effector T cells in both CLL patients and CMV-seropositive individuals (p<0.05). CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD160 and CD244 irrespective of CMV-serostatus (p<0.01), whereas increased PD1 expression on CD8+ T cells was limited to CMV-seronegative patients (p=0.002). CLL CD8+ T cells also showed functional defects in proliferation and cytotoxicity irrespective of CMV-serostatus, with the cytolytic defect caused by a combination of impaired granzyme B packaging into secretory vesicles and non-polarized degranulation. In contrast to virally-induced exhaustion, CLL T cells showed increased production of interferon-γ with increased T-BET expression (p<0.01), normal IL-2 production, and no downregulation of IL-7R. Therefore, while CLL CD8+ T cells exhibit some features of T-cell exhaustion, they show important differences (Table 1). These findings also exclude CMV as the sole cause of T cell defects in CLL.Lenalidomide has recently been demonstrated to have significant clinical activity in CLL. Its mechanism of action in this disease is not well understood, but it thought to act primarily by a combination of CLL cell and immune cell activation. We therefore examined the ability of lenalidomide to repair the observed T cell defects by investigating the impact of this agent on the gene expression profiles and function of CLL T cells. Treatment of CLL CD8+ T cells with lenalidomide increased the expression of 137 genes, while 34 genes were downregulated. The most prominent changes in expression were of genes involved in cytoskeletal signaling including WASF1 (Wiskott-Aldrich syndrome protein, family member 1), and TPM2 (tropomyosin 2). There was also upregulation of genes involved in lymphocyte activation, including TNFSF4 (Tumor necrosis factor ligand superfamily, member 4: OX40L), LAG3 (Lymphocyte-activation gene 3), and TNF, and genes involved in cell proliferation such as IKZF1 (Ikaros) and GRN (Granulin). Although lenalidomide treatment or anti-CD3 stimulation alone had no impact on T-bet expression, co-treatment with both anti-CD3 stimulation and lenalidomide resulted in significantly enhanced T-bet expression and increased production of interferon-γ. In contrast, lenalidomide treatment alone was able to improve T cell cytotoxic function, associated with repair of trafficking of granzyme B into the immunological synapse.In conclusion, T cells from CLL patients exhibit features of T-cell pseudo-exhaustion that are present irrespective of CMV serostatus. Treatment of CLL T cells with lenalidomide results in upregulation of genes involved in proliferation, activation, and cytoskeletal pathways, resulting in repair of the functional T cell defects.Table 1Comparison of the phenotypic and functional defects of T cells from CLL patients with T-cell “exhaustion” in chronic viral infectionsExhausted T cells in chronic viral infectionsT cells from CLL patientsIncreased expression of inhibitory receptorsYesYesAbnormal transcription factor profileYesYesReduced proliferative potentialYesYesDecreased expression of IL-7R (CD127)YesNoDecreased cytokine production ↓IL-2, ↓IFN-γYesNoImpaired cytotoxicityYesYes Disclosures:Riches:Celgene: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

T-Cells From Patients with CLL Exhibit Phenotypic and Transcription Factor Profiles of Exhaustion Independent of CMV Serostatus

Abstract 1780Cancer is associated with immune dysfunction, contributing to the failure to mount an effective anti-tumor immune response. T-cell exhaustion, a state of acquired T-cell dysfunction initially described in the context of chronic viral infections, has recently been described in human solid tumors. We have previously demonstrated alterations in gene expression and defects in function in T-cells from patients with chronic lymphocytic leukemia (CLL) and noted similarities to those described in exhausted T-cells in murine models. Therefore, we used multiparameter flow cytometry to determine if T-cells from patients with untreated CLL (n=23) had surface phenotypic and transcription factor profiles of exhaustion. When compared with healthy controls (n=10), an increased proportion of circulating CD8+ T-cells showed an effector-memory phenotype (p=0.018), with a shift towards a greater proportion of CCR7-CD45RA+ terminally differentiated cells. We found increased expression of markers of exhaustion including CD279 (PD-1) (p=0.0057), CD160 (p=0.0006), CD244 (p=0.0057), and CD57 (p=0.011), and decreased expression of CD28 (p=0.0058) on CLL CD8+ T-cells compared with healthy CD8+ T-cells. Increased expression of PD-1 (p=0.0019), CD160 (p=0.0011), CD57 (p=0.007), and LAG-3 (p=0.038) was also noted on CLL CD4+ T-cells. We next determined the level of the transcription factors T-bet and eomesodermin and the transcriptional repressor Blimp-1, as these proteins have been implicated in CD8+ effector-memory differentiation and development of T-cell exhaustion. Importantly, CD8+ T-cells from CLL patients showed increased expression of T-bet (p=0.038), eomesodermin (p=0.0037), and Blimp-1 (p=0.0002), compared with CD8+ T-cells from healthy donors. Furthermore, PD-1 expression identified a subset of exhausted CD8+ T-cells with high intranuclear staining of Blimp-1 that was markedly expanded in patients with CLL (p=0.0002).Previous studies have identified expanded populations of CMV specific CD8+ T-cells in CMV seropositive CLL patients. As chronic viral infection is a known cause of T-cell exhaustion, we determined whether our findings were limited to CMV seropositive patients. We observed that increased expression of T-bet and decreased expression of CD28 was seen only in CD8+ T-cells from CMV seropositive patients. T-bet represses expression of PD-1 and sustains CD8+ T-cell responses during chronic viral infection, and we noted relatively lower expression of PD-1 on CD8+ T-cells in seropositive compared with seronegative patients (p=0.049), although PD-1 expression was still higher than in healthy controls. However, CD8+ T-cells from both CMV seronegative and seropositive patients had significantly higher expression of CD160, CD244, and CD57 compared to CD8+ T-cells from healthy donors (Table 1). Furthermore, CD8+ T-cells from both CMV seronegative and seropositive patients had increased expression of Blimp-1 and eomesodermin.Table 1Surface phenotype/transcription factor profile of CLL according to CMV serostatusHealthyCLL: CMV IgG negative (n=8)CLL: CMV IgG positive (n=8)PhenotypePD-1LOWINCREASED**INCREASED*CD160LOWINCREASED**INCREASED**CD57LOWINCREASED*INCREASED**CD244LOWINCREASED*INCREASED***CD28HIGHNO CHANGEDECREASED**Transcription factorT-betLOWNO CHANGEINCREASED**EomesLOWINCREASED*INCREASED***Blimp-1LOWINCREASED***INCREASED****p<0.05,**p<0.01,***p<0.001In conclusion, T-cells from patients with CLL show phenotypic and transcription factor profiles of T-cell exhaustion that is not limited to the CMV-seropositive group. These findings may explain the acquired immune deficiency in patients with CLL, and provide potential therapeutic targets to reverse immune dysfunction in this disease. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

CD38 in Chronic Lymphocytic Leukemia: From Bench to Bedside?

Human CD38 is a cell surface molecule endowed with multiple functions. As an enzyme, it catalyzes the production of Ca2+ active metabolites, predominantly cADPR and ADPR. As a receptor, it regulates the activation of an intracellular signaling pathway, generally linked to lymphocyte activation and proliferation in physiological conditions. The finding that CD38 behaves as an independent negative prognostic factor in CLL patients was the starting point for investigations into the functional role of the molecule in the neoplastic context. Data accumulating in over a decade concur to define a model where CD38 is a central element of a large supramolecular complex that includes surface signaling receptors, chemokine receptors, adhesion molecules and matrix metalloproteases. Expression of CD38 within this supramolecular complex makes signal transduction as well as chemotaxis and homing more efficient, suggesting that the molecule is an integrator of proliferative and migratory signals. These data indicate that CD38 is not only a reliable disease marker but also a functional molecule in the CLL context. The next decade will likely tell whether it can also be a useful therapeutic target.

Expanded CLL CD8+ T-Cells Are Driven Into a Senescent KLRG1+ Effector Memory Phenotype

AbstractAbstract 913Altered numbers and functions of T-cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we determined numbers of blood lymphocyte subsets of CLL patients in a longitudinal manner. We found that dynamic expansions of the peripheral blood CD4+ and CD8+ T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Additionally, we performed gene expression profiling (GEP) of blood CD3+ T-cells of CLL patients and normal donors. We identified a list of 135 genes that had significantly increased expression and 11 genes that had significantly decreased expression in CLL T-cells. The up-regulated genes included killer cell lectin-like receptor familiy members KLRA1, KLRC2, KLRD1 (CD94), KLRK1 and KLRF1 as well as CD244 (NK-cell receptor 2B4), CD160 (NK cell receptor BY55), PRF1 (perforin 1) and CRTAM (class-I MHC-restricted T-cell associated molecule). These up-regulated genes are known to be preferentially expressed by CD8+ T-cells with an effector memory phenotype. We used Gene Set Enrichment Analysis (GSEA) to investigate whether CLL T-cell genes correlate with a previously published gene expression signature of effector memory CD8+ T-cells (Willinger et al., Journal of Immunolgy 175[9], 2005). This analysis revealed a highly significant enrichment of CLL T-cell genes within the effector memory CD8+ T-cell signature (p<0.0001, FDR q<0.001). Next, we studied the CLL CD8+ T-cell compartment using flow cytometry. As already implied by GEP and GSEA, the flow cytometric analysis revealed a relative shift of subsets within the CD8+ T-cell compartment. Compared to normal donors we observed a decreased proportion of naïve CD8+ T-cells (CD45RA+CCR7+) and an increased proportion of CD8+ effector memory cells (CD45RA-CCR7-) in the CLL cohort as compared to normal donor controls. When absolute cell numbers were calculated on the basis of these results it became evident that the elevation in the absolute number of overall CD8+ T-cells in CLL was primarily attributable to the expansion of the effector memory subset of CD8+ T-cells. Subsequently, we compared the killer cell lectin-like receptor G1 (KLRG1) surface expression of CLL and normal donor CD8+ effector memory T-cells. KLRG1 marks cells, which have undergone extensive proliferation and lack replicative potential. We observed that the absolute increments of effector memory CD8+ T-cells in human CLL patients were mainly due to the expansion of the senescent KLRG1 expressing subset of cells. In order to test whether the CD8+ effector memory expansion is a general biologic CLL phenomenon we studied the CD8+ T-cell compartment of a murine transgenic CLL model (7-month-old TCL1 transgenic mice). It was previously described that TCL1 CD5+CD19+ B-cell hyperplasia first emerges in the peritoneal cavity of TCL1 transgenic mice. Therefore, we specifically studied how CD8+ T-cell subsets respond to arising CLL in the peritoneal cavity of TCL1 transgenic mice. Strikingly, we found that our observation of effector memory shifted CD8+ T-cells in human CLL was phenocopied in the peritoneal cavity of TCL1 transgenic mice. The proportion of peritoneal naïve CD8+ T-cells (CD62L+CD44-) was significantly decreased while the proportion of CD8+ effector memory T-cells (CD62L-CD44+) was significantly increased. Moreover, we observed a more than two-fold increase of KLRG1+ effector memory CD8+ T-cells in the peripheral blood and spleens of TCL1 transgenic mice compared to wild-type controls. In summary, we were able to show that human as well as mouse CLL CD8+ T-cells are driven into a senescent effector memory phenotype. This might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment. Disclosures:No relevant conflicts of interest to declare.

Marrow-Infiltrating T Cells In Patients with Chronic Lymphocytic Leukemia Display Markers of Functional Impairment and Express PD-1

AbstractAbstract 2417Marrow is a major site of disease development and progression for chronic lymphocytic leukemia (CLL), as well as a priming site for antigen-specific T cells and a reservoir for memory T cells. To determine the extent to which T cells in the marrow microenvironment have an altered phenotype and function in CLL, we analyzed the immunophenotypic characteristics of marrow-infiltrating T cells of 18 CLL patients compared to 11 normal donors. Chemotherapy-naïve CLL patients (n=7) possessed comparable quantities of marrow T cells compared to normal donors (median CD8+ T cells/μl = CLL 904 vs normal 1247; median CD4+ T cells/μl = CLL 1975 vs normal 1110). However, we identified several aberrant characteristics among T cells infiltrating the marrow of CLL patients. First, the ratio of CD8+ to regulatory T cells (CD4+CD25+FOXP3+) was depressed (median ratio CLL 14 vs normal 41), indicating more regulatory T cells per effector T cells in CLL. Second, compared to normal marrow T cells, CLL marrow contained proportionally fewer functional effector CD8+ T cells (CD27+CD28+)(median normal 57%, CLL 48%) and more immunosenescent cells (CD27-CD28-)(median normal 21%, CLL 30%). Third, the T cell differentiation state of CLL CD8+ T cells was skewed to favor a phenotype of increased terminal differentiation (CD45RA+CCR7-)(median CLL 55% vs normal 40%), and decreased naïve (CD45RA+CCR7+) cells (median CLL 21% vs normal 31%) compared to normal donors. These differences were further accentuated in CLL samples collected within 4 months from treatment with conventional chemotherapy (n=11). Finally, by immunohistochemical staining of CLL marrow biopsies, we observed marrow-infiltrating lymphocytes to express PD-1 (mean of infiltrating T cells, untreated CLL 12%, treated CLL 35%, present even >6 months after therapy), a marker associated both with immuno-activation and inhibition. While the majority of PD-1+ CD8 T cells of normal donors (n=5) and treated CLL patients (n=4) were differentiated towards effector memory (CD45RA-CCR7-) cells (median normal 46% vs untreated CLL 16%, p=0.07; treated CLL 61%), the PD-1+ T cells from untreated CLL patients (n=5) were terminally differentiated (CD45RA+CCR7+)(median normal 23% vs untreated CLL 65%, p=0.04; treated CLL 24%). These results indicate an exhausted rather than an activated T cell phenotype in untreated patients. Paired immunophenotypic analysis on blood and marrow from the same individuals (n=9) demonstrated an increased percentage and intensity of PD-1 expression on T cells from marrow compared to blood (percentage CD8+ T cells BM vs blood p = 0.05). Interestingly, PD-1 was also detected on CLL cells (n=16) but not normal B cells (median normal 0%, vs CLL 17%, p = 0.004). The ligand for PD-1, PD-L1, was detected in the marrow vasculature by immunohistochemical staining of biopsies, suggesting that the marrow microenvironment plays a role in the induction of PD-1 associated immunosuppression. Ligation of blood PD-L1 on CLL-T cells led to a 2-fold decrease in activation (measured as CD69 expression) of CD3/CD28 stimulated patient T cells. In summary, we identify several phenotypic and functional alterations within marrow-infiltrating T cells of CLL patients. We speculate these together may contribute to impaired priming of host immunity against the tumor. The PD-1 pathway appears to be activated in CLL, especially in the setting of chemotherapeutic treatment. Since anti-PD1 antibodies are now clinically available, it may be possible to target this pathway to improve anti-tumor responses. Disclosures:No relevant conflicts of interest to declare.

Human CD38 Is a Potential Therapeutic Target for Sekected Chronic Lymphocytic Leukemia cases.

Chronic lymphocytic leukemia (CLL), the most common leukemia in Europe and North America, is characterized by a highly heterogeneous clinical behavior. Absence of mutations in the immunoglobulin variable (IgV) genes, expression of the surface receptor CD38 and the cytoplasmatic kinase ZAP-70 represent independent molecular markers of unfavorable prognosis.Recent data indicates that the proliferative core of the disease resides within peripheral lymphoid organs. There, the neoplastic cells have access to the antigen as well as to microenvironmental signals necessary to the maintenance and expansion of the clone. Our group has investigated the role of CD38 in CLL not only as a disease marker but also as a pathogenetic agent. We showed that CD38 ligation by agonistic monoclonal antibodies (mAbs) or the CD31 ligand is followed by proliferation and blast transformation of a subset of CLL cells. CD38/CD31 interactions occur predominantly in the lymph nodes and spleen where CD31 is abundantly expressed by stromal and nurse like cells (NLC). The signaling pathway initiated upon CD38 engagement relies upon tyrosine phosphorylation of ZAP-70 as a necessary step. CLL cells expressing CD38 and ZAP-70 also show the highest migration propensity towards CXCL12, a critical chemokine produced by NLC to attract CLL cells to lymph nodes. Collectively, these results point to a crucial role for the CD38/ZAP-70 pathway in directing CLL cells from the blood to peripheral lymphoid organs and suggest that targeting CD38 might impair CLL cells homing to sites favorable for neoplastic growth.In order to obtain a genome-wide signature of the transcriptional events taking place upon CD38/CD31 contacts CLL cells from 10 patients were co-cultured with CD31 transfected (L-CD31+) cells and L-mock controls for 5 days. Microarray analyses revealed 1,645 sequences selectively modulated as a consequence of CD38/CD31 interactions with 84 differentially regulated pathways: 12 of these pathways regulated cell adhesion/movement phenomena and 14 lymphocyte signalling.Attention was next focused on the impact of blocking anti-CD38 mAbs on CLL homing in vivo. To investigate the homing of CLL cells from blood to spleen and BM, PBMC were injected in the tail vein of female NOD/SCID mice and left to home for 24 hours. Next, mice were sacrificed and human cells tracked in spleen and BM by staining with specific reagents. Homing was significantly reduced when human cells were pre-incubated with anti-CD38 mAbs immediately prior to in vivo delivery. The in vivo data provided a rationale for a more in depth in vitro investigation of the effects of anti-CD38 mAbs on the CXCL12/CXCR4 signaling pathway. Results indicate that CXCL12-mediated chemotaxis can be blocked by anti-CD38 mAbs. Pre-treatment of CLL cells with anti-CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. These effects are specific for CD38+ samples, as no interference in migration was recorded when anti-CD38 mAbs were incubated with CD38− cells.The molecular explanation may be found at least in part in a physical proximity between CD38 and CXCR4 on the cell membrane, as shown by confocal microscopy and coimmunoprecipitation studies. In line with this working hypothesis, CXCL12 binding to CXCR4 is effectively inhibited following pre-incubation with anti-CD38 mAbs.In conclusion, these results offer biological evidence of a functional cross-talk between CD38 and CXCR4 signaling pathways. They also provide a preliminary rationale for the use of anti-CD38 ligands in therapy of a subset of CLL patients, representing a novel approach interfering with deleterious growth circuits and increasing the susceptibility of leukemic cells to conventional chemotherapy.

CD38 and ZAP-70 Mark Chronic Lymphocytic Leukemia (CLL) Cells with High Migratory Potential and Regulate Chemotaxis.

The absence of mutations in the IgVH genes along with the expression of the cell surface receptor CD38 and the cytoplasmic kinase ZAP-70 are independent molecular markers of unfavourable outcome in CLL patients. Our group has been interested in unravelling the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We have shown that CD38 can positively affect proliferation and survival of CLL cells through binding CD31, the ligand expressed by stromal, endothelial and nurse-like cells (NLC). Starting points of our current investigation are thati.NLC produce high levels of the attractant stromal cell-derived factor-1 (SDF-1α/CXCL12),ii.CD38 is an essential mediator of migration and homing in murine neutrophils, andiii.ZAP-70 is a key element in CXCR4 (SDF-1α receptor) pathway in T cells.The consequent working hypothesis is that CD38 and ZAP-70 intertwine to influence CLL cell migration. The analysis of a cohort of 56 clinically and molecularly characterized CLL patients demonstrated that CD38+/ZAP-70+ samples migrate significantly better towards CXCL12 than all the other CLL subgroups, including the CD38+/ZAP-70− one, suggesting a functional association between CD38 and ZAP-70. CD38 engagement by agonistic monoclonal antibodies (mAbs) leads to phosphorylation of the activatory tyrosines present in ZAP-70 in 26 CLL cell cases and in an ad hoc modified B line expressing a ZAP-70-myc construct. More importantly, ZAP-70 is required for CD38 signalling, as the CD38 pathway is impaired in CD38+/ZAP-70− CLL cells. Microarray analyses performed on a subgroup of 20 CLL patients indicate the existence of a genetic signature marking CLL samples endowed with a high migratory potential. These studies resulted in the identification of a panel of 24 differently expressed genes, many of which are known to be involved in adhesion and migration. Taken together, the above data point to a direct role for the CD38/ZAP-70 pathway in CLL migration towards CXCL12. Indeed, CXCL12-mediated chemo taxis of CLL cells and of the NALM-6 pre-B cell line can be efficiently blocked by anti-CD38 mAbs in a dose-dependent fashion. Out of the 9 different anti-CD38 mAbs tested, only the ones binding epitopes close to the C terminus of the molecule interfere with migration, while the ones binding closer to the plasma membrane do not. Pre-treatment of CLL and NALM-6 cells results in the almost complete block of the CXCR4 signalling pathway. This was shown by monitoring Ca2+ mobilization, actin polymerization and ligand-induced internalization. Further, upon exposure to CXCL12, CD38 and CXCR4 become physically associated, as shown using confocal microscopy and co-immunoprecipitation approaches. CD38 however, does not internalize upon CXCL12 biding to CXCR4. Collectively, these data suggest that the blocking effects of the anti-CD38 mAbs are due to steric hindrance. In conclusion, we believe that these results offer biological evidence explaining why the combination of CD38 and ZAP-70 results in a more efficient identification of CLL patients with aggressive disease. Further, it may provide a rationale for using anti-CD38 ligands or inhibitors as an adjunct tool for the therapy of aggressive CLL.

Regulation of CD38 in proliferating chronic lymphocytic leukemia cells stimulated with CD154 and interleukin-4

Chronic lymphocytic leukemia (CLL) cells, like normal B-cells, exist in two populations in vivo: quiescent cells in the peripheral circulation and proliferating cells in lymph nodes. The surface marker CD38 has roles in cell adhesion and signaling. Its expression correlates with poor clinical outcome and is associated with expression of the signaling intermediate ZAP-70, which is also a marker of poor prognosis. We investigated the regulation of CD38 and ZAP-70 in proliferating CLL cells. We cultured CLL cells on a stromal cell layer that maintains viability and also with some stromal cells expressing CD40 ligand (CD154) in order to measure changes in expression of CD38 and ZAP-70. We demonstrated up-regulation of CD38 expression by CD154. The degree of up-regulation did not correlate with clinical stage or mutational status. In addition in the majority of cases tested ZAP-70 expression increased in parallel with up-regulation of CD38 although discordant cases were also observed. Overall we demonstrated that regulation of CD38 in CLL is dynamic and dependent on signals from CD154 and a stromal cell layer. We speculate that CD38 and ZAP-70 are expressed in lymph node leukemic cells in both good and poor prognosis patients, but, in cases with good clinical outcome, these molecules are down-regulated in the peripheral blood whereas in cases with poor prognosis their expression is maintained.

Prognostic significance of combined analysis of ZAP‐70 and CD38 in chronic lymphocytic leukemia

The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL) poses critical questions concerning the identification of high risk patients. Unmutated IgV(H) genes, CD38 and ZAP-70 expression have emerged as the most useful tools in identifying aggressive CLL. The simultaneous expression of ZAP-70 and CD38 in 157 patients with CLL has been evaluated. Fifty-seven patients (36%) were positive for ZAP-70 and 46 patients (29%) were positive for CD38. Both molecules were highly correlated and predictive of the clinical course of the disease. According to the simultaneous evaluation of ZAP-70 and CD38, patients were divided into three groups. In 81 patients (52%), there was a negative concordance of both molecules (ZAP-70(-)/CD38(-)); in 27 patients (17%) there was a positive concordance (ZAP-70(+)/CD38(+)); in 49 patients (31%) there was a discordant expression (ZAP-70(+)/CD38(-) and ZAP-70(-)/CD38(+)). A comparison of the clinical and laboratory data showed in ZAP-70(+)/CD38(+) patients a significantly higher bone marrow and peripheral blood lymphocytosis, lower hemoglobin levels, more advanced clinical stage, and higher number of unmutated IgV(H) status with respect to the other two groups. Furthermore, ZAP-70(+)/CD38(+) patients displayed a much shorter treatment-free interval (median 12 months vs 42 months in discordant patients and not reached in ZAP-70(-)CD38(-) patients). These results prove that the concomitant evaluation of ZAP-70 and CD38 expression allows the separation of CLL patients in prognostic subgroups and suggest that their simultaneous assessment should become an integral component of the CLL diagnostic grid.

The expression and prognostic value of ZAP-70 and CD38 in chronic lymphocytic leukemia in a Chinese population

17538 Background: Advances in the biology of CLL have led to the identification of new prognostic markers,which may improve the accuracy of classic parameters in predicting the clinical course.For this purpose,we evaluated the prognostic value of 70-kD zata-associated protein (ZAP-70) and CD38 in B-chronic lymphocytic leukaemia(B-CLL) combined with clinical state, β2-MG and LDH. Methods: Mult-parameter flow cytometry were used to detect the expression of ZAP-70 and CD38 in 71 B-CLL patients. Results: ZAP-70 and CD38 were concordantly expressed in 70.4% of B-CLL. (P&lt;0.05) There was significant difference between ZAP-70+ and β2-MG(P&lt;0.05).But no significant difference between ZAP-70 and LDH(P&gt;0.05).In a subgroup of 15 consecutive patients analysis of CD38 and ZAP-70 expression were repeated in 2 years follow-up.The expression of CD38 was changed in 7 patients and ZAP-70 was not changed over time. Conclusions: Early prognostic parameters defected disease specificity. Using FCM to detect the expression of ZAP-70 and CD38 could be performed in clinical laboratory. The expression of ZAP-70 could be a novel prognostic indicators in B-CLL because it was not changed in whole course of disease. CD38 expression was an independent prognostic indicator and it could predict time to disease progression, as judged by treatment requirement. No significant financial relationships to disclose.

ZAP‐70 expression is low in normal precursor B cells or hematogones

Zeta-associated protein (ZAP-70) expression has been associated with a less favorable prognosis in chronic lymphocytic leukemia (CLL). The role of ZAP-70 in immature B-cells is not well understood. Immature or precursor B lymphocytes (hematogones) often occur in clinical samples and coexpress CD10, CD38, and CD19 lacking surface immunoglobulin expression. The present study was carried out in an attempt to further elucidate the role of ZAP-70 in the spectrum of B-cells seen in clinical specimens. ZAP-70 expression was evaluated on 25 patient samples that expressed greater than 2% of CD10(+)/CD38(bright)/CD19(+) coexpression during routine evaluation. In our sample set, the hematogone expression ranged from 2 to 18% of total leukocytes and occurred in a variety of conditions, including CLL, NHL, AML, MDS, Hodgkins Disease, and Multiple Myeloma. The method of ZAP-70 detection was that routinely applied to our clinical testing of CLL samples supplemented with the determination of ZAP-70 levels in the CD38(bright)/CD19(+) coexpressing cells. In all cases ZAP-70 was not expressed on the CD5 negative, CD38/CD19 coexpressing precusor B cells. In all cases hematogones were found to not express significant levels of ZAP-70. The presence of hematogones in clinical samples should be recognized so as to not adversely influence prognostic studies of ZAP-70 or CD38 in CLL.