Granular Lymphocytes In Patients Research Articles

Prevalence and significance of large granular lymphocytes in patients with immune thrombocytopenia

The association between T-cell large granular lymphocytes (T-LGL) and ITP is uncertain. The aims of this study were to determine the prevalence of T-LGL in patients with ITP and to describe its association with ITP disease severity. We analyzed flow cytometry results for T-LGL (using a threshold of 0.3 x109 or greater cells/L) or positive T-cell receptor clonality in patients with ITP and nonimmune thrombocytopenia. Descriptive statistics were used to characterize the association between T-LGL and ITP, response to ITP treatments (rituximab and splenectomy) and response to T-LGL treatment. Among ITP patients, 14.3% (13/91) had evidence of a T-LGL population compared to 10.3% (3/29) of patients with non-immune thrombocytopenia. ITP patients with T-LGL had lower nadir platelet counts (2 vs. 47 × 109/L) and received more ITP treatments (median 6 vs. 3) than ITP patients without T-LGL. Response to rituximab was observed in 14.3% (1/7) of ITP patients with T-LGL and 54.5% (6/11) without T-LGL. Response to splenectomy was observed in 25% (2/8) with T-LGL and 56.2% (9/16) without T-LGL. Four patients with ITP and T-LGL received treatment for T-LGL with methotrexate; none had an improvement in platelet count levels. T-LGL may appear in patients with ITP, and the meaning of this finding remains unclear; however, for some patients, the presence of abnormal T-LGL may indicate a more severe form of ITP that tends to be less responsive to therapy. In this cohort, treatment of T-LGL with methotrexate did not improve platelet counts in the few patients who were treated.

Clonal expansion of large granular lymphocytes in patients with spondyloarthritis and psoriatic arthritis treated with TNFα inhibitors.

To determine the prevalence of clonal T-large granular lymphocyte (T-LGL) cells in patients with spondyloarthritis (SpA) and psoriatic arthritis (PsA) and to define possible risk factors for this condition. We present a cross-sectional analysis with retrospective and prospective aspects. 115 SpA patients, 48 PsA patients and 51 controls were recruited between December 28, 2017 and January 23, 2019. Flow cytometry (FACS) was performed to screen for aberrant T-LGL cells. Molecular analysis was then employed to confirm the diagnosis in patients with suggestive FACS findings. Patients with clonal T-LGL populations were followed prospectively by FACS analysis. Electronic patient files were retrospectively analyzed to determine risk factors. Median age was 49years for SpA, 55.5years for PsA, and 54years for controls. Median disease duration of SpA and PsA was 15years and 11years, respectively. 79.8% of patients had received biologics at some point, 75.5% had ever received tumor necrosis factor (TNF) inhibitors. 59.5% were treated with TNF inhibitors at the time of study inclusion. We identified clonal T-LGL expansions in 13 individuals equaling a prevalence of 6% (13/214). T-LGL patients were taking TNF inhibitors more frequently at the time of study inclusion (p = 0.022) and were more likely to have ever been treated with TNF inhibition (p = 0.046). Clonal T-LGL expansions can be detected in patients with SpA, PsA and also in healthy controls. Confirming earlier results, exposure to TNFα-blocking agents appears to increase the risk of developing clonal expansions of T-LGL cells.

The raft marker GM1 identifies functional subsets of granular lymphocytes in patients with CD3+ lymphoproliferative disease of granular lymphocytes.

The raft marker GM1 is expressed at very low levels at the plasma membrane of resting T cells (GM1dull). In vitro T-cell activation induces synthesis of this lipid, which is then expressed at very high levels (GM1bright) at the membrane of activated/effector cells. By flow cytometry and confocal microscopy, we analyzed the expression and organization of GM1 in a series of 15 patients with CD3+ lymphoproliferative disease of granular lymphocytes (LDGL). We found that GM1bright GL were detectable in fresh blood samples obtained in all LDGL patients, although the range of brightly stained cells was extremely variable. This distinctive in vivo pattern has never been shown in T lymphocytes from healthy individuals or in patients with different chronic T or B lymphoproliferative disorders or active infectious diseases. The low number of cycling cells detected in LDGL patients was always included within the GM1bright GL population. Interestingly, GM1bright GL were demonstrated to contain a higher amount of IFN-gamma as compared to GM1dull GL. These findings allow to distinguish subsets of GL at different levels of activation within the monoclonal CD3+ population. The GM1bright GL subset is likely to be responsible for the renewing of GL and thus for maintaining chronic proliferation.

Interleukin-15 Triggers the Proliferation and Cytotoxicity of Granular Lymphocytes in Patients With Lymphoproliferative Disease of Granular Lymphocytes

The recently cloned cytokine interleukin-15 (IL-15) shares several functional activities with IL-2 in different cell systems. Although IL-15 does not show sequence homology with IL-2, it uses components of the IL-2 receptor (IL-2R) for binding and signal transduction, namely, p75 (β) and the p64 (γ) chains of IL-2R. To evaluate whether IL-15 is involved in the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), we evaluated the ability of IL-15 to stimulate GL proliferation, cytotoxic function, and the role of IL-2R β and γ molecules on relevant cells. Our results show that IL-15 stimulates cell proliferation and cytotoxic activity of GL in LDGL patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) and phenotypic analyses using the anti–IL-2R γ-chain–specific TUGh4 monoclonal antibody (MoAb) indicate that both CD3+ and CD3− GL express the p64 IL-2R, a result previously unknown. IL-15 activity was inhibited by antibodies against p75 and p64 IL-2R chains, while no inhibitory effects are detectable with anti-p55 IL-2R antibody. The association of anti-p75 and anti-p64 IL-2R MoAbs resulted in a nearly complete (95%) inhibition of IL-15–induced GL proliferation. Using RT-PCR analysis, we demonstrated that highly purified CD3+ and CD3− GL did not express mRNA for IL-15 or IL-2. By contrast, a clear-cut IL-15 mRNA signal was detected by RT-PCR in patients' peripheral blood mononuclear cells, with monocytes likely accounting for the source of IL-15 in LDGL patients. However, even in concentrated supernatants from enriched monocyte populations, we could not demonstrate the presence of IL-15 protein. Using anti–IL-15 specific MoAbs, a membrane-bound form of this cytokine was demonstrated both on CD3+ and CD3− LDGL cells. By RT-PCR analysis, purified GL from these patients were found to express the message for IL-15 receptor α chain. Taken together, these results indicate that both CD3+ and CD3− GL are stimulated by IL-15 and that this cytokine mediates its activity through the β and γ chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.

Interleukin-15 Triggers the Proliferation and Cytotoxicity of Granular Lymphocytes in Patients With Lymphoproliferative Disease of Granular Lymphocytes

AbstractThe recently cloned cytokine interleukin-15 (IL-15) shares several functional activities with IL-2 in different cell systems. Although IL-15 does not show sequence homology with IL-2, it uses components of the IL-2 receptor (IL-2R) for binding and signal transduction, namely, p75 (β) and the p64 (γ) chains of IL-2R. To evaluate whether IL-15 is involved in the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), we evaluated the ability of IL-15 to stimulate GL proliferation, cytotoxic function, and the role of IL-2R β and γ molecules on relevant cells. Our results show that IL-15 stimulates cell proliferation and cytotoxic activity of GL in LDGL patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) and phenotypic analyses using the anti–IL-2R γ-chain–specific TUGh4 monoclonal antibody (MoAb) indicate that both CD3+ and CD3− GL express the p64 IL-2R, a result previously unknown. IL-15 activity was inhibited by antibodies against p75 and p64 IL-2R chains, while no inhibitory effects are detectable with anti-p55 IL-2R antibody. The association of anti-p75 and anti-p64 IL-2R MoAbs resulted in a nearly complete (95%) inhibition of IL-15–induced GL proliferation. Using RT-PCR analysis, we demonstrated that highly purified CD3+ and CD3− GL did not express mRNA for IL-15 or IL-2. By contrast, a clear-cut IL-15 mRNA signal was detected by RT-PCR in patients' peripheral blood mononuclear cells, with monocytes likely accounting for the source of IL-15 in LDGL patients. However, even in concentrated supernatants from enriched monocyte populations, we could not demonstrate the presence of IL-15 protein. Using anti–IL-15 specific MoAbs, a membrane-bound form of this cytokine was demonstrated both on CD3+ and CD3− LDGL cells. By RT-PCR analysis, purified GL from these patients were found to express the message for IL-15 receptor α chain. Taken together, these results indicate that both CD3+ and CD3− GL are stimulated by IL-15 and that this cytokine mediates its activity through the β and γ chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.

IL-12 is involved in the activation of CD3+ granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes.

We investigated the effects of Il-12 on functional properties of CD3+ CD8+ granular lymphocytes (GL) of of patients with lymphoproliferative disease of granular lymphocytes (LDGL). To this aim, in 10 cases with clonal CD3+ GL proliferation (nine cases with an associated TCR alpha/beta receptor and one case with a TCR gamma/delta receptor) we studied the proliferative and cytotoxic activities of resting and alpha CD3 monoclonal antibody (mAb) activated cells in the presence of rIL-12 and anti-IL-12 blocking antibodies. Specific mRNA for IL-12 p40 subunit was also investigated. Our results showed that rIL-12 increased the proliferation of alpha CD3 pre-stimulated GL (2 to 6 times). Further, anti-IL-12 antibodies partially inhibited alpha CD3-induced cell growth, suggesting a role for this cytokine in the alpha CD3-mediated GL activation. The addition of antibodies blocking the p55 and p75 chains of IL-2 receptor (IL-2R) did not inhibit the rIL-12-mediated cell proliferation, indicating that the activity of rIL-12 is dependent of IL-2 in the in vivo expanded GL of patients under study. Concerning the cytotoxic activity, rIL-12 increased the alpha CD3-mediated NK activity against K-562 target cells and alpha CD3 redirected cytotoxicity against P815 target cells. Molecular analysis pointed out that, following alpha CD3 stimulation, patients' GL increased the expression of specific mRNA for the p40 subunit of IL-12 as compared to baseline conditions. Our data indicate that IL-12 is involved in the mechanisms of activation of clonal CD3+ GL in patients with LDGL; these features are consistent with the possibility that this discrete subset of GL might represent in vivo primed cytotoxic T lymphocytes.

Lymphocytosis of large granular lymphocytes in patients with Hodgkin's disease.

Clonal disorders of large granular lymphocytes (LGL) of either CD3- (NK cell) or CD 3+ (T-cell) phenotype have been described. B-cell malignancies such as hairy cell leukemia and non-Hodgkin's lymphoma have been observed in association with the T-cell type of LGL leukemia. Here we report the occurrence of LGL lymphocytosis in four patients with Hodgkin's disease. Immunophenotyping studies showed that these LGL were CD 3- in three patients and CD3+ in the other. LGL were polyclonally expanded in both patients in whom clonality could be assessed.

Lysis of autologous tumor cells by large granular lymphocytes in patients with acute leukemia in complete remission: Correlation between lytic activity and clinical outcome

In order to evaluate the effect of specific immune response on prognosis in acute leukemia, we investigated the correlation between the lysis of autologous tumor cells (ATC) by lymphocytes and prognosis. Peripheral mononuclear cells (PMC) from most patients with acute leukemia in complete remission (CR) do not exhibit cytotoxic activity against fresh-frozen ATC, although they have adequate cytotoxic activity against K562 cells. When the large granular lymphocyte (LGL) fraction was used in this study, we observed lysis of ATC in 17 (43.6%) of 39 patients with acute leukemia (12 (42.9%) of the 28 patients with acute myelogenous leukemia (AML) and 5 (45.5%) of the 11 patients with acute lymphocytic leukemia (ALL)). With regard to prognosis, the lytic activity of the LGL fraction did not reflect the duration of CR. The median CR duration in AML patients was 13 months for the lysis-positive group and 11 months for the lysis-negative group. No significant correlation was also found between lytic activity of the LGL fraction and overall survival in each patient. However, the lysis-positive group tended to have a longer survival, the median overall survival being 48 months for the lysis-positive group vs 12 months for the lysis-negative group. The prolonging of overall survival in the lysis-positive group was attributed to a high rate of induction of second remissions in this group. Long-term patient survival in the two groups did not differ.

A bispecific antibody detects cytotoxic T lymphocytes of unknown antigen specificity in patients with granular lymphocyte-proliferative disorders.

A simple and sensitive method for the detection of cytotoxic T lymphocytes (CTL) of unknown antigen specificity was investigated. Using anti-CD3 Fab' x anti-CD10 Fab' bispecific antibody or intact anti-CD3 monoclonal antibody, we induced cytotoxicity for CD10+, Fc gamma receptor-positive Daudi target cells in peripheral blood mononuclear cells (PBMC) obtained from patients with granular lymphocyte-proliferative disorders (GLPD). The results indicated that the bispecific antibody was much more efficient than the intact anti-CD3 monoclonal antibody in inducing cytotoxicity. Since CD3+CD4-CD8+ granular lymphocytes in patients with GLPD are considered to be in vivo-primed CTL of unknown antigen specificity, this bispecific antibody method may be useful for detecting in vivo-primed CTL within PBMC. By using this bispecific antibody, it will be possible to detect circulating in vivo-primed CTL in other clinical conditions.

Activation via the CD3 and CD16 pathway mediates interleukin-2- dependent autocrine proliferation of granular lymphocytes in patients with granular lymphocyte proliferative disorders

Granular lymphocytes (GLs) in patients with GL-proliferative disorders (GLPDs) are known to express the interleukin-2 receptor (IL-2R) beta chain (p70–75) constitutively and to proliferate in response to stimulation with IL-2 via the beta chain. In this report, we found that the anti-CD3 monoclonal antibody (MoAb) OKT3 could induce the proliferation of GLs from patients with T-cell lineage GLPDs (T-cell receptor-alpha beta+/CD3+16+), but not that of natural killer (NK) cell lineage GLs (T-cell receptor-alpha beta-/CD3–16+). In contrast, the anti-CD16 MoAb 3G8 that reacts with NK-lineage GLs could induce the proliferation of these GLs but not that of GLs with a T-cell phenotype. Furthermore, the anti-CD16 MoAbs CLB FcR gran1 (VD2) and OK-NK, which react with both T- and NK-lineage GLs, induced the proliferation of GLs with both T- and and NK-cell phenotypes. The proliferative response induced via the CD3 or IgG Fc receptor III (Fc gamma RIII: CD16) pathway was shown to be associated with the IL-2-dependent autocrine pathway by various findings, including the induction of endogenous IL-2 production, the coexpression of the IL-2R alpha chain (p55) and the IL- 2R beta chain, and the inhibition of GL proliferation by anti-IL-2 or anti-IL-2R MoAb. These results suggest that GL proliferation is mediated at least partly through the IL-2-dependent autocrine pathway, and that the TCR/CD3 complex in T-cell phenotype GLs and the Fc gamma RIII in both T- and NK-cell phenotype GLs play a role in their activation in GLPDs.

Activation via the CD3 and CD16 Pathway Mediates Interleukin-2-Dependent Autocrine Proliferation of Granular Lymphocytes in Patients With Granular Lymphocyte Proliferative Disorders

Granular lymphocytes (GLs) in patients with GL-proliferative disorders (GLPDs) are known to express the interleukin-2 receptor (IL-2R) beta chain (p70-75) constitutively and to proliferate in response to stimulation with IL-2 via the beta chain. In this report, we found that the anti-CD3 monoclonal antibody (MoAb) OKT3 could induce the proliferation of GLs from patients with T-cell lineage GLPDs (T-cell receptor-alpha beta+/CD3+16+), but not that of natural killer (NK) cell lineage GLs (T-cell receptor-alpha beta-/CD3-16+). In contrast, the anti-CD16 MoAb 3G8 that reacts with NK-lineage GLs could induce the proliferation of these GLs but not that of GLs with a T-cell phenotype. Furthermore, the anti-CD16 MoAbs CLB FcR gran1 (VD2) and OK-NK, which react with both T- and NK-lineage GLs, induced the proliferation of GLs with both T- and and NK-cell phenotypes. The proliferative response induced via the CD3 or IgG Fc receptor III (Fc gamma RIII: CD16) pathway was shown to be associated with the IL-2-dependent autocrine pathway by various findings, including the induction of endogenous IL-2 production, the coexpression of the IL-2R alpha chain (p55) and the IL-2R beta chain, and the inhibition of GL proliferation by anti-IL-2 or anti-IL-2R MoAb. These results suggest that GL proliferation is mediated at least partly through the IL-2-dependent autocrine pathway, and that the TCR/CD3 complex in T-cell phenotype GLs and the Fc gamma RIII in both T- and NK-cell phenotype GLs play a role in their activation in GLPDs.

Perforin Gene Expression in Granular Lymphocyte Proliferative Disorders

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin-dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

Perforin gene expression in granular lymphocyte proliferative disorders

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin- dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

Increase in OKM1+ granular lymphocytes in patients with rheumatoid arthritis.

Patients with very active rheumatoid arthritis that was being treated only with nonsteroidal antiinflammatory drugs had increased numbers of peripheral blood OKM1+ lymphocytes. In 3 patients, 90 degrees light scatter analysis revealed a double lymphocyte peak. When sorted, the high scatter peak contained a large percentage of granular lymphocytes. Patients with mild-to-moderately active rheumatoid arthritis had normal levels of OKM1+ lymphocytes, but when the drugs were discontinued, the activity of the disease and the numbers of OKM1+ cells increased. Administration of piroxicam was associated with clinical improvement and a decrease in levels of OKM1+ cells. OKM1+ granular lymphocytes are increased in some rheumatoid arthritis patients, and their numbers may correlate with clinical disease activity and/or therapy.