Abstract
T cells in B chronic lymphocytic leukaemia (CLL) have been reported to show qualitative and quantitative alterations, thereby regulating the antitumor immune response. We evaluated the absolute count, percentages of T cells and subsets and their association with disease parameters in Indian patients. 45 treatment naïve CLL cases and 10 healthy controls were evaluated for CD3 + Tcells, CD4 + T cells, CD8 + T cells percentage by flowcytometry. The absolute counts were obtained by multiplication with absolute lymphocyte counts obtained on cell counter. The clinical characteristics (age, sex, Rai stage, B symptoms, CD38 expression) were analysed for any association with alteration of these cells (percentages, absolute counts, ratio with monoclonal B cell count) and CD4: CD8 ratio. The mean absolute count of CD3 + T cell, CD4 + T cell and CD8 + T cells was significantly higher (p < 0.05) in CLL patients as compared to healthy controls. CD4:CD8 ratio was variable (normal, increased and decreased). The mean CD8 + T cells count was higher in advanced disease stage, and CD38 positive cases (p > 0.05). Younger CLL patients (< 55 years) had greater increase in CD8 + T cells (p > 0.05). Significant alterations in T cells and their subsets were observed in CLL. A trend towards advanced stage, CD38 expression, presentation an early age was seen with increase in CD8 + T cell counts.
Keywords: B-cell chronic lymphocytic leukemia, Chronic lymphocytic leukemia, T cells, T lymphocyte, CD4-positive T- lymphocytes, CD8-positive T-lymphocytes
Introduction
Chronic lymphocytic leukemia (CLL) is a clonal expansion of B cells with co expression of CD5 and CD23. Although CLL is the most common adult leukemia in west, in India it contributes only 3% of all adult leukemia. [1] The clinical course is highly variable. The accumulation of malignant cells in CLL is primarily due to alterations in regulation of apoptosis. There is postulated cross talk between the malignant B cell clone and T cells thereby regulating anti tumor immune responses. Several qualitative and quantitative abnormalities of T cells have thus been reported in CLL. These include oligo clonal expansion of T cells, inversion of CD4/CD8 ratio, enhanced FAS mediated apoptosis of CD4 T cells, altered cytokine expression. [1–4] The alterations in T cell number and function may affect the disease phenotype and prognosis as well. [5–8]
The biological characteristics of CLL in Asians differ from European population. [9] Hence we evaluated CD3 + , CD4 + and CD8 + T lymphocytes in peripheral blood of newly diagnosed untreated CLL patients and 10 healthy controls using flowcytometry. The CD4, CD8 T cells and CD4/CD8 ratio were then studied in relation to patient age, Rai stage at presentation and CD38 expression.
Materials and Methods
Forty-five untreated newly diagnosed patients of CLL were enrolled in this retrospective analysis. As this was a retrospective study ethical waiver was obtained from the institutional ethics committee. Ten age gender matched healthy volunteers were taken as control. Clinical characteristics of patients including age, sex, Rai stage, B symptoms, lymph node enlargement, and palpable liver and spleen were noted. Peripheral blood sample was collected in EDTA and processed within 4 h of collection. Automated complete blood count for haemoglobin concentration, platelet count and total leucocyte count, was done on ADVIA 2120 (Siemens diagnostics, New York, USA). The immunophenotyping was done on peripheral blood sample using stain lyse protocol and analysed using Cell Quest Pro software on BD FACS Calibur instrument. The gating was done using either CD45 or CD19 expression versus side scatter. The monoclonal antibodies were labelled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and peridin chlorophyll protein (PerCP). The markers studied were CD5, CD19, CD20, CD23, CD22, CD10, FMC7, lambda, kappa for CLL diagnosis, CD38 for prognosis, CD3, CD4, CD8 for identification of T cells. CLL was defined as lymphoid cells with expression of CD19, CD5, CD23, weak CD20, and clonal expression of kappa or lambda. CLL clone was regarded as CD38 positive if 30 percent of CLL cells stained positive with anti CD38 antibody HB-7 [10]. T cells were defined as lymphoid cells positive for CD3. CD8 + T cells (cytotoxic T cells) were defined as cells positive for both CD3 and CD8. CD4 + T cells ( T helper cells) were defined as cells positive for both CD3 and CD4. Monoclonal B cell (MBC) was obtained as percentage of lymphoid cells showing co expression of CD5 and CD19 on flowcytometry The percentage of T cells (CD3 +), CD4, CD8 cells were obtained by flowcytometry. The absolute counts were calculated by multiplying the specific subset by the absolute lymphocyte count obtained from the automated cell counter [11].
Results were presented as Mean + / − SD and percentages. Unpaired t test was used to compare means of two groups. One-way analysis of variance (ANOVA) was used to compare more than two means. Chi square test was used to compare the CD38 expression with CD4/CD8 ratio variations. The p-value < 0.05 was considered significant in all cases. All the analysis was carried out on SPSS 21.0 version (Chicago, Inc., USA).
Results
45 patients of CLL diagnosed in a period of 4 years were retrospectively analysed in this study. The mean age of patients was 59.71 year (37–80). 82.2% were male. Patient characteristics studied are as mentioned in Table 1. The control samples (Table 2) did not vary significantly (p > 0.05) with respect to age and gender. Mean TLC of cases was significantly higher (p < 0.05) as compared to control. The percentage, absolute count, and ratio with MBC of T cells, CD4 + T cells, and CD8 + T cells in cases is shown in Table 3. The mean absolute T cell count was significantly higher (p = 0.005) in CLL patients as compared to control. Mean T lymphocyte % in CLL patients was significantly lower (p = 0.001) in comparison to controls. Subsets of T cells (CD4 + , CD8 +) were analysed in 31 cases. The mean % of CD4 + T cells in CLL was significantly lower (p < 0.001) in comparison to controls. The mean absolute CD4 + T cell count in CLL was significantly higher (p = 0.01) in comparison to controls. The mean % of CD8 + T cells in CLL did not differ significantly(p = 0.084) in comparison to controls. The mean absolute CD8 + T cell count in CLL was significantly higher(p = 0.023) in comparison to controls. CD4: CD8 ratio in controls was 1.2 ± 0.6. CD4:CD8 ratio was variable in CLL cases. 11 out of 31 patients had reversed ratio(CD 8 increased). 9 had ratio in normal range(0.75–1.57). 11 had increased ratio(i.e.) CD4 increased in comparison to controls.
Table 1.
General Characteristics of CLL cases
| Sn | Characteristics | Statistics |
|---|---|---|
| 1 | Mean TLC ± SD (× 109/L) (Range) | 112.88 ± 102.49 (13.2–390.0) |
| 2 | Mean Hb ± SD (g/dL) (Range) | 10.36 ± 2.37 (5–15) |
| 3 | Mean Platelet count ± SD (× 109/L) (Range) | 143.87 ± 79.10 (20–367) |
| 4 | Rai stage | |
| 0 | 11 (24.4%) | |
| 1 | 4 (8.9%) | |
| 2 | 5 (11.1%) | |
| 3 | 9 (20.0%) | |
| 4 | 16 (35.6%) | |
| 5 | B-symptom positivity | 11 (24.4%) |
| 6 | CD38 Positive (n = 41) | 23 (56.1%) |
| 7 | Immunophenotypic abnormality | 12 (26.7%) |
Table 2.
Absolute count and percentage of T cells and subsets in controls
| Characteristics | Mean value ± SD |
|---|---|
| TLC (× 109/L) | 7.6 ± 1.89 |
| Absolute T cell count(× 109/L) | 0.7 ± 0.27 |
| T cells (%) | 9.3 ± 2.8 |
| CD4+ T cells (%) | 4.8 ± 2.3 |
| CD8+ T cells (%) | 4.3 ± 1.7 |
| Absolute CD4+ T cell (× 109/L) | 0.35 ± 0.14 |
| Absolute CD8+ T cell (× 109/L) | 0.31 ± 0.1 |
| CD4/CD8 ratio | 1.2 ± 0.6 |
T cells defined as CD3 + cells. CD4 + T cells defined as CD3 + , CD4 + ; CD8 + T cells defined as CD3 + , CD8 +
Table 3.
T cell, CD4 + T cell, CD8 + T cell: Percentages, absolute count and ratio with MBC
| Variable | Mean ± SD | ||
|---|---|---|---|
| Overall cases | CD38 + Cases | CD38 − Cases | |
| T cell %# | 4.80 ± 3.26 | 4.92 ± 4.05 | 4.56 ± 2.17 |
| CD4+ T cells%* | 1.87 ± 1.25 | 1.71 ± 1.17 | 2.38 ± 1.36 |
| CD8+ T cells%* | 2.36 ± 2.79 | 2.49 ± 3.42 | 1.61 ± 0.79 |
| Absolute T cell count (X109/L)# | 3.83 ± 2.78 | 3.57 ± 2.46 | 3.98 ± 3.28 |
| Absolute CD4 + T cell (X109/L)* | 1.63 ± 1.31 | 1.63 ± 1.49 | 1.79 ± 1.19 |
| Absolute CD8 + T cell(X109/L)* | 1.72 ± 1.55 | 1.54 ± 1.3 | 1.36 ± 1.07 |
| T: MBC ratio(X109/L)# | 0.06 ± 0.04 | 0.09 ± 0.10 | 0.09 ± 0.09 |
| CD4: MBC ratio (X109/L)* | 0.03 ± 0.02 | 0.03 ± 0.03 | 0.04 ± 0.02 |
| CD8: MBC ratio (X109/L)* | 0.04 ± 0.06 | 0.05 ± 0.08 | 0.02 ± 0.01 |
MBC: Monoclonal B cells defined by co expression of CD5 and CD19
CD38 + cases defined by a cut off of 30 percent or more CLL cells positive for CD38
#T cells defined as CD3 + cells, data for 45 patients
*CD4 + T cells defined asCD3 + , CD4 + , CD8 + T cells defined as CD3 + , CD8 + , data for 31 patients
Comparison of T Cell Subsets with Respect to Age and Gender
15 of 45 subjects were ≤ 55 years of age. Mean TLC(X109/L) in patients ≤ 55 years of age was 155.5 and in patients > 55 years was 91.57. Mean % of CD3 positive cells did not vary significantly amongst two age groups(p = 0.82). The absolute count (X109/L) was higher in patients ≤ 55 years of age (4.5) as compared to > 55 year age group (3.4). Mean % of CD4 + T cells was higher in age group > 55 years while mean CD8 + T cells % was higher in age group ≤ 55 years of age. The differences however were not statistically significant(p > 0.05). CD4:CD8 ratio also did not differ significantly amongst two age groups. No significant difference in T cell, CD4 + T cells, CD8 + T cells was seen amongst male and female cases.
Comparison of T Cell Subsets in Different Rai Categories and B Symptoms
Comparison of percentages, absolute count and ratio with MBC of T cell, CD4 + T cells, CD8 + T cells in different Rai stage categories is shown in Table 4. The mean % and absolute T cell count amongst different Rai stages did not show significant variation(p > 0.05). The mean % and mean absolute value of CD4 + T cells did not differ significantly amongst various Rai stages(p = 0.462). The mean % of CD8 + T cells was higher in Stage 3 and 4; however the difference was not statistically significant(p = 0.51). The mean absolute CD8 + T cell count also showed a similar trend.
Table 4.
T cell, CD4 + T cell, CD8 + T cell: Percentages, absolute count and ratio with MBC comparison amongst different Rai stage categories
| Rai stage | T cell %# | CD4+ T cells%* | CD8+ T cells%* | Absolute T cell count (X109/L)# | Absolute CD4 + T cell (X109/L)* | Absolute CD8 + T cell(X109/L)* | T:MBC ratio(X109/L)# | CD4: MBC ratio (X109/L)* | CD8: MBC ratio (X109/L)* |
|---|---|---|---|---|---|---|---|---|---|
| 0 | 5.2 ± 2.7 | 2.22 ± 1.04 | 1.61 ± 0.95 | 2.65 ± 2.59 | 1.19 ± 0.94 | 1.05 ± 1.41 | 0.11 ± 0.1 | 0.04 ± 0.02 | 0.02 ± 0.01 |
| 1 | 3.02 ± 2.1 (n = 4) | 1.92 ± 1.82 | 1.72 ± 1.3 | 3.21 ± 2.12 | 1.65 ± 1.76 | 1.69 ± 1.31 | 0.05 ± 0.04 | 0.03 ± 0.03 | 0.03 ± 0.02 |
| 2 | 4.4 ± 2.1 (n = 5) | 2.42 ± 2.08 | 1.32 ± 0.48 | 4.10 ± 0.79 | 2.94 ± 1.08 | 1.39 ± 1.13 | 0.06 ± 0.02 | 0.03 ± 0.02 | 0.01 ± 0.01 |
| 3 | 4.8 ± 3.1 (n = 9) | 1.07 ± 0.71 | 2.36 ± 2.31 | 3.06 ± 2.12 | 1.09 ± 0.99 | 1.97 ± 1.66 | 0.07 ± 0.06 | 0.01 ± 0.01 | 0.03 ± 0.03 |
| 4 | 5.1 ± 4.2 (n = 16) | 1.85 ± 1.16 | 3.72 ± 4.57 | 5.12 ± 3.37 | 1.98 ± 1.47 | 2.31 ± 1.83 | 0.08 ± 0.11 | 0.03 ± 0.03 | 0.07 ± 0.11 |
| P-value | 0.83 | 0.46 | 0.52 | 0.16 | 0.22 | 0.54 | 0.71 | 0.58 | 0.53 |
MBC Monoclonal B cells defined by co expression of CD5 and CD19
All values are mean ± SD
#T cells defined as CD3 + cells, data for 45 patients. Stage wise break up Stage 0: n = 11, stage 1: n = 4, stage 2: n = 5, stage 3: n = 9, stage 4: n = 16
*CD4 + T cells defined asCD3 + , CD4 + , CD8 + T cells defined as CD3 + , CD8 + , data for 31 patients. Stage wise break up Stage 0: n = 9, stage 1: n = 4, stage 2: n = 3, stage 3: n = 6, stage 4: n = 9
B symptoms were present in 8 of the 31 cases analysed for T cells and its subsets. No significant variation was observed with variation of T cell count(p > 0.05).
CD38 Expression and Comparison with T Cell Subsets (Table 3)
CD38 expression data was available in 41 cases of which 23 were positive. Mean and absolute T cell % did not differ significantly with CD 38 expression. The mean % CD4 + T cell was lower (1.7) in CD 38 positive cases as compared to CD38 negative cases (2.4). The mean % of CD8 + T cells was higher (2.5) in CD38 positive cases as compared to negative cases(1.6). The absolute counts also showed a similar trend. None of the above findings had any statistical significance.
Comparison of T Cell: MBC Ratio as Well as T cells Subsets with Stage and CD 38 Expression
The disease phenotype is primarily determined by the leukemic cell burden analysed as monoclonal B cell count(MBC) for CLL. Hence a comparison of ratio of residual T cells as well as their subsets with MBC was also done with respect to various variables (Table 4). In Rai stage, the mean T:MBC ratio progressively increased from stage 1 to 4. The mean CD4 + : MBC ratio did not differ significantly amongst various Rai stages. The mean CD8 + : MBC ratio was higher in stage 3 and 4, however the difference was not statistically significant.
Mean T cell and CD4 + : MBC ratio did not differ significantly amongst CD38 positive and negative cases. The mean CD8 + : MBC ratio was higher in CD38 positive cases but was not statistically significant.
Comparison of CD4:CD8 Ratio with Rai Stage and CD 38 Expression
CD4:CD8 ratio was altered in 22 out of 31 cases as compared to control. 11 cases had increased CD4:CD8 ratio while 11 had reversed ratio. All 9 cases with normal CD4:CD8 ratio were early stage CLL. Of the 11 cases with raised CD4:CD8 ratio, 6 were advanced stage of CLL. Majority patients(8/11) with reversed ratio were advanced stage CLL. The mean CD4:CD8 ratio was higher in CD38 negative cases but was not statistically significant. The proportion of cases with CD38 expression did not vary amongst cases with normal or increased ratio. However of the 11 cases with reversed ratio, majority(n = 7) had CD38 positive expression.
Discussion
Lymphocyte subsets CD4 + T cells and CD8 + T cells have been shown to vary in different geographical areas in India. Our values in control samples were comparable to the previous multicenter data from India. The mean CD4: CD8 ratio in our control group (1.2 ± 0.6) was comparable to the national reported mean ratio of 1.16 ± 0.41. [12] CLL is characterized by several immune dysfunctions contributing to disease pathogenesis. Investigations of T-cell alterations in CLL have gained importance with regard to disease phenotype and progression. Our study shows significant alterations in T-cells as compared to healthy controls. A reduction in mean percentage of CD 3 + T cells was seen in CLL. First, this can be attributed to clonal expansion of B cells. Secondly, the absolute number of T cells were increased which can be a part of antitumor response. Elevation in absolute T cell count was seen in 53.3% similar to previously reported proportion of 51%. The increase in CD8 + T cells (48%) was also proportional to previous report of 50.2%. Reversal of CD4: CD8 ratio seen in 35% is slightly lower than previous report of 40% [8]
CD4 and CD8 subsets of T cells may contribute by various mechanisms to the disease phenotype. In CLL cases there was significant reduction in mean percentage of CD4 + T cells, similar to the previous report. [5] The CD4 positive T cells have been shown to be susceptible to reduction due to Fas mediated apoptosis. CD4 + T cells in CLL have been reported to show up-regulation of Fas ligand. [3] The absolute CD4 + T cell count was raised in 49% of cases which is higher to the previous report of 42%. [8] CD4 + T cells comprise of helper T cells and regulatory T cells, an analysis for these subsets was not done separately in our study.
Clinical characteristics and outcomes of younger patients with CLL (≤ 55yrs) have been shown to differ from elderly patients. [13] 33.3% (15/45) of our patients were 55 years or less which is higher compared to previous report. [5, 13] Among these 9% (4/45) were ≤ 40 years. The mean TLC was higher as compared to elderly patients. There were equal proportion of patients distributed among higher Rai stage (III/IV), as well as lower Rai stage (0/I/II); again, differing from previous reports showing higher number of patients in early stage. [13] A higher percentage (73.3%) express CD38 positivity (11/15) as compared to previous report. [13] The percentage of elderly population with CD38 expression was lower i.e. 40% (12/30). These features point toward a biological difference among our CLL patients as compared to western population.
Since there were differences in the disease phenotype among young and elderly patients, we analyzed for variations in T cell subsets. The CD8 + T cell counts were higher in younger patients. A rise in CD8 + T cells leading to inversion of CD4:CD8 ratio has been associated with poor prognosis. [3, 6, 8] In younger age CLL patients, a shorter time to first treatment has been reported after adjustment of variations in Rai stage and IgHV mutation. [13] The early treatment requirement may hence be attributed to altered disease biology due to underlying rise in CD8 + T cells.
On evaluation of disease stage with respect to CD4 and CD8 T cells, we observe a higher mean CD8 + T cell count in advance stage (III/IV) in coherence with previous reports. [5, 7] The other reports however have shown no variation with Rai staging. [8] A similar pattern of increase in CD8 + T cells in advanced stage was seen even after comparison with leukemic cell burden (i.e. monoclonal B cell count). CD4 + T cells however did not show any specific pattern with Rai stage as previously reported. [8] Few authors have reported a decrease in absolute CD4 count with Rai stage. [7]
Reversal of CD4: CD8 ratio was seen to be associated with advanced stage as previously reported. [6, 7] However previous reports from India have shown no correlation. [5] Analysis of CD8 + T cells has shown up regulation of PD-1 (an indication of T cell exhaustion), differential methylation of immune regulatory genes (CCR6, KLRG1) and alteration of gene involving its cytotoxic potential resulting in inefficient CD8 cytotoxicity. [3, 6] Thus even though the CD8 + T count is higher in advanced stage, the cells are dysfunctional.
CD38 expression is a poor prognostic marker for CLL. A higher proportion of patients with reversal of CD4:CD8 ratio (7/19) showed CD38 expression as previously reported. [6] These findings suggest a rise in CD8 + T cells or a decrease in CD4 + T cells; in CLL is associated with poor immune response and prognosis. Of the 16 cases showing CD38 expression, 44% (7/16) had reversed ratio.
We have evaluated a small series of CLL patients, a larger sample size would be more useful.
To conclude significant alterations in T cells and subsets were seen in CLL in comparison to healthy controls. A trend towards increased CD8 + T cells in higher Rai stage and CD38 expression was seen. The increase in CD8 counts was more in younger CLL patients. Reversal of CD4:CD8 ratio was seen with higher Rai stage and CD38 expression.
Compliance with ethical standards
Conflict of interest
The author declared that there is no conflict of interest.
Ethical Approval
Approved by the institutional ethics committee.
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Informed consent taken.
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Informed consent taken.
Footnotes
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