Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3dimCD56+ natural killer T cells in fast treatment responders

H Veenstra; R Baumann; N M Carroll; P T Lukey; M Kidd; N Beyers; C T Bolliger; P D van Helden; G Walzl

doi:10.1111/j.1365-2249.2006.03144.x

. 2006 Aug;145(2):252–260. doi: 10.1111/j.1365-2249.2006.03144.x

Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3^dimCD56⁺ natural killer T cells in fast treatment responders

H Veenstra ^*, R Baumann ^*, N M Carroll ^*, P T Lukey ^¶, M Kidd ^‡, N Beyers ^†, C T Bolliger ^§, P D van Helden ^*, G Walzl ^*

PMCID: PMC1809688 PMID: 16879244

Abstract

The immune responses against pulmonary tuberculosis are still poorly defined. This study describes changes in leucocyte and lymphocyte subsets during treatment to find reliable immunological markers for the disease and treatment response. Flow cytometric peripheral blood immune phenotyping, routine haematology and sputum microbiology were performed on 21 HIV-negative adult tuberculosis (TB) patients with positive sputum cultures during therapy in comparison with 14 healthy purified protein derivative (PPD)-positive volunteers. Patients at diagnosis showed high absolute neutrophil and monocyte counts which fell during treatment but low lymphocyte subset counts which increased [except natural killer (NK) and NK T cells]. High counts of a population of CD3^dim/CD56⁺ NK T cells at diagnosis correlated significantly with negative sputum culture after 8 weeks of treatment. A multivariate classification technique showed improved correlation when NK cells were taken into account. In conclusion, peripheral blood white cell counts change significantly during treatment and counts at diagnosis, especially CD3^dim/CD56⁺ NK T cells, hold promise in predictive models of TB treatment response.

Keywords: immunophenotyping, NK T cells, treatment response, tuberculosis

Introduction

The mechanisms of protective immunity against Mycobacterium tuberculosis (Mtb) infection and disease in humans have not been fully clarified. Many reports have addressed the potential immunological defect(s) by comparing immune phenotypes in actively diseased patients to those with latent infection. Most of these investigations have focused on T lymphocyte subsets, particularly CD4⁺ and γδ T cells, generally reporting depressed CD4⁺ T cells in peripheral blood of tuberculosis (TB) patients [1–3], but results are discrepant for γδ T cells, where both elevated [4,5] and normal [6,7] numbers have been found. Only a few but inconclusive reports of B lymphocyte and natural killer (NK) cell numbers in TB patients exist [1,3,8,9] and NK T cells have, to our knowledge, not been investigated in TB patients. Generally, contributors to TB susceptibility remain unclear and follow-up data during therapy are scanty.

The aim of our study was to investigate immune parameters during therapy and this report describes a systematic follow-up of leucocyte counts and lymphocyte subsets in TB patients for the entire 26-week treatment period. Furthermore, due to the fact that the identification of high-risk patients for slow response to chemotherapy would have important clinical implications, we analysed peripheral blood immunophenotypes as potential surrogate markers of early TB treatment response and applied a multivariate classification technique to identify fast and slow responders to treatment by immunophenotype at diagnosis.

Materials and methods

Setting

This study was conducted in an epidemiological field site in metropolitan Cape Town, where the incidence of new smear and/or culture-positive TB was on average 313/100 000 population/year (1993–98) [10].

Patients and controls

The study was approved by the Ethics Committee of the Faculty of Health Sciences at Stellenbosch University and written, informed consent was obtained from all participants. Twenty-nine new smear-positive pulmonary TB patients were screened for this study. Inclusion criteria included: sputum culture-positive for Mtb, no multi-drug resistance, HIV-negative, taking at least 80% of prescribed doses during the intensive phase of treatment. Eight patients were excluded for the following reasons: non-compliance, multi-drug-resistant TB, negative sputum culture, refusal of HIV testing or incomplete follow-up visits. Twenty-one patients with first-time TB were enrolled and studied throughout treatment. Blood samples were taken at diagnosis prior to initiation of treatment and at weeks 1, 5, 13, and 26 after start of treatment (the last blood sample being taken on the last day of chemotherapy). Sputum smears and Bactec cultures were performed on days 1 and 3, and weeks 1, 2, 4, 8, 13 and 26 after start of treatment. A total white cell count (WCC) and differential blood count was performed on all blood samples using a Bayer Advia 120. The patients received standard therapy in accordance with the South African National Tuberculosis Program [based on World Health Organization (WHO) guidelines]. Therapy consisted of a fixed drug combination (depending on body weight) containing isoniazid (320–400 mg/day), rifampicin (480–600 mg/day), ethambutol (800–1200 mg/day) and pyrazinamide (1000–1250 mg/day) during the intensive phase (8 weeks) followed by rifampicin and isoniazid during the continuation phase (the remaining 18 weeks) under direct observation. Posterior–anterior and lateral chest X-rays (CXR) were taken at commencement of treatment allowing a 4-week time window on either side of diagnosis. The chest radiographs were evaluated using a standardized method [11] by a physician who had no prior knowledge of the patient’s condition. The extent of disease was estimated using a one-dimensional view of the upright posterior–anterior radiograph and by using the right upper lobe as reference area.

One blood sample was taken from each of 14 healthy HIV-negative, purified protein derivative (PPD) skin test-positive (> 15 mm) volunteers resident in the same community to serve as controls. These participants had no clinical or radiological signs of active TB.

Processing of sputum samples for Ziehl–Nielsen smear and culture

Sputum samples were processed for culture using standard methods [12], which included decontamination according to the Bactec 460TB System Procedure Manual (Becton Dickinson, Sparks, MD, USA) before inoculation into a Bactec 12B vial. The vials were incubated at 37°C and the growth index (GI) was read daily. Sputum smears, direct and concentrated, were examined for acid-fast bacilli using the Ziehl–Nielsen (ZN) stain and evaluated using the scoring system of the International Union against Tuberculosis and Lung Disease [13]. If multiple smears were performed the smear with the highest grade was recorded for that time-point.

Reagents

Fluorochrome-labelled monoclonal antibodies (mAb) anti-CD45-peridinin chlorophyll (PerCP), CD3-phycoerythrin (PE), CD3-PerCP, CD4-fluorescein isothiocyanate (FITC), CD8-FITC, CD19-FITC, CD56-FITC, γδ T cell receptor (TCR)-FITC, interferon (IFN)-γ-PE, interleukin (IL)-4-PE and rabbit anti-active caspase 3-FITC were from BD-Bioscience (Erembodegem, Belgium). A rabbit FITC control antibody was not available from the manufacturer. OKT3 anti-CD3 antibody was spent hybridoma medium. The hybridomas were from the American Type Culture Collection (ATCC, Rockville, MD, USA). Vα24-PE was purchased from Beckman Coulter (Johannesburg, South Africa), saponin from Sigma (Kempton Park, South Africa) and polyethylene glycol 4000 (PEG) from Merck (Cape Town, South Africa).

Immunophenotyping by flow cytometry

Whole blood (50 µl per test), anti-coagulated with sodium heparin, was washed once with phosphate-buffered saline (PBS), suspended in 100 µl of 0·1% bovine serum albumin (BSA), 0·05% sodium azide in PBS and added to the required antibody mixtures. After 20 min at 4°C, cells were washed and red blood cells (RBCs) lysed at the same time by diluting with 3–4 ml cold PBS containing 0·05% saponin, 0·05% sodium azide and 3% PEG. (We have noted previously that RBCs in whole blood from TB patients frequently failed to lyse when treated with commercial lysing solution and therefore used saponin as alternative lysis solution. The addition of 3% w/v PEG to the saponin buffer prevents damage and clumping of cells in blood obtained at diagnosis and also enhances the formation of antigen/antibody complexes [14]). After centrifugation at 700 g the cell pellets were fixed in 4% formaldehyde in PBS and stored at 4°C in the dark until flow cytometric analysis in a Becton-Dickinson fluorescence activated cell sorter (FACS)Calibur using CellQuest software. Lymphocytes were gated in a CD45-PerCP versus side scatter plot (10 000 events in this gate were acquired) and these were analysed further for expression of CD3 and CD4 (or CD8, CD19, CD56, γδTCR) in the FL1 and FL2 channels, respectively. The lymphocyte sums calculated were all between 95 and 100%. Isotype control antibodies were not used routinely as the background cell surface staining of ex vivo blood lymphocytes is very low (not shown).

Intracellular cytokine labelling

Briefly, whole heparinized blood was mixed 1 : 1 with RPMI-1640 medium with antibiotics in polypropylene tubes and incubated at 37°C with or without 0·1 µg/ml OKT3 antibody for 4 h, with 10 µg/ml Brefeldin A present during the last 3 h. After incubation the blood was diluted with cold PBS containing 0·05% saponin, 0·05% sodium azide and 3% PEG (lyse/wash buffer), centrifuged in the cold at 700 g, and the cells in the pellet were labelled with mAbs in the above buffer containing 0·1% BSA for 20 min in the cold. After one wash with cold lyse/wash buffer, the cell pellets were fixed in 4% formaldehyde in PBS and analysed in the flow cytometer.

Classification of patients into treatment response groups

In order to find possible differences between fast and slow responders to treatment, patients were divided into two responder groups according to Bactec culture status at week 8 after start of treatment. Of the 21 enrolled patients eight were culture-negative (fast responders) and 13 culture-positive (slow responders).

Statistical analysis

Data for patients at diagnosis and at the end of treatment were analysed for significant differences from those for healthy subjects by means of the Mann–Whitney test. The Friedman test with Dunn’s post-test was used to analyse longitudinal changes in parameters with respect to the diagnosis time-point values (* or #: P = 0·01–0·05, ** or ##: P = 0·001–0·01, *** or ###: P < 0·001; asterisks refer to the Mann–Whitney test and hashes to the Friedman test). The Pearson χ² test and Fisher’s exact test were used to analyse categorical CXR data.

To find the best combination of variables at diagnosis that may have potential for the prediction of early treatment response, as defined by the week 8 Bactec sputum culture, a support vector machines analysis was performed, a multivariate discriminant classification technique that has received much attention in the statistical literature in the past few years [15]. Combinations of up to a maximum of five variables were analysed and, using the variables included in the optimal classification model, a leave-one-out cross-validation table was constructed.

Results

Demographic data of study population

The 21 patients were all cured after 26 weeks of standard directly observed treatment short course (DOTS) therapy. Three patients were infected with an Isoniazid-monoresistant strain of mycobacteria. After 8 weeks of treatment 15 patients were smear-negative and six were smear-positive, while only eight were culture-negative and 13 culture-positive (two of these were Isoniazid-monoresistant). The week 8 Bactec culture was therefore used as the more sensitive indicator of early treatment response. No significant differences between fast and slow responders in CXR findings at diagnosis were found (including extent of disease and presence, number or size of cavities). The age and sex distribution of patients is given in Table 1.

Table 1.

Age and sex data of patients and controls.

	Patients

	Fast responders^a	Slow responders	Controls
Total (no.)	8	13	14
Male (no.)	3	9	3
Female (no.)	5	4	11
Age (years)	18–51	19–50	20–56

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As defined by negative sputum culture at week 8.

Longitudinal changes in total and differential WCC

The total WCC and absolute neutrophil counts were significantly elevated in patients at diagnosis relative to controls (Fig. 1a,b) but returned to normal levels by the end of treatment. The absolute monocyte counts were also significantly elevated at diagnosis but then dropped dramatically to significantly depressed levels at week 26 (Fig. 1c). The absolute lymphocyte count of patients at diagnosis was significantly depressed at diagnosis, but counts were no longer significantly different from controls at the end of treatment (Fig. 1d).

Lymphocyte subsets

Percentages of T lymphocytes and NK cells were not significantly different from those of controls at diagnosis or at week 26, while percentages of B lymphocytes were depressed in patients at diagnosis (P < 0·05) and recovered during treatment (not shown). The absolute lymphocyte subset counts were calculated from the subset percentages and absolute lymphocyte counts (Fig. 2). The absolute CD3⁺ T cell and absolute CD19⁺ B cell counts were significantly depressed in patients at diagnosis, but at week 26 these were not significantly different from those of control subjects (Fig. 2a,b). Absolute CD56⁺/CD3^– NK cell counts at diagnosis showed a trend towards lower numbers (P = 0·06) and remained depressed until week 26 (P < 0·05, Fig. 2c).

Fig. 2 — Absolute lymphocyte subset counts of healthy control subjects and tuberculosis (TB) patients, calculated from the absolute lymphocyte counts and the percentages of subsets determined by flow cytometric immunophenotyping. (a) T lymphocytes (CD3⁺), (b) B lymphocytes (CD19⁺), (c) natural killer (NK) cells (CD3^–CD56⁺). Box and whisker plots and statistical analyses as for Fig. 1 (Mann–Whitney test *P < 0·05, **P < 0·01, ***P < 0·001, Dunn’s post-test #P < 0·05, ##P < 0·01, ###P < 0·001).

T lymphocyte subsets

The percentages of CD4⁺, CD8⁺ and γδ T cells and the CD4 : CD8 ratio at diagnosis and at week 26 were not significantly different from those of control individuals and only small fluctuations were detected during follow-up. We detected two populations of NK T cells that differed in their levels of expression of CD3: a CD56⁺ cell population which expressed CD3 levels comparable to conventional T cells (CD3^bright/CD56⁺ NK T cells) and one that expressed reduced levels (CD3^dim/CD56⁺ NK T cells). The percentages of CD3^bright/CD56⁺ NK T cells in patients at diagnosis and at week 26 were not significantly different from those of controls (not shown) and CD3^dim/CD56⁺ NK T cells are described in detail below. Absolute numbers of T cell subsets, calculated from the absolute lymphocyte count and the percentages determined by immunophenotyping are illustrated in Fig. 3. CD4⁺ T cell numbers (Fig. 3a) were significantly depressed at diagnosis relative to control subjects (P < 0·01) and, while numbers increased significantly during treatment, they were still lower at week 26 than in controls (P = 0·06). CD8⁺ T cell counts were lower at diagnosis, although not significantly so (Fig. 3b, P = 0·13) and γδ T cell counts were significantly depressed (Fig. 3c, P < 0·05) but both subsets recovered during treatment to normal levels at week 26. Absolute numbers of CD3^bright/CD56⁺ NK T cells were lower at diagnosis (P = 0·06) and were significantly low at the end of treatment (P < 0·05, Fig. 3d).

Fig. 3 — Absolute T cell subset counts of healthy control subjects and tuberculosis (TB) patients, calculated from the absolute T cell counts and the percentages of the subsets determined by flow cytometric immunophenotyping. (a) CD4⁺ T cells, (b) CD8⁺ T cells, (c) γδ T cell receptor (TCR⁺) T cells, (d) CD3^bright/CD56⁺ natural killer (NK) T cells. Box and whisker plots and statistical analyses as for Fig. 1 (Mann–Whitney test *P < 0·05, **P < 0·01, ***P < 0·001, Dunn’s post-test #P < 0·05, ##P < 0·01, ###P < 0·001).

A CD3^dim/CD56⁺ NK T cell subset was more prominent in patients

We detected an unusual subset of lymphocytes more frequently in patients (nine of the 21 patients had ≥ 2% at diagnosis) than in controls (two of 14 had ≥ 2%). In the flow cytometric analyses, of which Fig. 4 is an example, these cells were weakly CD3⁺ (CD3^dim), CD4^–, weakly CD8⁺ or CD8^– and CD56⁺, shown in region R2 in Fig. 4d, and also γδTCR^– (not shown). The number of cells in region R2, as illustrated in Fig. 4, expressed as a percentage of the cells in the CD45 gate, was determined for all blood samples. Figure 5a shows increased percentages of CD3^dim/CD56⁺ NK T cells in patients at diagnosis relative to control subjects, although this was not statistically significant (P = 0·23). Very low or undetectable numbers remained so during follow-up, while higher numbers persisted and sometimes increased after start of treatment (shown for fast and slow responders in Fig. 5c); the highest recorded was 20·3% at week 1.

Fig. 4 — A representative lymphocyte subset analysis of flow cytometric data from a patient with a prominent CD3^dim/CD56⁺ natural killer (NK) T cell population. (a) Gating of the CD45^bright low side scatter total lymphocyte population; (b,c,d) the gated lymphocytes analysed for CD3 and CD4, CD8 and CD56 expression, respectively. Region R2 in (d) contains the CD3^dim/CD56⁺ NK T cells.

Fig. 5 — CD3^dim/CD56⁺ natural killer (NK) T cells. (a) Percentages in the lymphocyte gate in controls and patients at diagnosis compared with the Mann–Whitney test. (b) Percentages in the lymphocyte gate in patients at diagnosis grouped into slow responders to treatment [culture(+) at week 8] and fast responders [culture(–) at week 8], compared with the Mann–Whitney test. (c) Mean percentages of CD3^dim/CD56⁺ NK T cell counts with s.d. error bars in the slow and fast responder patient groups from diagnosis to end of treatment.

Differences between treatment response groups

When percentages and absolute numbers of each cell type at diagnosis in fast responders were compared to those at diagnosis of slow responders with a Mann–Whitney test, the percentages and absolute counts of CD3^dim/CD56⁺ NK T cells at diagnosis were the only parameters that correlated significantly with treatment response − they were significantly higher at diagnosis in fast responders (P = 0·01, Fig. 5b). The percentages of CD3^dim/CD56⁺ NK T cells did not change significantly during follow-up and are shown for the fast and slow responding patients in Fig. 5c.

As the CD3^dim/CD56⁺ NK T cell numbers at diagnosis did not correlate with treatment response in all patients, we used a multivariate classification technique to find combinations of variables that may classify patients more accurately into fast and slow responders. Differences between early response phenotypes were most prominent at diagnosis and the variables at diagnosis that were used for the analysis were the absolute numbers of leucocyte, lymphocyte and T cell subsets. In the support vector machines discriminant analysis the best classification of patients into the two treatment response groups could be obtained with just two variables: absolute CD3^dim/CD56⁺ NK T cells and absolute NK cells which correctly classified all 13 slow responders and five of eight fast responders in a leave-one-out cross-validation.

CD3^dim/CD56⁺ NK T cells produce IFN-γ and IL-4

To assess functional aspects of CD3^dim/CD56⁺ NK T cells we analysed flow cytometric data of intracellular IFN-γ and IL-4 measurements in saponin-permeabilized T cells after a 4-h stimulation of whole blood with anti-CD3 antibody. In samples from patients with a prominent CD3^dim/CD56⁺ NK T cell population, these cells are found in the CD3-PerCP versus side scatter plots used for gating the T lymphocytes. The CD3^dim and CD3^bright cells were analysed separately. IFN-γ was only produced by some patients and the CD3^dim and CD3^bright cells produced comparable low levels of this cytokine. All patients showed IL-4 production by both stimulated and unstimulated T cells and this tended to be higher in CD3^dim T cells (Fig. 6). The CD3^dim population contains more cells that express active caspase 3, an indicator of apoptosis, and this expression correlates with higher levels of intracellular IL-4.

Fig. 6 — Intracellular cytokine analysis of saponin-permeabilized lymphocytes from whole blood of two patients at diagnosis incubated for 4 h with or without stimulation with 0·1 µg/ml anti-CD3. (a) Gating of CD3^dim (R1) and CD3^bright (R2) T cells in a CD3-PerCP *versus* SSC plot. (b–e) Histograms of the gated cells of one patient showing IFN-γ (b,c) and interleukin (IL)-4 (d,e) expression. Overlaid histograms are: (–) stimulated, specific antibody, (...) unstimulated, specific antibody, (---) stimulated, control antibody. (f–g) Dot plots of similarly gated unstimulated T cells from another patient showing co-expression of caspase 3 and IL-4. The position of the quadrant markers was determined by a phycoerythrin-labelled control antibody (not shown).

Discussion

In this study we have shown significant changes in absolute numbers of neutrophils, monocytes and lymphocyte subsets during active TB. Our finding that these changes occur already during the first weeks of treatment is important, as it suggests strongly that TB patients tested at different time-points during their treatment should not be grouped together in the analysis of results. We detected a CD3^dim/CD56⁺ subset of NK T cells that is more prominent in TB patients and correlates with a faster treatment response. A multivariate classification technique identified CD3^dim/CD56⁺ NK T cells, in combination with NK cells, at diagnosis as variables indicating the likelihood of culture conversion early during TB treatment. NK T cells have, to our knowledge, not been reported in the context of TB disease and we believe that our findings support the future inclusion of these cells in the search for surrogate markers for treatment response.

The interesting subset of NK T cells found in this study expressed CD56 and reduced levels of CD3 and was either double-negative (DN) or weakly CD8⁺. NK T cells, which express CD3 and to a variable degree the NK cell markers CD56, CD57 and CD161 [16–18], are a heterogeneous population in mice and humans with several subsets that differ in phenotype, TCR repertoire, MHC restriction and cytokine profile, as reviewed in [18]. ‘Classical’ NK T cells express an invariant T cell receptor (TCR) with Vα24 (Vα14–Jα281 in the mouse, now Vα14–Jα18), are CD1d restricted and express the NK cell marker CD161 or NKR-P1A. Two subsets of non-classical NK T cells do not express this invariant TCR. Human CD56⁺ NK T cells are abundant in the liver, are predominantly CD8⁺ or DN and Vα24 TCR-negative, have cytotoxic capacity and produce Th1 and Th2 cytokines when stimulated in vitro [19].

As our detection of the CD3^dim/CD56⁺ NK T cells was unexpected, a Vα24 antibody was not included routinely in our panel, but some additional phenotypings with this antibody indicated that these cells did not express the invariant TCR (not shown). The possibility of artefactual CD3^dim staining of NK cells due to non-specific binding to Fc receptors must be considered but this is unlikely, as all the antibodies used were of the IgG1 isotype and the CD3^dim cells would be double-labelled with the CD4, CD19 and γδTCR antibody as well, which was not the case and, furthermore, NK cells do not express the high affinity Fcγ receptors CD32 and CD64 and can be seen as a clearly CD3-negative population in Fig. 4d.

The reduced expression of CD3 could be the result of TCR down-regulation [20] and the CD3^dim/CD56⁺ NK T cells could be an activated subset of CD3^bright/CD56⁺ NK T cells, but we found only a weak inverse correlation between the percentages of these NK T cell subsets (Spearman’s correlation coefficient −0·34, not shown). Takayama et al. [21] demonstrated that a CD122⁺ subset of human CD8 T cells with intermediate TCR expression in the peripheral blood produce high levels of IFN-γ and are also potently cytotoxic.

Peripheral blood CD56⁺ T cells are increased during the early phase of Plasmodium falciparum or P. vivax infections in humans [22], suggesting an important role in the immune response to intracellular pathogens. Slifka et al. [23] found that 90% of virus-specific CD8⁺ and CD4⁺ T cells from choriomeningitis virus-infected mice co-express one or more NK cells markers for more than 500 days post-infection. In our patients we did not detect much variation in the percentages of CD3^dim/CD56⁺ NK T cells over time and they could represent a similar persistent population specific for mycobacterial antigens.

Our observation of the often higher numbers and percentages of CD3^dim/CD56⁺ NK T cells in patients indicates that this cell population is expanded in the blood of some TB patients, and that these patients are able to clear the infection more efficiently after the initiation of chemotherapy. As CD3^dim/CD56⁺ NK T cells appear to produce variable IFN-γ and IL-4, we postulate that they are cells that have been activated, as could be indicated by their reduced CD3 expression, and are at variable stages between activation and apoptosis. This is supported by our finding that they contain a higher percentage of cells expressing active caspase 3 and that they produce more intracellular IL-4. Previous findings have associated intracellular IL-4 expression in lymphocytes with mitochondrial apoptosis markers [24]. Therefore CD3^dim/CD56⁺ NK T cells could be indicators of an active immune system in TB patients and would accelerate clearance of the infection by antibiotics.

The other variable that, together with CD3^dim/CD56⁺ NK T cells, had predictive value according to our multivariant discriminative analysis, was the absolute NK cell count. Interestingly, a higher NK cell count is partially indicative of a slow response to treatment. A higher NK cell count in the peripheral blood may be the result of an inability of these cells to migrate into infected tissues. In humans NK cells are present in tuberculous pleural effusions [25], and in mice infected with Mtb NK cell numbers in the lung increase over the first 21 days of infection, although their removal does not affect host resistance. A role of NK cells in the control of TB has been suggested by the results of in vitro studies with human NK cells and Mtb-infected monocytes [26–28].

Monocytes/macrophages are important components of the innate immune response to mycobacterial infections and the dramatic change in the absolute monocyte counts in our patients between diagnosis and week 26 should be noted. The surprising finding here is that their numbers are significantly depressed in fully treated patients and it is unknown what causes this depressed absolute monocyte count.

To determine whether the depressed absolute monocyte, NK cell and CD3^bright/CD56⁺ NK T cell counts at the end of treatment could contribute to increased susceptibility to TB relapse [10], phenotyping needs to be performed on larger numbers of blood samples taken after cessation of antibiotic treatment with subsequent long-term clinical follow-up.

A drawback of our study is that the patient numbers in the two treatment response groups are small and therefore the accuracy of the statistical classifications is limited. It is also not optimal that in our study, for logistical reasons, the week 26 blood samples were taken on the day of the last dose of antibiotics and not after cessation of drug therapy. It is unknown whether drug treatment directly affects cell counts.

In summary, peripheral blood white cell counts change rapidly during treatment and some counts at diagnosis hold promise as surrogate markers of treatment response. Further prospective studies with larger numbers of patients are now needed to evaluate the role of immunophenotyping in general and of CD3^dim/CD56⁺ NK T cells specifically, including their functional characterization. The role of these cells in predicting differential outcomes at month 6 and the development of recurrence after cure needs to be assessed.

Acknowledgments

We gratefully acknowledge the contributions by Erica Engelke, Marianna de Kock, Ilse Steyn and the clinical and administrative staff of the Department of Paediatrics and Child Health of the University of Stellenbosch, Professor Robert Gie for CXR analysis and the Department of Hematology at Tygerberg Hospital for performing full blood counts. Funding assistance for this study were provided by GlaxoSmithKline, Stevenage, UK and the DST/NRF Centre of Excellence for Biomedical and TB Research.

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PERMALINK

Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3dimCD56+ natural killer T cells in fast treatment responders

H Veenstra

R Baumann

N M Carroll

P T Lukey

M Kidd

N Beyers

C T Bolliger

P D van Helden

G Walzl

Abstract

Introduction

Materials and methods

Setting

Patients and controls

Processing of sputum samples for Ziehl–Nielsen smear and culture

Reagents

Immunophenotyping by flow cytometry

Intracellular cytokine labelling

Classification of patients into treatment response groups

Statistical analysis

Results

Demographic data of study population

Table 1.

Longitudinal changes in total and differential WCC

Fig. 1.

Lymphocyte subsets

Fig. 2.

T lymphocyte subsets

Fig. 3.

A CD3dim/CD56+ NK T cell subset was more prominent in patients

Fig. 4.

Fig. 5.

Differences between treatment response groups

CD3dim/CD56+ NK T cells produce IFN-γ and IL-4

Fig. 6.

Discussion

Acknowledgments

References

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3^dimCD56⁺ natural killer T cells in fast treatment responders

A CD3^dim/CD56⁺ NK T cell subset was more prominent in patients

CD3^dim/CD56⁺ NK T cells produce IFN-γ and IL-4