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Original articles
CVID patients with autoimmunity have elevated T cell expression of granzyme B and HLA-DR and reduced levels of Treg cells
  1. Clive R D Carter1,
  2. Ganesha Aravind2,
  3. Natuley L Smalle1,
  4. June Y Cole1,
  5. Sinisa Savic2,
  6. Philip M D Wood1,2
  1. 1Cellular Immunology Laboratory, Department of Immunology and Transplant Immunology, St James's University Hospital, Leeds, UK
  2. 2Department of Clinical Immunology, Beckett Wing, St James's University Hospital, Leeds, UK
  1. Correspondence to Dr Clive Carter, Cellular Immunology Laboratory, Dept of Immunology and Transplant Immunology, Level 09 Gledhow Wing, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK; clive.carter{at}leedsth.nhs.uk

Abstract

Aims Common variable immunodeficiency (CVID) is a primary antibody immunodeficiency with approximately 20% of patients reporting additional autoimmune symptoms. The primary aim of this study was to compare the levels of activated and regulatory T cells (Treg cells) in CVID patients in an attempt to clarify their possible interactions leading to the generation of autoimmunity.

Methods Immunophenotyping of T cells was performed by flow cytometry using a whole blood approach. Surface expression of human leukocyte antigen HLA class II DR and intracellular levels of granzyme B in T cell subsets were assessed; Treg levels were measured using CD4 CD25, FOXp3 and CTLA-4.

Results CVID patients had higher levels of granzyme B and HLA-DR on CD8+ T cells compared with control values (mean of 59% vs 30% and 45% vs 21%, respectively). Patients also had reduced levels of Treg cells compared with control values (con mean=3.24% vs pat=2.54%). Patients with autoimmunity (5/23) had a similar level of T cell activation markers to the rest of the patients but with lower Treg cells (mean of 1.1%) and reduced CD25 and CTLA-4 expression. Patients with autoimmunity had a higher ratio of activated to Treg cells compared with patients with no autoimmune symptoms.

Conclusions These results highlight that reduced levels of Treg cells were associated with elevated levels of activated T cells, suggesting that reduced Treg cells in these patients may have functional consequences in allowing exaggerated T cell responses.

  • Immunodeficiency
  • Immunology
  • Immunophenotyping
  • Lymphocytes

https://doi.org/10.1136/jclinpath-2012-201046

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    Introduction

    Common variable immunodeficiency (CVID) is the commonest of the primary antibody deficiencies requiring treatment and is characterised by reduced immunoglobulin levels and reduced specific antibody production, leading to recurrent sino-pulmonary and gastrointestinal infections.1 Due to its heterogeneous nature a single gene mutation is unlikely, although a number of candidate genes affecting small groups of patients have been identified.2 Patients with CVID are at risk of autoimmunity and inflammatory conditions with approximately 25% of patients affected3 ,4 although the reasons for this are unclear.

    A number of recent immunological investigations have focussed on B cell phenotypes in these patients leading to a number of disease classification.5 ,6 T cell defects have also been reported in CVID patients7 although their significance with regard to reduced immunoglobulin production is uncertain. Due to autoimmune involvement in CVID, there has been considerable interest in regulatory T cells (Treg cells) since they have an important role in maintaining peripheral tolerance.8–11 Studies have confirmed that CD4+ CD25hi Foxp3+ Treg cells were reduced in CVID patients compared with levels found in healthy controls.12–14 As Foxp3 alone may be insufficient to identify Treg cells in humans, we aimed to further characterise these important regulatory cells using additional staining to measure intracellular cytolytic T lymphocyte associated antigen 4 (CTLA-4) expression. CTLA-4 (CD152) is transiently expressed on activated T cells and provides a negative signal upon binding to its ligand (CD80/86)15 and is constitutively expressed in Treg cells.16

    The consequences of reduced Treg cell numbers or function on T cell activation in these patients remains uncertain. Since the balance between T cell activation and regulation may be important in the pathology of autoimmunity in CVID patients, we investigated the relationship between activated T cells (defined by their expression of HLA-DR or granzyme B) and Treg cells in CVID patients with particular emphasis on patients with autoimmunity.

    Material and methods

    Lymphocyte subset analysis

    Lymphocyte subsets, absolute numbers and frequency were determined using flow cytometry (single platform) with a standard panel of monoclonal antibodies and Trucount tubes (BD, Oxford, UK) following manufacturer's instructions.

    T cell expression of HLA-class II and granzyme B

    CD8+ T cells were assessed for expression of HLA-DR and granzyme B using whole blood staining and flow cytometry following the manufacturer's instructions. Briefly, whole blood was stained with CD3, CD8 and DR antibodies (or appropriate control antibodies); following lysis of erythrocytes, the cells were washed in buffer prior to flow cytometry. Levels of granzyme B were assessed by surface staining with antibodies against CD3 and CD8; the samples were lysed, washed and incubated with BD perm/fix solution followed by incubation with anti-granzyme B antibody. All samples were washed in buffer (BD) and analysed using a FACSCalibur flow cytometer (BD) with data analysed using CellQuest software. For all analyses, 25000 events were acquired, CD3+ CD8+ T cells were gated and investigated for expression of HLA-DR or granzyme B. Positive expression of markers was defined using the negative control antibody staining to define the negative/positive cut-off points and were consistently applied across the control and patient groups. Figures shown refer to the percentage of cells positive for each activation marker as indicated in the figures.

    Treg analysis

    Foxp3 and CTLA-4 expression in CD4+ CD25hi T cells was assessed by means of cell surface and intracellular staining of isolated peripheral blood mononuclear cells (PBMC) using anti-CD4-peridinin chlorophyll A protein (PerCP) and CD25-R- phycoerythrin (Pe) to surface stain and anti-Foxp3-Alexa Fluor 488 and CTLA-4 allophycocyanin (APC) for intracellular staining using Foxp3 buffer set (BD). A parallel staining procedure using appropriate isotype and fluorochrome control antibodies was included with each test. All antibodies used in this study were obtained from Becton Dickinson (Oxford, UK) and samples were stained according to the manufacturer's instructions as previously described.14 For the purpose of analysis, CD4 T cells were identified from the PBMC population and manually gated and subsequently investigated for the expression of CD25, CD25hi/ FOXp3+ and Foxp3+/CTLA-4+. Positive expression of markers was defined using the negative control antibody staining to define the negative/positive cut-off points. While the expression of CD25hi was arbitrary, it was consistently applied to all sample analysis.

    Statistical analysis

    Statistical analysis was performed using unpaired Student t test with a p value of less than 0.05 indicating significance.

    Results

    Patient characteristics

    In all, 23 patients (13 male and 10 female subjects, mean age=49) and 15 healthy controls (seven male and eight female subjects, average age=41) volunteered for the study. All patients were being treated with immunoglobulin replacement therapy and blood samples (from patients and controls) were taken at around the same time of day, prior to immunoglobulin infusions, and were processed immediately. No other confounding features were noted. Diagnosis was made according to European Society for Immunodeficiencies (ESID) guidelines and ethical approval (Bradford research ethics committee) was obtained for this study. All patients and controls gave informed consent prior to their inclusion in the study and individuals were well at the time of testing. Within the patient group, 5/23 had some form of autoimmunity as defined by clinical and laboratory criteria: Of these 4/5 had autoimmune thrombocytopaenia and 1/5 had autoimmune haemolytic anaemia (both diagnosis based on laboratory tests following exclusion of other possible causes). Two patients had biopsy proven inflammatory bowel disease and ultrasound studies indicated enlarged spleens in five patients, three of whom had autoimmune symptoms.

    Lymphocyte subsets

    The relative proportions and absolute number of T, B cells and NK cells were assessed (table 1). CD4+ T cells were reduced in patients compared with controls (mean absolute number of 638 vs 834 cells/μl) although 4/5 patients with autoimmunity had CD4+ absolute count within the normal adult range. CD8+ T cells were similar in both control and patient groups (mean absolute counts of 521 vs 519 cells/μl). In common with previous reports, B lymphocytes were considerably reduced in the patient group compared with controls with means of 169 and 296 cell/μl, respectively (p=0.003).

    View this table:
    Table 1

    Absolute cell numbers of T cell subsets and B cells (cells/μl)

    Patients had reduced percentages of CD4+ CD25+ T cells and CD4+ CD25hi Foxp3+ Treg cells compared with controls

    There was a significant difference in the percentage of CD4+ CD25+ T cells between patients and controls (means of 40% vs 50% of total T cells, p=0.04) (figure 1A). Patients with autoimmunity had significantly lower percentage of CD4+ CD25+ T cells than patients not affected by autoimmunity (25% vs 44%, p=0.007). The percentage of CD4+ CD25hi Foxp3+ Treg cells was also reduced in patients compared with healthy controls (means of 2.5% vs 3.2%, p=0.04) (figure 1B) and the decreased frequency of Treg cells was most notable in patients with autoimmunity (1.1% vs 3.0%, p=0.004). As intracellular expression of CTLA-4 in Treg cells has been reported, the intracellular levels of CTLA-4 in CD4+ CD25hi Foxp3+ Treg cells in patients and controls were measured (figure 1C). CTLA-4 expression was observed in a mean of 84% of control Treg cells; the level of expression was lower in the patient group (p=0.003) with a mean of 56% of Treg cells (29%–90%). There was no significant difference in CTLA-4 expression level between patients with and without autoimmunity (means of 54% and 61%, respectively).

    Figure 1
    Figure 1

    (A) CD25 expression on CD4+ T cells from patients (with and without autoimmunity) and controls. PBMC were stained with antibodies against CD4 and CD25. Results are expressed as percentage of CD4+ cells positive for CD25. Values for individual patients are shown along with the overall mean value for each group (indicated by the horizontal bar). (B) Expression of Foxp3 in CD4+ CD25hi Treg cells in patients and controls. PBMC were isolated and surface stained for CD4 and CD25. Cells were fixed and permeabilised and stained for expression of Foxp3. CD4+ T cells were gated and Foxp3 expression assessed in cells that had expressed high levels of CD25. Values for individual patients are shown along with the overall mean value for each group (indicated by the horizontal bar). (C) Expression of CTLA-4 in CD4+ CD25hi Foxp3+ T cell in patients and controls. PBMC were isolated stained as outlined above but with a CTLA-4 antibody included at the intracellular staining step. The percentage of CD4+ CD25hi Foxp3+ cells expressing CTLA-4. Values from individual patients are shown along with the overall mean value for each group (indicated by the horizontal bar).

    Patients’ CD8+ T cells have elevated levels of granzyme B and HLA class II (DR) compared with controls

    Since the levels of Treg cells were reduced in some patients the levels of activation markers HLA-DR and granzyme B on T cells were assessed. A statistically significant elevated percentage of CD3+ CD8+ granzyme B+ T cells was observed in patients compared with controls (means of 59% vs 30%, respectively, p=0.001) (figure 2A). CD8+ HLA class II (DR) was also expressed at a higher level in CVID patients than controls with respective means of 45% and 21% (p=0.0001) (figure 2A). There was no obvious link between expression levels and autoimmunity. The levels of GrB and DR were also assessed in CD3+ CD8− T cells. The mean percentage of CD3+ CD8− T cells expressing HLA-DR was 9% in the controls and 18% in the patients (p=0.03). Granzyme B was expressed in a mean of 6% of controls and 14% of patients (p=0.024, figure 2B). No correlation between elevated granzyme B and autoimmunity could be established.

    Figure 2
    Figure 2

    Expression of HLA-DR/granzyme B on T cells. Whole blood was stained with antibodies against anti CD3 and CD8 and DR/granzyme B. The percentage of cells expressing DR and granzyme B was assessed on CD3+ CD8+ (A) and CD3+ CD8− T cells (B). Values for individual patients are shown along with the overall mean value for each group (indicated by the horizontal bar).

    Ratio of effector to Treg cells

    To focus on the relationship between activated and Treg cells, we calculated the ratio of effector to Treg cells in control and patient groups comparing absolute numbers of activated CD8+ cells (granzyme B+/HLA-DR+) as well as activated (DR+) CD4+ T cells with absolute numbers of Treg cells (defined as CD4 CD25hi Foxp3+) (figure 3). The average ratio of CD8+ DR+ to Treg cells in the controls was 4. This increased to 13 in patients without autoimmunity and a further significant increase (to 92) was observed in patients with autoimmunity (p=0.03). A similar pattern was observed when CD4+ DR+ to Treg ratio was employed (cons=3; patients without autoimmunity=7 and patients with autoimmunity=29 (p=0.01)). The average ratio of CD8+GrB+ to Treg cells across the control group was 7; the ratio increased to 20 in patients with no autoimmunity and a further non-significant increase was seen in patients with autoimmunity (34, p=0.14).

    Figure 3
    Figure 3

    Ratio of activated to Treg cells using CD3+ CD8+ DR+/granzyme B+ and CD4+ DR+ as a measure of activation and CD4+ CD25hi Foxp+ as a measure of Treg cells. In all cases, the absolute number of relevant cell types was used in the calculations. Mean values with SD for each group are shown.

    Discussion

    The aim of this study was to investigate the relationship between activated and Treg cells with the presence of autoimmunity in patients with CVID. Using two measures of activation (HLA-DR and granzyme B expression), we observed an elevated level of activated CD8+ T cells in patients with CVID compared with levels in healthy controls. Similar findings have been previously reported in CVID patients17 ,18 although their relationship to autoimmunity and to the levels of Treg cells has not previously been defined. Patients with elevated levels of activated T cell have been reported to have reduced memory B cells6 and augmented cytotoxic capacity19 suggesting a possible direct role in B cell pathology. The enhanced level of granzyme B found in populations of CD3+ CD8− T cells from the whole patient group was a novel and unexpected finding although its significance is uncertain. Given that lymphocyte subset analysis showed that the CD3+ T cell compartment was made of up single positive CD4+ or CD8+ T cells, we assume that these cells were CD4 T helper cells although it was not clear if these cells were cytolytic or regulatory in function as granzyme B has been identified in both.20 Recent reports in murine systems suggest a possible role for Treg cells expressing granzyme B in delaying tumour clearance21 and in controlling lung inflammation22 although this work has yet to be confirmed in humans.

    Our results confirmed previous reports suggesting that the patient group had both lower total CD4 T cell CD25 expression13 ,14 (in contrast to the elevated levels of HLA-DR and granzyme B observed) and also reduced levels of Treg cells (affecting most notable patients with autoimmunity). These are important observations given the possible contribution of the high affinity IL-2 receptor as a regulatory mechanism employed by Treg cells.23 ,24 Furthermore, since IL-2 is important for the generation and maintenance of Foxp3 expression in Treg cells,25–27 reduced T cell IL-2 receptor expression may contribute to the reduced expression of Foxp3 in CVID patients.

    Treg cells are conventionally defined by their high expression of CD25 in association with intracellular expression of FOXp3 although in humans effector T cells have also been reported to be FOXp3+; additional staining using low expression of CD127 is subject to the same limitations. As Treg cells have been reported to express high intracellular levels of CTLA-4, which is important for the suppressor functions of these cells,28–30 we included this in our assessment of Treg cells in CVID patients and controls. The reduced levels of intracellular CTLA-4 noted in CVID patients could indicate recent or chronic activation in those individuals and is, to our knowledge, a novel finding. There was, however, no difference in CTLA-4 expression between patients with and without autoimmunity or between patients with low Foxp3 which suggests that any inefficiency on the part of Treg cells in patients with autoimmunity was not due to reduced CTLA-4 induced negative regulation of effector T cells.

    While measuring Treg levels alone is useful, comparing the levels of Treg cells and activated T cells may clarify the clinical significance regarding the relative balance between these subpopulations of T cells. This approach has been widely used in other studies31–35 and a key aim of this study was to investigate the usefulness of it in predicting the likelihood of autoimmunity developing in CVID patients. Patients with elevated levels of activated T cells but normal levels of Treg cells did not suffer from autoimmunity suggesting that T cell activation alone was not responsible for the autoimmune tissue damage. However, patients with autoimmunity had a consistent pattern of elevated activated T cells in conjunction with reduced levels of Treg cells highlighted by elevated activated to regulatory ratios. The true significance of these findings with regard to disease pathology is not clear and the findings could be epiphenomenal rather than causal although they highlight the complex immune dysfunction associated with this condition. These preliminary results in a small patient cohort suggest that the level of T cell activation should be assessed in conjunction with Treg cells in patients with CVID and this approach is worthy of further investigation in a larger study.

    Take-home messages

    ▸ Common variable immunodeficiency (CVID) patents have elevated levels of activated CD8+ T cells.

    ▸ Patients with autoimmunity have reduced regulatory T cell levels (defined as CD4+ CD25hi Foxp3+).

    ▸ Intracellular cytolytic T lymphocyte associated antigen 4 expression in Treg cells is also reduced in CVID patients compared with controls.

    ▸ CVID patients with autoimmunity have a higher ratio of activated to regulatory T cells compared with patients with no autoimmunity and control levels.

    ▸ We suggest that the balance between activated and regulatory T cells is important in determining the autoimmune pathological consequences of CVID.

    What this study adds 

    This paper describes a single centre study focussing on the relationship between activated and regulatory T cell subsets in patients with the antibody deficiency, common variable immunodeficiency (CVID), focussing on patients with additional autoimmune symptoms. Our results confirmed previous reports that showed CVID patients had increased T cell expression of the activation markers HLA-DR and granzyme B. We have added extra information to those reports by demonstrating that in some patients CD3+ CD8− T cells (believed to be CD4+ T helper cells) also expressed high levels of granzyme B although the significance of this finding is unclear. We also report that CVID patients had reduced Treg cells (as defined by their expression of CD4+ CD25hi and Foxp3+) which confirmed previous reports by ourselves and others. We additionally demonstrated that while intracellular cytolytic T lymphocyte associated antigen 4 (CTLA-4) expression was reduced in patients with CVID compared with healthy controls, there was no association between low CTLA-4 and autoimmunity. Finally, we show that increased T cell expression of granz B/HLA-DR in conjunction with reduced Treg levels in patients with autoimmunity leads to an elevated activated to regulatory T cell ratio. This suggested that reduced regulatory function in these patients may have functional consequences in allowing exaggerated T cell responses.

    Acknowledgments

    The authors wish to thank John Toolan and Kate Ford for their help in sample collection and patients and controls from St James's University Hospital for agreeing to take part in the study.

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    Footnotes

    • Contributors CC, SS and PW designed the study. GA recruited the patients. CC, GA, NS and JC performed the practical work. CC prepared the manuscript. All authors reviewed the manuscript.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.