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Keywords:

  • granzymes;
  • γ-interferon;
  • lung transplant;
  • natural killer T cell-like cells;
  • tumour necrosis factor-α

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Background and objective:  Natural killer T (NKT)-like cells are a small but significant population of T lymphocytes; however, their role in lung transplant and the effect of current immunosuppressive agents on their function is largely unknown. We have previously shown lung transplant rejection was associated with an increase in peripheral blood T cell γ-interferon (IFN-γ), tumour necrosis factor-α (TNF-α) and granzyme B. NKT-like cells are a source of these pro-inflammatory mediators and as such may be involved in lung transplant pathology.

Methods:  We analysed NKT-like cell numbers and cytokine and granzyme profiles in peripheral blood from a group of stable lung transplant patients and control subjects using multiparameter flow cytometry.

Results:  There was a significant increase in NKT-like cells in transplant patients compared with control subjects (6.8 ± 4.9 vs 0.8 ± 0.2% lymphocytes respectively). There was an increase in the numbers of NKT-like cells producing IFN-γ, TNF-α, IL-2 IL-17, granzyme and perforin in transplant patients compared with controls. Immunosuppressant drugs were less effective at inhibiting IFN-γ and TNF-α production by T and NKT-like cells than NK cells in vitro.

Conclusions:  Current therapeutics is inadequate at suppressing NKT-like cell numbers and their production of pro-inflammatory mediators known to be associated with graft rejection. Alternative therapies that specifically target NKT-like cells may improve patient morbidity.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Five-year survival after lung transplantation is less than 60%, clearly a disappointing statistic given the huge expenses involved in lung transplant, the demands of post-transplant care, and the critical shortage of donor organs.1 T-cell T-helper-type 1 (Th1) pro-inflammatory cytokines are involved in transplant rejection and are a target of current immunosuppression strategies.2 We have previously shown Th1 pro-inflammatory cytokines, IL-2 and tumour necrosis factor-α (TNF-α) were significantly reduced in peripheral blood CD4+ T cells in stable transplant patients consistent with some degree of efficacy of the anti-rejection regimens, however, production of γ-interferon (IFN-γ) by CD8+ T cells was not suppressed and therefore may have pathogenic significance.3 Recently we have also shown the pro-inflammatory mediator, granzyme B was increased in peripheral blood T cells in both stable and rejecting transplant patients4 and importantly, the combination of IFN-γ, TNF-α and granzyme B was increased in patients preceding a fall in lung function and diagnosis of chronic rejection, possibly providing an early marker of impending deterioration.5,6 These findings suggest current immunosuppression protocols are inadequate at preventing some pro-inflammatory mediators associated with transplant rejection that might, at least in part, explain the high incidence of chronic rejection in lung transplantation.

Natural killer (NK) and natural killer T (NKT)-like cells are also producers of these pro- and anti-inflammatory mediators and as such may play an important role in transplant rejection/tolerance.7 NK cells are divided into CD56bright (weakly cyotoxic but efficient cytokine producers) and CD56dim (strongly cytotoxic but poor cytokine producers).8 The role of NKT-like cells in solid organ transplant, particularly human lung transplant, is not well defined.7 One recent study showed an increase in NK cells in heart transplant patients whereas numbers of T and NKT-like cells were similar to controls.9 The mechanism of NKT cell-mediated transplant tolerance has been shown to be dependent on anti-inflammatory cytokines IL-1010 and transforming growth factor-β (TGF-β)11 in animal transplant models. However, the role of pro-inflammatory cytokines and granzymes by NKT-like cells has not been studied in human lung transplant patients. Although the function of NK cells has been shown to be altered in the presence of various immunosuppressive therapies,7 the effect of current immunosuppressive agents on NKT-like cell function is largely unknown.

We hypothesized that current immunosuppression protocols may be inadequate at preventing increased numbers of NKT-like cells producing these pro-inflammatory mediators.

To investigate this hypothesis, intracellular cytokine and granzyme profiles were determined in T, NK and NKT-like cell subsets from peripheral blood from stable lung transplant patients and healthy control volunteers using multiparameter flow cytometry. The effects of several commonly used immunosuppressants on T, NK and NKT-like cell production of IFN-γ, TNF-α and granzyme B were determined in vitro.

METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Patient and control groups

Ethics approval for the study was obtained from the Royal Adelaide Hospital Ethics committee (protocol 010711) in compliance with the Helsinki Declaration. Sixteen lung transplant recipients with stable lung function (FEV1) and no clinical evidence of current acute or chronic rejection or infection were invited to participate in the study. All patients were submitted to the same protocol and analysis performed retrospectively. All transplant patients were at least 6 months post-transplant (median = 37 months, range = 6–112 months).

Immunosuppression therapy comprised combinations of either cyclosporin A (CsA) or tacrolimus (Tac) with prednisolone, and azathioprine or mycophenolate mofetil. Trough plasma drug levels of either CsA or Tac were within or above recommended therapeutic ranges (range for CsA (80–250 µg/L) and Tac (5–15 µg/L). Fifteen healthy age-matched volunteers with no evidence of lung disease were recruited as controls. Venous blood was collected into 10 U/mL of preservative free sodium heparin (DBL, Sydney, Australia) and blood samples were maintained at 4°C until processing.

Leucocyte counts

Full blood counts, including white cell differential counts, were determined on blood specimens using a CELL-DYN 4000 (Abbot Diagnostics, Sydney, Australia). Blood films were stained by the May-Grunwald-Giemsa method and white cell differential counts checked by morphological assessment microscopically.

Absolute T, B, NKT-like and NK cell counts

One hundred and fifty microlitres of peripheral blood were stained with appropriately diluted fluorescently conjugated monoclonal antibodies to CD3 FITC (BD Biosciences, Sydney, Australia) (BD), CD56 PE (BD), CD45 perCP.Cy5.5 (BD) and CD19 Alexa 647 (BD). Samples were analysed by gating using forward scatter (FSC) versus side scatter (SSC) to exclude platelets and debris. Gated cells were analysed with CD45 perCPCy5.5 (BD) to ascertain that cells were of lymphoid origin as previously reported.3 A minimum of 5000 CD56 positive, low SSC events were acquired in list-mode format for analysis.

T, NKT-like and NK cell granzymes

One hundred and fifty microlitres of peripheral blood was added to FACS tubes (BD). T, NKT-like and NK onoclonal antcells were enumerated by staining with appropriately diluted fluorescently labeled mibodies, CD3 FITC (BD) and CD56 PC5 (Beckman Coulter, Sydney, Australia). T cells were defined as CD3+CD56-, NK cells as CD56+CD3- and NKT-like cells identified as CD56+CD3+ low side scatter events.

To enumerate T, NKT-like and NK cell granzyme A, granzyme B, granulysin and perforin, 150 µL of peripheral blood was added to FACS tubes. To lyse red blood cells, 2 mL of FACSlyse solution (BD) was added and tubes incubated for 10 min at room temperature in the dark. Tubes were decanted after centrifugation at 500 g for 5 min. Cells were permeabilized by addition of 0.5 mL 1:10 diluted FACSperm (BD) to each tube, mixed, and incubated a further 10 min at room temperature in the dark. Two mL 0.5% bovine serum albumin (Sigma, Sydney, Australia) in IsoFlow (Beckman Coulter) was then added and the tubes centrifuged at 300 g for 5 min. After decanting supernatant, Fc receptors were blocked with 10 µL human immunoglobulin (Intragam, CSL, Parkville, Australia) for 10 min at room temperature. For enumeration of granzyme A, 2.5 µL undiluted anti-human granzyme A (BD) was added for 15 min in the dark. Two mL of 0.5% bovine serum albumin in IsoFlow was then added and the tubes centrifuged at 300 g for 5 min. After decanting, 5 µL of rat anti-mouse IgG1 PE (BD) was added for 15 min in the dark. Two mL of 0.5% bovine serum albumin in IsoFlow was then added and the tubes centrifuged at 300 g for 5 min. Five microlitres of appropriately diluted anti-CD3 FITC (BD) and anti-CD56 PC5 (Beckman Coulter), PE-conjugated antibodies to granzyme B (BD), granulysin (eBioscience, San Diego, CA, USA), 10 µL undiluted perforin (BD) were added for 15 min in the dark at room temperature as shown in Table 1. Cells were analysed within 1 h on a FACSCalibur flow cytometer using CellQuest software (BD). Samples were analysed by live gating using FL3 staining versus side scatter (SSC). A minimum of 5000 CD56 positive, low SSC events were acquired in list-mode format for analysis.

Table 1.  Combinations of antibodies used to define T, natural killer T (NKT)-like and NK subsets and cytoplasmic molecules. All antibodies were directly conjugated except granzyme A (unlabelled) where a two-step stain using rat anti-mouse PE was performed
TubeFITCPEPC5
 1CD3Granzyme ACD56
 2CD3Granzyme BCD56
 3CD3GranulysinCD56
 4CD3PerforinCD56
 5CD3γ-InterferonCD56
 6CD3Tumour necrosis factor-αCD56
 7CD3IL-2CD56
 8CD3IL-17CD56
 9CD3IL-4CD56
10CD3Transforming growth factor-βCD56

T, NKT-like and NK cell cytokine production

Cytokine production was assessed as previously described for T cells.3 One-millilitre aliquots of blood and 1 mL RPMI 1640 medium were placed in 10 mL sterile conical polyvinyl chloride tubes (Johns Professional Products, Sydney, Australia). Phorbol myristate (PMA) (25 ng/mL) (Sigma) and ionomycin (1 µg/mL) (Sigma) were added to stimulate T, NK and NKT-like cell cytokine production. Brefeldin A (10 µg/mL) was added as a ‘Golgi block’ (Sigma) and the tubes reincubated in a humidified 5% CO2/95% air atmosphere at 37°C. At 16 h 100 µL 20 mM ethylenediaminetetraacetic acid/phosphate buffered saline was added to the culture tubes that were vortexed vigorously for 20 s to remove adherent cells. Cells were treated with FACSLyse and FACSPerm as for granzyme B enumeration and 5 µL of appropriately diluted anti-CD3 (BD), anti-CD56 PC5 (Beckman Coulter) and PE-conjugated anticytokine monoclonal antibodies to IL4, IL-2, IFNγ, TNFα (BD), IL-17A (eBioscience, Sydney, Australia) and TGF-β (IQ Products, Groningen, the Netherlands) (Table 1) were added for 15 min in the dark at room temperature. Events were acquired and analysed as described earlier.

Effect of immunosuppressants on T, NKT-like and NK cell granzyme B, IFN-γ and TNF-α production

The effect of therapeutic dose of commonly used immunosuppressants, CsA, Tac and prednisolone on T, NKT-like and NK cell granzyme B, IFN-γ and TNF-α production was determined in vitro using blood from five control subjects. One-millilitre aliquots of blood were placed in 10-mL sterile conical polyvinyl chloride tubes and incubated with 1 mL aliquots of RPMI containing a final concentration of 5 ng/mL cyclosporine A (Novertis Pharmaceuticals, North Ryde, New South Wales, Australia), 25 ng/mL Tac (Janssen-Cilag, North Ryde, New South Wales, Australia) or 1 × 10−6 M methylprednisolone (David Bull Laboratories, Melbourne, Australia) (diluted in RPMI 1640 medium) or RPMI medium as control for 24 h at 37°C in an atmospheric pressure of 5% CO2. Cells were then stimulated with PMA (25 ng/mL), ionomycin (1 µg/mL) and brefeldin A (10 µg/mL) for 16 h as described earlier, then 100 µL 20 mM ethylenediaminetetraacetic acid/phosphate buffered saline was added to the culture tubes, which were vortexed vigorously for 20 s to remove adherent cells. T, NKT-like and NK cells were stained for granzyme B, IFN-γ and TNF-α as described earlier.

Statistical analysis

Statistical analysis was performed using the non-parametric Mann–Whitney when comparing means of two groups and Pearson correlation tests using SPSS software (SPSS Inc., Chicago, IL, USA) and differences between groups of P < 0.05 considered significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

BLOOD T, B, NKT-like and NK cell counts

There was no significant difference in the absolute lymphocyte counts for controls and transplant patients (1.5 (1.4–1.9) and 1.6 (1.3–2.1) × 109/L, median and range for controls and patients, respectively, P > 0.05).

There was a trend for an increase in the percentage of T cells for transplant patients compared with controls (Table 2). There was a significant decrease in the percentage of CD19+ B cells for transplant patients compared with controls (P = 0.005) (Table 2). There was a trend for a decrease in the percentage of CD3-CD56+dim (P = 0.072) and CD3-CD56bright NK cells (P = 0.061) for transplant patients compared with controls (Table 2). Notably, there was a significant increase in the percentage of CD3+CD56+ NKT-like cells for transplant patients compared with controls (P < 0.001) (Table 2). There was a negative correlation between months post-transplant and the percentage of CD19+ B-cells in the patient group (R = −0.867, P = 0.012) but no correlation between other lymphocyte subsets. There were no correlations between lymphocyte subsets and age, drug type or dose (P > 0.05 for all).

Table 2.  Percentage of lymphocyte subsets for control and transplant patient groups (median and range)
SubjectsCD3CD19CD3-CD56+ dimCD3-CD56+ brightCD3+CD56+
Controls74 (61–81)12 (9–29)9 (4–17)0.4 (.3-.5)0.8 (.2–6)
Patients82 (66–91)3 (1–19)4 (1–19)0.2 (.1–1.0)5 (1–17)
P0.0530.0050.0720.061<0.001

Granzyme and perforin expression in T, NKT-like and NK cells

Granzyme and perforin expression in T, NKT-like and NK cells was analysed as a percentage of each lymphocyte subset and as a percentage of total lymphocytes.

As a percentage of each subset, there was an increase in granzyme A by NKT-like cells, granzyme B by T and NKT-like cells and a decrease in the percentage of NK lymphocytes expressing granzyme B (Fig. 1). There was no change in perforin expression by any cell subset (data not shown).

image

Figure 1. Box and whisker plots showing the percentage of T, Natural killer T (NKT)-like and NK cell subsets expressing granzyme B (GB), granzyme A (GA), granulysin (Gran), γ-interferon (IFNg) and tumour necrosis factor-α (TNFa) (as a percentage of each subset) (median ± interquartile range in box, data range in whiskers) from control (clear box) and transplant patients (shaded box).

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As a percentage of lymphocytes, there was a significant increase in the percentage of T cells and NKT-like cells expressing granzyme A, granzyme B and granulysin in transplant patients compared with the control group (Table 3). There was also a significant increase in the percentage of NKT-like lymphocytes expressing perforin in transplant patients compared with the control group. There was no change in the percentage of NK cells expressing granzymes or perforin in transplant patients compared with the control group. Data for T, NKT-like and NK expression of granzymes and perforin as a percentage of each subset and as a percentage of lymphocytes is summarized in Table 3. As the CD3+CD56+ bright population of NK cells was such a minor fraction of lymphocytes, NK cells were subsequently analysed as both CD3+CD56+ bright and dim populations combined. There was no correlation between months post-transplant, age, drug type or dose and granzyme or perforin expression in T, NKT-like or NK cells (P > 0.05 for all).

Table 3.  T, natural killer T (NKT)-like and NK cell granzyme A, granzyme B, granulysin and perforin expression in transplant and control groups (median and range) (as a percentage of lymphocytes) and as a percentage of T, NK-like and NK subsets (comparison with control)
  ControlTransplantPTransplant % lymphsTransplant % subset
  1. ([UPWARDS ARROW]), significant increase; ([DOWNWARDS ARROW]), significant decrease; NC, no change.

Granzyme AT0.03 (0.01–0.2)0.85 (0.1–10.2)0.005[UPWARDS ARROW]nc
NKT00.44 (0–1.3)0.001[UPWARDS ARROW][UPWARDS ARROW]
NK2.0 (1.2–3.8)0.8 (0.2–7.8)0.126NCNC
Granzyme BT3.6 (1.1–8.8)12.2 (0.3–39)0.020[UPWARDS ARROW][UPWARDS ARROW]
NKT0.2 (0–0.5)2.2 (0–9.3)0.001[UPWARDS ARROW][UPWARDS ARROW]
NK7.9 (2.3–14.5)2.8 (0.3–11.4)0.073NC[DOWNWARDS ARROW]
GranulysinT0.05 (0–0.10)0.19 (0.01-.22)0.020[UPWARDS ARROW]NC
NKT00.17 (0–1.9)0.003[UPWARDS ARROW]NC
NK1.4 (0.3–2.2)0.3 (0–5.7)0.364NCNC
PerforinT001.0NCNC
NKT00.02 (0–1.9)0.024[UPWARDS ARROW]NC
NK7.0 (3–16.3)2.2 (0.1–11.4)0.053NCNC

IFN-γ, TNF-α, IL-2 and IL-17 production by T, NKT-like and NK cells

IFN-γ, TNF-α, IL-2 and IL-17 production by T, NKT-like and NK cells was analysed as a as a percentage of each lymphocyte subset and as a percentage of total lymphocytes.

As a percentage of each subset, there was a significant decrease in IFN-γ and TNF-α production by T and NK cells and IFN-γ by NKT-like cells (Fig. 1). There was no change in IL-2 or IL-17 production in any cell subset (data not shown).

As a percentage of lymphocytes, there was a significant increase in production of IFN-γ and TNF-α by NKT-like cells in the transplant group compared with the control group (Table 4). There was a significant decrease in TNF-α and IL-2 and a trend for a decrease in IFN-γ in T cells in the transplant group compared with the control group. There was a significant decrease in IFN-γ and TNF-α in NK cells in the transplant group compared with the control group. Data for T, NKT-like and NK expression of cytokines as a percentage of each subset and as a percentage of lymphocytes is summarized in Table 4. Representative plots showing the expression of IFN-γ, TNF-α and granzyme B in NKT-like and NK cells in a transplant patients and control subjects are shown in Figure 2. There was a negative correlation between months post-transplant and the percentage of IL-17A producing NKT-like cells in the patient group (R = −0.900, P = 0.037) (Fig. 3). There were no correlations between the percentage of T, NKT-like or NK subsets producing IFN-γ, TNF-α, IL-2 or T or NK subsets producing IL17 and age, drug type or dose (P > 0.05 for all).

Table 4.  γ-Interferon (IFN-γ) and tumour necrosis factor-α (TNF-α), IL-2 and IL-17 production by T, natural killer T (NKT)-like and NK cells in transplant and control groups (median and range) (as a percentage of lymphocytes) and as a percentage of T, NKT-like and NK subsets (comparison with control)
  ControlTransplantPTransplant % lymphsTransplant % subset
  1. ([UPWARDS ARROW]), significant increase; ([DOWNWARDS ARROW]), significant decrease; NC, no change.

IFN-γT44 (28–57)33 (9–74)0.063[DOWNWARDS ARROW]trend[DOWNWARDS ARROW]
NKT0.5 (0.1–1.8)1.9 (0.3–11.5)0.027[UPWARDS ARROW][DOWNWARDS ARROW]
NK3.9 (1.5–13.1)1.1 (0.2–5.9)0.031[DOWNWARDS ARROW][DOWNWARDS ARROW]
TNF-αT61 (52–79)43 (2–83)0.032[DOWNWARDS ARROW][DOWNWARDS ARROW]
NKT0.7 (0.2–3.6)1.9 (0.1–14.9)0.037[UPWARDS ARROW]NC
NK3.7 (2–8.2)0.5 (0.1–3.8)0.001[DOWNWARDS ARROW][DOWNWARDS ARROW]
IL-2T31 (18–51)12 (6–36)0.032[DOWNWARDS ARROW][DOWNWARDS ARROW]
NKT0.12 (0.04–0.48)0.38 (0.20–4.90)0.015[UPWARDS ARROW]NC
NK001.0NCNC
IL-17T1.1 (0.9–2.1)1.7 (0.5–2.2)0.265NCNC
NKT0.02 (0–0.03)1.3 (0.03–0.64)0.015[UPWARDS ARROW]NC
NK001.0NCNC
image

Figure 2. Representative plots showing granzyme B (GB), γ-interferon (IFN-γ) and tumour necrosis factor-α (TNF-a) expression in CD3-CD56+ natural killer (NK) cells and CD3+CD56+ NKT-like cells in control (left column) and transplant subjects (right column). Cells were gated on CD56 PC5-positive low side scatter (SSC) events as shown in (A). Note the increase in the percentage of NKT-like cells in transplant patients expressing granzyme B, IFN-γ and TNF-α compared with control patients. Note also the decrease in the percentage of NK cells in transplant patients expressing IFN-γ and TNF-α (but not granzyme B) compared with control patients.

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image

Figure 3. Graph showing a negative correlation between months post-transplant and the percentage of IL-17A producing natural killer T (NKT)-like cells in the patient group (R = −0.900, P = 0.037).

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IL-4 and TGF-β production by T, NKT-like and NKT cells

There was no change in IL-4 or TGF-β production by T, NKT-like or NK cells between transplant and control groups (P > 0.05 for all) (data not shown).

Effect of immunosuppressants on T, NKT-like and NK cell granzyme B, IFN-γ and TNF-α production

The production of IFN-γ and TNF-α by T, NKT-like and NK cells was significantly inhibited in the presence of 5 ng/mL cyclosporine A, 25 ng/mL Tac or 10−6 M methylprednisolone compared with control without drugs (Fig. 4). Inhibition by all drugs was significantly greater in NK cells compared with both NKT-like and T cells (approximately two- to threefolds more). There was no difference in the inhibition of IFN-γ and TNF-α by NKT-like and T cells in the presence of all drugs (P > 0.05). Granzyme B production was increased in T, NKT-like and NK cells following stimulation without the presence of drugs, however, there was no change in production of granzyme B in any cell subset in the presence of any drug used compared with control without drugs (P > 0.05).

image

Figure 4. Graphs showing the inhibitory effect of immunosuppressants on T, natural killer (NK) and NKT-like cell granzyme B, perforin γ-interferon (IFNγ) and tumour necrosis factor-α (TNFα) (mean ± 2SD of five experiments). Clear bars: NK cells; light grey bars: NKT-like cells; dark grey bars: T cells. (*P < 0.05). CsA, cyclosporin A; Tac, tacrolimus, MP, methyl predisolone.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This is the first study to show that numbers of CD56+CD3+ NKT-like cells are increased in stable lung transplant patients and the first to identify these cells as producers of a range of pro-inflammatory cytokines and granzymes even in patients with ‘stable’ transplant status when compared with normal controls. Consistent with our current findings, we have previously shown that T-cell pro-inflammatory cytokines IFNγ, TNF-α and IL-2 were decreased in stable transplant patients consistent with an impact of immunosuppressant therapy although suppression of CD8+ T-cell production of IFNγ3 and T-cell granzyme B was less effective.4

Our gating technique used to define T cells in these previous studies did not distinguish between true T cells (CD3+CD56-) and NKT-like cells (CD3+CD56+). We now show most pro-inflammatory cytokines are reduced in true T-cells (CD3+CD56-) in stable transplant patients consistent with our previous findings.3 However, there was no change in (CD3+CD56-) T-cell IL-17 production between patient and control groups suggesting immunosuppression protocols are ineffective at reducing this pro-inflammatory cytokine shown to be associated with cardiac allograft rejection.12 Perhaps more importantly, we show that IL-17 production by NKT-like cells is increased in stable transplant patients suggesting that these cells may play an important role in lung transplant rejection. Furthermore, our findings of a negative correlation between the percentage of NKT-like cells producing IL-17A and time post-transplant, suggest a possible link between NKT-like cells and early rather than late graft rejection.

We also confirm our previous findings of increased T-cell granzyme B in stable lung transplant patients but now show that T-cell granzyme A and granulysin are also increased and perforin was unchanged in the patient group compared with controls suggesting therapeutics are also inadequate at reducing these cytotoxic mediators.

In general terms, data for the percentage of total lymphocyte subsets expressing pro-inflammatory mediators were similar to the percentage of individual T, NKT-like and NK subsets (or no changes were observed). Importantly, numbers of CD56+CD3+ NKT-like lymphocytes are increased in stable lung transplant patients and expression of a range of pro-inflammatory cytokines and granzymes are increased or unchanged in this subset suggesting immunosuppression protocols are also inadequate at inhibiting these pro-inflammatory mediators associated with graft rejection. However, a balance between over-immunosuppression of T, NKT-like and NK cell pro-inflammatory mediators resulting in inadequate response to infective organisms and malignancy on the one hand, and under-immunosuppression that may result in graft rejection on the other hand, is the ultimate goal of transplant therapeutics and future research is required to define these optimum physiological levels.

One of the very few studies on NKT-like cells in solid organ transplant showed an increase in NK cells but no alteration in T or NKT-like cell numbers in patients following heart transplantation9 although there was evidence of increased expression of the cytotoxicity effector molecule CD244 on T and NKT-like cells. In contrast, we found an increase in NKT-like cells, a trend for an increase in T cells and a decrease in NK cells in stable lung transplant group. These differences may have been due to a variety of causes such as the much longer post-transplant times of the heart transplant group (36 vs 67 months post-transplant for the lung and heart transplant groups, respectively). We further showed that there was no alteration in anti-inflammatory cytokines IL-4 or TGF-β by T, NK and NKT-like cells between patient and control groups suggesting that current immunosuppression therapy has little effect on these anti-inflammatory cytokines associated with graft tolerance following lung transplant.7,10

Further evidence for the ineffectiveness of current therapy was provided by our in vitro experiments that showed lack of modulation of granzyme B in T, NK and NKT-like cells in the presence of therapeutic doses of cyclosporine or Tac, prednisolone. Taken together our data suggests that other strategies are necessary to reduce these cytotoxic molecules. In this regard we have recently shown inhibition of T-cell granzyme B production in the presence of gabexate mesylate in vitro and this compound may also be a suitable therapeutic agent to reduce granzyme B production by NKT-like cells.4

Further, inhibition of pro-inflammatory cytokines in vivo was more effective for patient's T-cells than NKT-like cells suggesting possible resistance of NKT-like cells to therapy.

As we have previously shown increased peripheral blood T cell IFNγ, TNFα and granzyme B was associated with chronic lung rejection (bronchiolitis obliterans syndrome (BOS)),5 in several patients preceding BOS and in patients with lymphocytic bronchiolitis, a condition often leading to BOS,6 our current study raises several very important questions regarding the cell biology associated with lung transplant rejection. Are NKT-like cells some of the first cells to be activated following transplant? Are NKT-like cells the true instigators of graft rejection? Interestingly, a recent study showed a trend for an increase in NKT-like cells in bronchoalveolar fluid during acute lung transplant rejection.13 We are currently investigating longitudinal T, NK and NKT-like cell numbers and cytokine/granzyme profiles in blood, bronchoalveolar fluid and in intraepithelial lymphocytes derived from bronchial brushings14 in a group patients immediately following lung transplant to further elucidate these concepts. Our in vitro experiments showed that therapeutic doses of cyclosporine, Tac and prednisolone were significantly more inhibitory on IFN-γ and TNF-α production by NK cells than in T or NKT-like cells. However, these experiments do not explain why there was an increase in NKT-like cells compared with T cells producing these pro-inflammatory mediators in patients.

Our study also shows that there was a significant decrease in circulating B cells in these stable patients compared with healthy controls. To our knowledge this is the first report of B cell lymphopaenia in stable lung transplant patients. Long-term corticosteroid therapy has been shown to decrease B-cell counts,15 whereas calcineurin inhibitors have been shown to indirectly inhibit B cells by interfering with T cell signals,16 hence the action of these drugs may be responsible for our findings of decreased B cell counts in these patients. Furthermore, post-transplant hypogammaglobulinaemia has been reported as a frequent occurrence following lung transplant,17,18 which may be related to decreased B cell numbers. Our findings of a negative correlation between time post-transplant and B cell numbers may be due to action of these drugs, particularly prednisolone.15

In conclusion, current therapeutics is inadequate at suppressing NKT-like cell numbers and their pro-inflammatory mediators associated with graft rejection. Drugs that reduce NKT-like cell numbers and associated pro-inflammatory mediators may improve patient morbidity. Longitudinal monitoring of T, NK and NKT-like cell numbers, granzyme and pro-inflammatory cytokine profiles in the various blood/lung compartments in patients following lung transplant may identify the role of these cell subsets and associated pro-inflammatory mediators in transplant outcome and elucidate whether NKT-like cells have a significant role in lung transplant rejection.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The authors acknowledge the expert technical assistance of Miss Jessica Ahern. This study was funded by a National Health and Medical Research Council grant.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES