WO1999006811A2

WO1999006811A2 - Method for counting dendritic cells

Info

Publication number: WO1999006811A2
Application number: PCT/NZ1998/000116
Authority: WO
Inventors: Derek Nigel John Hart
Original assignee: Derek Nigel John Hart
Priority date: 1997-08-01
Filing date: 1998-07-31
Publication date: 1999-02-11
Also published as: WO1999006811A3; AU8653598A

Abstract

The invention provides a method for counting dendritic cells (DC). The method involves the use of antibodies capable of binding activated DC present in an activated mononuclear cell (MNC) population. Kits are also provided.

Description

METHOD FOR COUNTING DENDRITIC CELLS

FIELD OF THE INVENTION

This invention relates to a method of counting cells, specifically dendritic cells (DC) in human blood.

BACKGROUND OF THE INVENTION

DC originate from a bone marrow precursor, circulate in the blood en route to most tissues of the body, where they have a sentinel role in antigen surveillance. After acquiring antigen they migrate in the lymph to regional lymph nodes where they initiate T lymphocyte responses. No constitutional DC deficiency state has been described as yet in man. Likewise, although blood DC numbers may be reduced in HIV infection, there is little information on whether their number is either increased or decreased in other disease states. There is extensive evidence that DC turnover in the tissues is substantially increased by various inflammatory stimuli and that they are depleted following myeloablative treatment. It is likely that blood DC numbers or their precursors are raised to support this increased tissue turnover, unless some other source of DC is responsible for DC recruitment to these sites.

Knowledge of the number of DC present in an individual therefore has significance in both clinical and research applications, particularly those assessing the immune status of a patient. There is however no current method by which DC can be accurately counted. Although DC can be isolated from the blood in low numbers (typically estimated at < 1% of mononuclear cells (MNC)) accurate enumeration of DC counts after the use of complex isolation techniques inevitably introduces a range of potential errors, and is not applicable to the routine monitoring of the number of blood DC.

The object of this invention is to go some distance towards providing a method for accurately counting DC and their immediate precursors or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of counting DC comprising the steps of:

contacting a predetermined volume of activated mononuclear cells (MNC) obtained from a patient with antibodies capable of binding to activated DC; and

counting the number of antibody-DC complexes formed.

In preferred embodiments, the method includes preliminary steps selected from:

obtaining a sample of blood from a patient;

extracting and counting the MNC from said blood sample; and

activating the MNC extracted from said blood sample.

Preferably, the antibodies are selected from CMRF-44, CMRF-56 and CD83.

Conveniently, the antibodies carry a revealing label.

In the presently preferred embodiment, the step of counting the number of antibody-DC complexes formed involves flow cytometric analysis.

In a further aspect, the invention provides a method of monitoring DC numbers in a patient which includes counting DC by a method as defined above on at least two occasions.

The two occasions can be before and after administration of a compound to the patient, eg. a stimulatory compound such as a cytokine. In yet a further aspect, the invention provides a method of monitoring the health status of a patient which includes counting DC by a method as defined above on at least two occasions.

In still a further aspect, the invention provides a method of diagnosis of a disorder, disease or pathological state in a patient which includes counting DC cells by a method as defined above and comparing the result obtained with a predetermined reference database.

In yet a further aspect, the invention provides a kit for use in a method as defined above, said kit comprising:

a container including means for receiving a sample of MNC; and

a supply of antibody, which antibody binds activated DC.

BRIEF DESCRIPTION OF THE DRAWINGS

Although the invention is broadly as defined above, those persons skilled in the art will appreciate that it is not limited thereto and that it further includes embodiments of which the following description provides examples. In particular, a better understanding of the invention will be gained through reference to the accompanying drawings in which:

Figure 1 shows the gating and typical labelling dot plots for CMRF-50 (negative control) and CMRF-44 against CD14/CD19/PI staining;

Figure 2 shows typical time course labelling of percentage DC numbers of two subjects after blood MNC has been cultured for the indicated time period; Figure 3 shows the range and mean of DC numbers in normal individuals expressed as a percentage of MNC (left) and as an absolute number per ml of blood (right);

Figure 4 shows the effect of CD34⁺ stem cell mobilisation protocols on DC numbers; and

Figure 5 shows that mean DC number (percentage of MNC) of normal individuals and the patients during stem cell harvesting are significantly different (p < 0.05). The respective means are 0.44% (95% Cl = 0.38-0.52) and 0. 15% (95% Cl = 0.05-0.22).

DETAILED DESCRIPTION OF THE INVENTION

As defined above, the present invention provides a method of counting DC. The method has potential application in both clinical treatment and in research.

The first essential step of the method involves contacting a predetermined volume of activated MNC obtained from a patient with antibodies which bind activated DC. The antibodies can be selected from any of those antibodies which bind activated DC which are described in the literature. Examples include CMRF-44 (WO 95/ 12409), CMRF-56 (NZ 299537) and CD83 (Zhou L, Tedder TF. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J Immunol 1995; 154:3821). CMRF-44 is currently preferred.

The antibodies can be labelled if desired. Any conventional revealing label can be employed.

The requirement for the MNC to be activated is critical to the accuracy of the method to ensure binding of the antibodies to the DC within the MNC population. To date, any DC quantitation procedure involving activation of MNC was viewed as undesirable due to the assumed effect activation would have on DC numbers in terms of introducing artifactual variations. It has however been surprisingly found by the applicants that the activation step does not introduce such variations but instead allows accurate counts to be made.

A preliminary step is therefore activation of the MNC. This can be achieved by, for example, a brief period of in vitro culture, although other activation procedures (eg. exposure to cytokines or chemicals) are in no way excluded.

A further preferred optional preliminary step of the method is the separation of MNC from a blood sample obtained from a patient. Again, any conventional standardised procedure can be employed to achieve this.

The second essential step of the method involves the counting of the number of antibody-DC complexes formed. This can employ a number of immunological-based techniques depending upon the antibody selected and the revealing label used, if any. It is however preferred that the counting step employ a flow cytometrical analysis. This is conveniently automated using a FACS Vantage flow cytometer as described in the experimental section.

It is convenient to use established antibodies to simultaneously display other non- DC populations in MNC. For example, antibodies CMRF-50 and CMRF-75 (which reacts specifically with tetanus toxoid) can be used. Finally, it is a significant advantage to include propidium iodide or other agent to display dead cells so that they can be excluded from the analysis.

Application of the present method will allow a database of DC numbers to be established. This will include results from a range of patients, including those suffering from infections, diseases or immunocomprising disorders, as well as patients who are putatively normal. The database will allow a "normal" range to be established for the purpose of comparison and/ or diagnosis. For example, a decrease in the number of blood DC has been suggested for AIDS patients and for patients with advanced stages of breast cancer (Patterson S, Helbert M, English NR,Pinching AJ, Knight SC. The effect of AZT on dendritic cell number and provirus load in the peripheral blood of AIDS patients: a preliminary study. Res Virol 1996; 147: 109; and Gabrilovich DJ, Corak J, Ciernick IF, Kavanaugh D, Carbone DP. Decreased antigen presentation by dendritic cells in patients with breast cancer. Clin Cancer Res 1997;3:483).

The invention will now be better understood with reference to the following non- limiting experimental section.

EXPERIMENTAL

The presently preferred method uses flow cytometrical detection of DC after overnight culture of mononuclear cells (MNC) from a small specimen of blood and places a premium on minimising interventions which may led to cell loss during purification or cell death, thus artefactually altering results. Because of the low DC number the result is easily biased by relatively small changes in MNC composition.

Methodology

Blood samples are processed as follows: 5- 10 mis of blood is collected in standard EDTA tubes and a routine blood count performed on a small aliquot of blood using a Coulter STKS. The absolute number of MNC/1 blood is determined from this measurement. The remaining blood is diluted 1 : 1 with sterile PBS and underlayed with Ficoll/hypaque before contrifugation at 520 x g for 15 mins at room temperature. MNC are recovered from the low density interface and washed thrice before culture for 24 hours in RPMI/ 10% FCS and 5% C0₂. After culture cells are washed and their viability checked using trypan blue exclusion. Cells are then resuspended at 10⁷/ml and labelled during the following monoclonal antibodies: CMRF-44 (IgM) and the negative control CMRF-50 (IgM), (raised against a keratinocyte antigen and non-reactive with haemopoeitic cells). Phycoerythrin conjugated antibodies to CD14 (leuM3, IgG2a) and CD19 (leuM12,IgGl) are purchased from Becton Dickinson as is PE conjugated negative control. Fluoroscein isothyocyanate conjugated sheep-anti-mouse immunoglobulin (FITC-SAM) is purchased from Silenus (Hawthorne, Victoria, Australia) and propidium iodide (PI) from Sigma (St Louis, MO. USA). Cells are labelled using standard techniques, briefly, 50ul of cells (10⁷/ml) are placed into 5 tubes. Two tubes are labelled with the negative control CMRF-50 and three with CMRF-44 for 20 mins at 4°C, before being washed twice in PBS/ 1%BSA and labelling with FITC-SAM for another 20 mins. After further washing and blocking using 10% mouse serum PE conjugated CD 14 and CD 19 is added to each tube and incubated for 20 mins at 4°C. A final wash is followed by the addition of 5ul of lOuM PI to each tube. Flow cytometrical analysis is performed using a FACS vantage flow cytometer. 50,000 events are acquired from each tube and analysed using Cellquest software. Quadrants are established on the negative control dot plots such that the lower right quadrant contains a very small percentage of total events (typically <0.1%). This quadrant is then copied onto the three dot plots showing CMRF-44 vs CD 14/CD19/PI labelling and the percentage of events in the lower right quadrant noted. The negative control value is then subtracted in each case, to give triplicate measurements of DC number as a percentage of total MNC. The mean of these triplicate values is taken as the final result.

Results With this method it is possible to enumerate the percentage of DC present in cultured MNC populations as CMRF-44 positive/CD 14/CD 19 negative cells (Fig 1).

The optimal timing of the culture period required for this test was determined in time course experiments. Although CMRF-44 is one of the first activation antigens expressed by DC, a 4 hour culture period was insufficient to allow accurate detection of DC (Fig. 2). Culture times of 12 hours are inconvenient when applied to routine testing, although CMRF-44+ cells are clearly distinguished after this time. Culture times of 24 or 48 hours gave similar results by this method (Fig. 2) and although the strong expression of CMRF-44 at 48 hours enabled the cells to be clearly gated, the viability of the cultured cells was optimal at 24 hours and so this was the time point chosen for the current test.

The reliability of the test was considered based on the considerations below:

(i) The accuracy of the test was not assessed formally as there is no defined standard for comparison with our DC counts. The published data (Fearnley DB, McLellan AD, Mannering SI, Hock BD, Hart DNJ. Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen presenting cell function and immunotherapy. Blood 1997;89:3708; and Hock BD, Starling GC, Daniel PB, Hart DNJ. Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells. Immunology 1994;83:573) has established that the CMRF-44 positive population incorporates circulating DC or their precursors as defined by scientifically accepted criteria:

a) The ability to stimulate a primary T lymphocyte response (this may require differentiation /activation of the DC population);

b) Marked cell motility and the ability to extend and retract cell membrane processes freely at 37°C in vitro. The ability to migrate through tissues and track to the T lymphocyte dependent areas of lymph node;

c) Relatively limited phagocytic activity (in vitro) - DC uptake of extra-cellular material is probably greater than hitherto realised in vivo. Active fluid phase endocytosis is a feature of DC;

d) Spontaneous initial and rapid clustering with T lymphocytes at 37°C in vitro;

e) A cell surface Ag phenotype distinguishing it from other leucocytes, notably monocytes, macrophages and B lymphocytes;

f) Expression of certain DC associated antigens according to their differentiation / activation state ;

g) Cytochemical reactions, which differ from monocytes and macrophages.

Two additional features, "dendritic" morphology and high density membrane MHC class II Ag, are confirmatory only (particularly in man), as other cells, notably B lymphocytes and fibroblasts can adopt a very similar appearance. DC morphology is also influenced by temperature. Note that follicular DC, dendritic epithelial cells and lymphoid lineage DC (for the moment) are excluded by these criteria.

ii The precision of the test was ensured by using three separate labelling and flow cytometric analyses. The SD of each set of triplicates was calculated.

The mean SD of 33 tests was 0.06% MNC. The coefficient of variation was as expected relatively high at 15% due to the low percentage of DC within MNC. Three separate tests were performed on each of three individuals and the mean SD was 0.07% of MNC.

(iii) The specificity of the test depends on the properties of the monoclonal antibodies used. This is estimated by flow cytometry analysis to be extremely high. Further evidence of the specificity was provided by showing a strong correlation of absolute DC counts versus the %DC (r2=0.77) but no correlation of absolute DC number or the % DC with MNC as might occur with nonspecific labelling (r2 = 0.34 and 0.11 respectively).

(iv) The sensitivity of the test is down to 0.01% MNC and appears to extend below 1 x 10³/ml.

Analysis of reference population

The reference population comprised 35 normal individuals, some of whom were tested at three different times (with the interval between tests being 1 - 4 weeks) thus giving a total number of 38 measurements. The age range of this population was 20-50 years.

The mean number of DC as a percentage of MNC was 0.44 (95%C.l. = 0.38-0.51) the observed range was 0. 1 - 0.96. The mean absolute DC number was 11 x 10³/ml blood (95% Cl. = 9- 12) observed range 2 - 26 x 10 /ml (Fig. 3a) and these results appeared to follow a gaussian distribution (not shown) . A normal range was derived from these results, with 0.04-0.84% being the estimated normal range for DC as a percentage of MNC and 1 - 20 x 10³/ml blood as the range for the absolute DC number.

Analysis of patients treated with cyclophosphamide and G-CSF Eight patients undergoing stem cell mobilisation were tested either before or after the mobilisation and on day one of the harvest. Mobilisation caused a significant (p=<0.05) decrease in the percentage of blood DC and an almost significant (p=0.06) decrease in the absolute DC number compared to the pre-treatment values (paired students t test) . The mean DC numbers during harvest were also significantly lower than the mean of the normal population.

DISCUSSION /INDUSTRIAL APPLICATION

The potential significance of this invention can be summarised in three main areas. Firstly, the establishment of a reliable and practical method for counting blood DC, and the establishment of a normal range for this cell type will be of value to the general field of haematology. If data on the circulation time of DC can be added, the conclusions on DC numbers in different disease states will be considerably strengthened.

Secondly, if a clinical correlation between DC number and disease state can be made the implications for the understanding of the disease process and conceivably its treatment will be important. For example, the quantitation of DC in different stem cell sources for allogenic transplantation may provide insight into mechanisms of graft acceptance and graft versus host disease. Another example may be the requirement to monitor DC numbers after the administration of various immunosuppressive drugs.

Finally, this test may be directly useful for certain direct clinical interventions. Just as the enumeration of CD34+ cells is routinely used to optimise the mobilisation and the timing of the harvesting of DC or their precursors for stem cell transplantation, monitoring blood DC number may become important if blood DC are to be harvested for immunotherapeutic applications. Those persons skilled in the art that the above description is to be regarded as illustrative only and that variations may be made without departing from the invention, which is limited only by the lawful scope of the appended claims.

Claims

1. A method of counting dendritic cells (DC) comprising the steps of:

counting the number of antibody-DC complexes formed.

2. A method according to claim 1 which includes the preliminary step of activating the MNC.

3. A method according to claim 1 or claim 2 in which the antibodies are selected from CMRF-44, CMRF-56 and CD83.

4. A method according to any one of claims 1 to 3 in which the antibodies carry a revealing label.

5. A method according to any one of claims 1 to 4 in which the step of counting the number of antibody-DC complexes formed involves flow cytometric analysis.

A method of counting dendritic cells (DC) comprising the steps of:

contacting a predetermined volume of activated mononuclear cells (MNC) obtained from a patient which both primary and secondary antibodies, said primary antibodies being capable of binding to activated DC and said secondary antibodies being capable of binding to non-DC populations in MNC; and

counting the number of primary antibody-DC complexes formed.

7. A method according to claim 6 which includes the step of separating secondary antibody/non-DC complexes from primary antibody-DC complexes prior to counting the number of primary antibody-DC complexes.

8. A method according to claim 7 or claim 8 in which the primary antibodies are selected from CMRF-44, CMRF-56 and CD83.

9. A method according to any one of claims 6 to 8 in which the secondary antibodies are selected from CMRF-50 and CMRF-75.

10. A method of monitoring DC numbers in a patient which includes counting DC by a method as defined in any one of claims 1 to 9 on at least two occasions.

1 1. A method according to claim 10 wherein the first occasion is before administration of a compound to the patient, and the second occasion is after administration of said compound.

12. A method of monitoring the health status of a patient which includes counting DC by a method as defined in any one of claims 1 to 9 on at least two occasions.

13. A method of diagnosis of a disorder, disease or pathological state in a patient which includes counting DC cells by a method as defined in any one of claims 1 to 9 and comparing the result obtained with a predetermined reference database.

14. A kit for use in a method as defined in claim 1, said kit comprising:

a container including means for receiving a sample containing activated

MNC; and

a supply of antibody, which antibody binds activated DC.

15. A kit according to claim 14 in which the antibody is labelled.

16. A kit according to claim 15 in which the antibody is CMRF-44, CMRF-56 or CD83, or a mixture of these.

17. A kit according to any one of claims 14 to 16 which further comprises a supply of a secondary antibody which binds non-DC populations in MNC.