- Email: [email protected]
A preliminary study of serum class II levels in healthy individuals and bone-marrow transplant patients
45
Clinica Chimicu Acta, 185 (1989) 45-52 Elsevier
CCA 04578
A preliminary study of serum class II levels in healthy individuals and bone-marrow transplant patients S. Thompson ‘, M. Wareham ‘, A.D.J. L. Sviland 2 and G.A. Turner Departments
Pearson ’
2,
01 ’ Clinical Biochemistry and ’ Child Health, Medical School, Framlington University of Newcastle upon Tyne, Newcastle upon Tyne (UK) (Revision
received 30 June 1989; accepted
Key words: Blood; Bone-marrow:
Class II; MHC;
Place,
3 July 1989)
Transplantation
Summary A double-determinant ELISA has been developed to measure class II major histocompatibility complex molecules in blood from healthy individuals and patients who received either an allogeneic or an autologous bone-marrow transplant. Levels were higher in pre-transplant patients than in healthy individuals, and in the majority of patients, these levels rose even higher at 4 weeks post-transplantation. At later times after transplantation, it was difficult to draw firm conclusions, because the number of values available were too limited. Nevertheless, in the autologous group with no graft-versus-host disease (GVHD), levels were consistently lower at 6 months and one year; in the allogeneic group these long-term levels varied. As class II antigens are expressed on skin and rectal epithelia in GVHD, this study suggests that serial measurement of serum class II levels may be useful for monitoring GVHD in bone-marrow transplantation.
Introduction In recent years bone-marrow transplantation (BMT) has become increasingly important in the treatment of patients with leukaemias and lymphomas. Progress
Correspondence Place, University
0009-8981/89/$03.50
to: G.A. Turner, Department of Clinical Biochemistry, Medical School, of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K.
0 1989 Elsevier Science Publishers
B.V. (Biomedical
Division)
Framlington
46
has been retarded, however, by the adverse reactions which can occur between the graft and the host tissues [l]. Immunosuppressive therapy can be utilised to reduce these interactions, but the early diagnosis of graft-versus-host disease (GVHD) remains difficult as several other conditions can mimic the disease [2]. Major histocompatibility complex (MHC) class II antigens are expressed on heart [3] and renal transplants during rejection [4,5]. Class II levels are elevated lo-fold in the kidneys of mice with GVDH [6]. Similarly, histopathology shows MHC Class II molecules (HLA-Dr) are expressed on keratinocytes and enterocytes in human GVHD [7]. If these molecules are shed from the tissues into the blood stream then their measurement in sera could form the basis of a new test for monitoring GVHD. Recently a method has been reported for the measurement of soluble class II using a double-determinant ELISA [8]. We have adapted this method to assay class II levels in the blood of healthy individuals and BMT patients. Materials and methods Blood was obtained by venepuncture from patients (23 women and 22 men; median age 26 years) who received either an allogenic (20 patients) or an autologous (25 patients) BMT as part of their therapy and from 26 age- and sex-matched healthy individuals. The BMT group consisted of patients with acute lymphoblastic leukaemia, non-Hodgkin’s lymphoma, chronic myeloid leukaemia, acute myeloblastic leukaemia, multiple myeloma, aplastic anaemia and Ewing’s sarcoma. Specimens from the BMT groups were taken prior to transplantation (A), at 4 weeks after transplantation or as soon as GVHD was diagnosed clinically (B), at 6 months (C) and at 1 year (D). Because of the preliminary nature of this study, a complete set of specimens was not collected from all patients. Sera were separated by centrifugation at 600 x g for 10 min at 4O C and stored at - 20 o C until required for analysis. The onset of GVDH was determined using standard clinical methods [l], and the diagnosis was confirmed, in all cases, by histological examination of rectal biopsy specimens [9]. Blood specimens from patients with GVDH were collected within 24 h of the appearance of a characteristic skin rash and before any treatment was initiated. Antibodies A monoclonal rat anti-human class II (MAS 054C) was obtained as ascitic fluid from Sera-Lab, Crawley Dow, Sussex, UK. Pure IgG was prepared by taking the fraction that did not bind after passing through a column of Sephadex DEAE [lo]. A monoclonal mouse anti-human class II antibody (WR18) was obtained as tissue culture supernatant from Cymbus Bioscience Ltd., Southampton, UK. This was used at a 1 in 50 dilution in the ELISA. Affinity-purified, horse-radish peroxidase (HRP) conjugated polyclonal rat-anti mouse IgG (415-035-100, minimal cross-reaction with human proteins), was obtained from Jackson Immunoresearch Labs., West Grove, PA. This was used at a 1 in 4000 dilution in the ELISA.
47
ELISA Class II levels in sera were measured in a blind manner using a modified form of a recently reported double-determinant ELISA [S]. The precise conditions used were established in pilot experiments [ll] and these are given below. (1) The wells of a microtitre plate were coated with 100 ~1 of the purified IgG from rat anti-human class II monoclonal antibody (10 pg/ml in 12.5 mM borate-saline, pH 8.4) overnight at 4 o C. (2) The remaining active sites on the plastic were blocked and unbound material was washed away by washing the plate 6 times with phosphate-buffered saline (137 mmol/l NaCl, 8 mmol/l Na,HPO,, 2.7 mmol/l KCl, 1.5 mmol/l KH,PO,, 0.9 MgCl z, pH 7.4), containing 0.05% Tween-20 mmol/l CaCl 2, 0.5 mmol/l (PBS/Tween). (3) Sera (100 ~1, neat or : dilution) were added for 1.5 h at 37’C, then the wells were again washed 6 times with PBS/Tween. (4) 100 ~1 of diluted (in PBS/Tween) monoclonal mouse antibody (WR18), specific for a different class II determinant, were added for 1 h at 37 o C. (5) After another 6 washes in PBS/Tween, 100 ~1 of horse-radish peroxidase conjugated rat anti-mouse polyclonal antisera were added for 1 h at 37’C. (6) Unreacted material was washed away and bound horse-radish peroxidase was detected using o-phenylenediamine as substrate. (7) Colour development was read at 492 nm with a plate reader (Dynatech Ltd, Billinghurst, Sussex, UK). The presence of any interfering molecules was determined by control assays in which the class II specific antibody (WR18) was omitted from the assay. In 20% of the specimens assayed, a high absorbance value was obtained with this control. This was assumed to be due to interference in the assay by rheumatoid factors. Because of this, it was impossible to assign a reliable value to these specimens and they are not included in any of the presented data. In the other 80% of the specimens no interference was detectable with this or any other control (e.g., irrelevant 2nd layer antibody of same IgG subclass) and the ELISA value was assumed to accurately reflect the true class II level of the sample. The class II content of each sample was calculated relative to the absorbance level obtained in the ELISA for class II positive Daudi cell lysates. An arbitrary value of 1000 units/ml was assigned to the absorbance resulting from a Daudi lysate of 6 X 10’ cells/ml in its concentrated form. Thus, 100 units/ml and 10 units/ml corresponded to 1 in 10 and 1 in 100 dilutions (in PBS/Tween) of the lysate respectively. The preparation of class II standards Daudi cell lysates were prepared by pelleting cells from an actively growing culture (400 x g for 5 mm). The cells were then solubilised (at 6 x 10’ per ml) in PBS containing 0.5% Nonidet P40 with 50 mM iodoacetamide and soybean trypsin inhibitor (20 pg/rnl) as protease inhibitors. The solution was vortexed then left at 4” C for 15 min. After centrifugation at 4000 X g for 10 min to remove unsolubilised material the supematant contained the soluble class II molecules. This solution
48
remained stable for 6-8 weeks on storage appreciable loss in the class II levels.
at 4 o C. Freezing
and thawing
caused
an
Results The serum class II levels of the healthy individuals and the BMT patients are given in Fig. 1. Class II molecules were detectable in 6/22 healthy individuals (median = 0 U/ml) and 17/26 group A patients (median = 2.4 U/ml), and these groups were significantly different (p = 0.002, Mann-Whitney). After transplantation (groups B, C and D), the median concentrations of class II were 3.0, 1.6, 2.3 U/ml, respectively; none of these pooled groups were significantly different from the pre-transplant group (p > 0.05, Mann-Whitney). There was no relationship between levels and age or sex in any of these groups. Figure 2 compares class II levels on individual patients before (A) and after (B) transplantation. Because of the small numbers, data was pooled from the autologous 12-
. 10-
a-
6-
4-
2-
0
J
am HEALTHY
A??%?3 A
MA.
6
AAA.
C
a..
D
Fig. 1. Class II levels in sera from healthy individuals and cancer patients before grafting (A), 4 weeks (B), 6 months (C), and 1 year (D) after grafting. A, allogeneic graft; A, autologous graft.
49
AA
Posttronwlant
Pre transolant
(i)
(A;
transplantaFig. 2. A comparison of serum class II levels in 15 patients before and after bone-marrow tion. The two groups were significantly different using a paired Wilcoxon test (0.05 > p > 0.02). A, allogeneic graft; A, autologous graft.
and allogeneic groups. Levels were elevated in 12/15 patients after BMT. This difference between the groups was significant (0.05 > p > 0.02; paired Wilcoxon). This increase did not appear to be any greater in the allogeneic group than in the autologous group (see Fig. 2). Class II levels in serial sera collected prior to transplantation and up to 12 months post-transplantation from autologous and allogeneic patients are shown in
TABLE
I
The changes Patient
in serum class II levels in patients
No.
Serum class II antigen Aa
27
3.20
30 43 50 51 60
3.65 3.90 3.50 2.80 1.90
a A, before
who received
transplantation;
level (U/ml)
B
6.15 2.30 4.75 3.35 0.0
B, 4 weeks; C, 6 months;
autologous at various
grafts stages
C
D
0.0
_
4.30 1.80 3.35 1.30
0.0
and D, 1 year after grafting.
_ _ _
50 TABLE
II
The changes
in serum class II levels in patient
Patient No. 31
Serum class II levels (U/ml)
who received at various
allogeneic
B
C
3.40
2.60 GVHD,
2.70 GVHD
D
1.12 GVDH? B
44
3.70
4.85 Early GVHD h
3.40 GVHD
A
-death TR
TR
TR 0.0
and who had GVHD
stages
A
40
grafts
0.0 no GVHD
2.25
2.60 no GVHD
2.30
1.45 no GVHD
_
TR
TR 57
0.0
1.80 GVDH p TR
TR = treatment
given to reduce
the effects of the GVHD.
Tables I and II, respectively. It is difficult to draw firm conclusions from this data because the number of patients investigated is small; at the most, only four specimens were available from each patient; and frequently only 2 or 3 were collected. However, in the majority of the patients in the autologous group with no GVHD, the class II levels were lower in the specimens collected at 6 months or 1 year than in those collected at the early times. In the allogeneic group with GVHD, levels varied but insufficient samples were collected to determine if high levels were associated with GVHD. Discussion The results from this study suggest that measurement of serum levels of class II may be useful for monitoring GVHD in bone-marrow transplant patients. Why class II levels were elevated in the pre-transplant group and then increased further after transplantation is unknown. Presumably, the former change could be a reflection of the patient’s response to disease and/or the treatment received, whereas the latter could involve some non-immunological mechanism because it was independent of the type of graft given, i.e. allogeneic or autologous. There was a large overlap between the range of class II levels measured in the different groups. This suggests that single estimations are of no prognostic value. In order to assess this situation further it will be necessary to collect serial blood specimens more frequently and more completely in the future. Another problem, which tended to reduce the number of estimations that could be used for patient assessment, was interference by some non-specific factors. At the start of the study this was a serious problem affecting approximately 50% of the specimens, but by changing the 3rd~layer antibody, this was reduced to 20%.
51
Although this level of interference is still not totally acceptable for a routine assay, it does allow conclusions to be drawn if a reasonable number of specimens are collected. This problem may be overcome by using a different 3rd-layer antibody or missing out the 3rd-layer completely and coupling the indicator directly to the 2nd-layer antibody. Although this latter approach will reduce overall sensitivity, it may be possible to compensate for this by using a different amplification system such as biotin-avidin or enzyme amplification. Another approach could be to measure class II concentrations in urine. Using a two-antibody ELISA, it has recently been shown that urine interferes much less than serum with the assay of the class II molecules in Daudi cell extracts (personal communication, K. Moore). The potential value of measuring class II concentrations in body fluids for monitoring the condition of other types of grafts is clearly indicated by this preliminary study. As levels of class II are known to be markedly increased on kidney and heart tissue when they are rejected [3-51, predictive changes in the serum may be even more striking in these cases than with the bone-marrow transplants. Acknowledgements The authors gratefully acknowledge the Immunology Laboratory, Medical School, University of Newcastle upon Tyne for technical assistance in setting-up the assay, Dr. Anne Collins, Director of the Blood Transfusion Service, Newcastle upon Tyne, for help in obtaining blood specimens from healthy individuals, and the North of England Cancer Research Campaign for financial support. References 1 Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lemer KG, Thomas ED. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974;18:295-304. 2 Sale GE, Lemer KG, Barker EA, Shulman HM, Thomas ED. The skin biopsy in the diagnosis of acute graft-versus-host disease in man. Am J Path01 1977;89:621-636. 3 Milton AD, Fabre JW. Massive induction of donor-type class I and class II major histocompatibility complex antigens in rejecting cardiac allografts in the rat. J Exp Med 1985;161:98-112. 4 Benson EM, Colvin RB, Russel PS. Induction of Ia antigens in murine renal transplants. J Immunol 1985;134:7-9. 5 Hall BM, Bishop GA, Duggin GG, Horvath JS, Philips J, Tiller DJ. Increased expression of HLA-DR antigens on renal tubular cells in renal transplants: relevance to the rejection response. Lancet 1984;ii:247-251. 6 Wadgymar A, Urmson J, Baumal R, Halloran PF. Changes in Ia expression in mouse kidney during acute graft-vs-host disease. J Immunol 1984;132:1826-1832. 7 Sviland L, Pearson ADJ, Eastham EJ, Green MA, Hamilton PJ, Proctor SJ, Malcolm AJ. Class II antigen expression by keratinocytes and enterocytes is an early feature of graft-versus-host disease. Transplantation 1988;46:402-406. 8 Stevenson FK, George AJT, Walters MT, Hamblin TJ. Analysis of soluble HLA Class II antigenic material in patients with immunological diseases using monoclonal antibodies. J Immunol Methods 1986;86:187-190.
52 9 Sviland L, Pearson ADJ, Eastham El, Hamilton PJ, Proctor SJ. Malcolm AJ, the Newcastle upon Tyne Bone Marrow Transplant Group. Histological features of skin and metal biopsy specimens after autologous and allogeneic bone marrow transplantation. J Clin Path01 1988;41:148-154. 10 Bazin H, Beches A, Querinjean P. Three classes and four subclasses of rat immunoglobulins: IgM, IgA, IgE and IgG,, IgGs,, IgG,,, IgG,,. Eur J Immunol 1974;4:44-48. 11 Wareham M, Shed Membrane Antigens: possible Markers of Graft versus Host Disease. MSc Thesis, University of Newcastle upon Tyne, 1987.
Clinica Chimicu Acta, 185 (1989) 45-52 Elsevier
CCA 04578
A preliminary study of serum class II levels in healthy individuals and bone-marrow transplant patients S. Thompson ‘, M. Wareham ‘, A.D.J. L. Sviland 2 and G.A. Turner Departments
Pearson ’
2,
01 ’ Clinical Biochemistry and ’ Child Health, Medical School, Framlington University of Newcastle upon Tyne, Newcastle upon Tyne (UK) (Revision
received 30 June 1989; accepted
Key words: Blood; Bone-marrow:
Class II; MHC;
Place,
3 July 1989)
Transplantation
Summary A double-determinant ELISA has been developed to measure class II major histocompatibility complex molecules in blood from healthy individuals and patients who received either an allogeneic or an autologous bone-marrow transplant. Levels were higher in pre-transplant patients than in healthy individuals, and in the majority of patients, these levels rose even higher at 4 weeks post-transplantation. At later times after transplantation, it was difficult to draw firm conclusions, because the number of values available were too limited. Nevertheless, in the autologous group with no graft-versus-host disease (GVHD), levels were consistently lower at 6 months and one year; in the allogeneic group these long-term levels varied. As class II antigens are expressed on skin and rectal epithelia in GVHD, this study suggests that serial measurement of serum class II levels may be useful for monitoring GVHD in bone-marrow transplantation.
Introduction In recent years bone-marrow transplantation (BMT) has become increasingly important in the treatment of patients with leukaemias and lymphomas. Progress
Correspondence Place, University
0009-8981/89/$03.50
to: G.A. Turner, Department of Clinical Biochemistry, Medical School, of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K.
0 1989 Elsevier Science Publishers
B.V. (Biomedical
Division)
Framlington
46
has been retarded, however, by the adverse reactions which can occur between the graft and the host tissues [l]. Immunosuppressive therapy can be utilised to reduce these interactions, but the early diagnosis of graft-versus-host disease (GVHD) remains difficult as several other conditions can mimic the disease [2]. Major histocompatibility complex (MHC) class II antigens are expressed on heart [3] and renal transplants during rejection [4,5]. Class II levels are elevated lo-fold in the kidneys of mice with GVDH [6]. Similarly, histopathology shows MHC Class II molecules (HLA-Dr) are expressed on keratinocytes and enterocytes in human GVHD [7]. If these molecules are shed from the tissues into the blood stream then their measurement in sera could form the basis of a new test for monitoring GVHD. Recently a method has been reported for the measurement of soluble class II using a double-determinant ELISA [8]. We have adapted this method to assay class II levels in the blood of healthy individuals and BMT patients. Materials and methods Blood was obtained by venepuncture from patients (23 women and 22 men; median age 26 years) who received either an allogenic (20 patients) or an autologous (25 patients) BMT as part of their therapy and from 26 age- and sex-matched healthy individuals. The BMT group consisted of patients with acute lymphoblastic leukaemia, non-Hodgkin’s lymphoma, chronic myeloid leukaemia, acute myeloblastic leukaemia, multiple myeloma, aplastic anaemia and Ewing’s sarcoma. Specimens from the BMT groups were taken prior to transplantation (A), at 4 weeks after transplantation or as soon as GVHD was diagnosed clinically (B), at 6 months (C) and at 1 year (D). Because of the preliminary nature of this study, a complete set of specimens was not collected from all patients. Sera were separated by centrifugation at 600 x g for 10 min at 4O C and stored at - 20 o C until required for analysis. The onset of GVDH was determined using standard clinical methods [l], and the diagnosis was confirmed, in all cases, by histological examination of rectal biopsy specimens [9]. Blood specimens from patients with GVDH were collected within 24 h of the appearance of a characteristic skin rash and before any treatment was initiated. Antibodies A monoclonal rat anti-human class II (MAS 054C) was obtained as ascitic fluid from Sera-Lab, Crawley Dow, Sussex, UK. Pure IgG was prepared by taking the fraction that did not bind after passing through a column of Sephadex DEAE [lo]. A monoclonal mouse anti-human class II antibody (WR18) was obtained as tissue culture supernatant from Cymbus Bioscience Ltd., Southampton, UK. This was used at a 1 in 50 dilution in the ELISA. Affinity-purified, horse-radish peroxidase (HRP) conjugated polyclonal rat-anti mouse IgG (415-035-100, minimal cross-reaction with human proteins), was obtained from Jackson Immunoresearch Labs., West Grove, PA. This was used at a 1 in 4000 dilution in the ELISA.
47
ELISA Class II levels in sera were measured in a blind manner using a modified form of a recently reported double-determinant ELISA [S]. The precise conditions used were established in pilot experiments [ll] and these are given below. (1) The wells of a microtitre plate were coated with 100 ~1 of the purified IgG from rat anti-human class II monoclonal antibody (10 pg/ml in 12.5 mM borate-saline, pH 8.4) overnight at 4 o C. (2) The remaining active sites on the plastic were blocked and unbound material was washed away by washing the plate 6 times with phosphate-buffered saline (137 mmol/l NaCl, 8 mmol/l Na,HPO,, 2.7 mmol/l KCl, 1.5 mmol/l KH,PO,, 0.9 MgCl z, pH 7.4), containing 0.05% Tween-20 mmol/l CaCl 2, 0.5 mmol/l (PBS/Tween). (3) Sera (100 ~1, neat or : dilution) were added for 1.5 h at 37’C, then the wells were again washed 6 times with PBS/Tween. (4) 100 ~1 of diluted (in PBS/Tween) monoclonal mouse antibody (WR18), specific for a different class II determinant, were added for 1 h at 37 o C. (5) After another 6 washes in PBS/Tween, 100 ~1 of horse-radish peroxidase conjugated rat anti-mouse polyclonal antisera were added for 1 h at 37’C. (6) Unreacted material was washed away and bound horse-radish peroxidase was detected using o-phenylenediamine as substrate. (7) Colour development was read at 492 nm with a plate reader (Dynatech Ltd, Billinghurst, Sussex, UK). The presence of any interfering molecules was determined by control assays in which the class II specific antibody (WR18) was omitted from the assay. In 20% of the specimens assayed, a high absorbance value was obtained with this control. This was assumed to be due to interference in the assay by rheumatoid factors. Because of this, it was impossible to assign a reliable value to these specimens and they are not included in any of the presented data. In the other 80% of the specimens no interference was detectable with this or any other control (e.g., irrelevant 2nd layer antibody of same IgG subclass) and the ELISA value was assumed to accurately reflect the true class II level of the sample. The class II content of each sample was calculated relative to the absorbance level obtained in the ELISA for class II positive Daudi cell lysates. An arbitrary value of 1000 units/ml was assigned to the absorbance resulting from a Daudi lysate of 6 X 10’ cells/ml in its concentrated form. Thus, 100 units/ml and 10 units/ml corresponded to 1 in 10 and 1 in 100 dilutions (in PBS/Tween) of the lysate respectively. The preparation of class II standards Daudi cell lysates were prepared by pelleting cells from an actively growing culture (400 x g for 5 mm). The cells were then solubilised (at 6 x 10’ per ml) in PBS containing 0.5% Nonidet P40 with 50 mM iodoacetamide and soybean trypsin inhibitor (20 pg/rnl) as protease inhibitors. The solution was vortexed then left at 4” C for 15 min. After centrifugation at 4000 X g for 10 min to remove unsolubilised material the supematant contained the soluble class II molecules. This solution
48
remained stable for 6-8 weeks on storage appreciable loss in the class II levels.
at 4 o C. Freezing
and thawing
caused
an
Results The serum class II levels of the healthy individuals and the BMT patients are given in Fig. 1. Class II molecules were detectable in 6/22 healthy individuals (median = 0 U/ml) and 17/26 group A patients (median = 2.4 U/ml), and these groups were significantly different (p = 0.002, Mann-Whitney). After transplantation (groups B, C and D), the median concentrations of class II were 3.0, 1.6, 2.3 U/ml, respectively; none of these pooled groups were significantly different from the pre-transplant group (p > 0.05, Mann-Whitney). There was no relationship between levels and age or sex in any of these groups. Figure 2 compares class II levels on individual patients before (A) and after (B) transplantation. Because of the small numbers, data was pooled from the autologous 12-
. 10-
a-
6-
4-
2-
0
J
am HEALTHY
A??%?3 A
MA.
6
AAA.
C
a..
D
Fig. 1. Class II levels in sera from healthy individuals and cancer patients before grafting (A), 4 weeks (B), 6 months (C), and 1 year (D) after grafting. A, allogeneic graft; A, autologous graft.
49
AA
Posttronwlant
Pre transolant
(i)
(A;
transplantaFig. 2. A comparison of serum class II levels in 15 patients before and after bone-marrow tion. The two groups were significantly different using a paired Wilcoxon test (0.05 > p > 0.02). A, allogeneic graft; A, autologous graft.
and allogeneic groups. Levels were elevated in 12/15 patients after BMT. This difference between the groups was significant (0.05 > p > 0.02; paired Wilcoxon). This increase did not appear to be any greater in the allogeneic group than in the autologous group (see Fig. 2). Class II levels in serial sera collected prior to transplantation and up to 12 months post-transplantation from autologous and allogeneic patients are shown in
TABLE
I
The changes Patient
in serum class II levels in patients
No.
Serum class II antigen Aa
27
3.20
30 43 50 51 60
3.65 3.90 3.50 2.80 1.90
a A, before
who received
transplantation;
level (U/ml)
B
6.15 2.30 4.75 3.35 0.0
B, 4 weeks; C, 6 months;
autologous at various
grafts stages
C
D
0.0
_
4.30 1.80 3.35 1.30
0.0
and D, 1 year after grafting.
_ _ _
50 TABLE
II
The changes
in serum class II levels in patient
Patient No. 31
Serum class II levels (U/ml)
who received at various
allogeneic
B
C
3.40
2.60 GVHD,
2.70 GVHD
D
1.12 GVDH? B
44
3.70
4.85 Early GVHD h
3.40 GVHD
A
-death TR
TR
TR 0.0
and who had GVHD
stages
A
40
grafts
0.0 no GVHD
2.25
2.60 no GVHD
2.30
1.45 no GVHD
_
TR
TR 57
0.0
1.80 GVDH p TR
TR = treatment
given to reduce
the effects of the GVHD.
Tables I and II, respectively. It is difficult to draw firm conclusions from this data because the number of patients investigated is small; at the most, only four specimens were available from each patient; and frequently only 2 or 3 were collected. However, in the majority of the patients in the autologous group with no GVHD, the class II levels were lower in the specimens collected at 6 months or 1 year than in those collected at the early times. In the allogeneic group with GVHD, levels varied but insufficient samples were collected to determine if high levels were associated with GVHD. Discussion The results from this study suggest that measurement of serum levels of class II may be useful for monitoring GVHD in bone-marrow transplant patients. Why class II levels were elevated in the pre-transplant group and then increased further after transplantation is unknown. Presumably, the former change could be a reflection of the patient’s response to disease and/or the treatment received, whereas the latter could involve some non-immunological mechanism because it was independent of the type of graft given, i.e. allogeneic or autologous. There was a large overlap between the range of class II levels measured in the different groups. This suggests that single estimations are of no prognostic value. In order to assess this situation further it will be necessary to collect serial blood specimens more frequently and more completely in the future. Another problem, which tended to reduce the number of estimations that could be used for patient assessment, was interference by some non-specific factors. At the start of the study this was a serious problem affecting approximately 50% of the specimens, but by changing the 3rd~layer antibody, this was reduced to 20%.
51
Although this level of interference is still not totally acceptable for a routine assay, it does allow conclusions to be drawn if a reasonable number of specimens are collected. This problem may be overcome by using a different 3rd-layer antibody or missing out the 3rd-layer completely and coupling the indicator directly to the 2nd-layer antibody. Although this latter approach will reduce overall sensitivity, it may be possible to compensate for this by using a different amplification system such as biotin-avidin or enzyme amplification. Another approach could be to measure class II concentrations in urine. Using a two-antibody ELISA, it has recently been shown that urine interferes much less than serum with the assay of the class II molecules in Daudi cell extracts (personal communication, K. Moore). The potential value of measuring class II concentrations in body fluids for monitoring the condition of other types of grafts is clearly indicated by this preliminary study. As levels of class II are known to be markedly increased on kidney and heart tissue when they are rejected [3-51, predictive changes in the serum may be even more striking in these cases than with the bone-marrow transplants. Acknowledgements The authors gratefully acknowledge the Immunology Laboratory, Medical School, University of Newcastle upon Tyne for technical assistance in setting-up the assay, Dr. Anne Collins, Director of the Blood Transfusion Service, Newcastle upon Tyne, for help in obtaining blood specimens from healthy individuals, and the North of England Cancer Research Campaign for financial support. References 1 Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lemer KG, Thomas ED. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974;18:295-304. 2 Sale GE, Lemer KG, Barker EA, Shulman HM, Thomas ED. The skin biopsy in the diagnosis of acute graft-versus-host disease in man. Am J Path01 1977;89:621-636. 3 Milton AD, Fabre JW. Massive induction of donor-type class I and class II major histocompatibility complex antigens in rejecting cardiac allografts in the rat. J Exp Med 1985;161:98-112. 4 Benson EM, Colvin RB, Russel PS. Induction of Ia antigens in murine renal transplants. J Immunol 1985;134:7-9. 5 Hall BM, Bishop GA, Duggin GG, Horvath JS, Philips J, Tiller DJ. Increased expression of HLA-DR antigens on renal tubular cells in renal transplants: relevance to the rejection response. Lancet 1984;ii:247-251. 6 Wadgymar A, Urmson J, Baumal R, Halloran PF. Changes in Ia expression in mouse kidney during acute graft-vs-host disease. J Immunol 1984;132:1826-1832. 7 Sviland L, Pearson ADJ, Eastham EJ, Green MA, Hamilton PJ, Proctor SJ, Malcolm AJ. Class II antigen expression by keratinocytes and enterocytes is an early feature of graft-versus-host disease. Transplantation 1988;46:402-406. 8 Stevenson FK, George AJT, Walters MT, Hamblin TJ. Analysis of soluble HLA Class II antigenic material in patients with immunological diseases using monoclonal antibodies. J Immunol Methods 1986;86:187-190.
52 9 Sviland L, Pearson ADJ, Eastham El, Hamilton PJ, Proctor SJ. Malcolm AJ, the Newcastle upon Tyne Bone Marrow Transplant Group. Histological features of skin and metal biopsy specimens after autologous and allogeneic bone marrow transplantation. J Clin Path01 1988;41:148-154. 10 Bazin H, Beches A, Querinjean P. Three classes and four subclasses of rat immunoglobulins: IgM, IgA, IgE and IgG,, IgGs,, IgG,,, IgG,,. Eur J Immunol 1974;4:44-48. 11 Wareham M, Shed Membrane Antigens: possible Markers of Graft versus Host Disease. MSc Thesis, University of Newcastle upon Tyne, 1987.