Pollen systems to detect phytotoxicants in the environment: an introduction.

Peripheral blood leucocytes from chronic myeloid leukaemia patients in remission were tested for inhibition of migration in presence of solubilized membrane antigens from leukaemic cells in 15 cases. Eight out of 9 autochthonous combinations (88*8%) and 35/49 allogeneic combinations (7144%) showed inhibition of migration. Antigens prepared from relapse leukaemic cell samples in 4 cases showed inhibition of migration of autochthonous as well as allogeneic remission leucocytes. The same batch of CML antigens inhibited migration of normal leuco-cytes at the level of 22.2%. The difference between inhibition of migration shown by remission leucocytes and normal leucocytes in presence of CML antigens was statistically significant. Solubilized antigens, similarly prepared from normal leucocytes, showed inhibition of migration of remission leucocytes to the extent of 15% only. The difference between the reactivity of CML remission leucocytes to normal and CML antigens was also statistically significant. No enhancement of migration of remission leucocytes was seen with CML antigens. THE PRESENCE of lymphocytes sensi-tized to leukaemia-associated antigens has often been demonstrated in patients with acute leukaemia, using various techniques to assess cellular immunity such as lymphocyte blastogenesis, lymphocyte mediated cytotoxicity and delayed cuta-Attempts have been made to boost leukaemia-specific immunity (Powles et al., 1971 and Gutterman et al., 1973b), and to correlate the presence and degree of cellular immunity with the clinical stage of the disease (Gutterman et al., 1972a, 1973c; Char et al., 1973). Leukaemia-specific cellular immunity in chronic myeloid leukaemic patients (CML) has not, however, been reported All correspondence to S. G. Gangal. showed that 30%0 of CML patients specifically responded to low concentrations of solubilized autochthonous leuk-aemic cell membrane antigens in skin tests. In the present report attempts have been made to study leukaemia-specific reactivity in CML patients in remission to solubilized autochthonous and allo-geneic leukaemic cell membrane antigens by leucocyte migration inhibition tests. It was feasible to undertake these investigations because CML occurs frequently in India. MATERIALS AND METHODS Antigens.-Leukaemic cells were collected from the peripheral blood of 15 CML patients when they were first admitted to the Tata Memorial Hospital. They were diagnosed as CML on the basis of clinical evidence and bone marrow picture. They presented a high WBC count in the peripheral blood,

THE PRESENCE of lymphocytes sensitized to leukaemia-associated antigens has often been demonstrated in patients with acute leukaemia, using various techniques to assess cellular immunity such as lymphocyte blastogenesis, lymphocyte mediated cytotoxicity and delayed cutaneous hypersensitivity (Freedman and Kourilsky, 1969;Viza et al., 1969; and Leventhal, 1971;Santos et al., 1973;Anderson et al., 1974;Oren andHerberman, 1971 andLeventhal et al., 1972). Attempts have been made to boost leukaemiaspecific immunity (Powles et al., 1971 andGutterman et al., 1973b), and to correlate the presence and degree of cellular immunity with the clinical stage of the disease (Gutterman et al., 1972a(Gutterman et al., , 1973cChar et al., 1973).
Leukaemia-specific cellular immunity in chronic myeloid leukaemic patients (CML) has not, however, been reported All correspondence to S. G. Gangal. frequently. Oren and Herberman (1971) showed that 30%0 of CML patients specifically responded to low concentrations of solubilized autochthonous leukaemic cell membrane antigens in skin tests.
In the present report attempts have been made to study leukaemia-specific reactivity in CML patients in remission to solubilized autochthonous and allogeneic leukaemic cell membrane antigens by leucocyte migration inhibition tests. It was feasible to undertake these investigations because CML occurs frequently in India.

MATERIALS AND METHODS
Antigens.-Leukaemic cells were collected from the peripheral blood of 15 CML patients when they were first admitted to the Tata Memorial Hospital. They were diagnosed as CML on the basis of clinical evidence and bone marrow picture. They presented a high WBC count in the peripheral blood, ranging from 80,000 to 200,000. The bone marrow picture showed hypercellularity with granulocytes in all stages of differentiation and the ratio of M: E varying from 10: 1 to 20: 1.
Ten ml of blood was withdrawn in 4 ml of dextran-citrate mixture (6% Dextran, mol. wt. 110,000 with equal volume of 5% sodium citrate) from 15 CML patients. In three cases, one subsequent relapse sample each and in one case, two subsequent relapse samples were also collected. The RBCs were allowed to settle for 30 min at room temperature. The supernatant WBCs were washed three times and suspended in 10 ml saline. The cells were frozen and thawed rapidly six times in normal saline, with the help of dry ice. The lysis of cells was checked microscopically. The cellular extract was further subjected to membrane extraction procedure using sodium chloride solutions of graded molarity, as described by Oren and Herberman (1971). Supernatants from all the extracts were pooled, clarified by centrifugation at 500 g for 10 min, and finally centrifuged at 105,000 g for 60 min. The pellet obtained was suspended in MEM and passed through millipore filter (0-45 jtm). The protein was finally adjusted to 50 ,ug/0-1 ml of MEM before using for the experiment.
Leucocyte samples for testing.-Remission leucocytes from 5 CML patients from whom leukaemic cell antigens were prepared (autochthonous combinations), as well as from 14 other CML patients (allogeneic combinations) were tested for inhibition of migration. Remission patients were either on maintenance therapy with busulfan (2 mg twice a week), or free from any drug treatment when tested. They were tested between 2 months to 2 years of remission period. None of them had received any blood transfusions. The peripheral WBC count raniged from 8000 to 15,000 at the time of testing. They were clinically and haematologically free of the disease.

Leucocyte migration inhibition (LMI)
-The procedure utilized in these studies was as described by Cochran et al. (1973b), with slight modifications. Ten ml of peripheral blood from CML patients in remission and normal individuals was collected in dextran-citrate mixture. In some cases 6% Dextran was added to heparinized blood. RBCs were allowed to settle at room temp. for 30 min. The leucocyterich plasma was centrifuged. The leucocytes were washed 3 times with saline and finally suspended in MEM to contain approximately 10 x 106 leucocytes per 0-1 ml. The cell viability was checked with erythrocin B. Ten ,u of cell suspension was drawn in 20 ,ul micropets, so as to allow uniformity in cell number in every experiment. The capillaries were sealed and centrifuged at 1500 rev/min. They were cut at the cell-medium interface. Two capillaries were fixed in each migration chamber (perspex round chamber with outer diameter of 2 cm and inner diameter of 1-6 cm, depth 2 mm and capacity 0-5 ml) with silicon grease.
The chamber was then filled with 0-5 ml of medium containing MEM supplemented with 15% human AB group serum and antibiotics. In test chambers, the medium contained 50 jug antigen. The chambers were closed with coverslips and incubated at room temperature for 18-24 h. After incubation, the projected image of the migration field was traced on paper and measured by planimetry. The migration indices were calculated as described by Federlin et al. (1971). Migration index (MI) = area of migration in presence of antigen area of migration in absence of antigen Migration indices ranging from 0-8 to 1-2 were considered as within normal range (Federlin et al., 1971;Cochran et al., 1974), while indices below 0-8 denoted inhibition of migration and those above 1-2 indicated enhancement effect. The statistical significance of the reactions was assessed by chi-square analysis using 2 x 2 contingency tables. Yate's correction was applied.
Controls.-As controls, antigens extracted from leukaemic leucocytes were tested for inhibition of migration of leucocytes from normal individuals. Similarly, 4 leucocyte samples obtained from normal donors were subjected to similar antigen extraction procedures, and the solubilized membrane antigens from normal leucocytes were tested for inhibition of CML remission leucocytes obtained from 5 patients.

RESULTS
In all, 20 antigen samples, including 5 preparations from relapse blood, were tested on 19 remission leucocyte samples.
Each antigen was tested on 3-4 remission leucocyte samples. The total number of LMI tests performed with remission leucocytes was 57. Each of the 15 leukaemic cell membrane antigens prepared from untreated cases was tested on 5 to 6 leucocyte samples obtained from normal healthy persons. The total number of LMI tests performed with normal leucocytes was 81. Four antigeneic preparations of normal leucocytes were tested on 5 CML remission leucocytes. The total number of LMI tests performed was 20. In this group of experiments a positive control with leukaemic antigen was used to assess the ability of remission leucocytes to react.
Migration indices of all these tests plotted on scattergram are shown in Figs. 1 and 2. Each point represents the mean of indices from 2 to 4 replicates. Circled points represent autochthonous reactions.
The LMI pattern of leucocytes from CML patients in remission and normal individuals in presence of leukaemic anti-  Table I (Fig. 1). Fortythree out of 57 (75.4%) tests performed with remission leucocytes showed inhibition of migration. Eight of these tests represented autochthonous combination. t One of these represented autochthonous reaction.
The autochthonous reactions consisted of 2 tests using original antigen prepared from leukaemic cells from two untreated patients, 4 tests with original and first relapse sample antigens from two patients and 3 tests with original, first and second relapse sample antigens from one patient. The percentage of autochthonous positive LMI reactions was 88.8% and percentage of allogeneic positive LMI reactions was 71-4%. In one autochthonous combination, the migration was within the normal range. None of the tests showed enhancement in migration of leucocytes (migration of leucocytes in presence of antigen being more than migration of leucocytes in absence of antigen). In contrast, when normal leucocytes were allowed to migrate in the presence of leukaemic cell membrane antigen, only 18/81 (22-2%) tests showed inhibition of migration, while 58/81 (71.6%) tests showed migration within normal range. Enhancement was shown in 5/81 (6.2%) tests. The difference between inhibition pattern of normal leucocytes and CML remission leucocytes was statistically significant (X2 36-43 at 1 d.f., P < 0-001). The difference between enhancement of migration shown by normal leucocytes and remission leucocytes was not statistically significant. However, it is thought that enhancement of migration is a phenomenon of weak sensitization (Cochran et al., 1974). The number of positive reactions with normal leucocytes should therefore be considered as 23/81. Even then, the difference between positive reactions using remission leucocytes and normal leucocytes is highly significant (x2 2 27X29 at 1 d.f., P < 0 001).
Eliminating 9 autochthonous reactions from the total number of 57 tests carried out with CML remission leuco cytes (Table I), the difference between positive reactions using allogeneic remission leucocytes and normal leucocytes (inhibition + enhancement) is still significant (x2 23X53 at 1 d.f., P < 0.005). Table 2   OIne of these represente(l aoitochthoiiot)s reactioll.
frequency of enhancement, again, was not statistically significant. The difference between inhibition pattern with leukaemic and normal antigens was statistically significant (X2 -19 98 at 1 d.f., P < 0.005). Similarly the difference between positive reaction (enlhancement and inhibition together) with leukaemic and normal antigens was significant (X2 1391 at I d.f.,P < 0005).

DISCUSSION
Recently, leucocyte migration inhibition tests have been used to study tumourspecific cellular immunity in a variety of solidl tumours (Aindersoin et al., 1970;Mavligit et al., 1972;Braun et al., 1972;Cochran et al., 1973aCochran et al., , 1974and Kjaer, 1974). Santos et al. (1973) reported the in vitro production of MIF by remission leucocytes of acute leukaemic patients, which was tested indirectly on guinea-pig macrophages. G utterman (1 973a) used LMI test for studying cellular immunity in a few cases of leukaemias which included one CML in blast crisis.
The present report provides evidcence for autochthonous, as well as allogeneic, sensitization of CML patients in remission to solubilize(d membrane antigens of leukaemic cells. The autochthonotus reactivity was quite remarkable in that 8/9 tests showed inhibitioin of migrationi, ancd is most of them showed a very high degree of inhibition (Fig. 1, circled points). In four cases where antigens prepared from relapse leukaemic cells were tested on autochthonous remission le-ucocytes, 6/7 antigenic samples inhibited leucocyte migration, indicating similarity or constancy of leukaemic antigens during serial relapses.
When leukaemic cell antigens were tested on allogeneic CML remission leucocytes, 35/49 (714%) tests showed inhibition of migration. It has been suggested before that use of soluble antigens can discriminate between tumour-specific and HL-A reactivity (Gutterman et al., 1972b). However, recently, Dean et al. (1975) have reported positive blastogenesis of normal lymphocytes using solubilized tumour antigens. In our studies, the same batch of leukaemia antigens did not significantly inhibit the migration of normal lcucocytes (22.2%). Similarly, soluble antigens prepared from normal leucocytes did not significantlv inhibit migration of CML remission leucocytes (15%) These results, and the reports by Anderson et al. (1970) and Mavligit et al. (1972) using solid tumours, suggest that perhaps LMI is capable of distinguishing tumour-specific reactivity from the HL-A reactivity.
While studying tumour-specific sensitization with LMI test in breast canicer, Cochran et al. (1974) have noted enhancement of migration in a significant nutmber of cases. This was suggested to be indicative of weak sensitization. In the present series of experiments enhlancement to a siginificant level was not obtained with any of the antigein-leucocyte combination.