CELL TRANSFER STUDIES O N THE PERSISTENCE OF HOMOGRAFT SENSITIVITY IN THE MOUSE: I N VITRO A N D I N V I V O CORRELATES OF THE ACTIVITY OF SENSITISED LYMPH NODE CELLS

JUDITH WARREN AND G. GOWLAND University of Leeds, Department of Immunology,

Leeds General Infirmary, Leeds LSI 3EX

WARREN and Gowland (1970, 1972) have shown a relationship between time after grafting CBA mice and the ability of varying doses of their lymphocytes to transfer homograft sensitivity adoptively to normal CBA mice.

The object of the experiments reported here is to relate the activity of varying doses of CBA lymph node cells, removed at different times after sensitisation to allogeneic antigens, when transferred to different situations. One is an in vivo situation in which sensitised CBA lymph node cells are adoptively transferred to CBA mice test-grafted 1 day previously with A-strain skin; the second is an in vitro system in which sensitised CBA lymph node cells are placed in contact with ascites tumour cells from A-strain mice. In addition, any correlation with DNA synthesis was examined using 125I-IUdR.

MATERIALS AND METHODS

Experimental Animals Male animals from the following strains maintained by strict brother-sister mating were

used: A(H-2a), CBA(H-2k). Cell Transfer Experiment

The procedures for sensitisation of donor mice and preparation of suspensions of lym- phoid cells for passive transfer, along with the means of estimating survival time of test grafts are reported fully in a previous paper (Warren and Gowland, 1970).

Lymph node cell donors were mice sensitised to A-strain antigens by the lateral appli- cation of a full-thickness skin graft. At the following standard times after sensitisation (i.e., 8, 10, 12, 14, 18, 21 and 56 days), cell suspensions made from the pooled peripheral lymph nodes of donor mice were injected intraperitoneally into groups of 10 normal recipient CBA mice which carried a 24-hour-old graft of A-strain skin.

The test-grafted recipient mice were unhandaged 7 days after grafting and the grafts were inspected daily thereafter for signs of breakdown. Results are recorded as the Median Survival Time (MST) for each group of recipient mice.

In Vitro Cytotoxicity Experiment Medium used in cell cultures

The medium used throughout the in vitro experiments was tissue culture medium 199 with antibiotics, supplied as a concentrated solution (Glaxo Laboratories Ltd, Greenford, Middlesex, England). This was used as a 1 0 per cent. solution with further addition to 10

Received 20 Sept. 1973; accepted 3 Dec. 1973. J. PATH.-VOL. 114 (1974) 101

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102 JUDITH WARREN ARID G. GO WLAND

per cent. of sterile colostrum-deprived calf serum which had been inactivated at 56°C. The diluted culture medium had a final p H of 7.2.

All procedures prior to incubation of the cell cultures were carried out at 4°C and necessitated the use of bacteriologically sterile techniques.

Passage of target tumour cells The target tumour cell used, an ascites, Sarcoma I, native to A-strain mice, was maintained

by intraperitoneal passage in A-strain mice which were killed by cervical dislocation 7 days after inoculation with 10 x 106 Sarcoma I cells. The tumour cells in ascitic fluid were then removed aseptically and washed in tissue culture medium before further passage.

Preparation of Sarcoma I cell suspension Target cells were washed twice in tissue culture medium, resuspended in medium and

counted. Viability as determined by nigrosin exclusion was rarely below 99 per cent. at this stage.

Using bacteriologically sterile techniques, 106 tumour cells were dispensed into each of the required number of 50 ml capacity round glass bottles (3.5 cm diameter) and medium was added to a final volume of 5 ml. Overnight incubation was in a closed static water-bath operating at 23°C. In our experiments low temperature incubation was found to prohibit mitosis of target cells which occurred with incubation at 30°C and above, unless target cells were irradiated (cf. Wilson, 1965).

Preparation of lymph node cell suspensions Suspensions of pooled peripheral nodes from CBA mice presensitised as above, were

made according to the method of Billingham and Brent (1959), in tissue culture medium 199. Mesenteric nodes were not included because of possible bacterial contamination. After washing, cell suspensions were filtered through sterile, loosely packed, de-fatted cotton wool.

After the cells had been counted and their viability determined, 1 ml of the appropriate dilution was added to the relevant number of pre-incubated bottles containing ascites sarcoma. The final volume was made up to 10 ml with medium and the sealed bottles incubated in the covered water-bath at 23°C for 48 hr.

Measurement of cell killing On incubation, the cells settle under their own weight, thus allowing 8 ml of supernatant

fluid to be removed leaving the cells undisturbed in a volume of 2 ml. Two drops of an aqueous solution of 1 per cent. nigrosin were added to each of the bottles and mixed with the culture. One drop of the culture was then placed in a haemocytometer and a differential count of viable and dead cells was made. The relative sizes of ascites sarcoma cells and lymphocytes made them easy to distinguish in this counting system.

For the purpose of incorporation in figures, counts were expressed as the mean percentage tumour cells killed by any given dilution of lymphocytes :

] x l o o mean viable tumour cells in experimental bottles mean viable tunzour cells incubated alone

100- [ Controls

(i) 106 ascites sarcoma cells incubated alone. (ii) 106 ascites cells plus l o x 106 normal A-strain lymph node cells.

(iii) 106 ascites cells plus lox 106 normal CBA lymph node cells.

Radioactive tracer technique CBA male mice of 12 wk of age were sensitised by application of a full thickness skin

homograft (A-strain) or autograft (CBA). Control mice were CBA which had received no skin graft.

HOMOCRAFT SENSITIVITY 103

For 1 wk prior to commencement of the experiment and for its duration mice were supplied with iodinated water (40 ml of 5 per cent. KI solution per 10 1 of drinking water) to ensure elimination of label from organs not under investigation.

The radioactive tracer used was 125I-labelled 5-iodo-2’-deoxyuridine (125I-IUdR), (The Radiochemical Centre, Amersham, Bucks). This substitutes for thymidine in DNA synthesis and although its incorporation is less than X-C-thymidine, there is little incorporation in protein and almost no re-utilisation of this isotope (Fox and Prusoff, 1965).

Injection of Label Each mouse was injected intravenously into a tail vein with 7.5 x 10-7 mM of 125I-IUdR

with an activity of 1.5 pCi in a volume of 0.5 ml. The injection dose was also used as a standard solution for comparative estimation of y-emission of label recovered from lymph nodes.

Extraction of DNA Groups of 10 normal mice, or mice homografted or autografted at various times between

43 hr and 21 days previously, were injected with label. Four hours after injection of isotope the mice were killed by cervical dislocation and the peripheral lymph nodes of each individual mouse removed and weighed. DNA was then extracted as follows: lymph node tissue was macerated; washed 5 times in 5 per cent. ice-cold TCA (trichloracetic acid); centrifuged at 1500 r.p.m. after each wash and the supernatant was discarded. This process was followed by a 15 min. extraction in 5 per cent solution of TCA at 90°C.

The cold process eliminates acid-soluble nucleotides and in the subsequent heat extraction the nucleic acid goes into solution leaving extraneous protein as sediment.

Assay of 125I-IUdR incorporation One minute counts of standard solutions containing one injection dose (7.5 x 10-7 mM at

1.5 pCi), and extracted solutions were carried out on a Packard Series 3320 Auto-Gamma Spectrometer. Mean percentage counts and standard error of experimental samples compared with standard solutions were obtained using an Elliot 803 computer.

DNA estimation by ultraviolet spec trophotometry Samples extracted as above were diluted 1 : 300. DNA was measured at 260 mp and

protein at 330 mp on a Unicam S.P. 500 in 1 cm silica cells. After subtracting the reading for protein at 330 mp, the amount of DNA per ml of diluted sample was read from a table of optical densities of standard DNA solutions at 260 mp. The amount of DNA present in the whole sample extracted from the lymph nodes of each mouse was calculated.

Presentation of results in figure 4

mice used in this experiment were expressed as follows : The results of the two assays made on DNA from the pooled peripheral lymph nodes of

(a) The left hand ordinate and the solid line show the percentage of the i.v. injected dose of 1251-IUdR (7.5 x lO-7m~) which was incorporated (as a thymidine analogue) in the synthesis of new DNA in the lymph nodes. The actual percentage shown in figure 4 is:

HOMOGRAFTED MICE (A+CBA) AUTOGRAFTED MICE (CBA-ZBA)

x 1001 -XlOO] - [ 7.5 x 10-7 mM 125I-IUdR m~ 125I-IUdR in 1.n.

7-5 X 10-7 mM 125I-IUdR mM 1251-IUdR in 1.n.

(b) The right hand ordinate and broken line show the total amount of DNA which was obtained by TCA extraction from the lymph nodes of experimental mice. The actual amount shown in Figure 4 is the difference in total DNA (expressed as mg DNA/gm lymph node weight) between the lymph nodes of homografted (A-tCBA) and autografted (CBA-+CBA) mice.

104 JUDITH WARREN AND G. GOWLAND

Design of Experiments

Sensitised CBA mice

0-56 days

~ I r- I IUDR

I I

4 hr

1251 incorporation Total DNA

lymph node cell suspension

I .

I I I

.1 in vitro

cytotoxicity

.i adoptive transfer

In adoptive transfer experiments either 50 x 1 0 6 , 25 x 1 0 6 or 10 x 1 0 6 lymph node cells transferred intraperitoneally into normal CBA recipients test-grafted 24 hr previously with A-strain skin: MST of test grafts determined.

In cytotoxicity experiments varying concentration (ranging from 100 x 106 to 5 x 106) of lymph node cells incubated with 106 target cells and percentage tumour cell death determined.

RESULTS Figures 1 and 2 respectively show the effects of in vivo cell transfer of

syngeneic lymph node cells from sensitised to unsensitised CBA mice, and the effects of placing cells from similarly sensitised CBA mice in in vitro contact with target tumour cells.

Varying doses of lymph node cells from CBA mice previously sensitised to A-strain skin were transferred intraperitoneally into normal recipient CBA mice at various hmes after donor grafting (see Materials and Methods). The normal CBA recipients were test-grafted with A-strain skin. Each point in figure 1 shows the MST of a group of at least 10 A-strain test grafts on CBA recipients. The three lines on fig. 1 each represent the MST resulting in reci- pient CBA mice from a transfer of a particular dose of syngeneic lymph node cells (see legend to fig. 1). Agreement between the curves in fig. 1 is good at all points; in the period from 8-14 days after donor sensitisation sensitivity is being readily transferred by donor lymphoid tissue.

Cells transferred 14 days after donor sensitisation apparently have the greatest destructive effect on A-strain test homografts (Warren and Gowland, 1970); thereafter there is a gradual decrease in the effectiveness of transferred cells as measured by MST of test grafts.

Cells from the lymph nodes of similarly sensitised CBA mice, when placed in in vitro contact with A-strain target tumour cells in a’ratio of 25 x 106 CBA lymph node cells to 106 A-strain ascites cells, were responsible for the target cell death shown in fig. 2. This ratio of 25 lymph node cells to one target cell was chosen as it was equivalent to the middle dose of cells transferred by the in vivo method.

HOMOGRAFT SENSITIVITY 105

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Fie. 1.-Effect of intraperitoneal transfer of presensitised lymph node cells from CBA mice on the median survival time (MST) of A-strain skin on normal CBA mice. (Dose of cells transferred: - 50XlOa; *.*..25XlOa; - - - - -10~106.)

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F I ~ . 2.-Percentage target (A-strain, Sarcoma I) death after 48 hr incubation of 106 target cells with 25 x 106 lymph node cells taken from CBA mice at various times after presensitisation to A- strain skin grafts.

J. PATH.-VOL. 114 (1974) H

106 JUDITH WARREN AND G. GOWLAND

In fig. 2 the destructive effect of sensitised CBA lymph node cells on the ascites sarcoma cells increased with increasing time after grafting. Lymph node cells showed the maximum destructive effect at 14 days which compares well with the data in fig. 1, bearing in mind that the in vitro method is designed to prevent division of either target or effector cells (i.e., by incubation at 23°C).

The two figures also show similarity in that the power of lymph node cells from sensitised CBA mice to destroy skin homografts or to kill target tumour cells appears to decrease from 14 days after grafting. In fig. 1, after a 56-day

100

50

40- (d - 30.

FIG. 3.-Percentage target (A-strain, Sarcoma I) death after 48 hr incubation of 106 target cells with varying ratios of lymph node cells taken from CBA mice at various times after presensitisation to A-strain skin grafts. (N.B. Percentage target cell death is measured from the 20 per ccnt. baseline of the appropriate column. 0.4 cm = 10 per cent.)

transfer, the MST of test grafts is once again equivalent in time to that of a first-set rejection (i.e., 11.5 days in A-KBA), whilst in fig. 2 the percentage target cell death has decreased to 19-20 per cent.

In a series of controls carried out with each in vitro experiment, lox 106 lymph node cells from unsensitised CBA mice, when incubated with 106 A-strain ascites sarcoma cells, regularly caused an average of 20 per cent. tumour cell death. Any deductions as to the effectivity of cells from sensitised CBA mice were therefore made using a figure of 20 per cent. as a base-line.

Figure 3 is a three-dimensional graph drawn on isometric paper. Figure 2 showed the killing capacity, under our conditions, of a fixed ratio of sensitised CBA lymph node cells to A-strain tumour cells. Figure 3 shows the in vitro behaviour of lymph node cells from similarly sensitised CBA mice on various days after grafting when the lymphocytes were incubated with target cells in

HOMOGRAFT SENSITIVITY 107

several different ratios between 5 : 1 and 100 : 1. As described above, 20 per cent. is the starting-point for measuring tumour cell death.

Each of the columns in fig. 3 represents the percentage target cell death caused by a particular ratio of lymphocytes to tumour cells. For each day after donor sensitisation that an experiment was carried out there is a range of lymphocyte to tumour ratios. The height of each column from its appropriate base indicates the percentage target cell death (0.4 cm = 10 per cent. ascites sarcoma cell death).

. I I I 1 I I I I 1

A'

Days after grafting

_ _ _ _ _ .. -- - - - 0 2 6 10 14 18 22

FIG. 4 . 4 ) Synthesis of new DNA as a result of homografting as measured by 1251-IUdR uptake (solid line, left hand ordinate; results expressed as difference in uptake between the homografted and autografted group at each time interval). (ii) The effect of homografting on total lymph node DNA (broken line, right hand ordinate; results are given in mg DNA/g lymph node and expressed as the difference between the homografted and autografted group at each time interval).

The point which is made most obviously by fig. 3 is that by increasing the numbers of lymphocytes from sensitised mice incubated with a fixed number of tumour cells there is an increased percentage of target cell death. This fact is particularly apparent in that the higher ratios of lymph node cells cause 50-65 per cent. target cell death, in this system, as early as 6 days after donor sensitisa- tion. From 6 days onwards there is a progressive increase in the amount of target cell death caused by all concentrations of lymphocytes. This culminates in the highest overall effectiveness against the ascites sarcoma by cells removed from donors grafted 14 days earlier. Thereafter the now familiar pattern of decreasing effectiveness in the destructive capacity of lymph node cells is shown by the high numbers of lymphocytes required (ie., lOOx 106) to kill up to 50 per cent. of the inoculum of 106 tumour cells.

108 JUDITH WARREN AND G. GOWLAND

Incorporation of radioactive iodine as 125I-IUdR into the lymph nodes of homografted CBA mice injected with this label is shown by the solid line in fig. 4. The solid line indicates the difference in the amount of label incorporated in the lymph nodes of homografted (A-tCBA) mice as compared with autografted (CBA+CBA) mice.

The rise of almost tenfold in the percentage label incorporated from day 4 to 6 is very striking (cf. the marked increase in tumour cell death, shown in fig. 3, by lymphocytes from donors grafted 6 days previously). A " t " test on the mean uptake of 125I-IUdR in lymph nodes of homografted versus autografted mice on days 6 and 10 after grafting showed that the increased uptake in homografted mice was significant at 0.1 per cent. level. This represents the incorporation of the label as a thymidine analogue when new DNA is being formed. After day 12 the level of 125I-IUdR in homografted mice drops sharply and by day 18 the level of uptake of label is no greater than that in autografted mice.

The broken line in fig. 4 is to be considered quite separately (right hand ordinate). It represents the difference, between homografted and autografted mice, in the total amount (in mg/gm lymph node) of TCA-extracted DNA in the lymph nodes on various days after grafting. No differences appear in the total DNA obtainable by extraction from day 0 of the experiment until day 4. Thereafter there is a considerable increase in total extracted DNA in the lymph nodes of homografted as compared with autografted animals, with peaks at 6 and 10 days after grafting. No difference is again apparent 12 days after the mice were grafted but over the next 4 days the total DNA in lymph nodes of homografted CBA mice rises to 6.5 mg/gm higher than that in mice with autografts. The pattern is then repeated as there is no difference at 18 days but increased DNA in the lymph nodes of homografted mice at 21 days after sensitisation. No further estimations of TCA-extracted DNA were carried out so it is not known if this pattern is repetitive thereafter (but see Discussion).

DISCUSSION The capacity of lymphocytes from mice sensitised by a skin homograft,

either to confer sensitivity adoptively upon a normal mouse, or to destroy target tumour cells in vitro, is dependent both on the dose of lymphoid cells used in the experiment and on the time which has elapsed since the donor mice were presensitised.

Comparing figs. 1 and 2 suggests a relatively good correlation in the chronological nature of the results of passive transfer and in vitro cytotoxicity. The quantitative results of cell-killing in vitro are apparent however at a much earlier stage than test graft destruction due to passive transfer of cells (fig. 3). Large pyroninophilic or blast cells are present in increased numbers in the lymph nodes of mice 6 days after grafting. These cells might be able to destroy some target tumour cells when brought into intimate contact with them in vitro. Similar cells introduced into mice by intraperitoneal passive transfer may be less motile than small lymphocytes and this plus their relative bulk (1 5- 17 pm) could make more difficult their passage from the peritoneal cavity into the

HOMOGRAFT SENSITIVITY 109

circulation and thus to the site of a skin graft. If this situation existed it might explain the effectiveness in vitro of cells from 6 day grafted donors which is not mirrored in vivo. However, we recognise the obvious dangers in comparing in vivo and in vitro results especially as, in our in vitro experiments, lymph node cells and target cells were placed in immediate contact.

Figures 1-3 show a pattern of increasing effectiveness of lymph node cells from sensitised donors up to a peak at around 14 days after grafting followed by an overall decrease in activity both in vivo and in vitro from 18 days onwards. After the rejection of the donor’s homograft, therefore, the level of effector lymphocytes fell, which may indicate their demise in the destruction of the homograft.

It is also possible that a population of effector cells with a short life-span (cf. Gowans and Knight, 1964) may appear as a result of sensitisation. Either proposition would explain why we could transfer sensitivity only with very large doses of lymph node cells at later time intervals (see also Warren and Gowland, 1970).

A mouse will present a second-set rejection for more than a year after primary stimulation (Billingham, Brent and Medawar, 1954; Warren, 1970). This could be due to rapid multiplications of previously activated “ memory ” cells on second contact with antigen. Alternatively only a small number of the effector cells produced may be necessary to destroy the primary homograft and sufficient numbers could persist to cause accelerated rejection of a second graft. Either system could explain that sensitivity can be transferred at longer than 8 wk after homografting the donor mice but only with large numbers of cells from lymph nodes (Warren and Gowland, 1972); and also the relatively low percentage of target cell death demonstrated by 50-100 million lymph node cells 56 days after donor grafting.

Turk (1967) has argued that transferred cells are effective in destroying homografts because they have the opportunity to divide and increase in the recipient. Though no direct comparison is intended with events in vivo it may be noted that incubation of cells in vitro at 23°C prevents cell division but does not prevent early and effective destruction of target cells.

It is of interest to note the increase in the cytotoxic capacity of lymph node cells in vitro (fig. 3) 6 days after the grafting of donors, in relation to the evidence of 125I-IUdR uptake (solid line, fig. 4) in the lymph nodes of homografted mice on this day. The increased uptake of label in the lymph nodes of homografted as compared with autografted CBA mice is marked on both days 6 and 10 after grafting.

The increase in uptake of label on day 6 after grafting agrees well with the appearance of large pyroninophilic cells in the lymph nodes on days 6 and 8 after homografting of CBA mice (Micklem and Loutit, 1966; Warren, 1970), and can be taken as indication of DNA synthesis leading to blast cell formation. A second peak in uptake of 125I-IUdR in lymph nodes of homografted mice at 10 days may correlate with either or both of the phenomena of a second phase of production of large pyronin-positive cells and the production of large num- bers of small lymphocytes assumed to represent the effectors in homograft

110 JUDITH WARREN AND G. GOWLAND

rejection. The results are more difficult to interpret at 10 days because we also recognise that there is concurrent production of isoantibodies with accompany- ing germinal centre activity and production of plasma cells.

The mean percentage uptake of label is concurrent with high levels of DNA in lymph nodes at 6 and 10 days after the application of an A-strain homograft to CBA mice (broken line, fig. 4). Also the fluctuations in total DNA levels observed between 10 and 21 days after grafting are not a consequence of active synthesis of DNA as measured by 125I-IUdR uptake. In experiments published previously (Warren and Gowland, 1972, 1973) we have indicated the possibility of a periodicity of 3-4 days in the appearance and redistribution of sensitised cells in lymph nodes, spleen and peripheral circulation which might be interpreted as the perambulations of a sub-population of sensitised lymphocytes. Although there is no direct evidence on which to base a comparison, the apparently oscillatory nature of DNA levels shown in fig. 4 at times from 12 days after grafting may be some expression of the same phenomenon.

SUMMARY Complementary evidence is presented on the in vivo and in vitro behaviour

of cells from CBA mice sensitised to A-strain antigens by skin allograft. The in vivo system involved the rejection of test allografts following adoptive

transfer of sensitised cells; the in vitro test measured the ability of similarly sensitised cells to kill A-strain ascites target tumour cells in the absence of complement. The in vitro test did not allow replication of either target or effector cells. In both systems there was considerable agreement with regard to time after sensitisation of the donors of lymph node cells. Cells from 14 day donors were singularly effective in both systems.

Good correlation was also obtained with regard to the apparent decline in cellular reactivity with time.

Total DNA in lymph nodes and 12sI-IUdR incorporation into the lymph nodes of allografted, as opposed to isografted, mice was measured. It was possible to demonstrate a close relationship between DNA synthesis in the lymph nodes of sensitised donor mice, and the ability to both transfer sensitivity adoptively and exert in vitro cytotoxicity.

REFERENCES BILLINGHAM, R. E., BRENT, L., AND MEDAWAR, P. B. 1954. Quantitative studies on tissue

transplantation immunity. 11. The origin, strength and duration of actively and adoptively acquired immunity. Proc. Roy. SOC. Lond. B., 143, 58.

BILLINGHAM, R. E., AND BRENT, L. 1969. Quantitative studies on tissue transplantation immunity. IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease. Phil. Tram. Roy. SOC. B, 242,439.

Fox, B. W., AND PRUSOFF, W. H. 1965. Comparative uptake of 125I-labelled 5-iodo-2’- deoxyuridine and thymidine-H3 into tissues of mice bearing Hepatoma-1 29. Cancer Res., 25, 234.

GOWANS, J. L., AND KNIGHT, E. J. 1964. Route of recirculation of lymphocytes in the rat. Proc. Roy. SOC. B, 159, 257.

HOMOGRAFT SENSITIVITY 111

MICKLEM, H. S., AND LOUTIT, J. F. 1966. In Tissue grafting and radiation, American Inst. of Biological Sciences and US Atomic Energy Commission Monograph, Academic Press, New York and London, pp. 50-76. Radiation in relation to transplantation immunity.

TURK, J. 1967. In Delayed Hypersensitivity, North-Holland Research Monographs, Frontiers of Biology, edited by A. Neuberger and E. L. Tatum, Amsterdam, vol. 4, pp. 93-94.

WARREN, JUDITH 1970. Ph.D. Thesis, Faculty of Medicine, University of London. WARREN, JUDITH, AND GOWLAND, G. 1970. Cell transfer studies on the persistence of

homograft sensitivity in the mouse. J . Path., 100, 39. WARREN, JUDITH, AND GOWLAND, G. 1972. Cell transfer studies on the persistence of

homograft sensitivity in the mouse: The periodic redistribution of sensitised cells be- tween the circulation and lymphoid tissues. J. Path., 107, 225.

Homograft sensitivity in the mouse: The periodic redistribution of sensitised lymphoid cells. In Advances in Experimental Medicine and Biology, edited by Jankovic and Isakovic, vol. 29, pp. 553-9.

WILSON, D. B. 1965. Quantitative studies on the behaviour of sensitised lymphocytes in vitro. I. Relationship of the degree of destruction of homologous target cells to the number of lymphocytes and to the time of contact in culture and consideration of the effects of isoimmune serum. J. exp. Med., 122, 143.

WARREN, JUDITH, AND GOWLAND, G. 1973.

Vol. 114, No. 1, September 1974, was issued on 29.11.74.