Cellular und Tumor Immunology Section Laboratory
of Cell Biology National Cancer Institute Bethesda, Maryland 20014
Cellular immune reactions are generally thought to playa major
role in host resistance against tumor growth. Human acute leukemia
cells have been found to contain tumor associated antigens, and
it is possible to measure the cell-mediated immune response to these
antigens. In addition to specific reactivity, it is quite important
to evaluate the functional integrity of the cellular immune system
in leukemia patients. The disease process itself or the chemotherapeutic
agents could cause general depression in reactivity. The techniques
currently being used for such studies are summarized in Table 1.
In this paper, we will review the information available from each
of these approaches, and discuss their possible relevance to the
clinical state .
Table 1. Assays of Cellular Immunity in
Human Acute Leukemia
Competence of Cellular Immunity System
Three aspects of the competence of the cellular immunity system
in acute leukemia patients are of interest: a) the effect of the
tumor burden on reactivity; b ) the effect of chemotherapy; and
c) the inherent ability of patients to have cellmediated immunological
reactions against a variety of antigens. Unfortunately, it is difficult
to clearly study each of these factors. The ideal time to obtain
information on the patient's inherent reactivity would be prior
to development of disease. Based on the theory of immunological
surveillance ( 1, 2 ), one might anticipate that individuals developing
leukemia have depressed immunological competence. However , it is
not possible to obtain information at this time. It is practical
to examine patients at the time of diagnosis, when tumor is present,
and during clinical remission. Since chemotherapeutic agents are
used to induce remission, their effects must also be considered.
Skin tests for delayed hypersensitivity have been extensively used
to study cellular immunity in acute leukemia patients. In studies
before thereapy ( 3) and shortly after the onset of therapy ( 4)
, decreased reactivity of some patients to standard recall skin
test antigens was found. Hersh et al ( 4) found that depressed skin
reactivity was associated with poor response to therapy for six
months or longer had even less reactivity than that seen in initial
studies ( 5) .Chemotherapy may have had an important influence on
these results. Borella and Webster (6), in a study of children with
acute leukemia in remission, observed that long-term combination
chemotherapy had immunosuppressive effects on skin test reactivity.
Some treatment protocols appeared to be more immunosuppressive than
others. Many of the patients of Hersh et al ( 4, 5) received COAP,
which was noted to be very .. ImmunosuppressIve. Our laboratory
also studied the response of ALL (acute lymphocytic leukemia) and
AML (acute myelogenous leukemia) patients to a battery of standard
recall antigens (7). We performed almost all of our tests after
induction of remission, or at the time of bone marrow relapse. Patients
at the National Cancer Institute are usually treated with combination
hemotherapy, with repeated cycles of one week of treatment followed
by two-three weeks off. To reduce the possible effects of treatment
on the results, the patients were usually skin tested just prior
to a course of therapy. Table 2 gives a summary of our data. Ninety-six
percent of patients with ALL and all of the patients with AML reacted
with at least one of the antigens, when tested in remission or in
relapse. There were no significant correlations between the incidence
of reactivity to anyone of the particular antigens and clinical
state, time of test in relation to chemotherapy, or length of survival.
The reactivity of the ALL patients to PPD was high, due to immunotherapy
with BCG. There are several possible explanations for the differences
between our results and those of Hersh's group. Since they found
that patients with intact skin reactivity were more likely to go
into remission ( 4) , it is possible that our patients, initially
tested in remission, were a selected population. The type of chemotherapy
used, and the timing of tests in relation to therapy, may also have
been important differences. Stimulation of patients' lymphocytes
by mitogens, recall antigens and allogeneic leukocytes is another
widely used technique for assessing immune competence.
Table 2. Skin Reactions to Recall Antigens
Hersh et al ( 4) found that one-third of leukemia patients studied had decreased responses to phytohemagglutinin and streptolysin 0. As with the skin tests, most of these poorly reactive patients did not respond well to therapy. I t has been found the time of testing, in relation to chemotherapy, has an important influence on the results ( 8, 9, 10) .Reactivity was greatest 1 o~ 20 days after cessation of therapy, and in fact was sometimes higher than normal reactivity at this time.
Techniques have recently been developed which may allow enumeration of thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) in the peripheral blood. T cells ,appear to have receptors for sheep erythrocytes ( E ) , while B cells have receptors for the third com ponent of complement and can thereby bind E coated with antibody and complement (EAC). Rosette assays based on binding of E and EAC are easy to perform and may provide useful information in cancer patients. Many cancer patients have decreased percentages of circulating T cells (11,12). Sen and Borella (13) have found that longterm chemotherapy caused depression in lymphocyte counts, and B cells were decreased more than T cells. After cessation of therapy, the B cells returned to normal levels within two to three months, whereas recovery of T cells took longer . No systematic serial study of these cell populations at different phases of disease have been reported as yet.
Rosenberg et al ( 14) have recently found that the lymphocytes of most normal individuals have cytotoxic reactivity against human lymphoid cell lines. McCoy et al ( 15) found that many patients with solid tumors and patients with immune deficiency diseases had significantly reduced activity. Fig. 1 shows the results with leukemia and lymphoma. Many of the leukemia patients had low reactivity. Reactivity was found to vary at different times in relation to chemotherapy (16). However, no consistent pattern was seen among different patients. Patients who were off therapy for four to eight weeks (points labelled Rx in Fig. 1) had normal or high reactivity.
Cell-Mediated Immunity Against Leukemia Associated Antigens
Skin testing with membrane or soluble extracts of blast cells has
been used to measure delayed hypersensitivity reactions to leukemia
associated antigens (7, 17) . Table 3 is a summary of our tests
with autologous membrane extracts of blast and remission cells (7).
In patients with ALL who were in remission, positive reactions to
autologous blast extracts were obtained in 20 of 44 tests. ALL patients
tested in relapse gave only one positive reaction. In AML, there
was also a significant correlation of reactivity to blast extracts
and clinical state. Serial skin tests were performed in 29 ALL and
8 AML patients (7 ). In most cases, reactivity to autologous blast
cell extracts fluctuated in parallel with changes in clinical state.
Patients who had positive reactions in remission generally became
unreactive at the time of relapse. This represented specific depression,
in that reactions to recall antigens did not vary significantly.
In contrast to our results with autologous membrane extracts, it
has recently been reported that no positive reactions of acute leukemia
patients were elicited by autologous viable cells ( 17) or mitomycin-C
treated blast cells ( 3) .Gutterman et al ( 17) did find, however,
that 3M potassium chloride extracted soluble antigen produced positive
skin reactions. These studies indicated that the form of the antigen
used for skin testing may influence the results. Our group has performed
studies on antigens solubilized and separated from AML cells (18),
as indicated in Figure 2. Skin reactive antigens could be solubilized
by low frequency sonication, and then separated by Sephadex G-200
and DEAE-cellulose chromatography. Table 4 gives the representative
results of testing a patient with autologous DEAE fractions. Skin
reactions were elicited by two of the fractions from blast cells,
and not by the comparable fractions from remission cells. I t has
been possible to further separate the AML skin reactive antigens,
by gradient acrylamide gel electrophoresis. Tabel 5 shows a test,
in which only one region of the gel containing the blast extract
gave a positive reaction. No reactions were elicited by the comparable
remission cell fractions.
Fig. 2. Extraction procedure for solubilization
and separation of skin reactive antigens on cells from patients
with acute myelogenous leukemia.
Similar studies are now in progress with extracts of ALL cells and
of fetal thymus cells. Some positive results have been obtained
with the fetal thymu.'i cells, which are of particular interest,
since ALL blast cells appear to be T cells ( 12) . We have recently
also tested patients with membrane extracts of cells from allogeneic
leukemia patients (7 ). The results are summarized in Table 6. Positive
reactions were observed in both AML and ALL patients, in response
to extracts of blast cells from the same disease. Some of the extracts
produced as much reactivity as the autologous extracts, while others
were inactive. As with autologous extracts, reactivity to the allogeneic
preparations correlated with disease status. The antigens detected
on the allogeneic extracts appeared to be tumor associated, since
extracts of remission cells or of normal leukocytes were unreactive.
Membrane extracts of human lymphoid cell lines have also given positive
reactions in some patients with acute leukemia or lymphoma (19).
Most of the cell lines were derived from Burkitt's lymphoma. As
a control, patients were also tested with F-265, a cell line derived
from normal lymphocytes. The results are summarized in Table 7.
Patients with leukemia and lymphoma appear to be sensitized against
an antigen on the tumor derived cell lines, whereas carcinoma
Table 3. Skin reactions to Autologous
Table 4. Skin Tests with DEAE-Cellulose Fractions of Soluble
Autologous AML * Cells (Herberman, Char, and Hollinshead, 1973)
Table 5. Skin Tests with Acrylamide Gel Fractions (Herberman,
Char , and Hollinshead, 1973)
Table 6. Skin Reactions to Allogeneic Membrane Extracts
patients were unreactive. Reactivity in the ALL patients has correlated
well with clinical state. Thus far, only patients in remission have
been reactive. Several groups have found that remission lymphocytes
of patients with acute leukemia could be stimulated by autologous
blast cells (10,20-23). As with mitogens and common antigens, stimulation
by autologous blasts was found to correlate with the length of time
after cessation of a course of chemotherapy (10,16). Autologous
blast cells generally produced maximum stimulation at 10-20 days
after chemotherapy, but the peak response to blast cells did not
always occur at the same time as the peak response to mitogens or
allogeneic leukocytes (16). Positive stimulation has also been found
to correlate with good prognosis (23,24). Soluble extracts, prepared
by treatment of blast cells with 3M potassium chloride, have also
been shown to stimulate the autologous lymphocytes (17). We plan
to perform similar studies with the antigens solubilized by sonication.
Table 7. Skin Reactions to Extracts of
Lymphoid Cell Lines
Table 8. Cell-Mediated 51 Cr Release Assay -Autologous
The specificity of the antigens detected by lymphocyte stimulation
is not completely clear. Some experiments have been performed with
remission bone marrow as well as with relapse marrow (10). In some
experiments, both blast cells and remission cells produced some
stimulation. I t remains to be determined whether the stimulation
assay is detecting a differentiation antigen present on normal blast
cells as well as on leukemic blasts. In vitro cytotoxicity reactions
have been particularly stressed as likely analogues of the cell-mediated
defense against tumors (25 ). Our laboratory has tested the lymphocytes
of patients with acute leukemia and of normal individuals against
autologous and allogeneic target cells, by as I Cr release cytotoxicity
assay ( 10, 26) . Table 8 gives a summary of results obtained against
autologous target cells. There was no reactivity of normals against
their own lymphocytes. Both ALL and AML patients reacted against
their blast cells, but not against their remission cells. The observed
reactivity did not against their remission cells. The observed reactivity
did not correlate with clinical state. There was at least as much
reactivity during bone marrow relapse as there was during remission.
The results of tests against allogeneic target cells are given in
Table 9. Only a low incidence of reactivity was observed against
lymphocytes of normal individuals. In contrast, many positive reactions
against blast cells were seen; lymphocytes from leukemia patients
and also from normal controls had cytotoxic effects. Positive results
were also seen against remission lymphocytes of the leukemia patients.
The cytotoxicity assay appears to be detecting leukemia associated
antigens, but the specificity of the reactions may be different
from that of the skin tests. In the studies with autologous cells,
antigens were only detected on blast cells. In the allogeneic tests,
some results with remission cells were also positive. It is likely
that the remission cells contain antigens different from those on
blast cells, and which are undetectable by skin tests. There have
been few studies of leukemia patients thus far with the leukocyte
migration inhibition assay. However, using the assay of Rosenberg
et al ( 27) , Halterman et al ( 28) studied a pair of identical
twins, one with leukemia. Leukemic antigen extract, which gave a
positive skin reaction in the patient, also caused inhibition of
the migration of her leukocytes. The same extract did not affect
the migration of the normal twin's leukocytes. To determine the
possible relationships between skin testing, lymphocyte stimulation,
and 51 Cr cytotoxicity assays, the three tests were performed on
20 patients (10). The results are summarized in Table 10. As noted
above, results of skin tests correlated with clinical status. The
in vitro assays did not correlate with stage of disease, nor did
they correlate with each other. The reasons for the lack of correlations
are not clear. It is quite possible that each assay is measuring
different antigens. Leukemias in experimental animals have been
shown to have a complex variety of antigens (29,30). Studies with
isolated, soluble antigens are now feasible and should help to decide
this issue. The assays may also be measuring different phases of
the immune response, and different subpopulations of lymphocytes
may be responsible for the various effects. It is quite possible
that the lymphocyte stimulation assay measures the primary recognition
of foreign cell surface antigens.
Table 9. Cell-Mediated 51 Cr Release Assay
-Allogeneic Target Cells
Table 10. Results of three assays of cellular immune reactivity
in acute leukemia to autochthonous blasts cells
The lymphocyte cytotoxicity assays appear to detect the presence
of presensitized cells, capable of rapidly reacting with the target
cells. The skin tests are thought to depend on sensitized lymphocytes
capable of releasing migration inhibitory factor and other soluble
mediators, and also on the presence of adequate numbers of mononuclear
cells to accumulate at the reaction site.
Use of Immunological Assays to Monitor Immunotherapy
There has been much recent interest in the use of immunotherapy
in acute leukemia. Most of the studies already performed have been
empirical, without assessment of the antigenicity of the immunizing
cells and without immunological monitoring of the immune response
to the therapy. An immunotherapy study was performed on previously
treated ALL patients, in which allogeneic ALL blast cells plus either
BCG or methotrexate were given ( 24) . Immune responses were serially
determined, by skin testing and by lymphocyte stimulation. The most
dramatic change was in the response of the BCG treated group to
PPD. Skin tests with allogeneic blast extracts did not provide clear
evidence for immunization, even against the HL-A antigens of the
donor cells. There was, however, a correlation between skin reactivity
against the extract of donor cells and the duration of remission.
In the lymphocyte stimulation assays, there was also little evidence
for immunization by the donor cells. The use of these immunological
assays could help in designing future immunotherapy trials. Allogeneic
cells could be selected on the basis of their ability to elicit
skin reactions, thereby documenting that the donor cells possess
common antigens. In addition, monitoring with skin tests could be
used to determine the immunogenicity of different immunizing preparations
and schedules. It would be very helpful if autologous cells were
available for skin testing and for in vitro tests, since they would
permit a distinction between immunization to leukemia associated
antigens and immunization to normal histocompatibility antigens.
If an immunotherapy trial were clinically successful, it would provide
important information on the predictive value of each of the assays.
1. Burnet, F. M.; Brit. Med.J. 1: 779 and 841,1957.
2. Thomas, L.; In "Cellular and Humoral Aspects of the Hypersensitive
State" (H. S. Lawrence, ed.), p. 529, Hoeber, New York, 1959.
3. Santos, G. W., Mullins, G. M., Bias, W. B., Anderson, P. N.,
Graziano, K. D., Klein, D. L., and Burke, P. J.; Nat. Cancer Inst.
Monogr. 37: in press, 1973.
4. Hersh, E. M., Whitecar, J. P. Sr., McCredie, K. B., Bodey, G.
P. Sr., and Freireich, E. J.; New Eng. J. Med. 285: 1211,1971.
5. Hersh, E. M.; Proc. 26th Annual Symp. on Fundamental Cancer Research:
in press, 1973.
6. Borella, L., and Webster, R. G.; Cancer Res. 31: 420,1971.
7. Char, D. H., Lepourhiet, A., Leventhal, B. G., and Herberman,
R. B.; Int. J . Cancer: in press, 1973.
8. Cheema, A. R., and Hersh, E. M.; Cancer 28: 851,1971.
9. Harris, J. E., and Stewart, T. H. M.; In Proc. of the Sixth Leukocyte
Culture Conference (W. Schwarz, ed.), p. 555, Academic Press, New
10. Leventhal, B. G., Halterman, R. H., Rosenberg, E. B., and Herberman,
R. B.; Cancer Res. 32: 1820,1972.
11. Wybran,J., and Fudenberg, H. H.;J. Clin. Invest.: in press,
12. West, W., McCoy, J. L., and Herberman, R. B.; Unpublished observations.
13. Sen, L., and Borella, L.; Fed. Proc. 32: 980 Abs, 1973.
14. Rosenberg, E. B., McCoy, J. L., Green, S. S., Donnelly, F. C.,
Siwarski, D. F ., Levine, P. H., and Herberman, R. B.; J. Nat. Cancer
Inst., submitted for publication.
15. McCoy, J., Herberman, R., Perlin, E., Levine, P., and Alford,
C.; Proc. Amer. Assoc. Cancer Res. 14: 107, 1973.
16. Herberman, R. B., Rosenberg, E. B., Halterman, R. H., McCoy,
J. L., and Leventhal, B. G.; Nat. Cancer Inst. Monogr. 35: 259,1972.
17. Gutterman, J. U., Hersh, E. M., Freireich, E. J., Rossen, R.
D., Butler, W. T., McCredie, K. B., Bodey, G. P. Sr., Rodriguez,
V., and Mavligit, G. M.; Nat. Cancer Inst. Monogr. 37: in press,
18. Herberman, R. B., Char, D. H., and Hollinshead, A. C.; In preparation.
19. Herberman, R. B., McCoy, J. L., and Levine, P. H.; Nat. Cancer
Inst. Monogr.: in press, 1973.
20. Fridman, W. H., and Kourilsky, F. M.; Nature 224: 277, 1969.
21. Viza, D. C., Bernard-Degani, R., Bernard, C., and Harris, R.;
Lancet 2: 493, 1969.
22. Powles, R. L., Balchin, L. A., Hamilton-Fairley, G., and Alexander,
P.; Brit. Med. J. 1: 486,1971.
23. Gutterman, J. U., Rossen, R. D., Butler, W. T., McCredie, K.
B., Bodey, G. P., Freireich,E.J.,andHersh,E.M.;NewEng.J.Med. 288:
169,1973. 24. Leventhal, B. G., Lepourhiet, A., Halterman, R. H.,
Henderson, E. S., and Herberman, R. B.; Nat. Cancer Inst. Monogr.:
in press, 1973.
25. Hellström, I., Hellström, K. E., Sjögren, H. 0., and Warner,
G. A.; Int. J. Cancer 7: 1,1971.
26. Rosenberg, E. B., Herberman, R. B., Levine, P. H., Halterman,
R. H., McCoy, J. L., and Wunderlich, J. R.; Int. J. Cancer 9: 648,
27. Rosenberg, S. A., and David,J. R.;J. Immunol. 105: 1447,1970.
28. Halterman, R., Rosenberg, S. A., Rosenberg, E. B., and Herberman,
R. B.; Unpublished observations.
29. Herberman, R. B.; J. Nat. Cancer Inst. 48: 265, 1972.
30. Aoki, T., Herberman, R. B., Johnson, P. A., Liu, M., and Sturm,
M. J.; Virol. 10: 1208,1972.