CA2128424A1 - Method for determining cytolytic t cell precursors - Google Patents

Abstract

The invention relates to a method for determining precursors of cytolytic T cells and the frequency of these precursors, where the cytolytic T cells which develop therefrom are specific to antigens associated with or characteristic of the tumors. The method involves contacting peripheral blood monocyte cells (PBMC) containing sample to a source of antigen characteristic of or associated with the tumor, such as non viable tumor cells. The PBMCs, if precursors are contained therein, react and cytolytic T cells develop. These can then be determined by assaying for lysis of tumor cells introduced to the mixture. The methodology can be used for monitoring patient response to various therapeutic regimes.

Description

~93/14189 ~ PCT/US93/00083 ~T~OD FOR D~TERMINING CYT~LYTIC T CELL PRECUR80R8 FIELD OF THE INVENTION
This invention relates to methods for determining precursors of cytolytic T cells specific for antigens characteristic of or specific to tumors. More particularly, it relates to a non-obvious application and modification of the "limiting dilution" assay to determine the precursors discussed above.
The applications include the ability to monitor therapeutic regimes and subject response, to particular treatments, including immune responses.
BAC~GROUND AND ~RIO~ ART
The ability to measure and assay events that occur in an immunological response is of great interest and importance in studying the course of a pathological condition or disease.
Where a T-cell response is involved, of course, measurement and study of T cells is of concern.
T cells constitute a mixture of various types of lymphocytes, including cytolytic T cells, or "CTLs" as these will be referred to hereater. CTLs interact with a molecule presented by their target cell via the ~ cell receptor.
Following the interaction, the CTL causes the target cell to lyse.
The actual target recognized by the CTL is fre~uently referred to as an antigen, and this term will be used hereafter.
Prior work has established that various tumors can and do present antigens which can be the subject of a T-cell mediated response, leading to and rejection. Early work by Prehn et al., J~ Nat. Canc. Inst. 18: 769-778 (1957); Klein et al., Canc. Res. 20: 1561-1572 (1960); Old et al., Ann. N.Y. ~cad.
Sci. 101:
80-106 (1962); Kripke et al., J. Nat. Canc. Inst.l 53: 1333-1336 (1974~, and Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983) have established that the antigens expressed by these cells, now collectively referred to as "tumor rejection antigens", exist on murine tumors induced by viruses, WO93/14189 ~ PCT/~!S93/0~

chemicals, and ultra-violet ,adiation, as well as on spontaneous tumors. It ~as also been shown that, in some tumor systems, these tumor rejection antigens induce cytolytic T cell responses and highly specific CTLs, directed against murine tumor rejection antigens have been isolated. See Brunner et ~l., J. Immunol. 124: 1627-1634 (1980). The target antigen of these CTLs is relevant for recognition by a syngeneic host, since tumor cells which escape partial immune rejection in vivo have been found to have masked or to have lost the antigen CTLs recognize. See ~yttenhove et al., J.
Exp. Med. 157: 1040-1052 (1983). Adoptive transfer with cloned CTLs can eradicate tumor cells in animals bearing large tumors. See Kast et al., Cell 59: 603-614 (1989). The murine tumor P815 encodes a tumor rejection antigen, the gene for which has been identified and isolated, as per Van æe Eynde et al., J. Exp. Med. 173: 1373-1384 (1991). It was found that this gene is identical to one that can be expressed by normal mouse cells, but has little or no expression in normal adult mouse tissues.
Studies extended to humans have found that mixed lymphocyte tumor cell cultures ("MLTCs") frequently generate responder lymphocytes which lyse autologous tumor cells without lysing natural killer ("NK") cells, autologous EBV transformed B
cells or autologous fibroblasts. See Anichini et al., Int. J.
Cancer 35: 683-689 (1985). The response has been studied for melanomas in MLTC, using peripheral blood monocyte cells ~PBMCs) or tumor infiltrating lymphocytes (TILs) as reported by Mukherji et al., J. Exp. Med. 158: 240-245 (1983); Knuth et al., Proc. Natl. Acad. Sci. 86: 2804-2808 (1989~; Herin et al., Int. J. Canc. 39: 390~396 (1987~; Topolian et al., J.
Clin. Oncol. 6: 839-853 (198~). MTLCs derived from PBMC
generally contain responder cells which exert lysis on both tumor cells and on NK targets, when analyzed after two weeks of culture. After an additional two or three weeks, lytic activity on tumor cells increases, and that on NK targets disappears. See Herin et al., supra. It has been possible to derive CTLs from MLTC responders which seem to be completely ~93/14189 2 1 2 3 `1 2 ~1 PC~rtUSQ3tO0083 specific for tumors. The antigens recognized on tumor cells by these CTLs do not appear to be cultural artifacts, as they have been found to be present on fresh metastatic tumor tissue cells as well. See Mukherii, supra. Herin, supra; Knuth, su~ra. Panels of autologous CTLs permitted identification of four different stable antigens on human melanoma. See Van den Eynde et al., Int. J. Cancer 44: 634-640 (1989). Analysis of 70 additional CTLs from the patient on which the antigens were identified shows that all major stable antigens recognized by CTLs were in this set of four.
Assessment of the role of CTLs specific to tumor cells requires an ability to assay the fre~uency of their precursors in patients. This would permit the evaluation of, e.g., therapy protocols such as immune therapy approaches, and how these are affecting the CTL precursors (CTL-Ps), if at all.
The classic way to determine components of T cells is via a limit.ing dilution assay. This well known technique is described, e.g., by Sharrock et al., Immunol. Today 11(8):
281-285 (1990~, the disclosure of which is incorporated by reference. These assays are designed to determine the frequency of a particular type of cell in a mixed population.
A particular response is studied, in a number of different samples, and negative responses are used for further calculations. Negative responses are used because it is not immediately possible to know if a positive response can be associated with a particular cell. Essentially, a group of test cells, the "responder cells" are mixed with a second group, the "stimulator cells", and any reaction is then studied. The responder cells are used in a known or defined number, which can vary determining on the particular protocol being used. These assays have been carried out for CTLs, as reported by Sharrock, who discuss the importance of choice of an appropriate target cell, generally blast cells stimulated by concanvilin A or phytohemagglutinin. Epstein Barr virus transformants can also be used, if mixed with other targets.
The technique, while useful, is described as being in its infancy by Sharrock et al. Thisreference does teach the use WO93/14189 ~;l;'S t~/1 PCT/US93/00~

of PBNCs as responder cells, but shows a limited number of choices as stimulators.
It has now been found that one can in fact assay for CTL-P cells which are specific to tumor antigens. This type of assay is not ~hown by the art, and is not suggested by the references which constitute the prior art. Thus, a method for a~saying for CTL-Ps using a limiting dilution assay is the subject of the invèntion. The applications of this include the monitoring of subject response to therapeutic treatments, as explained infra.

BRIFF DE8CRI~TION OF T~E FIGURE~
Figure l is a depiction of lytic patterns and criteria for their classification.
Figure 2 presents levels of tumor cell lysis via limiting dilution microcultures and Poisson analysis of data.
Figure 3 shows tumor cell lysis via limiting dilution, when competing K562 is added.
Figure 4 shows results obtained for frequencies in anti-tumor lytic effectors, in presence and absence of K562.
Figure 5 presents frequencies of lytic effeGtors among PBMCs, CD4+ and CD8+ cells.

The examples that follow use different reagents and cells in a variety of contexts. To simplify the presentation, certain explanations are provided here.
When "medium" is used, unless otherwise noted, it is Iscove's medium, supplemented with 0.55 mM L-arginine, 0.~4 mM
L-asparagine, 1.5 mM L-glutamine and 5xl0 5 2-mercaptoethanol.
Human serum, abbreviated hereafter as "HS", refers to pooled types A, B and O serum obtained from healthy donors. The HS
had been decomplemented by treatment at 56C for 30 minutes, partially delipified via centrifugation (45 minutes, 17,000g) filtration and sterilization.
Fetal bovine serum is abbreviated as "FBS".
Interleukin-2 and Interleukin-4 are abbreviated as IL-2 and - 93/14189 ;'. 1 ,? ~ .i PCT/US93/~X~3 IL-4, respectively. In the following experiments, the recombinant, human form of the interleukins was used, although other forms would be expected to work as well. The concentrations used were 30 U/ml (IL-2), and 5 U/ml (IL-4) in all experiments. One unit/ml of IL-2 is defined as the concentration supporting half of maximal proliferation of CTLL-2 cell line. One unit/ml of IL-4 is that concentration which yields 50% maximal proliferation of human T cells previously treated with phytohemagglutinin-A ("PHA").
The cytokine "gamma interferon" is abbreviated as "IFN-~".
`~AMPLE ~
Peripheral blood mononuclear cells (PBMCs) or purified subsets were obtained from patients. The patient samples were subjected to density gradient centrifugation, and were cryopreserved prior to use in the limited dilution assays described infra. To separate CD4+ and CD8+ T cells from these samples, flow cytometric sorting was used, employing labeled anti-Leu 3 antibody (fluorescein), and anti-Leu 2 antibody (phycoerythrin). The particulars of the sorting are not of special relevance to the invention, and purifi~ation of PBMCs, CD4+ and CD8~ can easily be carried out via standard methods~
Sorted subpopulations were always more than 97.5% pure.
.

Limiting dilution assays were set up using various quantities of either-PBNCs or the subsets described supra.
The numbers ranged from about 200 to 10,000.
The particular amount, i.e., the defined number of PBMCstsubset chosen were seeded in 96 V~bottom microwells, together with 10~ irradiated autologous tumor cells (i.e., tumor cells taken from the same patient as the PBMC source)~
The tumor cells had previously been used to establish cell lines, and the irradiated cells were taken therefrom.
Irradiation was carried out using a Cs source (10000 rads).
` Irradiated tumor cells served as stimulator cells. The medium, as defined supra used to culture the mixture was WO93/14189 ~ PCT/US93/0~ ~

supplemented with l0~ HS and IL-4 as described su~ra. The wells were centrifuged for three minutes at l00g and incubated at 37OC in 8% Co2. On the third day of incubation, IL-2 as described su~ra was added.
On the seventh day of culture, fresh medium (l00 ul) was added, together with IL-2, IL-4, and 104 additional irradiated cells.
On the fourteenth day of culture, l00 ul of medium was discarded, the cells were transferred into flat bottom microwells, and IL-2, IL-4, and irradiated cells as above were added.
Over the course of culturing, the rate of proliferation of the CTL precursors in the sample were measured using radiolabeled thymidine and conventional methods. On those days, four aliquots of 40 ul of the cells were transferred into microwells and lytic activity was assayed. At the same time, the remaining samples were restimulated by adding 160 ul of ~edium, again containing the described IL-2, IL-4, and irradiated cells. At least l00 cultures were carried out for each number PBMC.
Stimulation of the type described suPra led to proliferation of sufficient cells to allow assay of lytic activity against tumor cell.
~X*MP~E 3 To determine the lytic activity of the proliferated cells, these were tested with autologous tumor cells in the manner described below.
The autologous tumor cells to be used were preincubated for 48 hours in medium containing 50 U/ml IFN-~. This enhances expression of histocompatibility molecules and adhesion molecules. When non-active n~tural killer cells ("K562", discussed infra) were added, these were not so treated~ IFN-~ does not modify susceptibility of these cells to lysis via NK-like effectors.
The tumor cells were labeled via suspending them at 107 cells/ml in medium supplemented with 10% HS, and i~cubated at 37C for 60 minutes, using 200m 5lCr Ci/ml. The labeled cells f~ 1 2 ~'J 1 ;'~, '',~
~93/14189 PCT/US93/~083 were washed three times with medium and 2% HS, and were then suspended in medium at 104 cells/ml. Following this, 60 ul of medium augmented with 2% HS or with an additional 5xl0~ K562 cells/well were added to wells containing the previously stimulated cells. After one hour of this incubation (37C), l000 tumor cells were added per well in l00 ul of medium. The mixtures were centrifuged (4 minutes, 200g) and then incubated for 4-5 hours at 37C in 8% CO2 atmosphere. Aliquots of supernatant (l00 ul) were collected and 5lCr specific release calculated. The following formula was used:
%RELEASE - (ER-SR)xl00(MR-SR) Where: ER is experimental 5lCr release, SR - spontaneous release (i.e., release by cells incubated in medium alone), and MR the "maximal release", i.e., that obtained by incubating the tumor cells in 0.15% TRITON X-l00. SR never exceeded 15% of MR.
Figure 2 shows results secured using samples taken from two patients (LB-33 & LB-30), using varied numbers of PBMCs and the percentage lysis of melanoma cells taken from the patient.
High degrees of lysis were observed in many microcultures. In control cultures, i.e., those where no tumor ~iells, and hence no antigen was presented, there was no lysis.
As the number of PBMCs decreased, the fraction of posi~ive microcultures also decreased, but the level of lysis did not decrease more than expected.
Analysis of the lysis pattern showed that a clear bimodal distribution allowing easy discrimination between positives and negatives did not exist. This is consistent with an earlier observation that different effector cells display different rates of proliferation, thus affecting the degree of lysis.
When the percentage of microcultures devoid of lytic activity is plotted as a function of PBMC number and using zero order terms of Poisson distribution, the logarithm of the fraction of negative wells decreased linearly when the quantity of PBMC increased. Figure 2 shows that this is true, regardless of whether 5% or l0~ lysis was chosen as threshold WO93/14t89 PCT/US93/~3 ~l~Si~"l 8 for positivity.
~AMP~E 4 In the past, stable lytic clones derived from responder cells have been reported to include anti-tumor CTLs, which show little or no lysis of the cell line K562, which is a natural killer (NK) cell target, as well as other clones which do lyse K562. (K562 is a human chronic myelogenous leukemia cell line, freely available from the American Type Culture Collection, e.g., under Accession Number ATCC CCL 243). ~his cell line has been described, e.g., at J. Nat. Canc. Inst. S9:
77-83 (1977), as a highly sensitive in vitro target in NK
assays. The K562 specific lytic clones are referred to as "NK
like", and it was important to determine if these were present.
To do this, the limited dilution materials described suDra were assayed for affect on K562, the NK target. Assays were run, in both the absence and presence of a 50 fold excess of non-viable K562 relative to the number of tumor cells in the sample. The assays used the chromium release methodology discussed suPra. The result showed that stimulated PBMC
samples fall into one of four patterns:
l. Some microcultures lyse tumor cells, but do not lyse K562. In such cultures, competition by unlabeled K562 never significantly reduced the tumor cell lysis.

2. Both tumor cells and K562 were lysed. In competition with "cold" K562, the lysis of labeled` K562 was nearly abolished, but tumor cell lysis was not. Such cultures show a mixture of tumor specific, and NK like clones.

3. Both tumor and K562 cells lysed, where competition with cold K562 abolishes all lysis. Su~h cultures appear to contain at least one NK like clone.

4. Lysis of only K562. Such cultures clearly contain NK
like clones, but not tumor specific cells.
The sum of "l", "2" and "3" corresponds to the total amount of anti-tumor lytic clones as described su~ra. Patterns "l"
and "2" indicate anti-tumor CTLs, and can be used to determine ~3/14189 ~ 1?,~,?~ 2 ~ PCT~US93/~

the frequency of CTL precursors. Table 1 summarizes the classification into one of the four types listed su~ra~ The abbreviation "LDA" used therein stands for "limiting dilution assay".
In the presence of competing K562, a decrease in PBMC number did not reduce tumor cell lysis more tban what would be expected from loss of cultures containing multiple CTL clones.
These results are shown in Figure 3.

093/14189 2 ~ 2 ~ 1 PCT/US93/~83 '11 ~XAMPL~ 5 The clones considered to be anti-tumor CTLs were examined for specificity. This was done by testing lytic cultures on various targets. Table 2, which follows, presents one set of these data, where PBMCs of a patient (LB-33) were tested against autologous tumor cells, K562, autologous PHA blasts, and tumor cells from patients LB-30 and LB-34. These cells were taken from cell line cultures as discussed su~ra. The lysis percentage was determined 28 days after setting up a lytic assay as described su~ra, using 1333 PBMCs per well. In the case of PHA-blasts, lysis was checked using ~butologous PBMCs activated for lO days with ~000 U/ml of IL-2.

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3~ æ E ~ o r ~93/14189 ~1~3 12~ PCT/~593/00083 Frequency of anti-tumor CTL precursor cells in various patients was evaluated. Limiting dilution cultures were set up using PBMCs, as described supra, using all of the indicated materials. Lytic activity was measured on the day indicated in the panels of Figure 4 ("d20", e.g.), and frequency of lytic effectors calculated. Anti-tumor and total of all lytic effectors are presented.
The relationship between number of PBMCs added and logarithm of fraction of microcultures without anti-tumor CTLs appear linear. This supports the point raised suPra, that tumor specific lysis appears to result from activity of single T
lymphocyte clones. Large variations were observed in different patients, both in frequencies of all tumor cell lysing cells, and in precursors of anti-tumor specific CTLs.
The ratios between these two frequently showed large differences as well.
EXAMPL~ 7 Analysis was carried out to determine frequency of NX-like effector clones, as detected around day 20.of the limiting dilution assays, as well as anti-tumor CTL precursor frequencies. These are shown in following Table 3.

WO93/14189 2 12 S i 2 ~1 14 PCI`/US~3/00' ~~

5T~ble 3. ~t~ti-~umoral CTL-P frcquencics of diffcrcn~ ~cl~om~ p~icnls.
~:rcqucncics of Pa~ient E~cp. Day spc~ efleclor ce~ls .
LB-33 11 20 I~229(1 1/860 2 18 l/lIB0 1/770 22 ~ 90 112560 3 2û ~L~;2Q 11780 4 ~8 1/910 1/102~

28 112100 ~J5û90 LB-34 21 116310 I/glO

L8-2~ 18 117120 . 1/250 MZ-2 1~ 26 1~1~3400 1/980 2 22 1/9640 1/2~60 LB-30 1~ 2~ 1/108û0 1/420 2 27 ~QQ 1/20S~

L~ 17 31 1/17900 1/830 LG-2 20 ~ 1/400 ~93/14189 2 1;?.(~Q)!t~ PCT/US93~00083 This table shows activity against autologous tumor cells, both with and without non-viable K562. The microcultures of pattern "l" or "2" were used to estimate CTL precursor (CTL-P) frequencies, and patterns "2", "3" or "4" used to estimate NK-like effector cell frequencies.
It is noted that constant CTL-P frequencies were observed even though there was some decrease with time in the number of microcultures with activity. The reason for this is that under the conditions used, most CTL clones do not proliferate indefinitely, most probably because of the absence of feeder cells.
Cultures with NX-like activity decreased more severely between days 14 and 30, which is consistent with prior observations by Hérin et al., Int. J. Cancer 39: 390-396 (~987).

Prior experiments leading to isolation of anti-tumor CTL
clones by limited dilution around day lS showed both CD4+ and CD8+ cells, but the vast majority of anti-tumor CTLs were CD8~. Studies were carried out to determine if this was because culture conditions favor production- of CD8+ cells~
Using the sorting meth~dologies described su~ra, it was found that most CTL-Ps are CD8~ ' Several features of the examples presented supra merit comment. Firstt the microcultures did not use feeder cells in the cultures~ For many patients, it is difficult to obtain the autologous PBMCs that are desirable feeder cells in sufficient a~ounts. It was also desired to present a generally applicable limiting dilution assay. In experiments performed by the inventors and not reported herein, when autologous, irradiated PBMCs were used as feeder cells, an increase in anti-tumor CTL frequency was observed, but t~is was coupled to higher NK-like frequency.
The experiments using the NK inhibiting material, i.e., non-viable X562 cells permits distinction between anti-tumor CTLs and NK like effectors which also lyse tumor cells. The NK
like effectors are inhibited by binding to the K562 cells, ~ i 2~ 16 which, since they are non viable, do not show 5lCr release.
Other methods to eliminate the effect of NK and/or NK like cells in the mixture, including other NK target ceils, can alio be used.
The frequencies of CTL-Ps specific for antigens characteristic of tumor cells ranged from l/33000 to l/900 PBMCs. The latter number, obtained with patient LB-33, may result from an autoimmune response against a tumor. The numbers observed should be compared to those secured by Borysiewicz et al., Eur. J. Immunol. l8: 269-275 (1988) of l/5000-20,000 for CMV, by Schmid et al., J. Immunol. 140:
3610-3616 (1988) of l/4000-8000 for HSV, or by Hickling et al., J. Virol. 61: 3463-3469 (1987), for varicella zoster of l/1600-90000. Sharrock, Immunol Today ll: 281-286 (l990) discusses alloreactive frequencies of l/500-500000.
The invention thus teaches a method by which cytotoxic T
cell precursors (CTL-Ps) specific for antigens characteristic of tumor cells can be determined via a limiting dilution assay. In the methodology, a defined number of peripheral blood mononuclear cells, which contain the CTL-PS, are contacted to an antigen which is characteristic of, charge for, i.e., is particularly associated with, a tumor type or types. This results in a mixture of materials which is then cultured. The stimulation from the tumor antigen causes the CTL-Ps to develop into the actual cytolytic T cells. This development is monitored via monitoring the lysis of tumor cells either in or added to the mixture.
The number of PBMCs used may vary, but preferably somewhere between about 200 and about l0000 are used per assay. The PBMCs may be added in a mixed sample~ or as a pure culture of PBMCs.
The antigen characteristic of the tumor is ideally added in the form of a tumor cell, generally non-viable. The tumor cell can be rendered non-viable via, e.g., irradiation. The tumor cells still present the antigen on their cell surfaces, an~ a proliferation linked reaction with CTL-Ps follows. The antigen can also be added, e.g., in pure form, but this is not ~93/14189 2 ~ 2 8 ~ PCT/US93/00083 the preferred mode.
It is especially preferred to administer an autologous sample of tumor cells - i.e., tumor cells taken from the same subject as the PBMCs. Ideally, the cell sample is as pure as possible to prevent reaction between CTL-Ps not directed against tumor cells and their targets. This is not always possible, however, and thus it is preferred to treat the mixture to eliminate natural killer ("NK") like cells contained therein. The term "natural killer like" includes natural killer cells, as well as cells which function in the same or an equivalent manner. One approach to doing this is by adding, e.q., an NK inhibitor.
The lytic determination can be accomplished by means well known in the art, including the 5lCr release method, described ~5 supra.
Desirably, when culturing the mixtures described herein, this is done without the use of feeder cells, however, this is not a requirement.
The ability to monitor CTL-Ps also enables one to monitor responses such as the immune response of a subject with respect to the subject's tumor. ~Changes in CTL-P levels are indicative of changes in the immune response, and serve an important diagnostic function in that changes over time,can indicate a worsening or improvement in the subject's health.
Various modifications to the method described herein have been shown. Others will be clear to the skilled artisan and are not repeated here.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

Claims

We Claim:
1. Method for determining a precursor of a cytolytic T cell specific for an antigen characteristic of a tumor, comprising consisting essentially of:
(i) contacting a defined number of peripheral blood mononuclear cells (PBMCs) taken from a subject to an antigen characteristic of said tumor to form a mixture, (ii) subjecting said mixture to a limiting dilution assay in the absence of feeder cells under conditions favoring development of cytolytic T cells specific for said antigen from said PBMCs, and (iii) determining lysis of tumor cells added to said mixture by said cytolytic T cells as an indication of said precursor in said subject.
2. Method of claim 1, wherein said defined number ranges from about 200 to about 10,000.
3. Method of claim 1, wherein said tumor specific antigen is a tumor rejection antigen.
4. Method of claim 1, comprising adding a sample containing tumor cells to said defined number of PBMCs of step (i) to form said mixture, wherein said tumor cells are derived from the same subject as said PBMCs.
5. Method of claim 4, comprising treating said mixture of step (iii) with non-viable natural killer target cells whereby said non-viable natural killer target cells compete with said tumor cells for lysis by natural killer cells.
6. Method of claim 5, wherein said natural killer cell target is K562.
7. Method for monitoring the immune response of an individual to treatment of a tumor related condition, consisting essentially of (i) determining precursor cytolytic T cells formed by said individual by subjecting a defined number of PBMCs taken from said individual to a limiting dilution assay in the absence of feeder cells and in the presence of tumor cells taken from said individual under conditions favoring development of cytolytic T cells specific for an antigen characteristic of said tumor cells at a first point in time, (ii) determining precursor cytolytic T cells formed by said individual by subjecting a defined number of PBMCs taken from said individual to a limiting dilution assay in the absence of feeder cells and in the presence of tumor cells taken from said individual under conditions favoring development of cytolytic T cells specific for an antigen characteristic of said tumor cells at a second point in time, and (iii) comparing values obtained in (i) and (ii), wherein a difference therebetween is indicative of a change in said immune response.

11. Method of claim 4, wherein said tumor cells are non-viable.
12. Method of claim 7, wherein said defined number PBMCs ranges from about 200 to about 1000.
13. Method of claim 7, wherein said tumor sample is a sample presenting a tumor rejection antigen.