Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 12, Num. 2, 2006, pp. 53-65

Indian Journal of Human Genetics, Vol. 12, No. 2, May-August, 2006, pp. 53-65

Invited Article

Tumor-associated antigen and antigen-presenting cells based vaccine therapy for cancer

Chakraborty NityaG

Department of Medicine, University of Connecticut Health Center, Farmington, CT
Correspondence Address:Department of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT-06030 - 1628, Email: chakrabortyCuchc.edu

Code Number: hg06010

Melanoma tumor specific cytotoxic T lymphocytes (CTL) were induced in melanoma patients with major histocompatibility complex (MHC) class I restricted peptide-antigen or melanoma tumor lysate-loaded autologous antigen-presenting cell (APC)-based vaccines. Along with clinical responses (if any), induction of tumor antigen specific CTL responses and the generation of helper / suppressor and/or regulatory T cells (T-reg) cell responses were monitored. We used antigenic peptide and MHC-I (here HLA-A2) complex as T cell receptor, binding component as tetramer staining and limiting dilution analyses as monitors of CTL responses. We detected a significant increase of antigen-specific CTL in peripheral blood lymphocytes (PBL) of the patients after vaccine therapy with the MART-127-35 peptide (AAGIGILTV)-pulsed autologous APC, by tetramer staining for HLA-A2 + patients. For HLA-A1 + patients where the MAGE-1 161-169 peptide (EADPTGHSY)-pulsed APC, or for those patients where autologous melanoma tumor lysate-pulsed APC, the technique of in vitro analysis of CTL response in limiting dilution was used. We observed that antigen-specific CTL reached the peak of expansion after 1 week and then declined to the pre-vaccine levels within a month. The decline of antigen-specific CD8 + CTL number was associated with the increase of CD + CD25 + cells, and those cells were found to secrete interleukin-10 (IL-10), a cytokine known to suppress immune response. In vitro cultures with PBL from patients after vaccination reveal that CD8 + CTL could be generated in cultures too. In vitro induced CTL from PBL also declined with concomitant expansion of CD4 + regulatory T (T-reg) cells. A continuous presence of Th1 condition with IL-12 (250 u/ml) and anti IL-4 antibody (lug/ml) in the culture blocked the activation and expansion of CD4 + T-reg cells, keeping the CTL activity for a prolonged period. Our observations prove that tumor antigen specific CTL generation is possible in cancer patients, but keeping those CTL active for a significantly extended period could only be possible if the emergence of CD4 + regulatory T cells is blocked.

The region of short arm of chromosome 6 (6p21.3) has a high gene density, many genes with immune system functions, large sequence variations and diversity, duplications and redundancy and a strong association with more than 100 different diseases. It determines the expression of MHC.[1] T lymphocytes are activated against any antigen when such antigen is presented in association with self-MHC.[2] In cancer and/or in autoimmune disease, MHC molecules or HLA A, B, C or DP, DQ DR expression and the degree of such HLA expressions by antigen-presenting cells or by the tumor cells might play a major role in controlling the disease.[2],[3] Although initial immune reaction is provided by the innate immunity of the host, adoptive immunity is the longer-lasting immunity, and self-MHC class I and II play the most important role involving the APC or dendritic cells (cells), CD8 + cytotoxic T lymphocytes and CD4 + helper/suppressor cells (or regulatory T cells). It has been shown that immunization with tumor antigen derived specific peptides, DNA or tumor lysate-based vaccines can induce CTL responses against cancer cells in vivo.[4],[5],[6],[7],[8],[9],[10],[11] Clinical trials for tumor vaccines using tumor-associated antigens and APC or dendritic cells (DC) are under way for the treatment of different types of cancers. Different groups, including our laboratory, have shown that in human melanoma such vaccinations can induce specific CD8 + cytotoxic T-cell responses followed by some clinical responses success of clinical trials for immunotherapy of cancer with patients using the method of inducing CTL is somehow modest.[11] Since cancers grow from within the body and caner antigens are also normal gene products, it is true that body's immune systems accept that growth or tolerate such growth. Suppression of even cancer, a vaccine-induced CTL, by the induced T-reg cells is a strong indication of active tolerance. In this paper, we show that tumor-reactive CTL could be generated in a patient, but expansion of such CTL would not be robust enough unless the tolerance-inducing T-reg cells are controlled in addition to induction of CTL response.

Materials and Methods

Patients

Patients with metastatic melanoma and patients with no active disease but who were considered at high risk of recurrence were included in the trial with informed consent. The details of the trial design and the patient monitoring systems have been described previously.[4],[10] Briefly, the study consisted of three groups of patients receiving either a vaccine made of synthetic peptides MAGE-1161-169 (for human leukocyte antigen HLA-A1) or MART-127-35 (for HLA-A2) pulsed APC.[4],[9],[10] In MAGE-1161-169 A1 group, 3 of the 5 patients completed the study. Of the 9 patients enrolled in MART-127 35 A2 group, 7 completed the study. In the last group 17 patients of 20 completed the study. The immunization protocol consisted of four intradermal injections with 1 x 10 ⁷ autologous myeloid APC (grown in granulocyte macrophage colony stimulating factor [GM-CSF]) pulsed with synthetic peptide or with the tumor lysate at monthly intervals. The desired numbers of antigen-loaded APC were taken in 0.1 ml normal saline and were injected intradermally, close to a peripheral lymph node bearing region on a rotating basis. Laboratory monitoring of immune response to the vaccine was performed by detailed phenotypic and functional analyses of lymphocytes, in circulation and infiltrating the vaccination sites or tumor sites, expanded in vitro in IL-2.

Peptide and antigens

Details about tumor-associated antigenic peptide MART-127-35 and MAGE-1161-169 have been described.[10],[12]

Preparation of tumor lysate as a source of tumor-associated antigens

The procedure for the preparation of the tumor cell lysate has been described.[9] Fresh tumor tissues were homogenized without using any enzyme to obtain homogenates of single melanoma cells or melanoma cells in clumps. The resulting homogenate was washed and cell concentration was adjusted to approximately 106 cells/ml in phosphate-buffered saline. A cellular lysate was then prepared from the homogenate by six repeated freezings (at -180°C) and thawing to room temperature. Total lysis of all the cells was verified by trypan blue dye exclusion staining, cellular debris was removed by centrifugation (1,000 g for 10 min) and the protein concentration of the lysate was determined.[9] Aliquots of the homogenates were then frozen at -80°C for loading onto the cultured APC for vaccination or for in vitro assays.

Tissue culture

Tissue culture technique and the procedure of isolating fresh tumor cells from tissue explants have been described earlier.[4],[6] Briefly, tissue cultures were performed in Iscove medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Gemini Bio-products, Inc., Calabasas, CA), L-arginine (0.55 mM), L-asparagine (0.24 mM) and L-glutamine (1.5 nM) (Life Technologies, Inc.), henceforth described as complete medium (CM). Fresh tumor cells were isolated by mechanical fine mincing of tumor tissues in serum-free medium.

APC culture

APC used in this study were peripheral myeloid APC expanded in vitro as described earlier.[6],[9] Briefly, monocytes or macrophages were isolated as adherent cells from Ficoll-Hypaque gradient-derived mononuclear cell populations. The adherent cells were then cultured in CM containing 1,000 U ral of GM-CSF (Immunex Corp, Seattle, WA) for 14 days. The non-adherent and loosely adherent cells were harvested by vigorous washing.

Peripheral blood lymphocytes

PBL were isolated on a Ficoll-Hypaque gradient as described earlier.[7] All cultures and experiments were performed in CM.

In vitro expansion of infiltrating lymphocytes or PBL

Tumor-infiltrating lymphocytes (TIL) and vaccine-infiltrating lymphocytes (VIL) were expanded, ex vivo, as previously described.[6],[9] Briefly, homogenates of the excised tissues were prepared mechanically and the minced homogenates containing the infiltrating lymphocytes were then cultured in IL-2 (50 U/ml) in the presence of the appropriate immunogen (melanoma cell lysate-loaded or peptide-loaded autologous APC). PBL were stimulated in culture with antigen (peptide or lysate)-pulsed autologous APC. The cultures were re-stimulated every 7 to 10 days with the respective immunogen. Phenotypic and functional analyses were carried out after sufficient numbers of lymphocytes became available. Some of the experiments shown in this communication were performed either with the ex vivo expanded infiltrating lymphocytes that were stimulated at regular intervals and were maintained in culture or with freshly isolated and directly frozen before any in vitro treatment.

Human monoclonal antibodies

CD4-FITC, CD8-FITC, CD25-PE, IL- 10-APC and purified anti-IL-10 were purchased from BD Biosciences, San Jose, California.

Phenotypic analysis

The immunofluorescence procedure for phenotypic analysis by flow cytometry has been described.[6],[9]

Microcytotoxicity assay

The 51Cr release microcytotoxicity assay was performed as described.[9] Freshly prepared or cryopreserved target cells were labeled with 5lCr with good efficiency. The spontaneous release of the radioactivity from the 51Cr-labeled target cells was usually below 15%.

Cytokine synthesis assay

The ex vivo expanded VIL or TIL (1 x 10 ³ to 2 x 10 ³ cells/well), PBL or in vitro co-culture with PBL were stimulated against the appropriate antigen (e.g., autologous melanoma cells if available, lysate or peptide-loaded APC) in individual wells of a 48-well cluster plate (Costar, Cambridge, MA) in 0.5 ml media for 24 h. The supernatants, harvested after 48 h, were assayed for relevant type 1 or type 2 cytokine, depending on the experimental design. Cytokine, interferon-g, IL-4 and IL-10 were assayed in appropriate enzyme-linked immunosorbent assays as per manufacturer's (Coulter Corp, Miami, FL) protocol.

Tetramer assay

Analysis for the MART-127-35 antigen-specific T-cell response bearing CD8 + cells has been described previously.[12],[13],[14] Briefly, effector cells were washed twice in phosphate-buffered saline and then incubated with 1 ml MART-127-35 peptide phycoerythrin-conjugated HLA-A0201 tetramer (Beckman Coulter, Inc., Fullerton, CA) at room temperature for 30 min. The effector cells were again washed twice and resuspended in FAGS buffer. Thereafter, the number of tetramer-positive cells was determined by flow cytometry using FACS Calibur (Becton Dickinson, Mountain View, CA) and the acquired cytofluorographic data were analyzed using Cellquest software (Becton Dickinson).

Limiting dilution assay of autologous melanoma-specific CTLp frequency

The limiting dilution analyses of CTLp frequency were carried out as described previously.[15] Briefly, cryopreserved PBLs obtained before and after immunization were seeded in 96-well round-bottom plates (Corning, NY) at concentrations ranging from 80,000 to 62 cells/well, using a limiting dilution factor of 1/1 in the presence of a constant number (10,000 cells/well) of the MAGE-1161-169 nonapeptide-pulsed or tumor lysate pulsed APCs and IL-2 (50 units/ml) in a total volume of 0.2 ml. Forty-eight replicate wells of each PBL dilution were set up. The culture medium was partially replaced every 3 days and the cultures were re-stimulated with the same number of stimulator cells on day 7. After 14 days of culture, half of the contents of each well was removed and tested for the presence of autologous melanoma-reactive cytolytic effector cells in the 4-hour microcytotoxicity assay in the presence and absence of anti-MHC class I monoclonal antibody W6/32 (a gift of Soldano Ferrone, Buffalo, NY). The cytotoxicity assay was performed in the presence of a 50-fold excess of K-562 cells to eliminate nonspecific lysis. A positive test was defined as one showing MHC class I-restricted (i.e., cytolysis inhibitable, equal to or in excess of 50%, by the anti-MHC class I monoclonal antibody) chromium release in test wells above three standard deviations of the mean of the spontaneous release. This usually amounted to specific lysis of 15% or higher. The fractions of negative wells were plotted against the number of cells seeded, and a linear regression analysis of the best fit was carried out using Statworks (Cricket Software, Philadelphia, PA).

Intracellular cytokine staining

The method for intracellular analysis of cytokine production by T cells has been described.[6],[16] Briefly, the effector cells (CD4 + / CD8 + T cells) were stimulated with IL-2 (50 U ral) for 6 h, after which the cells were incubated with nonstimulatory doses of phorbol myrastic acid (PMA) (0.5 ng/ml) and ionomycin (4.0 ng/ml; Sigma Chemical Co., St. Louis, MO) for 4 h at 37°C. Brefeldin A (1 mg/ml; Sigma Chemical Co. ) was added 2 h before harvest. The cells were washed and stained with anti-CD4-FITC and anti-CD25-PE for 30 min on ice. They were then fixed in cytofix and permeabilized by cytosperm (BD Biosciences). Cells were then stained with anti-IL-10 APC and analyzed by flow cytometry using FACS Calibur (Becton Dickinson), and the acquired cytofluorographic data were analyzed using Cellquest software (Becton Dickinson).

Th1 conditioning of PBL

For both normal donors and patients, polarization of PBL with Th1 conditioning was done with rIL-12 (250 U/rr R and D Systems), anti IL-4 antibody (1 mg/ml; BD Biosciences, San Diego, CA) for 7 days. For Th1 or Th2 polarizing, conditions of CD4 + cells (human recombinant IL-4 [1,000 U/ml] plus neutralizing mAb to human IL-12 [1 mg/ml; R and D Systems]) along with IL-2 (50 U ral, R and D Systems) was used for 3 days before CTL generation, according to the methods described by Sallusto et al.,[17] with some modification.[18]

Results

Characteristics of APC and biological response to vaccine

Phenotypic features of the APC used in the vaccine preparation are shown in [Figure - 1]. The APC exhibited all the essential features of professional APC. Those cells expressed high level of MHC-I and MHC-II and B7.1 and B7.2 and ICAM-1 [Figure - 1]A. An example of the skin reaction is shown in [Figure - 1]B. The reaction consists of erythema and induration characteristics of DTH response. Histological examination of the vaccine site [Figure - 1]C revealed perivascular infiltrates of mononuclear cells and disruption of collagen bundles, suggesting edema, a characteristic feature of DTH.
The summary of patient characteristics and relevant clinical results of vaccine therapy are shown in [Table - 1]. It represents 17 patients in the tumor lysate group and 8 in the peptide group. Out of 17 patients in the lysate group, 10 had active disease and 7 had no detectable metastatic disease but were considered at high risk for recurrence. In the peptide group, only 1 patient had active disease, but the other 7 patients did not have any detectable metastatic disease but were also considered at high risk for recurrence.

The vaccinations were well tolerated. No patient developed fever in excess of 37.80°C or showed significant constitutional symptoms other than mild transient fatigue.

Induction and expansion of antigen-specific CTL in vivo

In the group of patients receiving the MART-127-35 epitope-loaded APC as vaccine, significant increases in the number of HLA-A2/MART-127-35 tetramer-positive cells by day 7 after first vaccine and after the fourth vaccine were observed [Figure - 2]A. However, the number of epitope-specific CTL declined to the baseline levels within 1 month [Figure - 2]B. In the HLA-A1-positive group of patients who received the MAGE-1161-169 peptide loaded APC as vaccine, limiting dilution analysis showed significant increases in number of CTL capable to lyse MAGE-1161-169-positive autologous or allogenic tumors or the MAGE-1161-169 peptide (EADPTGHSY)-pulsed targets [Figure - 2]C, RM, HS. Increase of tumor-specific CTL was also detected in the group of patients who received autologous tumor lysate pulsed APC as vaccine [Figure - 2]C, except RM and HS. In fact, significant increase in the number of antigen-specific CD8+ T cells could be detected in the circulation in the majority of the cases we studied. Interestingly, the increase in the number of specific CTL did not persist for long; the numbers declined to the baseline levels within 28 to 30 days after vaccine therapy.

Induction and expansion of CTL in cultures using PBL taken from patients after vaccination Antigen-specific CTL responses could also be generated from PBL taken from vaccinated patients, in cultures; such ex vivo -generated CTL responses could not be maintained in long-term culture even with repeated stimulations.[6],[9],[10] Most of the time, the decline in the CTL responses in culture corresponds with the emergence of an IL-10-producing CD4 + regulatory T-cell population. We examined the dynamics of the CTL and regulatory T-cell responses ex vivo in the vaccinated patients. PBL from patents who received all three forms of the vaccines were stimulated with the antigen-loaded APC or with autologous tumor cells in the presence of exogenous IL-2, and the nature of the responses generated in the cultures were assessed before and after several rounds of re-stimulation. [Figure - 3] shows the collective result of three case studies. As shown, there was a remarkable shift in the nature of the responses. By day 14, the number of CD8 + T cells and the specific target cell killing showed a steady increase. By day 28, the number of CD8 + T cells and lytic activities significantly declined, whereas the number of CD4 + T cells increased. By day 60, the cultures contained mostly CD4 + T cells, with no detectable lytic activity. Interestingly, when the same cultures were maintained in the presence of anti-IL-10 antibody, the number of CD8 + T cells remained high and they maintained high cytolytic activity [Figure - 3].

Emergence of the regulatory T cells in PBL following vaccine therapy

The observations of the decline of the number of CD8 + CTL from TIL and VIL despite antigenic stimulation provided through APC,[7] expressing all the essential co-stimulatory molecules, and the emergence of the Th2 type (specificity unknown) CD4 + regulatory T cells suggest that robust expansion of vaccine-induced CTL against tumor antigens might be a difficult task. As we have previously seen, that with TIL and VIL[6] the CTL activity in culture could be prolonged by anti-IL-10 antibody, we wanted to have a general idea of the amount of IL-10 produced by PBL from patients before and after vaccination with or without in vitro stimulation. We used directly frozen PBL samples of patients collected at different time points of the vaccine protocol. We analyzed prevaccine, 7 days postvaccine and 28 days postvaccine status (after each vaccination x 4) of PBL for their ability to produce IL-10 in native state or after stimulation with IL-2. We found that PBL cultured in medium alone did not secrete any significant amount of IL-10, but when stimulated with IL-2 (50 U/ml), a significant amount of IL-10 production was detected [Table - 2]. In this connection, it should be noted that no matter whether the antigen is MAGE-1161-169 or MART-127-35 or tumor lysate, an increase of IL-10 production by PBL was observed with most of the cases studied. It should be noted that patient 4 in the table is one of those exceptional cases where no IL-10 was detected from PBL, whether stimulated with IL-2 or not. Interestingly, we could not detect any significant amount of transforming growth factor beta (TGF-β ) in the culture supernatants.

Our observations indicate that IL-10 could be an important factor that plays a down-regulatory role against the expansion of the antigen-specific CTL. The possible source of IL-10 that could initially be seen in the culture might come either from APC or from CD4+ T cells or even from CD8 + T cells.[6],[19],[20] In our previous report,[19] we showed that DC/APC from some donors (healthy normal or patients) secrete IL-10 spontaneously in culture without any stimulation. Those IL-10-producing DC might be inhibitory in mixed lymphocyte reaction, but they did not completely block CTL generation in co-culture when they were used to generate CTL response after pulsing with peptide antigens. It is possible that IL-10 detected in the cultures may also come either from CD8 + T cells or CD4 + T cells. To determine the type of cells in PBL that secrete IL-10, we tested PBL from patients (frozen at different time points of vaccine protocol) or from normal donors after short-term stimulation with IL-2. Then an intracellular cytokine assay was carried out using specific conjugated antibody against IL-10, after the surface-staining with the conjugated antibodies of CD4, CD8 and T-cell activation marker CD25 (IL-2R). Our observation reveals that, indeed, CD4 + CD25 + T cells express intracellular IL-10 in the PBL [Figure - 4]. CD8 + T cells did not show any intracellular IL-10 expression (data not shown). Significant increase in the number of CD4 + CD25 + IL-10 + T cells was observed in patients'PBL collected 28 days postvaccination. [Figure - 4] shows the data of intracytoplasmic staining. [Figure - 4]A shows a representative data with PBL from a patient before vaccine. [Figure - 4]B represents data from postvaccine PBL from a patient. The average of CD4 + CD25 + IL-10+ in normal donors was found to be 1.02%.

Presence of Th1 cytokines in the cultures and life of CTL

We studied the effect of Th1 conditioning in in vitro cultures. We first put the PBL from patients and normal donors in Th1-conditioning medium as described under the heading 'Material and methods'for 7 days.[18] Then the conditioned PBL (PBLC) were used in IVCs with autologous tumors or autologous DC pulsed with MART-127-35 peptide under the following conditions: IVC-1 = in medium with 50 U/ml IL-2, IVC-2 = 1 + anti-IL-10 antibody 1 µg/ml and IVC-3 = 1+ continuous presence of Th1 conditioning (anti-IL-4 + rIL-12). PBLC with only IL-2 and not with tumor was used for another control of IL-2-stimulated cells. The cultures were fed every other day with appropriate cytokine and antibodies and were re-stimulated with appropriate stimuli every 7 days. Phenotype and functional analyses were done on day 7, day 14, day 21 and day 28. We present the results of assays done on day 14 and day 28. The statistical comparison was done within the different conditions of the cultures with PBLC, and these were then further compared with IVC with PBL. [Table - 3] represents the data with PBLC stimulated with autologous tumor cells. After the initiation of the cultures on day 14 and day 28, some of the cells were taken from the cultures and washed (x 3) in PBS to remove cell surface bound cytokines. These cells were then counted and stimulated separately in 96-well plates (1 x 10 ⁵ cells/well) with autologous or allogenic tumors and also with autologous or allogenic APCs (responder: stimulator = 10:1). After 24 h, the supernatants from the 96 wells were collected and cytokine ELISA for IFN-γ and L-10 were done.

As can be seen in [Table - 3], day 14 and day 28 represent the data of cytokine assay with cells taken from control IVC with PBLC. We have observed that both on day 14 and day 28, the IVC with continuous presence of Th1 conditioning generated a higher number of tumor-specific IFN-γ-producing cells but fewer of the IL-10-producing cells from whole PBL. Intracellular cytokine analysis revealed that the IFN-γ-producing cells are CD8 + (data not shown). When these results were compared with results of other culture conditions, it was found to be highly significant ( P < 0.03). When the data from IVC-3 were compared with unconditioned PBL-IV, where within 28 days CTL activity was totally lost, it was also found to be very significant ( P < 0.05).

Consistent with our previous observations, we have found that Th1-conditioned PBL-IVCs also generate a better tumor antigen specific (MART-127-35) CTL and maintain a higher number of CD8 + cells in the cultures for longer period as compared with control (medium) in an auto APC/DC-based stimulation system [Figure - 5]. We have found significant elevation of the mean fluorescence intensity of MART-127-35 tetramer expression on CD8-gated population in those cultures with persistent Th1 conditioning with PBL as compared to cells in medium and with cells transferred from Th1 culture to medium (Th1 > medium) [Figure - 5].

Discussion

Specific immunotherapy for cancer continues to focus on a robust induction of antigen-specific CTL because most tumor cells only express MHC class I molecules. It is clear that vaccination with defined peptides, tumor cell lysates and DNA, alone or loaded onto APC or DC, can induce antigen-specific CTL response of varied magnitude.[4],[5],[9],[21],[22],[23] This study shows that the CTL responses in circulation contract by day 28. Multiple factors may explain the decline of the antigen-specific CTLs in circulation. For example, the decline might be a result of the activated CTL leaving the circulation and homing into extravascular sites. It might also be a reflection of programmed cell death or activation--induced cell death as physiologic homeostatic processes.[24] Our data suggest that the contraction could also have resulted from an IL-10-producing T-reg cell mediated regulatory process. It should, however, be mentioned that a precise phenotypic definition of T-reg cells is by no means complete and the CD4+CD25+IL-10+ T cells described here fulfill some of the attributes of T-reg cells.

The data also point out a potentially major constraint in inducing a robust CTL response in tumor immunotherapy with epitopes or antigens that are essentially 'self'-antigens. Clearly, CTL precursors for these types of 'self' but tumor-associated antigens are not centrally deleted. However, it is now equally clear that a peripheral mechanism for 'tolerance induction'exists, and T-reg cells are increasingly implicated in the peripheral tolerance induction process.[20] Although the traditional views of peripheral tolerance to self-antigens have been 'anergy,'[19] exhaustion,[25] ignorance[26],[27] or peripheral deletion by apoptosis of T cells through a Fas/Fas-L interaction,[28] the activation and expansion of CD4+ regulatory T cells after vaccination, especially with APCs made to present as self-antigens, cannot be ignored. Thus, although it might be feasible to 'break tolerance' for self-antigens (i.e., generate a CTL response of some degree against a bona fide 'self'-antigen), at least temporarily and to a degree with vaccinations, as in the case of active, specific, anti-tumor immunotherapy, the ultimate success might depend on the balance between the capacity of the immunogenic stimulus to 'break tolerance' and on the inherent physiologic constraint against activation of 'self'-reactive T cells. Our observation of a vaccine-induced CTL response followed by the activation of a regulatory T -cell response suggests that a better understanding of how the regulatory response gets activated and expanded will be useful.

The concept of T cell mediated 'suppression' of cellular immune response has been controversial. However, functional dichotomies with CD4+ T cells into Th1 and Th2 types exhibiting opposite effects (Th2 type acting as regulatory cells in some situations) are fairly well established.[29] Indeed, in some animal models, the ultimate nature of the immune response to certain types of infection (i.e., protection versus infection) is often determined by whether the animal mounts a Th1- or Th2-type response.[30],[31] The decline of CTL response in vivo [Figure - 2a, b, and c] despite antigenic stimulation provided through APC and the emergence of the T-reg type CD4 + T cells in vitro [Figure - 3], suggests that the window of opportunity to expand a CTL response to these types of 'self'-antigens might be quite narrow. The emergence of the T-reg type response after a CTL response might represent a form of inherent regulatory mechanism. An interesting point is that the amount of IL-10 produced by IL-2-stimulated PBL, as seen in [Table - 2], does not really match with the number of CD4 + CD25 + IL-10 + cells as shown in [Figure - 3]. In our judgment, the highly active T-reg cells in PBL, in addition to secreting IL-10, might be activating some other cells also (e.g., macrophage/monocyte, natural killer) to produce IL-10 in the culture. Understanding the mechanism of regulation by these T-reg cells has become a matter of great interest. The T-reg cells were found to suppress T-cell activation directly as evidenced by their suppressive effect on the lectin or T-cell response driven activation of fresh T cells (data not shown). This is in line with our earlier study in which we showed that CD4 + regulatory T cells can exert an inhibitory effect on other T cells by down-regulating the expression of IL-2 receptor.[8],[32] In addition to affecting T-cell activation directly, these cells can also down-regulate the expression of co-stimulatory molecules on the APC.[33] The T-reg cells can exert their regulatory function through a cytokine, which seems to be IL-10 or TGF-b[33],[34],[35], and as seen in [Table - 2], admittedly the relationship between the different subsets of regulatory T cells remains complex and unclear. In our present observation, neutralization of IL-10 in the cultures maintained the CTL activity for a considerable length of time. In this context, it should be pointed out that IL-10 and TGF-3 have been reported to be remarkable immunoregulatory cytokines.[34],[35] As others and we have shown, an increase of IL-10 or TGF-β production down-regulates CTL response in vitro;[6],[36] therefore, a routine analysis of IL-10 and TGF-β in serum of the immunized patients will be important to correlate with the clinical response.

We have previously shown that the regulatory T cells from melanoma patients up-regulated IL-10 production in response to autologous MHC-II molecule.[5] We have also shown that a CD4 + regulatory T-cell clone up-regulated their Tac receptor (CD25) on stimulation with tumor antigen pulsed autologous APC;[8] whereas in this study, we find T-reg cells secrete IL-10 simply after stimulation with IL-2 [Table - 2], [Figure - 4]. In this connection, the recently published information by Wang et al.[37] is important. This group has clearly demonstrated the tumor-antigen specificity of a suppressor-type CD4 + T-reg cell clone expanded from TIL from a patient. Hence it appears that the question of specificity of the regulatory T cells, although remaining unresolved, could possibly be resolved with clonal analyses of T-reg cells.

To date, more than 1,000 tumor vaccines have been reported[38] since our first published report of an APC- and peptide-based cancer vaccine study in 1995.[4] The collective results remain encouraging. The field of cancer vaccine trials with antigen and APC has reached a crossroads. Our earlier observations [L, 6 and 7] and the result of this study support the notion that a vaccine-induced activation/expansion of this type of regulatory T cells might have physiologic relevance in the peripheral tolerance induction process and suggest that strategies designed to 'suppress' or 'silence'the suppressors (regulatory T cells) could be useful.

References

Copyright 2006 - Indian Journal of Human Genetics

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