(Slovin, 2003). Therefore, it is clear that optimal construction of DC vaccines will require further investigation.

DC that present foreign peptides in the context of high levels of co-stimulatory molecules to naïve and memory T cells promote both CD4 and CD8 T cell adaptive immunity (see Section D). These immune effectors mediate protection from infection and control of tumor progression and metastasis. DC that present self-peptide can induce Treg cells and tolerance and immunosuppression (Steinman et al, 2000) and play a critical role in prevention of autoimmune disease (Von Herrah and Harrison, 2003) and promotion of transplant tolerance (Sakaguchi and Wood, 2003). In addition, DC are mediators of innate immunity through secretion of the cytokines IL12 and type I interferons (Münz et al, 2005). DC have a bidirectional interaction with natural killer T cells (NKT) and gd T cells promoting their activation and destruction of specific target cells, while the innate effectors feedback to trigger DC maturation thereby, furthering both adaptive and innate immunity (Mocikat et al, 2003).

The appropriate source of immunogen to load DC for vaccines protocols also remains to be determined. Many protocols have used peptide-pulsed DC matured in vitro and found promising results in animal models of cancer (Steinman and Dhodapkar, 2001). However, this approach is limited by the requirement to tailor the peptide to the HLA-type expressed by the patient and the limited number of known tumor epitopes (Pardoll, 2000; Trombetta and Mellman, 2005). Moreover, cell surface expression of the peptide-MHC complex may be rapidly lost and can only induce an immune response that is limited to the T cell clones specific for the selected epitope (Trombetta and Mellman 2005). Since tumor cells frequently lose expression of antigens, even successful expansion of tumor-reactive T cell clones will be unable to lyse cells that have lost the target antigen. One approach, long advocated by our group (see Section I), has been the use of whole tumor cells killed or rendered apoptotic for DC loading. Immature DC can ingest the apoptotic or killed tumor cells and through a mechanism known as cross-priming express peptides derived from tumor proteins in MHC class I molecules (Chow and Mellman, 2005). Cross-priming of exogenous proteins into the class I pathway distinguishes DC from other APC that can present only endogenous peptides in class I molecules (Trombetta and Mellman 2005). This pathway provides crucial access for ingested peptides derived from proteins of engulfed killed tumor cells to class I MHC molecules where they may be presented and can stimulate a CD8 anti-tumor response (Albert et al, 1998).

The limitations that preclude effective DC immunotherapy are manifold and include a lack of understanding of the optimum protocol for maturation, loading and antigen type for induction of successful adaptive anti-tumor immunity, as well as the means for overcoming the rather alarming counter measures that can be deployed by malignancies (see Section H). The goal of successful DC immunotherapy is induction of the major mediators of anti-tumor immunity CD4 and CD8 T cells.

C. The role of CD4 and CD8 T cells in immunity

The effector cells of adaptive immunity are the two major T cell subsets defined by their expression of either CD4 or CD8 accessory molecules. CD4 T cells are divided based on cytokine profiles into two major subtypes the TH1 and TH2 cells, where TH1 cells are helper cells that promote cell mediate immunity, secrete IFN-g and TNFa and carry a TCR that recognizes antigens displayed in class II MHC molecules on an APC. TH2 cells secrete IL 4, 5, 10 and 13 are responsible for humoral immunity and may limit immune responses through IL10 production (Kalinski and Moser, 2005).

CD4 T cells comprise the helper T cell subset and promote B cell immunoglobulin production (Kalinski and Moser, 2005) and under appropriate circumstances license CD8 T cell effector functions (Bevan, 2004). CTCL cells are mature CD4 T cells that have been shown to function as helper T cells (Berger et al, 1979; Broder et al, 1997) an observation that appears to conflict with their recently described T-regulatory (Treg) nature (Berger et al, 2005). However, other studies have demonstrated that this dichotomy may be consistent with results obtained with cloned normal Treg cells (Kitani et al, 2000).

CD8 T cells are stimulated by peptides displayed in class I MHC molecules on an APC and can mediate tumor cytolysis through production of cytokines such as TNF-a and the secretory granules perforin and granzymes (Raja, 2003). Tumor immunotherapy has been focused in large degree towards induction of a CD8 T cell anti-tumor response that can effectively destroy malignant cells. Although a number of approaches including transfer of ex vivo expanded cytotoxic T cells as well as a spectrum of DC therapies have been designed to optimize the host anti-tumor response, the overall success of these strategies has been disappointing (Banchereau and Palucka, 2005). Recently, the importance of expanding helper CD4 T cells for the development of effective CD8 T cells has garnered more attention and may improve the efficacy of the induced CD8 T cell response. The emergence of an additional subset of CD4 T cells, the immunosuppressive Treg cells, may limit anti-cancer immunity and the effects of these cells may have to be circumvented before effective immunotherapy can be achieved.

D. T regulatory cells

T regulatory cells have been recently identified as CD4⁺, CD25⁺ T cells that inhibit immune responses, prevent autoimmune disease (Von Herrah and Harrison, 2003) and maintain transplant tolerance (Sakaguchi and Wood, 2003). At least two types of Treg cells are recognized, those that arise in the thymus and circulating inducible Treg precursors that comprise 5-10% of the peripheral blood CD4 T cell compartment (Von Herrah and Harrison, 2003). Cell surface markers used to characterize the Treg population include the IL2 receptor (CD25), the inhibitory member of the co-stimulatory family cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNF receptor (GITR) and the forkhead transcription factor, Foxp3. Disruption in the Foxp3 gene product, in humans, has been correlated with the disease immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). IPEX in males is associated with lymphocyte activation autoimmune disorders and fatality due to overproduction of inflammatory cytokines. A similar disease in the scrufy mouse has been related to a mutation in the Foxp3 gene (Khattri et al, 2003). In the mouse, Foxp3 expression is confined to Treg cells and has been proposed as a specific marker restricted to Treg cells (Coffer and Burgering, 2004).

Treg cells are anergic and do not proliferate in response to TCR stimulation (Burg et al, 1978; Dalloul et al, 1992). However, Treg cells can be driven to proliferate in vitro in the presence of mature antigen-loaded DC in the absence of added cytokines (Yamazaki et al, 2003). Mature DC present antigen to CD4 T cells and can drive their conversion into Treg and thereby play a role in the regulation of autoimmunity and effector T cell expansions. The mechanism of Treg cell suppression remains controversial and appears to vary based on the culture conditions and the model of immunosuppression monitored (Wang and Wang, 2005). Secretion of the inhibitory cytokines IL10 and TGF-b as well as direct cell contact possibly mediated through CTLA-4 engagement of B7-1 and 2 on the APC have been described in several models. In addition, since Treg express high levels of membrane CD25 depletion of IL2 which is a required growth factor for both antigen responsive CD4 T cells and Treg cells may also serve to inhibit immune responses (Antony and Restifo, 2005).

The association of Treg cells with cancers including melanoma (Vignier et al, 2004), lung, ovarian (Woo et al, 2002), pancreatic and breast cancer (Liyanage et al, 2002) has suggested that Treg may play a role in suppressing anti-tumor immune response and perpetuating the malignancy. This observation may explain the limited efficacy of many anti-tumor immunotherapies where induction of an immune response is short-lived and abrogated by the tumor milieu. Therefore, improved immunotherapy will require a means for the depletion or inactivation of the Treg response.

E. Danger signals and the immune response

A major paradox in immunology is the ability of the immune system to discern danger while ignoring self tissues thereby, permitting destruction of pathogens without harm to normal tissues. A number of theories have been offered that partially explain this dichotomy including the clonal selection theory which proposes that lymphocytes in the thymus early in the organisms’ life die rather than proliferate when they encounter self antigens (Burnet 1957) eliminating potentially autoreactive clones. This explanation of central tolerance, however, did not address how the adult develops tolerance to exogenous antigens. An explanation for this part of the puzzle was provided by the two signal paradigm for T cell activation, whereby, the T cells need to see MHC displayed antigen and co-stimulation to initiate an immune response (Lafferty and Cunningham, 1970). Signal 1 (antigen stimulation) in the absence of signal 2 (co-stimulation) is proposed to lead to T cell anergy and non-responsiveness. It was then left to Janeway to explain how APC discriminate between self and foreign molecules (Janeway Jr, 1989). Janeway predicted that the APC would express pattern recognition receptors (PRR) that could detect conserved common molecules displayed by pathogens but not found in the human body. The identification of Toll-like receptors (TLR) on APC that can bind molecules derived from bacteria, viruses and parasites and the demonstration that signaling through Toll receptors leads to DC stimulation and induction of immunity has lent support for this theory (Janeway Jr and Medzhitov, 2002).

An alternative point of view was suggested by Matzinger, who reasoned that rather than discriminating between self and non-self antigens the immune system senses danger due to not just microorganisms but any form of stress or damage (Matzinger, 1994). Evidence has accumulated indicating that damaged or dying cells are able to activate DC to promote immunity and that self molecules that represent danger signals include heat shock proteins (HSP), uric acid and nucleotides (Pulendran, 2004). This hypothesis has been of particular relevance to our studies of CTCL since we have demonstrated that CTCL cells display a cell membrane HSP (Berger et al, 1997) that is highly homologous to the ER chaperone BiP (binding protein) or GRP78 (glucose regulated protein). Whether HSP up-regulation in tumor cells is a negative sign that correlates with a poor clinical outcome (Mintz et al, 2003) or whether HSP enhance anti-tumor immunity (Feng et al, 2001) is a yet another enigma that waits to be unraveled and may have significant ramifications for tumor immunotherapy.

F. Heat shock proteins

HSP are essential for many of the house-keeping functions of the cell including regulation of protein folding, the removal of misfolded proteins, the stress response and protection from caspase-mediated apoptotic cell death (Nicchitta 2003). Due to the essential nature of their role in the cell, HSP are highly conserved throughout evolution. HSP are up-regulated by the cell under stress conditions including fever, starvation and exposure to pro-inflammatory cytokines such as TNF and IFN-g, oxidative stress and infection (van Eden et al, 2005). The HSP are divided into a number of families based on molecular size. At least three members of the HSP families, HSP60, 70 and 90 appear to play a major role in inflammation and immunity. Since we have found that the HSP70 family member GRP78 or BiP is abundantly expressed on the cell membrane of CTCL cells (Berger et al, 1997), we propose that BiP may serve as a source of autoantigen that drives CTCL Treg conversion when it is presented in class II MHC molecules on DC to the CTCL cell TCR.

BiP is an endoplasmic reticulum chaperone that is responsible for the folding and transport of polypeptide chains (Hendershot, 2004). The expression of BiP on the cell membrane has been confirmed in other cancers and may relate to a chronic stress response and has been associated with a poor prognosis (Triantafilou et al, 2001; Mintz et al, 2003). BiP up-regulation appears to play a role in protection of malignant cells from apoptosis, thereby, potentiating tumor cell growth and survival (Reddy et al, 2003).

Studies in autoimmune disease have indicated that HSP are a major source of autoantigen(s) that trigger HSP-reactive T cells with an immunoregulatory phenotype which can suppress immune responses in inflammatory conditions such as rheumatoid arthritis, type 1 diabetes and potentially atherosclerosis and allergy (van Eden et al, 2005). The role of HSP in autoimmune disease may relate to their highly conserved homology to bacterial HSP. Both HSP60 and 70 are immunodominant proteins that induce specific cellular and humoral immune responses after infection with bacteria, protozoa, fungi and parasites (van Eden et al, 2005). It appears that HSP-responsive cells escape central thymic tolerance and enter the periphery where they may be controlled by HSP-reactive Treg cells (van Eden et al, 2005).

HSP also serve as immunogens presented by class I MHC molecules and are targets for cytotoxic T cells (Huang et al, 2000; Feng et al, 2001). HSP70 specific cytotoxic T cells have been generated without the assistance of helper T cell responses (Huang et al, 2000). HSP are also efficiently presented by MHC class II molecules (Anderton et al, 1995) and HSP70 related peptides comprise a substantial proportion of the peptides eluted from class II MHC molecules (Newcomb and Cresswell, 1993). When APC were stressed by heat shock, the responding CD4 T cells displayed a Treg cytokine profile secreting IL10. HSP have been shown to be protective in a variety of experimental disease models independent of the source of the disease inducing antigen (van Eden et al, 2005). Based on this evidence it has been proposed that HSP are involved in the control of inflammatory disease through the induction of Treg cells. Therefore, the inflammation that accompanies malignant transformation may provide an ideal milieu for the up-regulation of HSP and the induction of regulatory T cells which could suppress anti-tumor immunity.

These current concepts in immunology have had direct impact on our understanding of CTCL and enabled us to decipher many of the clinical clues that were provided by the malignancy. The ability to culture CTCL cells derives directly from an understanding of their relationship to Langerhans cells in the pautrier microabcess (Figure 1) and was dependent on the description of methods for the cultivation of DC.

G. In vitro growth of CTCL

CTCL cells isolated from the peripheral blood of patients are anergic and respond poorly to mitogen, antigen and alloantigen stimulation (Burg et al, 1978; Dalloul et al, 1992). Investigation of the immunobiology of the malignancy has been severely hampered by the inability to routinely culture the malignant T cells in vitro and the absence of suitable animal models.

We have found that CTCL cells proliferate in the presence of autologous DC cultured with the supportive T cell cytokines IL2 and IL7 and for the DC granulocyte-monocyte colony stimulating factor (GMCSF) and IL4 (Berger et al, 2002a). In this system, co-cultivated CTCL cells and DC proliferate for up to three months while both cell types die within one week when cultured individually (Berger et al, 2002a). These studies also demonstrated that proliferating CTCL cells secrete IL10 and TGF-b two immunosuppressive cytokines that maintain DC immaturity and also inhibit immune responses. The ability to grow CTCL cells has enabled us to obtain sufficient malignant T cells for further evaluation that interpreted in the light of the bourgeoning information about Treg cells led to an explanation for the immunosuppressive nature of CTCL.

Since cultured CTCL cells proliferate, they were rapidly rendered apoptotic by antibodies that bound to the TCR or the associated CD3 complex, in the presence of DC bearing co-stimulatory molecules (Berger et al, 2005). When we challenged freshly isolated cultured CTCL cells with DC pulsed with apoptotic malignant T cells, we found that the CTCL cells up-regulated the phenotype and function of Treg cells. Treg CTCL cells express CTLA-4, an inhibitory member of the co-stimulatory family, enhanced levels of membrane CD25 and down-regulate the CD4 T cell TCR indicating engagement by DC class II presented peptides. Treg CTCL cells also up-regulate cytoplasmic FoxP3, a specific marker for Treg cells. Anti-class II antibodies and an inhibitor of the class II pathway were shown to prevent adoption of a Treg profile in CTCL cells confirming the requirement for class II MHC molecules in the induction of a Treg conversion in CTCL.

Treg CTCL cells upon stimulation by apoptotic cell loaded DC were found to secrete the immunosuppressive cytokines IL10 and TGF-b. Addition of Treg CTCL cells to normal T cells responding to recall antigen or allostimulation resulted in suppression of the normal T cell proliferative or cytokine response (Berger et al, 2005). Further investigation revealed that the mechanism of suppression was not related to the release of immunosuppressive cytokines nor was it mediated by cell contact, indicating that Treg CTCL cells do not employ conventional means for inhibition of normal immune responses. We have recently demonstrated that the suppression of normal immunity mediated by Treg CTCL cells can be partially reversed through the addition of a neutralizing antibody to CTLA-4 as well as with the administration of high doses of IL2 (unpublished results). In addition, sera collected from patients with varying stages of CTCL contained elevated levels of soluble CTLA-4 which correlated with the severity of the disease (unpublished results). Therefore, one means through which CTCL cells suppress immunity may be through secretion of soluble CTLA-4 which could act through interference with effector T cells’ access to B7-1 and 2 co-stimulatory molecules. In addition, depletion of growth factors such as IL2 may inhibit normal T cell antigen-driven proliferative responses. These two scientific observations provide obvious avenues for therapeutic exploitation (Section J), through the use of anti-CTLA-4, recombinant IL2 or IL2-binding toxins. In addition, current studies are in progress to determine if there is a role for heat shock proteins as autoantigens in CTCL. These studies may identify opportunities for selective targeting of Treg cells to improve the efficacy of immunotherapy (Section J).

Although, the interactions of CTCL cells with DC suggest that immunotherapy using DC loaded with apoptotic cells might be precluded in this disease, our clinical experience argues that this approach is not just feasible but efficacious. We have found that a standard therapy, extracorporeal photopheresis (ECP, 65) and a simple modification Transimmunization (TI, Berger et al, 2001) both operate through the induction of differentiating DC derived from monocyte precursors and simultaneous DC loading with apoptotic malignant T cells.

H. ECP and TI

ECP is a widely used form of immunotherapy that is FDA approved for the treatment of cutaneous T cell lymphoma (Zic, 2003). ECP derived from a combination of therapeutic leukapheresis, a cyto-reductive treatment (Edelson et al, 1974) and psoralen and ultraviolet light therapy (PUVA) used in the treatment of psoriasis and early stage CTCL limited to the epidermis (Gilchrest et al, 1976). In the ECP therapy, a photactivatable drug 8-methoxypsoralen (8-MOP) is added to a therapeutic leukapheresis which is passed through an ultraviolet A (UVA) exposure field. The drug intercalates in the DNA helix where after photoactivation it forms cross-links between pyrimidine bases (Berger et al, 1985). The cross-links prevent replication and are poorly repaired leading to gradual apoptotic cellular death over a 6 day period.

We have demonstrated that the combination of the physical perturbation initiated by the passage of the leukapheresis through the large plastic plate, permitting adherence and release of monocytes, when combined with the apoptotic death of the malignant T cells has profound consequences for the reinfusate returned to the patient (Berger et al, 2001). The monocytes become activated and begin to transition into aggressively phagocytic immature DC while the malignant T cells are rendered apoptotic and are ingested by the DC. Therefore, some semi-mature DC loaded with apoptotic T cells are returned to the patient along with a large number of transitioning DC and apoptotic CTCL cells that may interact inefficiently in vivo.

We have developed a modification of ECP which we term Transimmunization (TI, Figure 3) to indicate the transfer of immunogenic peptides from apoptotic cells to DC that can effectively present them to the immune system displayed with the full complement of co-stimulatory and accessory molecules (Berger et al, 2002b).

Since the TI procedure allows access to all the components of the immune response the addition of exogenous agents such as drugs, antibodies or cytokines is facilitated and can potentially be used to modify the reinfusate and tailor it to the patient. In addition, the level of apoptotic cells reinfused can be controlled and since high levels of apoptosis appear to correlate with induction of a Treg response, it may be possible to extend the therapy to other disorders due to a failure of immunoregulation. Other types of malignancies may also be treated by TI since the nature of the apoptotic cells added to the overnight culture can be simply modified through the use of other mediators of programmed cell death (such as irradiation of isolated solid tumor cell suspensions) and co-incubation with TI generated transitioning DC overnight.

Dissection of the basic science aspects of CTCL has allowed us to identify targets for immunotherapy and also explained why many approaches are limited in efficacy. Strategies to overcome these limitations readily suggest themselves and include anti-CTLA-4 antibodies, toxin conjugated-IL2, inhibition of heat shock proteins and maximizing DC differentiation and antigen presentation. Some of these approaches have already been tested clinically (Table 1) and will be reviewed in the following sections.

I. Targeting Treg cells

The expression of CTLA-4 on the surface and in the cytoplasm of Treg cells has suggested that it might provide a logical target for depletion of these immunosuppressive cells that may limit cancer immunotherapy. The first clinical trial focusing on anti-CTLA-4 therapy was conducted with nine patients who had previously been immunized against their ovarian cancer or melanoma. Each of these nine patients received a single IV injection of CTLA-4 monoclonal antibody. In these patients, just over half (55%) experienced some degree of tumor cell death as determined histopathologically or via stabilization of biochemical tumor markers (Hodi et al, 2003). In this pilot study, the only notable side effect was the appearance of a transient rash on nearly all patients, which was easily controlled with antihistamine therapy. In a later trial by Phan and colleagues in 2003, 14 patients with metastatic melanoma were given a peptide pulsed melanoma vaccine along with a standardized dose of anti-CTLA-4 antibody. All patients enrolled in the trial developed T cell reactivity against the immunizing peptides given in conjunction with the anti-CTLA-4. Notably, three patients of the fourteen (21%) experienced objective cancer regression, with two patients having a complete tumor response at one year and one patient having a partial response, while two others experienced mixed responses (regression of certain metastases with growth of others). A significant percentage (43%) of patients experienced grade III/IV autoimmune disease, including three with dermatitis, two with colitis and one with hypophysitis and hepatitis. Each of these patients was treated with supportive care and/or steroid therapy and all patients experiencing autoimmunity recovered from the acute toxicity without later relapse (Phan et al, 2003). A number of other small studies have been conducted looking at various dosing regimens and co-administration of anti-CTLA-4 with peptide vaccines and these studies also provide similar encouraging results in both objective tumor regression and clinical response (Korman et al, 2005). While the overall regression rates remains somewhat low, these trials do provide proof-of-principle that substantiates the proposition that depletion of the T-regulatory population can result in an enhanced tumor vaccine effect and that Treg cells control autoimmunity. As with many existing immunotherapeutics, there remains a profound need for further research into applying these principles to human subjects, with focus on proper dosing schedules and the use of anti-CTLA-4 as a possible therapeutic adjuvant to other cancer vaccines. Institution of anti-CTLA 4 therapy in CTCL may reverse the immunosuppressive consequences of the malignancy and allow induction of more effective anti-tumor immune responses.

Aside from anti-CTLA-4 therapy, there are a number of other options that exist for selective targeting of T regulatory cells to enhance the effectiveness of cancer immunotherapy. GITR, a member of the tumor necrosis factor-nerve growth factor receptor family, has been described on the surface of the CD4⁺ CD25⁺ T cells and is thought to play a role in regulating suppression. Stimulation of this receptor via an activating antibody has been shown to reverse the induction of suppression and removal of GITR⁺ cells resulted in organ-specific autoimmunity in murine models (McHugh et al, 2002; Shimizu et al, 2002). Other options include cell signaling molecules such as TNF-related activation induced cytokine (TRANCE) and receptor activator of NF-kB (RANK), which have been shown to be involved in activating signaling pathways of CD4⁺CD25⁺ T cells (Green et al, 2002). Depletion of these molecules resulted in a rapid onset of diabetes in murine models.

Another alternative for Treg inactivation lies in the close association of T regulatory cells and a number of solid tumors. It has been demonstrated that high levels of CD4⁺CD25⁺ T cells are present in lung, ovarian, breast and pancreatic tumor samples (Liyanage et al, 2002; Curiel, et al, 2004) and in ovarian cancer, there appears to be an inverse relationship between the number of T regulatory cells and survival (Curiel, et al, 2004). Researchers have demonstrated that the chemokine CCL22 and the chemokine receptor CCR4 are vital to the migration of T regulatory cells to the tumor site and inhibition of this chemokine resulted in decreased migration of Treg cells (Lee et al, 2005). Therapies that selectively deplete Treg in tumor sites while maintaining the Treg population that controls autoimmunity would enable eradication of immunosuppression while preserving inactivation of autoreactive T cell clones.

J. Cytokine therapy

Cytokines also play a major role in the development and functional capacity of T regulatory cells. As previously described, T regulatory cells produce a number of soluble, inhibitory cytokines, such as IL-10 and TGF-b. Selective inhibition of these cytokines has been shown to reverse generalized immunosuppression in a number of murine models and in humans (Khoo et al, 1997; Nakamura et al, 2004). In the realm of cancer immunotherapy, T-cell-specific blockade of TGF-b allows the generation of an immune response capable of eliminating tumors in murine model systems (Gorelik and Flavell, 2001). Phase I clinical trials looking at the efficacy of anti-TGF-b therapy in the setting of glioblastoma and non-small-cell lung cancer, are in progress (Lahn et al, 2005). Blockade of IL-10 has demonstrated enhanced tumor destruction in murine models and may also offer some clinical utility (Piccirillo and Shevach, 2000).

Perhaps the best studied cytokine for tumor immunotherapy is IL-2. First investigated in the 1980’s, IL-2 has been found to enhance the potency of immunotherapy due to its role as activator/expander of tumor-specific T cells. Early clinical success with IL-2 used as monotherapy demonstrated durable regression in 20% and complete response in 9% of renal cell carcinoma patients. Furthermore, in the case of metastatic melanoma, a regression rate of 17% with complete response of 7% was noted (Gaffen and Liu, 2004). These early successes have translated into a continued role for IL-2 today as a vaccine adjuvant in metastatic melanoma and renal cell carcinoma. IL-2 has also been used with good clinical results in CTCL patients (Marolleau et al, 1995).

Current studies are underway to determine whether IL-2 can be used synergistically with IL-12 to enhance the anti-tumor effects of a given vaccine (Rook et al, 2003). In vitro studies have shown that a synergism between these two cytokines can augment the immune response in CTCL patients (Zaki et al, 2002). Recent results indicate that although IL-2 appears to be necessary for T cell activation and growth, it also supports the growth and differentiation of T regulatory cells and this may limit the beneficial aspects of this cytokine. However, our preliminary studies suggest that despite the requirement for IL2 to support Treg cell growth, the addition of recombinant IL2 to co-cultures of Treg CTCL cells and antigen-stimulated normal T cells reverses immunosuppression of IFN-g production (unpublished results). Therefore, a role for IL2 in restoration of anti-tumor immunity may be feasible.

Similarly, a number of newer studies have looked at the use of alternative cytokines, such as IL-15 and cytokine combinations with vaccines in hopes of more specifically activating the tumor infiltrating lymphocytes without expanding the T regulatory lymphocyte population (Antony and Restifo, 2005).

Members of the naturally occurring T regulatory cell subpopulation are CD25⁺. Given the relative specificity of CD25 to T regulatory cells, antibody dependent cell death via CD25 may serve as a means to eliminate T regulatory cells. Recently, the FDA approved ONTAK, a recombinant cytotoxic protein composed of diphtheria toxin conjugated to the IL-2 binding domain, for use in CTCL patients. Due to its specificity for the IL2 receptor, this antibody should ideally be able to deplete the CD4⁺CD25⁺ cell population in vivo in hopes of enhancing the efficacy of adjunct immunotherapy. One caveat, however, is that CD25 is also expressed on newly activated CD4⁺CD25^- T cells as well as effector CD8⁺ T cells (Annacker et al, 2001). Thus, destruction of the naturally occurring T regs via a CD25 specific mechanism may potentially destroy some of the effector T cells capable of mounting a clinically salient immune response. Appropriate timing of anti-CD25 therapy appears crucial, as CD25⁺ depletion before vaccination was more effective than CD25⁺ depletion after vaccination (Sutmuller et al, 2001). There continues to be great difficulty in distinguishing an actual T regulatory cell from an effector T cell, especially during the midst of an immune response, therefore, the dosing and time course of therapy may be vital to success.

An alternative approach to selectively removing the CD25⁺ population prior to introduction of a tumor vaccine is total lymphocyte ablation in vivo, followed by introduction of the appropriate immunotherapeutic. In a key study conducted at the National Cancer Institute, 35 melanoma patients had their tumors harvested ex vivo for selection of appropriate tumor-infiltrating lymphocytes. Prior to re-infusion of the expanded tumor-specific lymphocyte population, cyclophosphamide and fludarabine were administered to deplete the lymphocyte population in vivo. Marked expansion of tumor-specific T cells was observed and 51% of the melanoma patients achieved objective responses (Dudley et al, 2002; Rosenberg and Dudley, 2004).

K. Monoclonal antibody therapy

A prevalent modality in the quest for effective immunotherapeutics is the use of monoclonal antibodies (mAB) as adjuvant to immunotherapy. First discovered in the late 1970’s (Kohler and Milstein, 1975), monoclonal antibodies have become standard therapy for certain malignancies given their enhancement of the anti-tumor response, relatively safe toxicity profiles and high selectivity. Overall, these antibodies can be divided into a number of specific classes, based on the mechanism by which they exert their effect. The initial classes of antibodies exerted their anti-tumor effects via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC, Maloney et al, 1994). However, newer classes of antibodies are being studied that are directed against a variety of cellular targets, including growth factors, such as vascular epidermal growth factor (VEGF), epidermal growth factor receptor (EGFR), a number of specific cell signaling molecules and receptors on the cells of the immune system in hopes of enhancing the cellular immune response against cancer (Gutheil et al, 2000; Ciardiello and Tortora, 2001; Gordon et al, 2001).

The first anti-tumor antibody approved by the FDA was rituximab in 1997, which was quickly followed by tastuzumab in 1998. These were approved for the treatment of refractory non-Hodgkin lymphoma and HER2/Neu+ breast cancers respectively. Since that time, six additional monoclonal antibodies have been approved for clinical use. These antibodies include anti-CD52 alemtuzumab for refractory B-cell chronic lymphocytic leukemia, anti-CD33 gemtuzumab ozogamycin conjugated to calicheamicin for refractory acute myeloid leukemia, anti-CD20 radioisotope conjugates ibritumaomab and tositumomab for refractory non-Hodgkin lymphoma, to target VEGF bevacizumab for metastatic colon cancer in combination with chemotherapy and anti-EGFR cetuximab for metastatic colon cancer (Lin et al, 2005). As previously described, these antibodies all work either through stimulation of ADCC and CDC or inhibition of specific growth factors/growth factor receptors essential for tumor proliferation. The results of clinical trials using these drugs have recently been reviewed (Harris, 2004) and ongoing clinical trials for other indications are continuing. This limited selection of monoclonal antibodies has enjoyed huge success in their respective oncologic fields and there are now over 400 clinical trials currently underway to determine the efficacy of a huge variety of other anti-cancer antibodies (Gura, 2002).

As described earlier, the newest class of monoclonal antibodies is being used in an attempt to augment immunomodulation in conjuction with other immunotherapies, of which only one has entered clinical trials. A primary target is suppression of the aforementioned T regulatory cell, but a number of other potentially beneficial targets exist as well. Anti-4-1BB (CD137) is a surface glycoprotein in the TNF receptor family and it is expressed by activated T and NK cells (Murillo et al, 2003). Treatment of tumor-bearing mice with anti-4-1BB caused tumor regression and addition of anti-4-1BB mAbs help potentiate the effectiveness of adoptive immunotherapy, likely through preventing programmed cell death in lymphocytes (Melero et al, 1997; Guinn et al, 1999; May Jr, et al, 2002). CD40 is a TNF receptor family member expressed on B cells, DC and macrophages and is essential in mediating both cellular and humoral immune responses (Grewal and Flavell 1998). Treatment of B cell malignant mice with anti-CD40 has lead to complete cure in some cases (French et al, 1999). Very early clinical trial data has been published showing a 37.5% disease stabilization rate and 6% partial response rate when anti-CD40 was used for high grade NHL or solid tumors (Vonderheide et al, 2001). In the case of CTCL, use of CD40 ligand has been used to restore IL-12 and TNF-a production in the peripheral mononuclear cells of cancer patients (French et al, 2005), further confirming the possible therapeutic potential involved in the manipulation of CD40 in a number of malignancies. Other directions that antibody research is headed include the use of immuno-modulators, such as IL-2, along with monoclonal antibody administration, in hopes of boosting immune effector function, as well as the development of novel immunoconjugates in order to improve efficacy. Each of these new avenues is undertaken with the goal of combining the best aspects of various parts of immunotherapy in order to create the most efficacious treatment.

L. Heat shock proteins as immunotherapy

A newer approach to producing an effective cancer vaccine is through the use of highly immunogenic heat shock proteins as tumor-associated antigenic adjuvants. This dichomatous role of HSP as both immunostimulant and immunosuppressor has been the subject of much critical debate in recent years. The ability of HSPs to elicit an immune response was first demonstrated in 1986 by Srivastava and colleagues, who showed that mice immunized with gp96 produced tumor-specific immunity against the tumors that were used to isolate the HSP, but not against other tumors (Srivastava et al, 1986). This early work in the field was done using the HSP gp96, but it was further validated with later results in which a number of other HSP, including HSP70, HSP90, calreticulin, HSP110 and GRP170 (Udono and Srivastava, 1993; Tamura et al, 1997; Basu and Srivastava, 1999; Wang et al, 2001), were used. In each of these studies, it was shown that the HSP isolated from cancer cells elicited immunity while those HSP isolated from normal tissues did not. This initial work led to subsequent discoveries of the immunostimulatory effects that HSP have on nearly all cells of the immune system, including T cells, B cells, macrophages and dendritic cells (Quintana and Cohen, 2005). Aside from the immunogenicity of HSP, they provide an exciting target for cancer vaccination because of their ability to function as carriers of self and non-self peptides. Given their role as intracellular chaperone, HSP bind a wide variety of peptides in vivo, albeit for a very short time (Reits et al, 2003). These short-lived HSP-peptide complexes are useful immunologic targets because they provide an up-to-date reflection of the internal environment of the cell. In the case of a malignant cell, a number of the HSP-peptide complexes presented on the surface of the cell are unique tumor-associated antigens and thus can be attacked by the immune system of a vaccinated patient. This is supported by initial research which showed that the anti-tumor response generated by HSP-associated tumor cells was derived from peptides bound to the HSP and not directed against the HSP themselves (Li and Srivastava, 1993; Udono and Srivastava, 1993). When alone, neither the HSP nor peptides were immunogenic; only the HSP-peptide complex was able to elicit the desired CD8⁺ cytotoxic T cell response (Blachere et al, 1997). Thus, the specific immunogenicity of each HSP-peptide preparation is due to the inherent antigenic variety found in the malignancy of interest (Menoret et al, 1999).

These discoveries have opened up a new door in anticancer therapy, whereby cancer vaccines can be created using tumor-derived heat shock protein-peptide complexes (HSPPC) as effective immunologic targets. HSP could be loaded in vitro with synthetic peptides and injected back into the patient to achieve a desired immune response. In order to achieve immunity by HSP vaccination, both APC and CD8+ T cells are required, as depletion of either cell type diminishes the protective effects of the vaccine (Udono et al, 1994). Interestingly, it has also been shown that HSP can mediate efficient cross-presentation into the Class I pathway after being endocytosed by APC, via the recently identified CD91 receptor, making them powerful adjuvants and rendering them more effective at generating the desired cytotoxic T cell response (Udono et al, 1994; Jung et al, 2002). In addition to the direct effects on antigen presentation, the interactions of HSP with APC leads to secretion of pro-inflammatory cytokines such as TNF-a, interleukin-1b, IL-12 and GM-CSF; maturation, migration of dendritic cells; and translocation of nuclear factor-kappaB (NF-kB, Basu et al, 2000; Singh-Jasuja et al, 2000; Somerson et al, 2001). Given the homology of HSP and their generalized immunogenicity, this allows for the immunization of the host against a wide variety of host-specific tumor-associated antigens. This was initially supported in a number of murine models, which demonstrated that HSP immunization slowed the progression of primary tumors and reduced the overall metastatic burden as well (Tamura et al, 1997). Given its success in murine models, the vaccine was ready for study in human trials. The initial autologous HSP vaccine to be tested in a clinical trial was HSPPC-96 (Oncophage), a gp96 HSP-peptide complex, made from resected tumor tissue and given back to the patient via intradermal or subcutaneous injection. Since 1995, there have been ten phase I or II clinical trials looking at the safety profile of this vaccine as well as the optimal route of administration and dosing schedule (Lewis, 2004). Throughout these studies, the most common side effects noted were transient and included injection site inflammation and low-grade fever. More encouraging was that HSPPC-96 treatment resulted in a better quality of life during treatment when compared to those patients undergoing chemotherapy or high-dose cytokine immunotherapy (Cohen et al, 2002). In addition, no clinical signs of autoimmunity have been documented in the more than 500 patients treated to date.

The encouraging safety profile of the HSPPC-96 vaccine has led to a number of phase II and III clinical trials to determine whether or not the vaccine can have a beneficial effect on cancer patients. In the initial pilot trial, 6 of 12 patients with miscellaneous, advanced cancers had a CD8⁺ response against autologous tumor following HSPPC-96 vaccination (Janetzki et al, 2000). Another trial was conducted in patients with renal cell carcinoma. In 16 patients with stage 4 renal cell carcinoma given a 25μg HSPPC-96 vaccination dose, 3 patients had observable partial responses while on the vaccine and three additional patients showed disease stabilization at one year (Srivastava and Amato, 2001). A phase II study involving melanoma patients showed an increase in blood IFN-g levels following vaccination and 11 of 23 of these patients showed an increase in melanoma-specific T cells (Belli et al, 2002).

The frequency of these measurable immune responses has appeared to correlate well with favorable clinical responses. In the melanoma trial mentioned previously, the majority of the clinical responders were also those that had an immune response to the tumor cells while those without clinical responses also frequently lacked demonstrable immunity to the malignant melanocytes (Srivastava and Amato, 2001; Belli et al, 2002). In this study, five patients of twenty eight with measurable disease post-vaccination showed either objective response or disease stabilization. In a phase II trial looking at patients with colorectal carcinoma, patients clinically free of disease post-surgery had better overall survival if they developed an immune response following HSPPC-96 vaccination than if they did not (Lewis, 2004).

Currently, there is sufficient evidence to support the idea that cancer vaccination with HSPPC can produce measurable anti-tumor responses in cancer patients. Why these measurable immune responses are not always translated into an observable clinical response may relate to the role of HSP as autoantigens that generate a Treg response (van Eden et al, 2005). If high levels of HSP are present in apoptotic cells engulfed by DC, peptides derived from the HSP may be presented in class II MHC molecules and induce Treg cell stimulation. As in other forms of immunotherapy, the immune system may both potentiate and limit the success of the treatment and as previously discussed a combination approach using anti-Treg modalities to supplement immunotherapy may allow us to circumvent the difficulties while preserving the benefits.

As a whole all of these approaches argue for careful scientific investigation and attention to clinical signs and signals so that the optimal form of immunotherapy can be developed, tested and employed. Since we have better tools and an improved understanding of the immune system, we should be able to pursue therapies that can be rapidly translated from the laboratory to the patient in a safe, efficacious fashion.

M. Dendritic cell immunotherapy

Since the publication of the first peptide-pulsed DC cancer vaccine clinical trial in 1995 by Mukherji et al. hundreds of clinical trials have been completed. This great interest in vaccines grew from the widespread success of vaccines in preventing widespread viral diseases, ease of administration and minimal side effects. As previously described, cancer vaccines are designed to treat growing tumors by inducing tumor-specific effector T cells that reduce the tumor mass and via induction of tumor-specific memory T cells to control tumor relapse (Banchereau and Palucka, 2005). Thus, when properly prompted, the immune system's innate, antigen non-specific, immunity and the adaptive, antigen specific, immunity synergize to eradicate pathogens as well as cancers. The induction, coordination and regulation of the adaptive immune systems is ultimately controlled by DC (Steinman, 1991; Banchereau et al, 2000).

Although, multiple vaccination models are under investigation such as peptide vaccines, viral vector vaccines and tumor cell vaccines, dendritic cell vaccines have the best response rate (Rosenberg et al, 2004). The completed trials utilizing dendritic cell vaccines have been numerous and varied success observed (Slovin, 2003). By 2003 there were 98 published peer-reviewed articles describing over 1000 patients vaccinated for over two dozen cancer types (Slovin, 2003): adenocarcinoma, bladder, breast, lung, colorectal, CML, duodenal, esophageal, GI carcinoma, glioblastoma, astrocytoma, glucogonoma, gynecologic carcinoma, head and neck, hepatocellular, multiple myeloma, lymphoma, neuroblastoma, ovarian, pancreatic, parathyroid, prostate, sarcoma stomach, thyroid, wilms, neuroendocrine. Nevertheless, the ideal vaccine has yet to be worked out, for example, the source of DC, method for tumor antigen presentation to DC, effectiveness of artificially maturing DC, site of vaccination, number of administrations, vaccine preservation, number of DC necessary and timing of vaccinations remains to be determined. The most effective antigen for use in loading DC is not clear, whether peptide, whole tumor, nucleic acids (Caruso et al, 2005) or viral. The adverse effects associated with DC vaccinations were minor, and mostly self-limited; the most common side effects were fever, injection site reactions and adenopathy. Autoimmunity was uncommon and only a handful of patients experienced conversion to positive ANA, RF, anti-dsDNA and antithyroid titers. Fortunately the autoimmunity was generally not clinically significant.

Some conclusions about the best methodology have been reached based on previous research. Vaccine treatments, when successful, are most effective in patients with predominately lymphatic or cutaneously restricted disease (Timmerman et al, 2002; Rosenberg et al, 2004). One explanation for this observation is that in contrast to solid tumors, lymphoid tumors and possibly cutaneous tumors allow direct circulatory access with the best results seen when the vaccines are given subcutaneously or intradermally, rather than intravenously (O’Neill et al, 2004). In almost all cases the DC were collected from the patient’s peripheral blood. Some investigators are exploring the possibility of using bone-marrow derived DC in vaccines, since early animal models have been promising ( Mayordomo et al, 1997). Neverthless, DC from whole blood are easily accessible in large numbers and can be subsequently cultured to an even greater number (Dillman et al, 2004). Adjuvants have also been employed to boost the reaction towards vaccines. One very common adjuvant is Keyhole Limpet Hemocyanin (KLH, Höltl, 2005). KLH is an inducer of strong CD4⁺T cell helper responses as a highly immunogenic neo-antigen. When given in conjunction with tumor antigen, it amplifies the immune response via the production of cytokines in the lymph node microenvironement causing an enhanced CD8⁺ response.

There are currently 22 active clinical trials involving the use of dendritic cells to treat cancers (http://cancernet.nci.nih.gov/clinicaltrials). The types of cancer under investigation include AML, Brain, Breast, Colon, Renal, Lung and Melanoma. All trials are in the first or second phases of testing with 32% in Phase I, 36% in Phase II and 32% in phase I/II of testing. The vaccine composition is rather varied employing autologous dendritic cells paired with a range of potential antigenic substances. Common substances employed in the vaccine design include whole-tumor lysates, tumor antigens, recombinant transfection viruses, RNA and tumor cell-dendritic cell fusion-type combinations. Adjuvants are commonly added to the vaccines in an attempt to amplify antigenicity and to cause the maturation of monocytes into immature dendritic cells. IL-4 and GM-CSF are often used to dedifferentiate monocytes into immature dendritic cells, that are very potent APCs. The uptake of tumor antigen causes the shift of immature dendritic cells to antigen presenting cells mature dendritic cells. DC vaccines are frequently pulsed with an individual peptide creating CTL that recognize a single tumor epitope. However, tumors by nature are thought to accumulate gene mutations and the antigenic genes are no exception. Thus it is possible that over time the antigens change their structure or may not be expressed at all (Trefzer et al, 2005). Thus, limiting vaccines to one antigen alone may not be as efficacious as a whole tumor lysate that could provide innumerable antigens for the induction of multiple CTL clones.

Future vaccines will need to overcome the immune systems regulatory balances. The immune systems has naturally evolved robust suppressor systems to prevent detrimental antigen-specific responses to self and environmental antigens thereby averting excessive damage to host tissue (Smits et al, 2005). Knowledge regarding the biology of these regulatory mechanisms will become crucial for the successful application of cancer immunotherapy.

II. Concluding comments

Several themes suggest themselves as the leitmotiv of this review of cancer immunotherapy. First, a bidirectional knowledge flow between clinical observations and research investigation are crucial for successful immunotherapeutic protocols. The clinical clues form the springboard from which a well planned laboratory study can be launched to dissect and resolve previously mysterious physical signs and symptoms. Once a model has been built in the laboratory, it can be used to identify therapeutic regimens that may be the most beneficial and the least toxic.

This paradigm has been exemplified by our current studies in CTCL where our understanding of the immunobiology of the malignancy has elucidated previous cryptic physical manifestations and led us to identify new therapeutic approaches that can be translated into clinical trials. Our in vitro model system of the disease will allow us to screen new therapeutics before they enter the clinic saving time, expense and potential patient harm. In the future, further advances in our understanding of the functioning of the immune system will have immense significance for clinical medicine allowing us to develop new and improved therapies and approaches for disease management. Finally, it is becoming clear that the immune system is a finely balanced interconnected web and that pulling one string may have unforeseen consequences for the entire fabric. It is therefore crucial that we continue to test and learn from our mis-steps so that our future interventions can become ever more sure footed.

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Modality	Therapy	Disease Targeted
Photochemical
Psoralen and ultraviolet light resulting in apoptotic cell loaded dendritic cells	Photopheresis and Transimmunization	T cell mediated diseases: Cutaneous T Cell Lymphoma, Graft-versus Host Disease, autoimmune disease, transplant rejection
Vaccine
Dendritic Cell Vaccines	Pulsed with peptides, tumor lysates, hybrid formation, ingestion of apoptotic cells, nucleic acids and viral transfection	Numerous cancers including: renal cell carcinoma, melanoma, colon cancer, lung cancer, neuroblastoma, and prostate cancer.
Monoclonal Antibodies
	Alemtuzumab	B-Cell CLL
	Anti-CD40	Non-Hodgkin’s Lymphoma, solid tumors
	Bevacizumab, Cetuximab	Colon Cancer
	Gemtuzumab	AML
	Ibritumaomab, Tositumomab	Non-Hodgkin’s Lymphoma
	Rituximab, Tastuzumab	Non-Hodgkin’s Lymphoma and Breast Cancer
Cellular Therapy
Cytokine Therapy	Antibodies to TGF-b, IL10	Glioblastoma, Non-small Cell Lung Cancer
	IL2	Melanoma, CTCL
	ONTAK	CTCL
T Regulatory Cell Control	Antibodies to: CTLA-4, GITR, TRANCE, RANK and chemokine receptors	Variety of cancers
Heat Shock Proteins	HSPPC-96	Variety of cancers

Review Article

Received: 27 October 2005; Accepted: 11 November 2005; electronically published: March 2006

I. Introduction

A. Cutaneous T cell lymphoma

B. Dendritic cells