Main

Ever since the notion of tumor antigenicity emerged as an irrevocable concept,1, 2, 3, 4, 5, 6, 7 the failure of immunocompetent hosts to reject antigenic cancers has been a longstanding conundrum in the tumor immunology field.8, 9, 10, 11 Despite the troubling failure of the host to reject antigenic tumors, evidence of an antigen-specific immune response is undeniable.12, 13 Lymphocytes infiltrate many murine and human tumors, yet to the consternation of many immunologists, spontaneous regression rarely occurs. Even the significance of inflammatory cells within or surrounding solid tumors has been the subject of conflicting reports.14, 15, 16, 17

Several studies have demonstrated that elevated levels of tumor-infiltrating lymphocytes (TILs) are associated with better prognosis.14, 15, 17 However, a sweeping assumption that the influx of lymphocytes to the tumor site is invariably beneficial to the patient may be inappropriate. Recent studies suggest that the type, not the quantity, of tumor-infiltrating cells may be a more critical determinant for the prognosis.16 For example, infiltrating regulatory CD4+ cells can be more detrimental than favorable.16 Specifically, this subpopulation of TILs may impair the host's ability to defend against malignant cells.18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Likewise, antitumor lymphocytes migrating to the tumor site may become compromised once within the tumor milieu, or may adversely adapt to the suppressive environment to promote growth instead of regression. This review will rigorously examine the subtypes of T lymphocytes that behave either as friends or as foes in antitumor immunity. A detailed understanding of the functional peculiarities of individual T lymphocyte subtypes may explain the paradox that the presence of TILs does not always correlate with improved prognosis and may also allow the development of targeted approaches that specifically augment antitumor immune responses.17, 30, 31, 32, 33, 34, 35, 36, 37, 38

Antitumor functions of T lymphocytes

The mature T-cell population is composed of αβ T cells expressing CD4 or CD8, and the CD4−/CD8− γδ T-cell receptor (TCR)-expressing cells. The unique function of CD4+ and CD8+ T cells is dictated by the expression of these coreceptors, CD4 or CD8, for which the ligand is the β2 domain of the major histocompatibility complex (MHC) class II molecule39 and the α3 domain of MHC class I molecules,40 respectively. Due to these specificities, the αβ TCR of CD8+ T cells is restricted to the recognition of antigens presented by MHC class I molecules and the αβ TCR of CD4+ T cells to antigens presented by MHC class II molecules. However, relatively little is known about the function of γδT in antitumor immunity so far; thus, this review will mainly focus on the αβ T cells.

The important role of T cells as effectors in antitumor immunity was first shown in numerous murine models. For instance, UV light-induced tumors are rejected regularly by normal mice, but grow progressively in the absence of T cells.41, 42, 43 In human melanoma patients, a brisk TIL response is also of prognostic significance. It has also been demonstrated convincingly that T-cell-mediated immunity is essential for the rejection of virally and chemically induced tumors.5, 7, 44, 45, 46, 47 For example, with murine model tumors induced by the chemical carcinogen methylcholanthrene (MCA), intravenous injection of immune cells, but not of immune serum, can transfer systemic tumor-specific immunity into sublethally irradiated mice.5 These results are consistent with studies showing that the protective immunity against a plasma-cell tumor was abolished by prior depletion of T-lymphocytes via anti-T-cell antibodies and complement.47

The importance of T cells in tumor immunity has also been implicated in human studies, albeit with discernable limitations. While most murine tumor models have the advantage of utilizing antigen-specific T cells generated from tumor-free syngeneic mice, generating a human homolog is not feasible. Thus, in human studies, T lymphocytes are isolated from peripheral blood48, 49 or from the tumor50 of cancer patients. Such T cells can react in vitro with autologous cancer cells.51 Utilizing adoptive transfer of in vitro expanded TIL in combination with chemotherapy, recent clinical trials have shown up to 50% positive response rates in selected patients with late-stage aggressive cancers.52, 53, 54, 55

CD8+ T cells in tumor immunity

While the significance of lymphocytes in tumor immunity is rarely disputed, the relative importance of various T-cell subsets in tumor rejection is the subject of great controversies.56 Broadly speaking, since cancer is a disease caused by an array of mutations in various types of cells, differences in the T-cell subsets required for mediating tumor rejection are not altogether surprising. One such subset is the CD8+ cytotoxic T lymphocytes (CTLs). Most tumors are positive for MHC class I but negative for MHC class II, and CTLs are able to induce tumor killing upon direct recognition of peptide antigens, presented by the tumor's MHC class I molecules. Thus, the initial attention to antitumor immune responses was preferentially given to CD8+ T cells. That the CD8+ T cells are critical effectors against tumor cells is further supported by numerous studies in murine models. For instance, in UV light-induced tumors the CD8+ cytolytic T-cell subset appears to be required for rejection.41 Murine CTLs that kill tumor targets in vitro can be freshly isolated from mice after repeated intraperitoneal injection of antigenic tumor cells, or can be generated ex vivo in a 7-day mixed lymphocyte-tumor cell culture. Elimination of CD8+ T cells from mice—either via depleting antibodies or using genetic knockout mice—can, at least partially, abrogate the antitumor immunity induced by most cancer vaccines.57, 58, 59, 60 Consistent with the murine models, clinical data from cancer patients uphold the importance of CD8+ T cells in bringing forth an antitumor response. Adoptive transfer studies of in vitro stimulated CD8+ T-cell lines and CD8+ clones specific for tumor antigens effectively mediate antitumor immunity when transferred back into tumor-bearing hosts.10, 13, 52 Furthermore, recent reports suggest that immunization, using either adjuvant or dendritic cells (DCs) with pure tumor peptides, can result in productive antitumor immunity that is restricted by MHC class I.61, 62 Taken together, CD8+ T cells in tumor immunity can be unquestionably heralded as one of the principal subsets of T cells that constructively mediate an effective antitumor response (see Figure 1).

Figure 1
figure 1

Proposed functions of T cells in tumor immunology. The CD8+ cytotoxic lymphocyte (CTL) is a well-documented effector of tumor immunity, interacting with tumor cells through the MHC-I receptor (not shown). CD4+ T-helper cells can help prime CTL, both directly and through stimulation of professional antigen-presenting cells (APC). The APC, in turn, present tumor antigens to the CTL. Both effects of CD4+ T-helper cells promote the host immune response to tumors. An additional proposed effect of CD4+ helper cells is the maintenance of a subpopulation of CD8+ T cells as circulating ‘memory’ cells and possibly enhanced conversion of memory cells back into functional CTL upon re-exposure to tumor antigens. However, a population of CD4+CD25+ Treg cells is capable of downregulating the action of CTL. These Treg cells may reside within the tumor microenvironment. The role of tumor-induced inflammation or recruitment of CD4+ T cells from the circulation in generating Treg cells is unclear. Regardless, TIL may induce both CTL and Treg cells, making assessment of TIL a critical clinical issue.

Full size image

Helper and effector role of CD4+ T cells

Undeniably, CD4+ T cells are an integral part of adaptive immunity, but the specific role they play in mounting an antitumor response remains a subject of debate. The critical function of CD4+ T cells in promoting immunity has been consistently demonstrated by vaccine and challenge experiments employing antibody-mediated depletion of CD4+ T cells, or by using CD4-knockout mice.57, 58, 59, 60, 63, 64, 65, 66, 67, 68 More importantly, similar to CD8+ T cells, tumor-specific CD4+ T cells that can recognize tumor antigens do exist and data show that these T cells migrate to the tumor site in both murine and human cancers.69, 70, 71 However, complications arise when the accumulation of CD4+ T cells within the tumor microenvironment during tumor progression seemingly hinders the effector function of CD8+ T cells.18, 72, 73, 74, 75 The hallmark phenotype of the CD4+ T cell that impedes the antitumor response seems to be attributable to a regulatory function.16, 76, 77, 78, 79 This paradoxical dualism of CD4+ T cells obligates further differentiation of this subtype into helper and regulatory CD4+ T cells.

Although CD4+ T cells have been shown to be sufficient to eliminate tumor cells in the absence of CD8+ T cells in some tumor models,70, 80, 81, 82, 83 it is more often the case that both CD4+ and CD8+ are required for effective tumor rejection to occur.84 In part, this is due to a substantial portion of tumor cells expressing only class I MHC molecules, not class II molecules, thereby limiting direct recognition by the CD4+ T cells. Moreover, the predominant effector mechanism in tumor immunity is direct lysis of tumor cells by the MHC class I recognizing CD8+ CTL. The role of CD4+ T cells, in antitumor responses is often to aid in the activation of CD8+ T cells, leading to the destruction of the tumor by CD8+ CTL. The CD4+ T-cell help of the CD8+ CTLs in tumor immunity can be divided into three phases: early induction, effector maintence, and memory.

CD4+ T cells help CTL induction

It has been debated for decades if CD4+ T cells are required for the priming of CTL. Accumulating evidence has indicated that for the induction of tumor-specific CD8+ T-cell responses, cross-presentation of antigens that have been captured by professional antigen-presenting cells (APCs) such as DCs plays a dominant role.85, 86, 87, 88, 89 CD4+ T-cell help has been hinted to be essential for such cross-priming in the induction of CTL immunity. This requirement for CD4 help is believed at least in part to activate APCs,90 which in turn express costimulatory molecules such as ICAM-1,91 CD80, and CD86,92 or to secrete cytokines including interleukin (IL)-12.92, 93 These factors are essential for better CD8+ T-cell activation. Most T-cell help for CTL priming is dependent on the interaction between CD40 ligand (CD40L) expressed by CD4+ T cells and CD40 on APC.94, 95, 96, 97 The CD40–CD40L interaction has also been proven to be important in the generation of protective T-cell-mediated tumor immunity.98, 99 The requirement for CD4+ T cells to ‘license’ APC for the priming of CTL helps explain some scenarios in which the induction of CTL can be achieved in the absence of CD4+ T cells. The typical CTL priming that is independent of CD4+ T cells is via the direct activation of DCs by virus that provides the optimal inflammatory signal to activate DCs,100 which can subsequently prime antigen-specific CTL responses in the absence of CD4+ T cells.101 CD4-independent CTL induction via activated DC is further demonstrated by data showing CD40-mediated activation with soluble ligand, or activating antibodies which ‘license’ DC for cross-priming in the absence of CD4+ T cells.98, 99

However, these observations have not been confirmed in all experimental models. One study has shown that injection of MHC class I-deficient tumor cells into MHC class II knockout mice resulted in the induction of tumor cell-specific CTL responses,102 demonstrating that cross-priming can occur even in the absence of CD4+ T cells, albeit with decreased efficiency. Hence, it is still possible that the requirement for CD4 help was bypassed in this model because DCs were activated by cellular components released during tumor cell apoptosis, which can serve as adjuvants.103

Other evidence seems to suggest that high levels of antigen can bypass the requirement for CD4 help in CTL induction. For example, RMA-S cells loaded with an MHC Class I-restricted peptide can induce CTL responses in vitro. The observed in vitro CTL priming was independent of CD4+ T cells or MHC class II-expressing cells, but was dependent upon the level of MHC class I expression on the RMA-S cells.104 A high expression level of MHC class I peptide complexes on the peptide-presenting cells was the decisive requirement for the induction of CD4+ T-cell-independent CTL response in this model. This hypothesis would explain other observations in the vaccination experiment using MHC Class I-restricted peptides emulsified in noninflammatory incomplete Freund's adjuvant (IFA), whereby robust CTL function was induced in mice depleted of CD4+ T cells.105

Even with high level of antigen provided for CTL priming, other conditions may be required for CTL induction in the absence of CD4 help. We have shown that CD4 help is dispensable for cross-priming of CTL when intact draining lymph nodes are available.88 However, in the absence of draining lymphoid tissue, this additional ‘help’ becomes essential for the proliferation of naïve CD8+ T cells.88

Other observations appropriately illustrate that the CD4+ help even in the presence of activated DCs cannot be altogether excluded or ignored, suggesting that CD4+ T cells may play roles other than ‘licensing’ DCs in CTL induction. For example, in experiments utilizing vaccinations with OVA-transduced CD40-activated DCs, only in the presence of CD4+ T cells was there protection against formation of ovalbumin (OVA)-expressing tumors.106 Furthermore, in some studies, vaccination with transduced DCs or DCs pulsed with peptides, tumor lysates, or tumor cell-derived exosomes has been shown to be at least partially CD4+ T cell-dependent, although it is possible that DCs were not properly activated prior to injection.107, 108, 109, 110

In summary, the available data suggest that CD4+ T cells are generally required for CTL priming when DCs are not activated through another mechanism. However, other conditions, such as the level of antigen provided or the integrity of draining lymphoid tissues, may also impact on the requirement for CD4 help. Nevertheless, CD4+ T-cell help has been considered essential for the induction of CTL responses against tumors in most cases. Given the noninflammatory condition of the majority of cancers in addition to the unavailability of tumor antigens for cross-presentation,89 DCs require activation by CD4+ T cells before they can induce the full activation and differentiation of naïve CD8+ T cells into CTLs.

CD4+ T cells help maintain a CTL response

While the necessity of helper CD4+ T cells in the induction of CTL remains a controversy, it is generally accepted that CD4+ T cells are critical for the maintenance of CTL in both virally directed and tumor-specific immune responses.111, 112, 113, 114 As clearly demonstrated in human cytomegalovirus (CMV) infection, the survival of adoptively transferred anti-CMV CTLs is dependent on the presence of CMV-specific CD4+ T cells persisting in the host.115 For the host to perpetually sustain an effector CTL function to counter a persisting virus infection, help from CD4+ T cells may be required either through cytokine secretion or stimulation that is independent of DC help. In certain reports, the need for helper CD4+ T cells can be replaced by exogenously provided IL-2,116, 117, 118 suggesting that the requirement for CD4+ T cells may simply be to supplement CD8+ T cells with IL-2, a cytokine traditionally thought to be essential for promoting growth and proliferation of T cells. Whether IL-2 is the primary mechanism or a byproduct utilized by CD4+ to provide help for the maintenance of CTL remains to be determined. However, the contribution of CD4+ help in sustaining a tumor-specific CTL response is unequivocally recognized as an important means to an end. There is no shortage of corroborating data from therapeutic vaccination and adoptive transfer studies that implicate CD4+ help in sustaining viable CTL function.

In some murine models, while tumor growth was prevented by vaccinations with peptide-pulsed DC-mediated CTL induction, the same therapeutic immunization against an established tumor (a scenario mimicking the chronic persistence of antigen) required CD4+ T-cell help.114 Similarly, while adoptive transfer of tumor-specific CTLs prevented tumor formation in CD4+ T cells depleted or MHC class II knockout mice, systemic metastases (a condition that may require persistence of effector CTLs) could not be cured unless the hosts bear CD4+ T cells.119 Thus, as the tumor matures, CD4+ help becomes vital to the persistence of the effector response. Published reports on clinical trials of adoptive immunotherapy further support this concept.55 Cotransfer of CD4+ T cells with CD8+ T cells expanded from autologous tumor-infiltrating T cells prolonged the survival of the adoptively transferred T cells.55 Taken together, it is evident that the longevity of a tumor-specific CTL response is enhanced by the presence of CD4+ T cells.

Role of CD4+ T cells in the induction and maintenance of CD8+ T-cell memory responses

After the clearance of antigen the majority of effector CTLs undergo apoptosis, while a minor portion convert into lymphocytes with a memory phenotype. Thus, upon a second antigen encounter, CTL response propagates with alacrity and potency to provide the host with ‘anamnestic’ protection. It is generally accepted that memory cells persist in circulation subsequent to an effector response. However, the role of CD4+ T cells in the induction and maintenance of memory CTLs has been controversial. While recent publications seem to have reached a consensus that CD4+ T cells are indispensable for an intact CTL memory response,120, 121, 122, 123, 124 several studies have suggested that CD4+ T cells are required in the primary antigenic encounter in order to ‘program’ the CD8+ T cells to differentiate into long-lived, functional memory cells.120, 121 Others have argued that CD4+ T cells are required after antigen is eliminated to maintain the number or normal functions of CD8+ memory T cells.123, 124

In regard to achieving an anamnestic CTL response, one earlier study reported a CD4+ T- and B-cell-independent persistence of memory H-Y-specific CTLs.125 Two other groups also found that virus-specific peptide-tetramer-positive memory cells persisted in CD4 or MHC class II knockout mice using viral infection models.100, 126 However, despite the clear presence of virus-specific CD8+ memory T cells, both groups were unable to revert memory T cells into effector T cells in the absence of CD4+ T cells.100, 126 In other words, the memory phenotype CD8+ T cells were nonfunctional. Similar results were obtained in the previously described mouse H-Y model, whereby exogenous cytokine addition was required to revert memory cells back into CTLs in the absence of CD4+ T cells.125 These earlier findings suggest that CD4+ T cells were essential for the conversion of memory CTLs into effector CTLs upon secondary antigen encounter. Other groups have expanded on the aforementioned studies by utilizing adoptive transfer experiments to emphasize the necessity of CD4+ in sustaining the memory cells in the circulation. They complemented earlier studies with MHC class II knockout mice in adoptive transfer of memory T cells, by illustrating that, in the absence of CD4+ help, reactivation into effector CTLs could not occur.119 While these earlier studies did not address whether CD4+ T cells were required in the priming phase of CLT to ‘program’ them to later develop into long-lived functional memory cells, recent studies by Bevan's group revealed that CD4+ T cells are not required during the priming of CTL, but only are required at a later stage for the maintenance of memory CD8+ T cells.123

However, two recent publications by Shedlock et al121 and Janssen et al120 supported the idea that CD4+ T cells are only essential during the primary antigen encounter to ‘program’ the CD8+ T cells to differentiate into long-lived, functional memory cells. They show that adoptively transferred CTLs can survive and function properly in an environment without CD4+ T cells after being primed in their presence.120, 121 In addition, CD4+ T cells were not required for the secondary activation of CTLs. Taken together, the data available suggest a general requirement for CD4 help to generate a healthy CD8 memory. However, the stage at which CD8 T-cell responses require CD4 help and the nature of such help remain unresolved.

Th1 vs Th2 responses for anti-tumor immunity

CD4+ T-cell responses can be divided into different types depending upon their cytokine profile.127 The defined Th1 cells are characterized especially by the production of interferon (IFN)-γ, whereas Th2 cells produce IL-4, IL-5 and other cytokines. The balance between Th1 and Th2 cytokines has definite influence on the outcome of various immune responses, as Th1 preferentially induces cellular immunity and Th2 tends to elicit humoral immunity.128 The cytokine IFN-γ impacts positively on antigen processing and presentation because MHC class I and II—and the expression of several other molecules such as transporter associated with antigen processing (TAP) and proteasome components—are under the control of this cytokine.129 Therefore, the Th1 response is generally correlated with a better cellular and CTL response. Since a cellular immune response is preferable for tumor destruction, a Th1 response has been proposed to be beneficial for antitumor immunity. Several reports have supported this idea and demonstrated the parallelism between the generation of a Th1 response and a stronger antitumor immunity.130, 131, 132, 133, 134 A Th1 response has even been shown to be essential for antitumor immunity, and Th2 cytokines downregulate antitumor immunity in some reports.134, 135, 136 The concept of immune deviation, namely, a shift from a Th1 to a Th2 cytokine profile, has been hinted to be one of the major contributors to the failure of T-cell-mediated immunity against tumors. Indeed, immune deviation to Th2 cytokine production has been reported in progressive cancer patients.137 On the contrary, an immunization-evoked Th2 to Th1 change was shown to induce tumor rejection in a murine tumor model.138 Additionally, Th2 cytokines have been shown to promote tumor growth in several experimental models.138

However, as in most immunological situations, one can never irrevocably conclude one way over another. There are plenty of data supporting the opposite: Th2 cytokines have been shown to be helpful for cancer gene therapy,139, 140 and tumor-specific Th2 clones have been demonstrated to exhibit strong antitumor activity in vivo.141, 142, 143, 144 The mechanisms of how Th2 helper T cells destroy tumors are not yet established, but there is some evidence to suggest that the antitumor effect is mediated through the activation of innate immune cells such as eosinophils and macrophages by Th2 CD4+ T cells, which in turn secrete superoxide and nitric oxide.145 Although an effective antitumor immunity would be preferentially mediated through a Th1 response resulting in a direct killing by CTL, the results published up to this point seems to be directed towards a cooperative balance between the Th1 and Th2.

CD4+ regulatory T cells within the tumor environment

Succinctly stated, the immune system is constantly working to find a balance between Th1 and Th2, activation and apoptosis, and proinflammatory and anti-inflammatory conditions. It has recently come to light that during tumor progression the tumor microenvironment becomes host to such a balancing event, specifically the balance between the effector and the regulatory response. Although not much is known regarding T-cell regulatory function pertaining to the tumor immunity, there is some evidence to suggest that the subtype of T cells responsible for ‘regulating’ the effector immune response within the tumor site is similar to the now well-characterized regulatory CD4+ T cells involved in autoimmunity.16, 18, 75, 76, 77, 78, 79

Existence of different types of CD4+ regulatory T cells in vivo

During the past decades there has been much speculation and some evidence suggesting the existence of suppressor T cells, but these cells were only recently identified phenotypically. A major advance resulted from the discovery by Sakaguchi and colleagues, and later confirmed by others, that depletion of this small subset of CD4+ T cells, distinguished from CD4+ helper T cells by the expression of high levels of CD25 in naïve mice, could induce organ-specific autoimmunity. Adoptive transfer of CD4+ CD25+ T cells could prevent the development of organ-specific autoimmunity.146, 147, 148 These data established the regulatory nature of these cells, now defined as naturally occurring CD4+ CD25+ T regulatory cells. This subset of cells has been established to be a powerful regulator of T-cell responses in organ-specific autoimmunity and chronic infections.149, 150, 151, 152, 153 However, it is becoming increasingly clear that, in many situations, CD4+CD25− T cells are as effective as CD4+CD25+ T cells in controlling T-cell-mediated disease.154, 155, 156 There are other subsets of CD4+ T cells that exhibit regulatory phenotypes, including CD4+CD45Rblow suppressor cells that secrete large quantities of IL-10 and IL-4 (termed Tr1 cells) and CD4+ suppressor T cells that secrete large quantities of transforming growth factor (TGF)-β (termed Th3 cells).157, 158 The immune-regulatory potential and functional significance of these cytokine-secreting CD4+ T cells are supported by the findings that TGF-β-deficient mice develop autoimmune disease159 and administration of neutralizing antibodies to IL-4 or TGF-β abrogates the in vivo prevention of autoimmunity or tolerance-inducing activity of CD4+ T cells in tumor models and some autoimmunity models.18, 160, 161 It remains to be determined, however, to what extent these subtypes of CD4+ cells diverge from naturally occurring CD4+CD25+ T cells in regulatory function and significance.

Markers to identify CD4+ regulatory T cells in vivo

There are no known cell surface molecules that uniquely distinguish the CD4+ regulatory T cells (Treg) from conventional activated CD4+ cells. For example, the CD25 molecule, which is the α-chain of the IL-2 receptor, is expressed on all peripheral antigen-reactive CD4+ T cells from one to several days following antigen activation. Moreover, many of the other cell-surface molecules in addition to CD25, including the tumor necrosis factor (TNF)-family member glucocorticoid-induced TNF receptor (GITR) and cytotoxic T-lymphocyte antigen-4 (CTLA4)162, 163 that seem to distinguish CD4+CD25+ from CD4+CD25– effector cells, are upregulated on CD4+CD25– T cells following antigen activation. In this regard, it is of great interest that a recently cloned transcription factor, termed Foxp3, a member of the forkhead family of DNA-binding transcription factors, is not expressed in naïve CD4+CD25– cells, but is highly expressed in the naturally occurring CD4+CD25+ regulatory cells. More importantly, mutational defects in the Foxp3 gene result in the fatal autoimmune and inflammatory disorder of the scurfy mouse and in the clinical and molecular features of the immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX syndrome) in humans.164, 165, 166 In Foxp3-overexpressing mice, both CD4+CD25– and CD4− CD8+ T cells show suppressive activity, which suggests that expression of Foxp3 is linked to suppressor functions.167, 168, 169 Taken together, these data strongly support the idea that Foxp3 may uniquely define the subset of CD4+ Treg in mice167, 168, 169 and humans.170 However, the recent findings that Foxp3 can be expressed in CD4+CD25– cells following activation and is also expressed in activated CD8+ T cells suggest that Foxp3 is linked to functional suppression, but is not necessarily a specific lineage marker.170, 171, 172, 173 Whether a specific lineage marker even exists for these CD4+ regulatory cells awaits to be determined, but what can be concluded with little doubt is that a subset of CD4+ T cells exist that regulate an inflammatory immune response to cancer.

Tumor-induced CD4+ Treg

Cancers generally develop over a long period of time. In addition, the major pathophysiologic characteristics of malignant cancer, invasion across natural tissue barriers and metastasis, are often associated with the disruption of normal tissue architecture leading to the initiation of inflammatory responses. In this regard, cancers can very much resembled to a chronic inflammatory response. In view of this, one can speculate that the anti-inflammatory mechanisms that are turned on at the beginning of invasion coupled with the internal mechanisms controlled by malignant cells to produce cytokines like TGF-β set off a regulatory reaction, which may inhibit antitumor immunity. Given such events, the loss of regulatory function by depletion of tumor-induced CD4+ Treg may enhance the effector response, resulting in tumor rejection.

Recent findings specifically attest to possible negative regulatory roles by CD4+ T cells within the tumor environment. In some spontaneous tumor models, the presence of CD4+ T cells seems to promote cancer development instead of inhibiting it.174 One study by Schreiber's group has shown that active immunization with antigen-specific CD4+ T cells in cancer-prone mice carrying a germline mutant ras oncogene resulted in immune responses that fail to eradicate mutant oncogene-expressing tumor cells, and instead induced a remarkable enhancement of tumor growth.175 Similarly, the studies by North73, 74 have shown that suppression by CD4+ T cells led to the progressive growth of an immunogenic tumor and intravenous depletion of these cells reversed the suppression and elicited CD8+ T-cell-mediated antitumor immunity.72 While the presence of CD4+ T cells in some models has been shown to be deleterious to the onset of tumor immunity, the necessity of CD4+ T-cell help in mounting an effector immune response cannot be negated. In some studies, the depleting antibody given during the early stages of tumor growth was damaging to the generation of an immune response against the tumor, especially if T-cell help was obligatory.19, 21 These obsevations strongly suggest the existence of functionally distinct CD4+ T-cell subsets.

CD4+CD25+ Treg in mice and humans

The above studies have suggested that CD4+ CD25+ suppressor cells are relevant to tumor immunology, albeit more detrimental than favorable for the host. Most studies up to recent times have utilized depletion of the entire CD4+ population, resulting in enhancement of immunity against tumors via the depletion of the suppressive CD4+CD25+ T-cell population. In vivo experiments in several murine tumor models demonstrate that more specific depletion of CD4+CD25+ T cells by anti-CD25 antibody treatment prior to tumor challenge significantly enhances the efficacy of vaccine-induced antitumor immunity.20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Another study revealed that splenic cells depleted of CD4+CD25+ T cells can mediate tumor regression, presumably through promoting autoreactivity because autoimmune diseases were also induced.23 These studies suggest that Treg may inhibit initial priming of CD8+ T cells, some of which recognize tumor antigens. One study demonstrated more clearly that the CD4+CD25+ T cells prevented priming of CTL, as depletion of CD25+ T cells had to be performed within the first 2 days after tumor inoculation, suggesting that suppression was ineffective once the priming was initiated.24 It was also shown that elimination of these Treg, despite causing increased autoreactivity in some cases, could increase immune responses to tumors such as melanomas overexpressing self-antigens.21, 22, 23, 26 Even when the host bears a poorly immunogenic cancer, concomitant immunity can be rescued by systemic depletion of the CD4+CD25+ regulatory T-cell subset.19 These observations in the tumor models are consistent with the features that have been defined for CD4+ CD25+ Treg in other disease models.

It has been demonstrated that the equivalent of CD4+CD25+ Treg identified in mice also exists in humans. These cells, CD4+CD25+CD45RO+ T cells, represent about 6% of CD4+ T cells and are present in the blood of healthy human adults.76 Prevalence of Treg is increased in peripheral blood and the tumor microenvironment of patients with pancreas or breast adenocarcinoma.77 The CD4+CD25+ T cells possessing regulatory properties have also been reported to be among the tumor-infiltrating T cells in different types of human lung, ovarian, pancreas, breast and gastrointestinal cancers, and lymphoma.78, 79, 176, 177 Antigen-specific activation and cell–cell contact were required for these clones of Treg cells to exert suppressive activity on CD4+ effector cells. The presence of CD4+ Treg cells at tumor sites suggests that they could have a profound effect on the inhibition of T-cell effector responses against some human cancers.16, 18, 75

Immune suppression occurrs inside tumor tissues

It is possible that different subsets of CD4+ T cells, either by providing ‘help’ or ‘regulation’, predominate at various stages of tumor progression. A recent study by our group18 has shown that suppression of immunity against tumors mainly occurs in the effector phase at the tumor site and depletion of the Treg at the late-stage of tumor progression did not mitigate the possible T-helper function. We found that a highly antigenic tumor that expresses a strong antigen on the surface, yet fails to regress in the host, induced an accumulation of CD4+ Treg within the tumor microenvironment. With tumor persistence, there was inhibition of CD8+ T-cell function. In this model, local intratumoral depletion of these CD4+ Treg unmasked the immunogenicity of tumor and reversed the CTL tolerization, leading to the rapid rejection of well-established tumors. From this study, we propose that CD4+ cells predominantly play an enhancing helper role during the initial stages of tumor progression, but once tumors become chronically persistent, the increased accumulation of CD4+ Treg inhibits CD8+ cell function and mask the immunogenicity of tumor. In fact, depletion of Treg unveils the immunogenicity of tumor cells and provides long-term protection against re-challenge of even poorly immunogenic parental tumor cells. This result suggests that the depletion of Treg promoted immunity against previously poorly immunogenic tumor antigens and expanded the tumor-reactive CD8+ T-cell repertoire. Our study revealed that the population of tumor-infiltrating cells is skewed to favor regulatory CD4+ T cells over the helper CD4+ T cells within the tumor tissue, especially as tumor progresses and becomes established in the host.

Origins of tumor-induced CD4+ Treg

It is not clear thus far whether CD4+CD25+ T cells inhibiting antitumor immunity in mice and humans are naturally occurring Treg or are generated in the periphery. It is possible that the tumor microenvironment preferentially recruits naturally occurring CD4+CD25+ T cells. One study published recently suggested that the chemokine CCL22, abundantly expressed in ovarian cancer tissues and tumor ascites cells, preferentially attracts the CD4+CD25+ T cells identified in the ovarian cancers.16 These cells were positive for the transcriptional factor Foxp-3 and exhibited regulatory function similar to the naturally occurring CD4+CD25+ T cells. It is not yet clear whether these tumor-infiltrating Treg can be defined as the counterpart of the naturally occurring CD4+CD25+ T cells in the mouse. Another possibility is that the tumor microenvironment converts CD4+ T cells to CD4+CD25+ Treg or expands naturally occurring CD4+CD25+ T cells. There is evidence to indicate that tumor-specific CD4+ T cells change their phenotype from effectors to suppressors during cancer progression.178 Conversion from effector cells coincided with a substantial reduction in the antigen expression level, resulting in tumor persistence that ultimately led to T-cell tolerance. The authors have evidence to suggest further that these antigen-specific T cells became CD4+CD25+ Treg.178

It may therefore be that the processes of immunosurveillance and tumor editing coexist with a process in which the functional tumor-specific T-cell repertoire is also edited by the tumor environment, to the ultimate benefit of tumor progression. Besides anti-inflammatory cytokines, the CD4+CD25+ Treg inside the tumor may suppress antitumor immunity via other mechanisms. For example, these ‘Tregs’ may inhibit the immune response through their ability to control T-cell numbers because they have been shown to regulate T-cell proliferation in vitro.152, 153 Whether the regulatory cells that accumulate in the tumor site are ones that naturally exist in the host, or whether they initially arrive as helper CD4+ T cells, but convert to regulatory cells by encountering the suppressive tumor environment is not altogether clear. It would be beneficial to better characterize the nature of CD4+ T cells isolated from or present in the tumor tissues by surface markers and cytokine profiles, in order to utilize the CD4+ helper T cells instead of CD4+ Treg for adoptive transfer immunotherapy and potentially improve the prognosis of cancer patients.

Targeting tumor tissues to recruit and train T cells

Tumors often form a barrier that limits T-cell infiltration and reduces drainage of tumor antigens to lymph nodes. Since positive roles are played by CD8+ T cells and CD4+ helper T cells in antitumor immunity, it is reasonable to speculate that infiltration of T lymphocytes and initiation of antitumor immune responses inside tumor tissue at an early phase are highly favorable. Specifically, it could increase the T-cell repertoire and provide more antigens to stimulate T cells inside tumor, and could therefore be a strategy for cancer immunotherapy. Several cytokines or chemokines have been used to attract and activate T cells at tumor sites. In this section, we will focus on our recent finding that stimulation of lymphotoxin (LT) β receptor (LTβR) inside tumor tissues promotes strong infiltration of immune cells, leading to tumor rejection.

LTβR plays an important role in the formation of lymphoid structures.179, 180, 181 LTβR is activated by two members of the TNF family, membrane LT αβ and the LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT).182 Signaling via LTβR regulates the expression of chemokines and adhesion molecules within secondary lymphoid organs. For example, such chemokines and adhesion molecules control the migration and positioning of DCs and lymphocytes in the spleen.183, 184 Therefore, it is possible that enhanced LTβR signaling inside tumor tissues may promote the formation of lymphoid-like structures for direct T-cell sequestration. TNF receptor (TNFR) signaling may also play a similar, but less effective role in regulating chemokine expression.184 However, TNFR signaling may have a more toxic effect, as has been seen in other systemic TNF treatments without extra costimulation.185 Soluble LTα can signal through the TNFR, resulting in the upregulation of chemokines. To avoid such a toxic effect, recombinant LTα has been conjugated with antibody targeted specifically to the tumor tissues, resulting in an effective antitumor immune response associated with the induction of peripheral lymphoid-like tissue.186 However, LTα lacks a costimulation function, which would result in a less effective activation of recruited naïve T cells in the lymphoid-like structure inside tumor tissues.

LIGHT is a ligand for LTβR and herpes virus entry mediator (HVEM).182, 187 LIGHT is predominantly expressed in lymphoid tissues, especially on the surface of activated DCs and T cells. LIGHT is a strong costimulatory molecule.182, 188, 189 Our data indicate that the interactions between LIGHT and LTβR restore lymphoid structures in the spleen of LTα−/− mice. In addition, upregulation of LIGHT causes T-cell activation and migration into nonlymphoid tissues and formation of lymphoid-like structures.190, 191 Therefore, a LIGHT-mediated microenvironment inside a tumor could be effective in both recruiting and activating naïve T cells. The expression of LIGHT in the tumor environment induces a massive infiltration of naïve T lymphocytes that correlates with an upregulation of both chemokine production and expression of adhesion molecules. Activation and expansion of these infiltrating T cells leads to the rejection of established, highly progressive tumors at local and distal sites.192 Our study indicates that induction of T lymphocytes and initiation of antitumor immune responses inside the tumor tissue may be an effective strategy for cancer immunotherapy.

Conclusion: incorporating two strategies to promote tumor regression

The balance of antitumor effector T cells vs Treg may be critically important in determining the outcome of immune responses within tumors. Our recent study has demonstrated that rapid recruitment of naïve lymphocytes and expansion of CD8+ T cells inside tumors may be a way of creating a dominant proinflammatory environment, leading to tumor rejection at local and distal sites.192 We further demonstrated that the depletion of Treg inside the tumor is another efficient way of converting the local anti-inflammatory environment to a proinflammatory one.18 From a clinical-therapeutic point of the view, local treatment to eliminate Treg has certain critical advantages over systemic treatment. First, local treatment may avoid side effects induced by systemic depletion of CD4+ T cells, which may abrogate T helper-mediated protective immunity against pathogens. Second, treatment of the local tumor environment would not hinder effective priming of CD8+ T cell in lymphoid tissues by helper CD4+ T cells, since depletion remains local. Third, the local treatment would be expected to be more effective if suppression of CD4+CD25+ Treg resides inside the tumor. CD4+CD25+ T cells have been shown to be present in a variety of human cancer tissues78, 79, 176, 177 and these cells are negatively associated with the prognosis of the ovarian cancer patients in addition.16 Therefore, Treg within the tumor environment represent an attractive target, and their depletion may lead to improvements in the current immunotherapy protocol in the future clinical trials. Finally, intratumoral treatment would reduce the dose of depleting agent, for example, antibody, required. It is likely that a combination treatment which would rapidly expand the effector cells at the tumor site, while locally depleting the regulatory cells, could constitute a potent strategy for enhancing antitumor immunity and promote a clinically desirable outcome for cancer patients.

It is increasingly clear that some TILs may be friends, while others may be foes. An increase in TIL may not always be associated with a better prognosis. Accumulation of Treg inside tumor tissue both in animal models and in human patients suggests that an aberrant immune response can occur inside growing tumors. Strategies to effectively reverse the immunologically suppressive environment (eg, depleting Treg inside tumors) may be a new way to enhance the effectiveness of immune responses against tumors at local and distal sites.

Further laboratory investigation is critical for us to better understand the role of TIL. A hypothesis to be further tested is if cancer tissue contains few TIL, local treatment to increase TIL in tumor tissue, especially CTL, may be advantageous. On the other hand, depletion of T regulatory cells may be important for patients with cancer tissues containing abundant T regulatory cells. Pathologists of the future may play critical diagnostic roles in determining the type of TIL infiltrates, thereby providing both prognostic information as well as guidance in selecting an appropriate immunotherapeutic strategy for each patient.