Journal of Hepatology
Volume 45, Issue 2 , Pages 178-181, August 2006

T cell infiltration and prognosis in HCC patients

Units of Immunotherapy of Human Tumors and of Immunobiology of Human Tumors, Istituto Nazionale Tumori, Milan, Italy

published online 16 June 2006.

Article Outline

 

Hepatocellular carcinoma (HCC), the fifth most frequent tumor in the world [1], and the third most common cause of cancer-related death [2], is an aggressive tumor with poor prognosis. HCC incidence has been increasing over the last two decades in Europe, the United States, and Japan [3], and will likely continue to increase due to the world-wide diffusion of HBV and HCV infections. Available treatments include surgical resection, liver transplantation [4], and local ablative therapy. The latter approaches include percutaneous ethanol injection, thermal ablation, and intra-arterial chemoembolization, but such treatments are effective only in localized tumors [5], and 3-year survival in patients with moderate disease is less than 10% [6]. Only in highly selected patients with small tumors (<5cm in diameter or fewer than three tumors <3cm in diameter), liver transplantation has achieved an 83% 4-year recurrence-free survival [7].

In spite of the severe prognosis in most HCC patients, increasing evidence suggests that immune response to HCC, in at least a fraction of the patients, may impact on the clinical evolution of the disease and even on survival. Early evidence indicated that small tumors with marked T cell infiltrate were associated with a lower recurrence rate and higher 5-year survival rate compared to a large set of tumors without such T cell infiltration [8]. In that study, most of the lymphocytes infiltrating the tumor tissue were identified by immunohistochemistry as T cells with a predominance of the CD8+ over the CD4+ subset [8]. These initial results were consistent with a protective effect against tumor growth of T cell-mediated immune response directed to HCC-associated antigens. This hypothesis has been subsequently corroborated by the identification of antigen-specific T cells (at tumor site and in periphery) in HCC patients, with specificity for different classes of tumor-associated antigens including cancer testis [9], [10], self antigens as AFP and SART-1 [11], [12], as well as viral epitopes [13], [14].

Two papers in this issue of the Journal [15], [16] provide further evidence for the contrasting roles of the immune system in HCC. In the first study Unitt and co-workers show that lymphocyte infiltration of the tumor and a high CD4+:CD8+ T cell ratio are associated with a reduced risk of tumor recurrence following liver transplantation. By multivariate analysis the authors found that a high CD4+:CD8+ T-cell ratio had a predictive value greater than the Milan criteria [7], or tumor grade, and was equivalent to vascular invasion for predicting tumor recurrence. As the authors point out, these results are consistent with the hypothesis that CD4+ HCC-specific T cells, with a “T Helper”, rather than with a “T regulatory” phenotype, may play a significant protective role against tumor recurrence, possibly by providing help for functional differentiation of HCC-specific CD8+ T cells. In the same study Unitt and co-workers also looked at two markers of Treg cells (CD25 and Foxp3). In contrast with the significant role for tumor recurrence of lymphocyte infiltration and of the CD4:CD8 ratio, FoxP3+ lymphocytes were only found associated with the presence of vascular invasion. CD25+ lymphocytes did not follow the pattern of FoxP3 cells and the authors explained this apparent anomaly by the small sample size used and subsequent lack of power and, quite correctly, by the fact that CD25 subset was not analyzed together with the CD4 cells, thus preventing a correct counting of the double positive CD4+ CD25+ cells. However, an analysis of the presence and the role of CD8+ T cell subpopulations (e.g., central/effector memory cells) and their localization to the tumor site was not studied or discussed. In fact, previous evidence has indicated the presence of CD8+ T cells with defective expression of granzyme β and perforin in HCC patients [9], [17]. A lack of adequate CD4+ T cell help at tumor site may explain the defective CD8+ T cell maturation to effector and/or to effector memory stages. Additional studies will be needed to evaluate whether the presence of differentiated CD8+ T cells at tumor site may be a significant predictor for survival and/or of tumor recurrence in HCC patients, in agreement with reports in early colorectal and ovarian cancer patients [18], [19].

In the other study of this issue, Yang and co-workers [16] have evaluated the nature of T cells infiltrating the primary HCC, and, to this end, analyzed the presence and function of CD4+CD25+ FoxP3 expressing lymphocytes. The authors found an increase of such cells in the marginal region of the tumor, but not in unaffected areas of the same liver, or in the peripheral blood. Likewise, no increase of Tregs was observed in the liver of cirrhotic or chronic hepatitis patients. Functional experiments corroborated the authors’ conclusions inasmuch as Tregs isolated from the marginal region of HCC were able to inhibit autologous CD8+ T cell proliferation. By considering also previous reports of other groups, Yang and co-workers concluded that Tregs can inhibit the activation of tumor-specific CD8+ T cells, thereby contributing to the uncontrolled growth of HCC cells. This is an important observation that, along with those already reported for other human tumors like melanoma, NSCLC, gastrointestinal and ovarian cancer, and non-Hodgkin’s lymphomas [20], [21], [22], [23], [24], [25], [26], [27], emphasizes the potential role of Tregs in determining the prognosis of cancer patients and, therefore, the crucial function of other different tumor cytotoxic CD8+ T cell subsets in limiting the growth and progression of the neoplastic disease (see [28]).

Thus, it is the balance between CD8+ T cells and Tregs that is likely to be important for the outcome of immunotherapy, though this remains to be demonstrated in HCC patients. This conclusion is supported by studies in animal models suggesting that removal or functional blocking of Tregs in tumor-bearing hosts can result in the rejection of tumors by unleashed cytotoxic CD8+ T cells [29]. However, since subpopulations of Tregs have been described, a more detailed analysis appears necessary before making strong conclusions, as suggested by recent findings in colorectal cancer patients where the obvious correlation between Tregs and prognosis was not apparent [30]. In order to better define the role of Tregs in controlling tumor growth, we need to understand the mechanisms of their recruitment to and activation in the context of tumor microenvironment. Results obtained in ovarian cancer patients [23] indicate that Tregs expressing the CCR4 chemokine receptor can be recruited at tumor site by chemokines as CCL22, released by neoplastic cells. Expression of chemokine receptors on TIL in HCC has been reported [31], but the contribution of chemokines produced in the tumor microenvironment to Treg recruitment in HCC tissue remains to be elucidated. This issue is not addressed by these papers, thus leaving unsolved the problem of whether CD4+CD25+ T cells accumulate at the margins of HCC and of other tumors due to recognition of tumor antigens (perhaps self TAA, see [32]), or to a reaction triggered by non-specific factors (inflammation?), and which may or may not be relevant in down-regulating an activated CD8+ memory/effector tumor-specific cytotoxic T cells. Moreover, in case of HCC, none of the two articles addresses the potential role of pre-existing HBV/HCV infection (frequently present in the studied patients) in the generation of the lymphocyte infiltrate and of Treg in particular. Available evidence indicates that chronic HCV infection is associated with expansion of Treg cells that suppress CD8+ T-cell responses to different viral antigens, thus contributing to viral persistence [33]. Similarly, chronic HBV patients have been shown to have a higher percentage of Treg in peripheral blood compared to controls and such Treg can suppress proliferation of CD4+ cells to HBV core antigen [34]. It would be interesting to evaluate whether Tregs that mediate negative regulation of anti-tumor response, upon HCC development, are (or not) the same Treg previously induced by chronic HCV/HBV infection.

Likewise, no mention is made by the authors of the potential role of NKT lymphocytes (a population enriched in the liver which is a site of both T cell tolerance and effective T cell response; [35]) in determining Treg activation, a circuit described in murine but not yet in human tumors and mediated by TGFβ/IL-13 interactions [36]. Moreover, one should consider the complexity of the HCC recurrence in transplanted and immunosuppressed patients in terms of effect of the therapeutic immune suppression which may alter the physiology of activation and expansion by tumor antigens of different T cell populations, including Tregs.

What impact this new information may have on the design of diagnostic and/or immunotherapy protocols in human cancer? What therapeutic implications? If these early results are confirmed on a large number of patients by appropriate trials, the nature of infiltration of early lesion by cytotoxic CD8+ T cells vs. immune suppressive Tregs may serve as an independent prognostic factor in a variety of human tumors as suggested by the articles of Yang et al. and Unitt et al. and by other already published reports [18], [37], [38]. Moreover, the removal/deletion of Tregs should be attempted in order to improve the clinical outcome of immunotherapy trials. Early evidence that depletion of Tregs can increase at least the level of T cell response to vaccine has been recently reported for RCC patients though the consequences of such a modulation on the clinical response still need to be investigated [39]. Since immunization with autologous, formalin-fixed HCC has been shown to increase significantly the disease-free and overall survival of vaccinated patients [40], it is entirely possible that a similar or different immunization approach can be further improved by deleting Tregs in these HCC patients.

In conclusion, the two papers published in this issue of the Journal provide further evidence for the potentially contrasting roles of the host immune response in the clinical evolution of HCC patients. Moreover, they offer new insights for better defining the crucial role of T cells in the spontaneous control of human tumor growth which could be further strengthened by appropriate immunotherapeutic strategies.

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PII: S0168-8278(06)00290-X

doi:10.1016/j.jhep.2006.06.005

Journal of Hepatology
Volume 45, Issue 2 , Pages 178-181, August 2006

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