Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.     

Characteristic Immune Response in Peyer’s Patch Cells Induced by Oral Administration of Bifidobacterium Components

We demonstrate immunomodulatory effects, especially those involving murine intestinal IgA secretion, in Peyer's patch cells following oral administration of Bifidobacterium immunomodulator (BIM) derived from sonicated B. pseudocatenulatum 7041. BALB/c mice were administered BIM orally for 7 consecutive days. The PP cells demonstrated upregulated secretion of total IgA including BIM-specific IgA following BIM administration. In observing the response of PP cells co-cultured with BIM, we found enhanced secretion of interferon-γ (IFN-γ) and interleukin (IL)-6 in the CD4⁺ T cells. In contrast, IL-12 secretion by Thy1.2⁻ PP cells was enhanced, but secretion of IFN-γ, IL-5, and IL-6 was not significantly affected. Furthermore, the population of CD4⁺ CD45RB^high T cells in PP increased following oral administration of BIM. These data suggest that CD4⁺ T cells were affected by BIM administration. Overall, the results show that oral administration of BIM induced CD4⁺ PP cells to change their expression of cell surface antigen and cytokine production.     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Characterization of a murine thymic CD4+ T cell subset-TCRαβ+ 3G11-6C10- CD4+ CD8-thymocytes

The presence of a relatively mature CD4⁺ CD8⁻ (SP) T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69^+/−, HSA^med/lo and heterogeneous for Qa-2 expression. The proliferation capability of TCRαβ⁺ 3Gl l⁻ 6C10⁻ CD4⁺ CD8⁻ thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4⁺ subset of 3G11⁻ 6C10⁻ NK1.1⁻ phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from Th0 to Th2, with a propensity to Th2. Project supported by the National Natural Science Foundation of China (Grant No. 39730410).     

Identification and characterization of a tumor infiltrating CD56+/CD16− NK cell subset with specificity for pancreatic and prostate cancer cell lines

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56⁺) with few T cells (12% CD3⁺). A majority (88%) of the NK cells were CD56^brightCD16⁻. TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56⁺CD16⁻CD3⁻ subset showed reactivity similar to TIL-2742 while the CD56⁻CD16⁻CD3⁺ cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56^brightCD16^dim) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56⁺CD16⁻ NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent.     

Reciprocal effects of Th1 and Treg cell inducing pathogen-associated immunomodulatory molecules on anti-tumor immunity

We have addressed the hypothesis that pathogen-associated immunomodulatory molecules may influence anti-tumor immunity through their pro- and anti-inflammatory activities and abilities to induce effector and regulatory T (Treg) cells. We found that CpG oligonucleotides (CpG) and cholera toxin (CT), which promote Th1 or Th2/Treg cell biased responses, respectively, had differential effects on tumor growth. Therapeutic peritumoral administration of CpG significantly reduced subcutaneous tumor growth and prolonged survival, whereas CT enhanced tumor growth and reduced survival. Peritumoral administration of CpG enhanced the frequency of IFN-γ-secreting and reduced IL-10-secreting CD4⁺ and CD8⁺ T cells, in the tumor and in the draining lymph nodes, whereas, CT significantly enhanced the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells, but reduced IFN-γ-secreting T cells infiltrating the tumor. In contrast to the beneficial effect of CpG in mice with subcutaneous tumors, CpG or CT had no protective effect against tumor growth in the lungs when given therapeutically by the nasal route. However, prophylactic intranasal administration of CpG significantly reduced the number of lung metastases and this was associated with an enhanced frequency of IFN-γ-secreting CD8⁺ T cells in the draining lymph node and enhanced tumor-specific CTL responses. Our findings demonstrate that pathogen-associated molecules can either inhibit or enhance anti-tumor immunity by selectively promoting the induction of effector or regulatory T cells, and that the environment of the growing tumor influences the protective effect. Joanne Lysaght and Andrew G. Jarnicki contributed equally.     

A B-cell lymphoma vaccine using a depot formulation of interleukin-2 induces potent antitumor immunity despite increased numbers of intratumoral regulatory T cells

Therapeutic vaccination holds great potential as complementary treatment for non-Hodgkin’s lymphoma. Here, we report that a therapeutic whole cell vaccine formulated with IL-2 adsorbed onto aluminum hydroxide as cytokine-depot formulation elicits potent antitumor immunity and induces delayed tumor growth, control of tumor dissemination and longer survival in mice challenged with A20-lymphoma. Therapeutic vaccination induced higher numbers of tumor’s infiltrating lymphocytes (CD4⁺ and CD8⁺ T cells and NK cells), and the production of IFN-γ and IL-4 by intratumoral CD4⁺ T cells. Further, strong tumor antigen-specific cellular responses were detected at systemic level. Both the A20-derived antigenic material and the IL-2 depot formulation were required for induction of an effective immune response that impacted on cancer progression. All mice receiving any form of IL-2, either as part of the vaccine or alone as control, showed higher numbers of CD4⁺CD25^+/highFoxp3⁺ regulatory T cells (Treg) in the tumor, which might have a role in tumor progression in these animals. Nevertheless, for those animals that received the cytokine as part of the vaccine formulation, the overall effect was improved immune response and less disseminated disease, suggesting that therapeutic vaccination overcomes the potential detrimental effect of intratumoral Treg cells. Overall, the results presented here show that a simple vaccine formulation, that can be easily prepared under GMP conditions, is a promising strategy to be used in B-cell lymphoma and may have enough merit to be tested in clinical trials.     

CD4+CD25?Nrp1+ T Cells Synergize with Rapamycin to Prevent Murine Cardiac Allorejection in Immunocompetent Recipients

Besides CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4⁺CD25⁻Nrp1⁺ T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4⁺CD25⁻Nrp1⁺ T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4⁺CD25⁻Nrp1⁺ T cells suppressed the proliferation of naive CD4⁺CD25⁻ T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4⁺CD25⁻Nrp1⁺ T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-γ, IL-17 and increased IL-10, TGF-β, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4⁺CD25⁻Nrp1⁺ T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4⁺CD25⁻Nrp1⁺ T cells in preventing allorejection. CD4⁺Nrp1⁺ T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3⁺ Tregs.     

Human CD80/IL2 lentivirus transduced acute myeloid leukaemia cells enhance cytolytic activity in vitro in spite of an increase in regulatory CD4<Superscript>+</Superscript> T cells in a subset of cultures

Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4⁺ T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8⁺ T cells (P = 0.046) CD3⁻CD56⁺ NK cells (P = 0.028) and CD3⁺CD4⁺CD25^highFoxp3⁺ Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8⁺ T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8⁺ T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-γ and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.     

Active hexose correlated compound enhances tumor surveillance through regulating both innate and adaptive immune responses

Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals derived from cocultured mycelia of several species of Basidiomycete mushrooms. AHCC has been implicated to modulate immune functions and plays a protective role against infection. However, the potential role of AHCC in tumor immune surveillance is unknown. In this study, C57BL/6 mice were orally administered AHCC or water, followed by tumor cell inoculation. We showed that compared to pure water-treated mice, AHCC treatment significantly delayed tumor development after inoculation of either melanoma cell line B16F0 or lymphoma cell line EL4. Treatment with AHCC enhanced both Ag-specific activation and proliferation of CD4⁺ and CD8⁺ T cells, increased the number of tumor Ag-specific CD8⁺ T cells, and more importantly, increased the frequency of tumor Ag-specific IFN-γ producing CD8⁺ T cells. Interestingly, AHCC treatment also showed increased cell number of NK and γδ T cells, indicating the role of AHCC in activating these innate-like lymphocytes. In summary, our results demonstrate that AHCC can enhance tumor immune surveillance through regulating both innate and adaptive immune responses.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4⁺ memory T cells, whereas CD4⁺ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4⁺ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α. A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Circulating and tumor-infiltrating Foxp3⁺ regulatory T cell subset in Chinese patients with extranodal NK/T cell lymphoma

Foxp3⁺ regulatory T lymphocytes (Tregs) usually act as an immune suppressor and correlate with poorer survival in malignancies. This study aims to investigate the distribution and characterization of Foxp3⁺ subset in peripheral blood mononuclear cells (PBMCs) and tumor tissues from extranodal NK/T cell lymphoma (ENKTL). Our study showed the percentage of Foxp3⁺ subset from PBMC was significantly higher than that of healthy individuals (P<0.001). The Foxp3⁺ subset from PBMCs expressed CD45RO, CTLA4, GITR, CCR7, and had an IL-10^highIFNγ⁺TGFβ⁺IL-2^lowIL-17^low cytokine secreting phenotype. Interestingly, the existence of EBV antigen-specific CD8⁺Foxp3⁺ Tregs was discovered in ENKTL. Furthermore, the high density of Foxp3⁺ TILs was associated with improved progression-free survival (PFS) in ENKTL patients (P<0.05). Collectively, our study implicates that EBV antigens could induce antigen-specific CD8⁺Foxp3⁺ Tregs in ENKTL, and Foxp3⁺ TILs is an independent factor for PFS in ENKTL.     

Induction of specific T cell immunity in patients with prostate cancer by vaccination with PSA146–154 peptide

T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4⁻CD8⁺ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.     

Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

Vγ9Vδ2 T cell-mediated recognition of human solid tumors. Potential for immunotherapy of hepatocellular and colorectal carcinomas

Introduction Vγ9Vδ2 T lymphocytes are reported to participate in the anti-tumor immune surveillance in human. They are known to recognize phosphoantigens and molecules expressed on cells undergoing neoplasic transformation. In this study, we investigated phenotype and anti-tumor cytotoxicity of ex vivo expanded Vγ9Vδ2 T cells in view of adoptive immunotherapy. Materials and Methods Experiments were performed with peripheral blood samples from eleven patients [six colorectal carcinoma (CRC), four hepatocellular carcinoma (HCC), one sarcoma] and sixteen healthy donors. Results/Discussion Ex vivo expansion of Vγ9Vδ2 T cells could be achieved by a single dose of phosphoantigen, either bromohydrin pyrophosphate or zoledronate, and supported by exogenous IL-2. After 2 weeks, expanded Vγ9Vδ2 T lymphocytes acquired the effector memory phenotype CD45RA⁻CD45RO^highCD27⁻. They expressed NKG2D and CD161 and the proinflammatory CXCR3 and CCR5 chemokine receptors. Vγ9Vδ2 T cells displayed a strong lytic activity toward a broad panel of tumor cell lines or primary cultures. Interestingly, HCC and CRC primary cells could be lysed by autologous Vγ9Vδ2 T cells whereas autologous normal cells were not sensitive to the lysis. mAbs blocking assays demonstrated that TCR was the most important receptor involved in the lysis of tumor cells. However, NKG2D receptor could deliver a costimulatory signal enhancing the lysis of HCC and CRC tumors expressing MICA/B. Treatment of tumor cells by the mevalonate pathway inhibitor, zoledronate, enhanced the killing of both HCC and CRC. Expansion index of Vγ9Vδ2 T cells was in similar levels in healthy donors or in cancer patients and total expansion was suitable for adoptive immunotherapy. Conclusion These results provide a rationale for the clinical evaluation of Vγ9Vδ2 T lymphocytes in HCC and CRC.