Circulating T regulatory cells migration and phenotype in glioblastoma patients: an in vitro study

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4⁺/CD25^bright) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4⁺/μL or Treg/μL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.     

Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^? cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2‐like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2‐like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4^? cells, metastasized to lymph nodes which contained CCL22 producing M2‐like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2‐like macrophage‐dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.     

NK depletion results in increased CCL22 secretion and Treg levels in Lewis Lung Carcinoma via the accumulation of CCL22‐secreting CD11b+CD11c+ cells

Tumor‐induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T‐cells (Tregs). Previous studies demonstrated that NK‐dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). To extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK‐derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC‐bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK‐depleted LLC‐bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22‐producing cellular fractions in LLC‐bearing lungs. A novel CD11b⁺CD11c⁺ cell population was identified that accumulates in large numbers in NK‐depleted LLC‐bearing lung tissue. These CD11b⁺CD11c⁺ cells secreted large amounts of CCL22 that may overcompensate for the loss of NK‐derived CCL22 in the lungs of NK‐depleted LLC‐bearing mice. Taken together, these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC‐bearing lungs.     

Increased intratumoral regulatory T cells are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma patients

Immunosuppression mediated by regulatory T cells (Tregs) is a key facilitator of tumor immune evasion, but the source of these Tregs and their contribution to human cancer progression remains unclear. This study investigated the properties of FoxP3⁺ Tregs, their prognostic value in patients with hepatocellular carcinoma (HCC) and the underlying mechanisms of FoxP3⁺ Treg intratumoral accumulation. In addition to an increased number of circulating FoxP3⁺ Tregs, the results also showed that FoxP3⁺ Tregs gathered in the tumor site, where they suppressed tissue‐derived CD4⁺CD25^? T‐cell activation (p < 0.001), promoting disease progression and poor prognosis in HCC patients (< 0.01). The intratumoral prevalence of FoxP3⁺ Tregs was associated with a high density of macrophages (Mφ) (p < 0.001). Depletion of tissue Mφ thus attenuated the increase of liver FoxP3⁺ Treg frequency attributed to in vivo inoculation with hepatoma (p = 0.01), whereas Mφ exposed to tumor culture supernatants from hepatoma‐derived cell lines increased FoxP3⁺ Treg frequency in vitro (p < 0.001). This increase was partially blocked by antiinterleukin‐10 antibody (p < 0.01). In conclusion, tumor‐associated Mφ may trigger a rise of the intratumoral FoxP3⁺ Treg population, which in turn may promote HCC progression. © 2009 UICC     

Regulatory T cells in tumor immunity

Recent studies have revealed that Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self‐tolerance, play critical roles for the control of antitumor immune responses. For example, a large number of Foxp3⁺Tregs infiltrate into tumors, and systemic removal of Foxp3⁺Tregs enhances natural as well as vaccine‐induced antitumor T‐cell responses. Tregs are recruited to tumor tissues via chemokines, such as CCL22 binding to CCR4 expressed by Tregs. They appear to expand and become activated in tumor tissues and in the draining lymph nodes by recognizing tumor‐associated antigens as well as normal self‐antigen expressed by tumor cells. These results indicate that cancer vaccines targeting tumor‐associated self‐antigens may potentially expand/activate Tregs and hamper effective antitumor immune responses, and that tumor immunity can therefore be enhanced by depleting Tregs, attenuating Treg suppressive function, or rendering effector T cells refractory to Treg‐mediated suppression. Recent attempts have indeed demonstrated that combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity and are more effective than a single monoclonal antibody therapy. Combination therapy targeting a variety of molecules expressed in antigen‐presenting cells, effector T cells and Tregs is envisaged to be a promising anticancer immunotherapy.     

Immune correlates of clinical benefit in a phase I study of hyperthermia with adoptive T cell immunotherapy in patients with solid tumors

Abstract

Purpose: To characterize the T cell receptor (TCR) repertoire, serum cytokine levels, peripheral blood T lymphocyte populations, safety, and clinical efficacy of hyperthermia (HT) combined with autologous adoptive cell therapy (ACT) and either salvage chemotherapy (CT) or anti-PD-1 antibody in patients with previously treated advanced solid tumors.

Materials and methods: Thirty-three (33) patients with ovarian, pancreatic, gastric, colorectal, cervical, or endometrial cancer were recruited into the following therapeutic groups: HT?+?ACT (n?=?10), HT?+?ACT?+?anti-PD-1 inhibitor (pembrolizumab) (n?=?11) and HT?+?ACT?+?CT (n?=?12). Peripheral blood was collected to analyze TCR repertoire, measurements of cytokines levels and lymphocyte sub-populations before and after treatment.

Results: The objective response rate (ORR) was 30% (10/33), including three complete responses (CR) (9.1%) and seven partial responses (PR) (21.2%) and a disease control rate (DCR?=?CR?+?PR?+?SD) of 66.7% (22 of 33). The most common adverse reactions, blistering, subcutaneous fat induration, local heat-related pain, vomiting and sinus tachycardia, were observed in association with HT. IL-2, IL-4, TNF-α, and IFN-γ levels in peripheral blood were significantly increased among the clinical responders (p?<?0.05) while IL-6 and IL-10 were elevated among those with progressive disease (p?<?0.05). Peripheral blood CD8⁺/CD28⁺ T cells increased (p?=?0.002), while the CD4⁺/CD25⁺/CD127⁺Treg cells decreased after therapy (p?=?0.012). TCR diversity was substantially increased among the clinical responders.

Conclusions: Combining HT with ACT plus either CT or anti-PD-1 antibody was safe, generated clinical responses in previously treated advanced cancers, and promoted TCR repertoire diversity and favorable changes in serum IL-2, IL-4, TNF-α, and IFN-γ levels in clinical responders.     

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice

Ex vivo expansion of CD8⁺ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8⁺ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8⁺ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8⁺ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2×10⁶ CD8⁺ T-cells and achieved the same level of tumor growth inhibition as that of 5×10⁶ CD8⁺ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8⁺ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8⁺ T-cells which also stay longer.     

Specific antitumour immunity of HIFU-activated cytotoxic T lymphocytes after adoptive transfusion in tumour-bearing mice

Purpose: The aim of this study was to investigate the specific anti-tumour immunity of cytotoxic T lymphocytes (CTL) activated by high-intensity focused ultrasound (HIFU) after adoptive transfer in a murine tumour model. Materials and methods: H22 tumour-bearing mice were treated by either HIFU or sham-HIFU, while naïve syngeneic mice were used as controls. They were sacrificed and the spleens were harvested 14 days after HIFU. T lymphocytes were obtained from the spleens, and then adoptively transferred into 40 mice each bearing a 3-day implanted H22 tumour. On day 14 after adoptive transfer, 10 mice were sacrificed in each group for assessment of the number of tumour-infiltrating T lymphocytes and interferon-gamma (IFN-γ) secreting cells. The remaining 30 mice were continuously observed for 60 days, and tumour growth, progression and survival were recorded. Results: HIFU significantly increased peripheral blood CD3⁺, CD4⁺ levels and CD4⁺/CD8⁺ ratio (P?<?0.05), CTL cytotoxicity (P?<?0.01) and IFN-γ and TNF-α secretion (P?<?0.01) in H22 tumour-bearing mice. Adoptive transfer of HIFU-activated T lymphocytes into the autologous tumour-bearing mice induced a significant increase of tumour-infiltrating T lymphocytes and IFN-γ-secreting cells (P?<?0.001). Compared to the control and sham-HIFU groups, HIFU-activated lymphocytes elicited significant inhibition of in vivo tumour growth (P?<?0.01) and progression (P?<?0.0001), and longer survival time in the tumour-bearing mice (P?<?0.001). Conclusions: HIFU could enhance CTL’s specific antitumour immunity. Adoptive transfer of HIFU-activated T lymphocytes could increase local antitumour immunity, and elicit stronger inhibition on tumour growth and progression, with more survival benefit in the autologous tumour-bearing mice.     

CCL21-Ser expression in melanoma cells recruits CCR7+ naïve T cells to tumor tissues and promotes tumor growth

CCL21-Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21-Ser-expressing melanoma cells and the Ccl21a-deficient mice, we demonstrated the functional role of cancer cell-derived CCL21-Ser in melanoma growth in vivo. The B16-F10 tumor growth was significantly decreased in Ccl21a-deficient mice compared with that in wild-type mice, indicating that host-derived CCL21-Ser contributes to melanoma proliferation in vivo. In Ccl21a-deficient mice, tumor growth of melanoma cells expressing CCL21-Ser was significantly enhanced, suggesting that CCL21-Ser from melanoma cells promotes tumor growth in the absence of host-derived CCL21-Ser. The increase in tumor growth was associated with an increase in the CCR7⁺ CD62L⁺ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell-derived CCL21-Ser expression. These results suggest that CCL21-Ser from melanoma cells promotes the infiltration of CCR7⁺ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.     

Evaluation of improvement of onychomycosis in HIV‐infected patients after initiation of combined antiretroviral therapy without antifungal treatment

Onychomycosis in HIV‐infected patients has a prevalence of 20–44% and is more frequently seen with CD4⁺ T cell counts ≤450 cel μl^?1. There are case reports of improvement in onychomycosis after initiation of combined antiretroviral therapy (cART), but there are no prospective studies that prove the existence and frequency of this phenomenon. The aim of this study was to evaluate if HIV‐infected patients with onychomycosis who begin cART improve and/or cure without antifungal treatment. We included HIV‐infected patients with onychomycosis who had not started cART and nor received antifungal therapy during 6 months prior to the study. We evaluated affected the nails with the Onychomycosis Severity Index (OSI); nail scrapings were collected and direct microscopy with potassium hydroxide (KOH) as well as mycological culture were performed. We repeated these procedures at 3 and 6 months to assess changes. CD4 T cell counts and HIV viral load were obtained. A total of 16 patients were included, with male gender predominance (68.7%); distal and lateral subungual onychomycosis (DLSO) was the most common form (31.3%). Trichophyton rubrum was the most frequently isolated microorganism. OSI decreased 21.5% at 3 months and 40% at 6 months after initiation of antiretrovirals (P = 0.05). We found a non‐significant tendency towards improvement with higher CD4⁺ T cell counts and with viral loads <100 000 copies ml^?1. This could be due to the increase in CD4⁺ T cells, decreased percentage of Treg (CD4⁺CD25⁺) among CD4⁺ Tcells and/or a decreased viral load; further studies are necessary to prove these hypothesis.     

Functional heterogeneity of circulating T regulatory cell subsets in breast cancer patients

Background

Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women.

Methods

The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4⁺CD25⁺FoxP3⁺CD45RA⁺ N nTregs, CD4⁺CD25⁺FoxP3⁺CD45RA^? EM nTregs, and CD4⁺IL-4^?IL-10⁺ Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry.

Results

Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25⁺ and FoxP3⁺ Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25⁺ Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I–III).

Conclusions

Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25⁺ Tr1 cells represent prospective markers for assessing disease prognosis.

     

Skewed immunological balance between Th17 (CD4+IL17A+) and Treg (CD4+CD25+FOXP3+) cells in human oral squamous cell carcinoma

Background

Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer.

Methods and results

We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p?<?0.0001) higher proportion of both Th17 (CD4⁺IL17A⁺) and Treg (CD4⁺CD25⁺FOXP3⁺) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8⁺ subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4⁺T and CD8⁺T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4⁺T and CD8⁺T cells.

Conclusions

Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.     

Systemic immune suppression in glioblastoma: the interplay between CD14+HLA-DRlo/neg monocytes, tumor factors, and dexamethasone

Patients with glioblastoma (GBM) exhibit profound systemic immune defects that affect the success of conventional and immune-based treatments. A better understanding of the contribution of the tumor and/or therapy on systemic immune suppression is necessary for improved therapies, to monitor negative effects of novel treatments, to improve patient outcomes, and to increase understanding of this complex system. To characterize the immune profile of GBM patients, we phenotyped peripheral blood and compared these to normal donors. In doing so, we identified changes in systemic immunity associated with both the tumor and dexamethasone treated tumor bearing patients. In particular, dexamethasone exacerbated tumor associated lymphopenia primarily in the T cell compartment. We have also identified unique tumor and dexamethasone dependent altered monocyte phenotypes. The major population of altered monocytes (CD14⁺HLA-DR^lo/neg) had a phenotype distinct from classical myeloid suppressor cells. These cells inhibited T cell proliferation, were unable to fully differentiate into mature dendritic cells, were associated with dexamethasone-mediated changes in CCL2 levels, and could be re-created in vitro using tumor supernatants. We provide evidence that tumors express high levels of CCL2, can contain high numbers of CD14⁺ cells, that tumor supernatants can transform CD14⁺HLA-DR⁺ cells into CD14⁺HLA-DR^lo/neg immune suppressors, and that dexamethasone reduces CCL2 in vitro and is correlated with reduction of CCL2 in vivo. Consequently, we have developed a model for tumor mediated systemic immune suppression via recruitment and transformation of CD14⁺ cells.     

The CC ligand chemokine family members CCL17/CCL22 predict the survival and response to immune checkpoint blockade therapy of patients with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is considered an immunosuppressive malignancy. Cross-talk between cancer cells and immune cells is modulated in part by CC ligand (CCL) chemokines, having a major effect on tumor progression. However, the predictive value and function of CCL family members in HNSCC have not been elucidated. Here, the predictive value of CCL members in cancer prognosis and Immune checkpoint blockade therapy response was investigated. CCL17 and CCL22 were screened as the key CCL chemokines in HNSCC through co-expression analysis. Further, the correlation between CCL17/CCL22 expression and cancer immune infiltration were evaluated based on TIMER and were validated by a set of scRNA-seq data. Moreover, the expression level of CCL17/CCL22 we evaluated to predict the response to Immune checkpoint blockade therapy in a panel of cancer types by using the TIDE database. Results indicated that CCL17/CCL22 had a high co-expression correlation and had a marginally statistical significance with the overall survival in HNSCC patients (P value = 0.057 and 0.055, respectively). Our findings showed high expression of CCL17/CCL22 was positively correlated with CD4⁺ T cell infiltration levels in HNSCCs and activate mTORC1 signaling pathway in CD4⁺ T cells. Further analysis from TIDE showed the high expression of CCL17/CCL22 might predict favorable responses to immune checkpoint blockade therapy in HNSCC patients. These findings provide an insight into the predictive roles of CCL17/CCL22 in HNSCC.     

Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

Specific thermal ablation of tumor cells using single‐walled carbon nanotubes targeted by covalently‐coupled monoclonal antibodies

CD22 is broadly expressed on human B cell lymphomas. Monoclonal anti‐CD22 antibodies alone, or coupled to toxins, have been used to selectively target these tumors both in SCID mice with xenografted human lymphoma cell lines and in patients with B cell lymphomas. Single‐walled carbon nanotubes (CNTs) attached to antibodies or peptides represent another approach to targeting cancer cells. CNTs convert absorbed near‐infrared (NIR) light to heat, which can thermally ablate cells that have bound the CNTs. We have previously demonstrated that monoclonal antibodies (MAbs) noncovalently coupled to CNTs can specifically target and kill cells in vitro. Here, we describe the preparation of conjugates in which the MAbs are covalently conjugated to the CNTs. The specificity of both the binding and NIR‐mediated killing of the tumor cells by the MAb‐CNTs is demonstrated by using CD22⁺CD25^? Daudi cells, CD22^?CD25⁺ phytohemagglutinin‐activated normal human peripheral blood mononuclear cells, and CNTs covalently modified with either anti‐CD22 or anti‐CD25. We further demonstrate that the stability and specificity of the MAb‐CNT conjugates are preserved following incubation in either sodium dodecyl sulfate or mouse serum, indicating that they should be stable for in vivo use. © 2009 UICC     

Persistence and differentiation of HTLV-I-transformed immature T-cells in HTLV-I carrier rabbits

Two T-cell lines with immature phenotypes, CD4⁻8⁻ and CD4⁺8⁺, were found among HTLV-I-transformed T-cell lines obtained during experiments using a rabbit model of adult T-cell leukemia. Persistence and differentiation of these clones in vivo were examined by retrospectively analysing a series of cell lines from individual animals and by adoptive transfer of these cells into syngeneic hosts. The double negative cell line did not differentiate and remained double negative in adoptive transfer into neonates and in long-term in vitro culture, while the double positive cell line differentiated both in vivo and in vitro into CD8⁺ cell, as if the fate of this cell line had been predetermined. Cells of the same clone as this double positive cell line persisted for more than one year in the peripheral blood of the animal. Long-term persistence was not observed for five cell lines of CD4 single positive phenotype, which were obtained from another carrier rabbit. These results suggested that HTLV-I-transformed immature T-cells persist in neonatally infected carriers, continuously giving rise to mature T-cells harboring HTLV-I.