Primed tumor-reactive multifunctional CD62L<Superscript>+</Superscript> human CD8<Superscript>+</Superscript> T cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8⁺ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L⁺, CCR7⁺, CD28⁺). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.     

BCR/ABL-specific CD8<Superscript>+</Superscript> T cells can be detected from CML patients,but are only expanded from healthy donors

The BCR/ABL p210 fusion protein has long been considered an ideal target antigen for the development of immunotherapeutic strategies in chronic myeloid leukaemia (CML) due to its central role in malignant transformation and to its unique novel amino acid sequence solely expressed in leukaemia cells. However, the feasibility to expand BCR-ABL-specific T cells remains still controversial. Using BCR/ABL peptide/MHC tetramers, significantly higher frequencies of tetramer positive cells were detected in the peripheral blood of HLA-A*0301 (mean 0.38%) and HLA-B*0801 (mean 0.28%) CML patients than in healthy donors (P = 0.0025 and 0.0026, respectively). However, following stimulation with autologous peptide-pulsed DCs, BCR/ABL-specific T cells were only expanded from some healthy donors, suggesting that CML patients may have a specific immune deficit with respect to the BCR/ABL antigen. Professor A. I. Dodi passed away recently. This paper is an original contribution from the meeting which took place 28–29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. A. I. “Tony” Dodi (29 January 2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Functional CD8<Superscript>+</Superscript> T cells infiltrate into nonsmall cell lung carcinoma

Infiltration of CD3(+)CD8(+) cytotoxic T cells was analyzed by multiparameter confocal laser microscopy in a panel of 16 randomly selected stage I nonsmall cell lung carcinomas. T-cell infiltration was observed in the stroma (range 57-2,093 T cells/mm(2)) but also in the tumor epithelium (range 21-892 T cells/mm(2)) and showed wide variation between individual tumors. Interestingly, a significantly higher percentage of CD3(+)CD8(+) T cells was detected in the tumor epithelium compared to the stroma illustrating that cytotoxic T cells may preferentially migrate into tumor epithelium. Aberrant HLA class I antigen expression was observed in 69% of the nonsmall-cell lung carcinoma (NSCLC) tumors. One tumor of a squamous cell lung carcinoma patient with the highest number of tumor infiltrating CD3(+) and CD3(+)CD8(+) cells was studied in detail and the majority (90%) of these cells were shown to be functionally activated granzyme B-positive cytotoxic T cells. DNA oligotyping of a lung carcinoma cell line established from this tumor revealed loss of one HLA haplotype corresponding with a translocation involving chromosome 6, as observed by COBRA-FISH. HLA class I-restricted tumor specific T cells could be isolated from PBMC. One further characterized cytotoxic CD8(+) T cell clone, that released TNF-alpha, IFN-gamma, and granzyme B upon co-incubation with the autologous tumor cells, was shown to be restricted by the remaining HLA-A11 allele, which was also shown to be expressed in the tumor tissue. Our data indicate that, despite HLA-haplotype loss a vigorous antitumor immune response mediated by CD8(+ )T-cells can be present in NSCLC offering possibilities for specific immunotherapy.     

TNK cells (NKG2D<Superscript>+</Superscript> CD8<Superscript>+</Superscript> or CD4<Superscript>+</Superscript> T lymphocytes) in the control of human tumors

Innate and adaptive immune responses have many interactions that are regulated by the balance of signals initiated by a variety of activatory and inhibitory receptors. Among these, the NKG2D molecule was identified as expressed by T lymphocytes, including most CD8⁺ cells and a minority of CD4⁺ cells, designated TNK cells in this paper. Tumor cells may overexpress the stress-inducible NKG2D ligands (NKG2DLs: MICA/B, ULBPs) and the NKG2D signaling has been shown to be involved in lymphocyte-mediated anti-tumor activity. Aberrant expression of NKG2DLs by cancer cells, such as the release of soluble form of NKG2DLs, can lead to the impairment of these immune responses. Here, we discuss the significance of NKG2D in TNK-mediated anti-tumor activity. Our studies demonstrate that NKG2D⁺ T cells (TNK) are commonly recruited at the tumor site in melanoma patients where they may exert anti-tumor activity by engaging both TCR and NKG2D. Moreover, NKG2D and TCR triggering was also observed by peripheral blood derived T lymphocyte- or T cell clone-mediated tumor recognition, both in melanoma and colorectal cancer (CRC) patients. Notably, heterogeneous expression of NKG2DLs was found in melanoma and CRC cells, with a decrease of these molecules along with tumor progression. Therefore, through the mechanisms that govern NKG2D engagement in anti-tumor activity and the expression of NKG2DLs by tumor cells that still need to be dissected, we showed that NKG2D expressing TNK cells are a relevant T cell subtype for immunosurveillance of tumors and we propose that new immunotherapeutic interventions for cancer patients should be aimed also at enhancing NKG2DLs expression by tumor cells. This paper is a focused research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany, 15–16 May 2008.     

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.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b<Superscript>+</Superscript>Gr-1<Superscript>+</Superscript> myeloid cells without compromising CD8<Superscript>+</Superscript> T cell immune responses

Background

Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.

Methods

We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.

Results

We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b⁺Gr-1⁺ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8⁺ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.

Conclusions

Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

     

Simultaneous ex vivo quantification of antigen-specific CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell responses using in vitro transcribed RNA

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.     

LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

Identity of mysterious CD4<Superscript>+</Superscript>CD25<Superscript>-</Superscript>Foxp3<Superscript>+</Superscript>cells in systemic lupus erythematosus

Various abnormalities in CD4⁺CD25⁺ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3⁺ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4⁺ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25^-Foxp3⁺ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3⁺CD4⁺CD25^dim rather than CD25^- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3⁺ T cells in SLE have protective suppressive activity.     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Prognostic value of the CD4<Superscript>+</Superscript>/CD8<Superscript>+</Superscript> ratio of tumour infiltrating lymphocytes in colorectal cancer and HLA-DR expression on tumour cells

The purpose of this study was to clarify whether HLA-DR expression of colorectal tumour cells or the CD4+/CD8+ ratio of the tumour infiltrating lymphocytes is significantly associated with the prognosis of colorectal cancer. Using flow cytometry, we studied the tumour cell expression of the HLA class II in 70 enzymatically dissociated colorectal cancers and the phenotype of tumour infiltrating lymphocytes (TILs) in 41 cases. There was no trend in 5-year survival between three levels (low, medium, high) of HLA-DR expression on the tumour cells. Patients with low CD4+/CD8+ ratios had a better clinical course, with significantly higher 5-year survival, p=0.046, independent of the Dukes stage and age. Our results have implications for tumour immunology; colorectal cancer cells might be a target for cytotoxic T-lymphocytes, however the tumour cells are not able to initiate an immune response. Stimulation of the immune system could possible be obtained using dendritic cells cultured in vitro and loaded with tumour antigens.     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> immunoregulatory T cells may not be involved in controlling autoimmune arthritis

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4⁺CD25⁺ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4⁺CD25⁺ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4⁺CD25⁺ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4⁺CD25⁺ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4⁺CD25⁺ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4⁺CD25⁺ T cells, were highly resistant to PGIA. These findings indicate that the CD4⁺CD25⁺ regulatory T cells may not play a critical role in controlling PGIA.     

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8<Superscript>+</Superscript> T-cell immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8⁺ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8⁺ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

Actin integrity is indispensable for CD95/Fas-induced apoptosis of HIV-specific CD8<Superscript>+</Superscript> T cells

We have recently provided data suggesting a potential role for mitochondria and Bcl-2-family molecules in apoptosis sensitivity of HIV-specific CD8⁺ T cells. Here, we report on the role of filamentous (F) actin in this process. Disruption of actin by cytochalasin D (cytD) or lantrunculin A remarkably reduced CD95/Fas-induced apoptosis of HIV-specific CD8⁺ T cells while their spontaneous apoptosis was unaffected. This inhibition cannot be attributed to changes of CD95/Fas distribution or levels in these cells. Furthermore, cytD treatment reduced CD95/Fas-induced apoptosis of CD8⁺ T cells from HIV⁺ patients independently of their differentiation status. CD95/Fas-induced apoptosis of both CD38⁺ and CD38⁻ HIV-specific CD8⁺ T cells was inhibited by cytD treatment indicating that actin mediates this apoptotic process independently of the activation level of these cells. CytD was found to reduce the activation of caspase-8 induced by short treatment of purified CD8⁺ T cells from HIV⁺ patients with anti-CD95/Fas. Our data reveal actin as a critical mediator of HIV-specific CD8⁺ T cell apoptosis; further analysis of the molecular mechanisms governing this process may potentially contribute to design new therapies targeting the enhancement of the immune system in HIV infection.     

A paragon of self-tolerance: CD25<Superscript>+</Superscript>CD4<Superscript>+</Superscript>regulatory T cells and the control of immune responses

The interest in naturally arising regulatory T (T_R) cells as a paradigm for maintaining immunological self-tolerance has undergone an explosive re-emergence in recent years. This renaissance was triggered by several key experimental observations and the identification of specific molecular markers that have enabled the isolation and experimental manipulation of these cells. Although their existence was once controversial, a large body of evidence now highlights the critical roles of T_R cells in maintaining immunological self-tolerance. Furthermore, abnormality of natural T_R cells can be a primary cause of autoimmune and other inflammatory diseases in humans.     

Breast cancer cells expressing stem cell markers CD44<Superscript>+</Superscript> CD24<Superscript>lo</Superscript> are eliminated by Numb-1 peptide-activated T cells

Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen⁺ (ESA) CD44^hi CD24^lo, CD44^hi CD133⁺, and CD133⁺ CD24^lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM^Res MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM^Sens MCF7. The levels of Numb, and Numb-L-[P]-Ser²⁶⁵ were similar in GEM^Res and GEM^Sens MCF7 cells. Only the levels of Numb-L (long)-Ser²⁹⁵ decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEM^Res MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112–2120) and Numb-1 (87–95) eliminated NICD^positive, CD24^hi CD24^lo MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.