T Lymphocytes from Chagasic Patients Are Activated but Lack Proliferative Capacity and Down-Regulate CD28 and CD3ζ

Background

Chronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls.

Methodology/Principal Findings

Forty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζ^bright/CD3ζ^dim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζ^bright/CD3ζ^dim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ.

Conclusions

CD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.     

Unprimed CD4+ and CD8+ T cells can be rapidly activated by a CD3×CD19 bispecific antibody to proliferate and become cytotoxic

We previously reported that a CD3×CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3×CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3×CD19 bsAb. Within the same time spaancytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3×CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.This work was supported by grant IKMN 90-10 from the Dutch Cancer Society. M.C. was supported by a grant from the UK Medical Research Couneil     

T Cell Receptor Engagement Triggers Its CD3ε and CD3ζ Subunits to Adopt a Compact,Locked Conformation

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3ε subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3ε and CD3ζ subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3ε and CD3ζ, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.     

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-��1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.The CD2 family of cell surface receptors is differentially expressed on immune cells (1, 2) and is involved in regulating both innate and adaptive immunity (3). These receptors have related extracellular immunoglobulin superfamily domains and interact either homophilically or heterophilically within the CD2 family (1, 2). The CD2 family contains a subgroup of receptors termed the SLAM family that have a conserved tyrosine signaling motif in their cytoplasmic region TXYXX(I/V) referred to as an immunoreceptor tyrosine-based switch motif (ITSM).² The SLAM family of receptors include CD244 (2B4), NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9). Defects in signaling and aberrant expression of these receptors have been implicated in several immunodeficiency and autoimmune disorders in humans and mice (4–8). Within the SLAM family, CD244 is unusual in that it shares its ligand CD48 with the receptor CD2 in rodents, whereas in humans CD2 has evolved to interact with CD58 (9). The affinity of CD244 for CD48 in rodents is 6–9-fold higher than the still functionally relevant CD2/CD48 interaction (10). CD244 and CD2 have different cytoplasmic regions comprised of tyrosine motifs or proline-rich motifs, respectively.CD244 is predominantly found on NK cells and cytotoxic T cells and primarily characterized as an activating receptor (11–15). CD2 is found on the same cells as CD244 but is also expressed on all T cells, both activated and resting, and has an activating or costimulatory function upon engagement of ligand (9). The tyrosine motifs found in the cytoplasmic tail of CD244 have been shown to bind the SH2 domains of cytoplasmic adaptor proteins SAP and EAT-2 and FYN kinase (16–18) and are important to its function (5, 19–21). In contrast to SH2 interactions of CD244, several SH3 domain-mediated interactions have been reported for the cytoplasmic region of CD2 including CD2AP/CMS, CIN85, FYN, and LCK (22–26).The activating function of CD244 was called into question when a study using cells from a CD244 knock-out mouse showed that CD244 had an inhibitory effect as loss of CD244 resulted in enhanced NK killing of target cells (27). This suggested that previous results in mice where positive effects were seen may have been due to blocking CD244 ligand engagement as opposed to cross-linking with antibodies against CD244 (27). This has led to proposals that there are differences in function between mouse and human CD244 as there is more evidence to suggest that human CD244 is a positive regulator enhancing cytotoxicity and cytokine production (13, 15, 28). However, other more recent studies have shown the mouse CD244/CD48 interaction to be important for cytokine production and effector functions such as cytotoxicity against tumor targets in CD244-deficient mice (29). Long and short forms of CD244 have been cloned from mice with the short form being described as activating and the long form inhibitory (27, 30). Only the long form of CD244 is present in humans and is regarded as activating (14).Positive signaling by CD244 has been attributed to the recruitment of SAP (18), which is a signaling adaptor molecule comprised of a single SH2 domain encoded by the SH2D1A gene and has the ability to recruit the kinase FYN by binding its SH3 domain (31, 32). Loss of the SAP/FYN interaction can lead to X-linked lymphoproliferative disease in humans (17). The molecular basis of in vitro inhibitory effects observed with CD244 in mice on ligation with mAb or ligand remains elusive (33). Protein tyrosine and inositol phosphatases have been reported to associate with CD244 (18, 19, 34) but our studies using surface plasmon resonance found them to be very weak and unlikely to bind competitively compared with the SAP family of adaptors or FYN (16). The SAP-related adaptor EAT-2 has been reported to have an active inhibitory effect that is dependent on tyrosine motifs in the tail of EAT-2 (35) but its mechanism is not understood. The only interaction reported for the tail of EAT-2 is with FYN kinase and studies overexpressing EAT-2 in a T cell hybridoma resulted in increased IL-2 production upon antigen stimulation (16).The conservation between mouse and human CD244 cytoplasmic regions and associated adaptors suggests that both function in a similar way. We have explored the main difference between mouse and human CD244, which is the extracellular interaction through CD48 ligation in the mouse. This has revealed that inhibitory effects of CD244 ligation in mice can be due to competition between CD244 and CD2 for CD48. We have also found that the adaptor protein EAT-2 binds PLCγ1 providing a molecular basis for the important role CD244 plays in regulating cellular cytotoxicity (13, 36). We demonstrate that there is a potentially shared signaling mechanism through the FYN kinase that links CD2 and CD244 intracellularly even though in humans CD2 and CD244 no longer share a cell surface ligand.     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

CD3γ-independent pathways in TCR-mediated signaling in mature T and iNKT lymphocytes

Antigen recognition by T-lymphocytes through the T-cell antigen receptor, TCR–CD3, is a central event in the initiation of an immune response. CD3 proteins may have redundant as well as specific contributions to the intracellular propagation of TCR-mediated signals. However, to date, the relative role that each CD3 chain plays in signaling is controversial. In order to examine the roles of CD3γ chain in TCR signaling, we analyzed proximal and distal signaling events in human CD3γ^−/− primary and Herpesvirus saimiri (HVS)-transformed T cells. Following TCR–CD3 engagement, certain early TCR signaling pathways (ZAP-70, ERK, p38 and mTORC2 phosphorylation, and actin polymerization) were comparable with control HVS-transformed T cells. However, other signaling pathways were affected, such TCRζ phosphorylation, indicating that the CD3γ chain contributes to improve TCR signaling efficiency and survival. On the other hand, CD3γ^−/− primary invariant NKT cells (iNKT cells) showed a normal expansion in response to alpha-galactosylceramide (α-GalCer) and TCRVβ11^bright iNKT cells were preferentially selected in this in vitro culture system, perhaps as a consequence of selective events in the thymus. Our results collectively indicate that a TCR lacking CD3γ can propagate a number of signals through the remaining invariant chains, likely the homologous CD3δ chain, which replaces it at the mutant TCR.     

The CD9/CD81 Tetraspanin Complex and Tetraspanin CD151 Regulate α3β1 Integrin-Dependent Tumor Cell Behaviors by Overlapping but Distinct Mechanisms

Integrin α3β1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3β1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3β1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3β1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3β1 association with other tetraspanins and impairs α3β1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3β1 functions is unclear. To begin to clarify which aspects of α3β1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3β1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3β1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3β1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3β1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3β1 integrin function. Importantly, some of CD9/CD81''s α3β1 regulatory functions may not require CD9/CD81 to be physically linked to α3β1 by CD151.     

Sequential binding of αVβ3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.     

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

Proteomic analysis of CD44(+) and CD44(−) gastric cancer cells

CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(?) gastric cancer cells to understand characteristics of CD44(+) and CD44(?) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(?) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(?) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(?) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(?) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(?) cells and one protein (prohibitin) showed increased expression in CD44(?) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(?) cells in gastric cancer.     

Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.