Anti-CD44 induces apoptosis in T lymphoma via mitochondrial depolarization.

A blockade of CD44 can interfere with haematopoietic and leukemic stem cell homing, the latter being considered as a therapeutic option in haematological malignancies. We here aimed to explore the molecular mechanism underlying the therapeutic efficacy of anti-CD44. We noted that in irradiated mice reconstituted with a bone marrow cell transplant, anti-CD44 exerts a stronger effect on haematopoietic reconstitution than on T lymphoma (EL4) growth. Nonetheless, in the non-reconstituted mouse anti-CD44 suffices for a prolonged survival of EL4-bearing mice, where anti-CD44-prohibited homing actively drives EL4 cells into apoptosis. In vitro, a CD44 occupancy results in a 2-4-fold increase in apoptotic EL4 cells. Death receptor expression (CD95, TRAIL, TNFRI) remains unaltered and CD95 cross-linking-mediated apoptosis is not affected. Instead, CD44 ligation promotes mitochondrial depolarization that is accompanied by caspase-9 cleavage and is inhibited in the presence of a caspase-9 inhibitor. Apoptosis becomes initiated by activation of CD44-associated phosphatase 2A (PP2A) and proceeds via ERK1/2 dephosphorylation without ERK1/2 degradation. Accordingly, CD44-induced apoptosis could be mimicked by ERK1/2 inhibition, that also promotes EL4 cell apoptosis through the mitochondrial pathway. Thus, during haematopoietic stem cell reconstitution care should be taken not to interfere by a blockade of CD44 with haematopoiesis, which could be circumvented by selectively targeting leukemic CD44 isoforms. Beyond homing/settlement in the bone marrow niche, anti-CD44 drives leukemic T cells into apoptosis via the mitochondrial death pathway by CD44 associating with PP2A. Uncovering this new pathway of CD44-induced leukemic cell death provides new options of therapeutic interference.

CD44 variant isoforms promote metastasis formation by a tumor cell-matrix cross-talk that supports adhesion and apoptosis resistance.

CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44v(kd)) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASML(wt)) were established. ASML-CD44v(kd) clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44v(kd) cells (ASML-CD44v(rsc)) restores the metastatic potential. The following major differences in ASML(wt), ASML-CD44v(kd), and ASML-CD44v(rsc) clones were observed: (a) ASML(wt) cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44v(kd) cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASML(wt) cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44v(kd) cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44v(rsc) cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation.

Thymocyte expansion and maturation: crosstalk of CD44v6 on thymocytes and panCD44 on stroma cells.

Re-acquisition of immunocompetence after allogeneic bone marrow cell (BMC) transplantation depends on intrathymic maturation of the allogeneic T progenitor cells. We recently reported that CD44 promotes progenitor homing into the thymus and T-cell maturation and now elucidate the molecular mechanisms of CD44-supported thymocyte maturation. Lethally irradiated, tumor-bearing mice, allogeneically reconstituted with T-cell-depleted BMC and a small number of common lymphoid progenitor 2 cells (CLP2) from transgenic (TG) mice, that express ratCD44v4-v7 under the Thy1 promoter, showed accelerated immunocompetent T-cell recovery compared with mice reconstituted with non-transgenic (NTG) CLP2. In addition, graft-versus-host disease was strongly reduced after tumor vaccination. TG, but not NTG double-negative (DN) thymocytes showed high proliferative potential, accompanied by constitutive association of lck with CD44. Importantly, when thymocyte adhesion was strengthened by anti-CD44, co-cultures of DN thymocytes with thymic stroma supported DN thymocyte maturation. The close contact between DN thymocytes and thymic stroma promoted persisting activation of lck and ERK1/2, particularly in CD44v6(+) DN thymocytes. Thus, intrathymic T-cell maturation in allogeneically reconstituted, leukemia-bearing hosts can be considerably accelerated by high CD44v6 expression in early thymocytes, in which proliferation-supporting signals are initiated by a crosstalk between CD44v6 on thymocytes and panCD44 on the thymic stroma.

CD44 promotes progenitor homing into the thymus and T cell maturation.

Regain of immunocompetence after myeloablation and bone marrow cell (BMC) reconstitution essentially depends on T progenitor homing into the thymus and intrathymic T cell maturation. CD44 facilitates progenitor homing and settlement in the bone marrow and is known as a T progenitor marker. In search for improving regain of immunocompetence after BMC reconstitution, we explored whether the CD44 standard (CD44 s) and/or variant isoforms CD44v6 and CD44v7 contribute to thymus repopulation and thymocyte maturation. Antibody-blocking studies and cells/mice with a targeted deletion of CD44v6/7 or CD44v7 revealed that CD44s, but not CD44v6 and CD44v7, has a major impact on progenitor cell homing into the thymus. Instead, CD44v6 strengthens apoptosis resistance and expansion of early thymocytes. CD44v6-induced apoptosis resistance, most strong in double-negative (DN) thymocytes, is accompanied by Akt activation. CD44v6-induced proliferation of DN cells proceeds via activation of the MAPK pathway. At later stages of T cell maturation, CD44 acts as an accessory molecule, initiating and supporting TCR/CD3 complex-mediated signal transduction in double-positive and single-positive thymocytes. Thus, CD44 plays a major role in thymus homing. In addition, CD44v6 is important for survival and expansion of early thymocytes. These findings suggest that strengthening CD44v6 expression on lymphoid progenitors could well contribute to accelerated regain of immunocompetence.

CD44 and EpCAM: cancer-initiating cell markers.

Embryonic stem cells are immortal, can self renew, and differentiate into all cells of the body. The adult organism maintains adult stem cells in regenerative organs that can differentiate into all cells of the respective organ. Virchow's hypothesis that cancer may arise from embryonic-like cells has received strong support, as it was demonstrated that tumors contain few cells, known as cancer stem or cancer-initiating cells (CIC), that account for primary and metastatic tumor growth. CIC are mostly defined by expression of CIC-markers that are associated and correlated with the potential of CIC to grow in xenogeneic mice. CIC marker profiles have been elaborated for many tumors, with several markers as CD24, CD44, CD133, CD166, EpCAM, and some integrins, being expressed by tumors of different histological type. Their function in promoting CIC maintenance and activity is largely unknown. The fate of stem cells, determined by their position, is minutely regulated by few adjacent cells creating a niche. CIC also require a niche, mostly for settlement and growth in distant organs. This so called pre-metastatic niche is initiated by the primary tumor before metastasizing cell arrival. How do CIC prepare the pre-metastatic niche? Cancer cells secrete a matrix that serves a cross-talk with surrounding tissues. Additionally, cancer cells can abundantly deliver exosomes, which function as long-distance intercellular communicators. Studies on a rat pancreatic adenocarcinoma support our hypothesis that tumor-derived matrix and exosomes are the main actors in forming the pre-metastatic niche with CIC markers being engaged in matrix preparation and/or exosome delivery.

Opposing effects of fibrosarcoma cell-derived IL-1 alpha and IL-1 beta on immune response induction.

There is evidence that cell-associated IL-1 alpha supports immune response induction. Here we explored the impact of malignant cell-derived IL-1 on immunogenicity, immune response induction and tumor-induced immunosuppression using 3-methylcholanthrene-induced fibrosarcoma lines derived from wild-type (wt), IL-1 alpha-, IL-1 beta- or IL-1a beta-knockout (IL-1 alpha(-/-), IL-1 beta(-/-), IL-1 alphabeta(-/-)) C57BL6 mice. The wt, IL-1 alpha(-/-), IL-1 beta(-/-) and IL-1 alphabeta(-/-) fibrosarcoma lines express MHC class I molecules at a high level. The lines do not differ in their susceptibility toward NK cells, macrophages, and allogeneic CTL, or in their capacity as stimulators of an allogeneic response. However, IL-1 beta(-/-) tumors rarely grow in the syngeneic host, which is the consequence of a strong T helper and CTL response induction by IL-1 alpha-competent, IL-1 beta(-/-) tumors. On the other hand, IL-1 beta-competent, IL-1 alpha(-/-) tumors strongly assist CD11b(+)Gr-1(+) myeloid-derived suppressor cell and regulatory T cell expansion, which both suppress with high efficacy activated T helper cell proliferation and CTL lysis. In IL-1 alphabeta(-/-) tumors, the absence of IL-1 alpha becomes decisive, i.e. despite reduced suppressor cell recruitment, tumor growth was unimpaired due to inefficient immune response induction. Thus, sarcoma cell-derived IL-1 alpha and IL-1 beta do not act in concert. Induction of a strong immune response by IL-1 alpha demands therapeutic exploitation, which may become more efficient if systemic induction of immunosuppression by IL-1 beta can also be circumvented.

Anti-CD44-mediated blockade of leukocyte migration in skin-associated immune diseases.

CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed-type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair-follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti-CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform-specific antibodies. Anti-panCD44 and anti-CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen-treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti-panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease-dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM-1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti-panCD44 and anti-CD49d inhibit T cell, anti-CD11b, and anti-CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44-CD49d-bispecific antibody.

Safety Profile and Immunologic Responses of a Novel Vaccine Against Shigella sonnei Administered Intramuscularly, Intradermally and Intranasally: Results From Two Parallel Randomized Phase 1 Clinical Studies in Healthy Adult Volunteers in Europe.

BACKGROUND: Approximately 164,000 deaths yearly are due to shigellosis, primarily in developing countries. Thus, a safe and affordable Shigella vaccine is an important public health priority. The GSK Vaccines Institute for Global Health (GVGH) developed a candidate Shigella sonnei vaccine (1790GAHB) using the Generalized Modules for Membrane Antigens (GMMA) technology. The paper reports results of 1790GAHB Phase 1 studies in healthy European adults. METHODS: To evaluate the safety and immunogenicity profiles of 1790GAHB, we performed two parallel, phase 1, observer-blind, randomized, placebo-controlled, dose escalation studies in France ("study 1") and the United Kingdom ("study 2") between February 2014 and April 2015 (ClinicalTrials.gov, number NCT02017899 and NCT02034500, respectively) in 18-45years old subjects (50 in study 1, 52 in study 2). Increasing doses of Alhydrogel adsorbed 1790, expressed by both O Antigen (OAg) and protein quantity, or placebo were given either by intramuscular route (0.059/1, 0.29/5, 1.5/25, 2.9/50, 5.9/100µg of OAg/µg of protein; study 1) or by intradermal (ID), intranasal (IN) or intramuscular (IM) route of immunization (0.0059/0.1, 0.059/1, 0.59/10µg ID, 0.29/5, 1.2/20, 4.8/80µg IN and 0.29/5µg IM, respectively; study 2). In absence of serologic correlates of protection for Shigella sonnei, vaccine induced immunogenicity was compared to anti-LPS antibody in a population naturally exposed to S. sonnei. FINDINGS: Vaccines were well tolerated in both studies and no death or vaccine related serious adverse events were reported. In study 1, doses ≥1.5/25µg elicited serum IgG median antibody greater than median level in convalescent subjects after the first dose. No vaccine group in study 2 achieved median antibody greater than the median convalescent antibody. INTERPRETATION: Intramuscularly administered Shigella sonnei GMMA vaccine is well tolerated, up to and including 5.9/100µg and induces antibody to the OAg of at least the same magnitude of those observed following natural exposure to the pathogen. Vaccine administered by ID or IN, although well tolerated, is poorly immunogenic at the doses delivered. The data support the use of the GMMA technology for the development of intramuscular multivalent Shigella vaccines.

A chronic contact eczema impedes migration of antigen-presenting cells in alopecia areata.

Long-lasting allergen treatment is the most efficient therapy in alopecia areata (AA). The underlying mechanism is unknown. We here asked whether treatment with a contact sensitizer influences leukocyte migration such that dendritic cell (DC) migration or the recruitment of activated T-cells towards the skin become hampered. Allergen treatment of AA mice was not accompanied by a decrease in skin-infiltrating leukocytes or draining lymph node cells (LNC). However, the distribution of leukocyte subsets was changed with a dominance of monocytes in the skin and a reduced percentage of DCs in draining nodes. Chemokine and chemokine receptor expression in skin and draining nodes was strikingly increased and LNC from untreated and allergen-treated AA mice showed high migratory activity in vitro and readily homed in draining nodes and skin after intravenous injection. However, FITC labelling of the skin and subcutaneous transfer of dye-labelled DC revealed that allergen treatment created a chemokine milieu severely hampering DC migration from the skin towards the draining node. An allergic eczema-induced reduction in DC migration and antigen transfer could well contribute to insufficient T-cell activation and the recovery of hair follicle in AA and possibly be of relevance for other skin-related autoimmune diseases.

CD44v6 promotes proliferation by persisting activation of MAP kinases.

CD44v6 is transiently expressed during T cell activation, and constitutively CD44v4-v7 expressing transgenic T cells show accelerated responses towards nominal antigens. The underlying mechanism is unknown. The mouse thymoma EL4 was transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA (EL4-s, EL4-v6). Only EL4-v6 cells proliferated at an over 10-fold higher rate than untransfected cells, displayed up-regulated expression of CD69, CD25, and IL-2, and were protected from apoptosis by CD44v6 cross-linking. In the absence of any stimulus, ERK1/2 was partly phosphorylated, and phosphorylation was significantly increased by CD44v6 cross-linking. The same accounted for JNK, c-jun, and IkappaBalpha. Moreover, NF-kappaB was partly translocated into the nucleus. Instead, CD44s cross-linking induced ERK1/2, JNK, c-jun, and IkappaBalpha phosphorylation only in the context of TCR engagement. No selectively CD44v6 associated transmembrane proteins were uncovered in EL4 cells. However, CD44v6, as opposed to CD44s, did not colocalise with the TCR/CD3 complex after CD3 cross-linking. Furthermore, a CD44-associated 85-kDa protein became hypophosphorylated only after CD44v6 cross-linking. Threonine hypophosphorylation of this protein coincided with the activation of MAP and SAP kinases, which was prohibited in the presence of a phosphatase inhibitor. Thus, CD44v6, distinct to CD44s, stimulates autonomously growth and IL-2 secretion of a thymoma line and rescues cells from apoptosis.

CD44v7 interferes with activation-induced cell death by up-regulation of anti-apoptotic gene expression.

Blockade of CD44v7 was described to cure trinitrobenzene sulfonic acid-induced colitis, a disease not developed by mice with targeted deletion of the CD44v7 exon. There was evidence for a reduction in activation-induced cell death on lamina propria lymphocytes of control as compared with CD44v7-deficient mice. To elucidate the mechanism underlying the relative apoptosis resistance of CD44v7-competent as compared with CD44v7-deficient lymphocytes, T cell activation and induction of apoptosis were analyzed on mesenteric lymph node cells and Peyer's patch lymphocytes of CD44v7-deficient and CD44v4-v7-transgenic mice, which overexpress rat CD44v4-v7 on T lymphocytes. CD44v7 deficiency was characterized by an increase in the percentage of apoptotic cells after stimulation, increased numbers of CD95L- and CD152-positive cells, low levels of the anti-apoptotic proteins Bcl-2 and Bcl-Xl, and decreased phosphorylation of the pro-apoptotic protein BAD. Also, lymphocytes from CD44v4-v7-transgenic mice displayed reduced levels of CD95L, low numbers of apoptotic cells, and constitutively elevated levels of Bcl-Xl. When stimulating lymphocytes by CD3 cross-linking, CD44v7 was not recruited toward the immunological synapse and preferentially associated with the cytoskeletal-linker protein ezrin. Thus, as opposed to the CD44 standard isoform, CD44v7 does not function as an accessory molecule; instead, it supports survival of activated T cells by interfering with activation-induced cell death.

Immunogenicity and safety of the Vi-CRM197 conjugate vaccine against typhoid fever in adults, children, and infants in south and southeast Asia: results from two randomised, observer-blind, age de-escalation, phase 2 trials.

BACKGROUND: Typhoid vaccination is a public health priority in developing countries where young children are greatly affected by typhoid fever. Because present vaccines are not recommended for children younger than 2 years, the Novartis Vaccines Institute for Global Health developed a conjugate vaccine (Vi-CRM197) for infant immunisation. We aimed to assess the immunogenicity and safety of Vi-CRM197 in participants of various ages in endemic countries in south and southeast Asia. METHODS: We did two randomised, observer-blind, age de-escalation, phase 2 trials at two sites in Pakistan and India (study A), and at one site in the Philippines (study B), between March 2, 2011, and Aug 9, 2012. Adults aged 18-45 years, children aged 24-59 months, older infants aged 9-12 months, and infants aged 6-8 weeks were randomly assigned (1:1) with a computer-generated randomisation list (block size of four) to receive either 5 µg Vi-CRM197 or 25 µg Vi-polysaccharide vaccine (or 13-valent pneumococcal conjugate vaccine in children younger than 2 years). Both infant populations received Vi-CRM197 concomitantly with vaccines of the Expanded Programme on Immunization (EPI), according to WHO schedule. With the exception of designated study site personnel responsible for vaccine preparation, study investigators, those assessing outcomes, and data analysts were masked to treatment allocation. We specified no a-priori null hypothesis for the immunogenicity or safety objectives and all analyses were descriptive. Analyses were by modified intention-to-treat. These studies are registered with ClinicalTrials.gov, numbers NCT01229176 and NCT01437267. FINDINGS: 320 participants were enrolled and vaccinated in the two trials: 200 in study A (all age groups) and 120 in study B (children and infants only), of whom 317 (99%) were included in the modified intention-to-treat analysis. One dose of Vi-CRM197 significantly increased concentrations of anti-Vi antibody in adults (from 113 U/mL [95% CI 67-190] to 208 U/mL [117-369]), children (201 U/mL [138-294] to 368 U/mL [234-580]), and older infants (179 U/mL [129-250] to 249 U/mL [130-477]). However, in children and older infants, a second dose of conjugate vaccine had no incremental effect on antibody titres and, at all ages, concentrations of antibodies increased substantially 6 months after vaccination (from 55 U/mL [33-94] to 63 U/mL [35-114] in adults, from 23 U/mL [15-34] to 51 U/mL [34-76] in children, and from 21 U/mL [14-31] to 22 U/mL [14-33] in older infants). Immune response in infants aged 6-8 weeks was lower than that in older participants and, 6 months after third vaccination, antibody concentrations were significantly higher than pre-vaccination concentrations in Filipino (21 U/mL [16-28] vs 2.88 U/mL [1.95-4.25]), but not Pakistani (3.76 U/mL [2.77-5.08] vs 2.77 U/mL [2.1-3.66]), infants. Vi-CRM197 was safe and well tolerated and did not induce any significant interference with EPI vaccines. No deaths or vaccine-related serious adverse events were reported throughout the studies. INTERPRETATION: Vi-CRM197 is safe and immunogenic in endemic populations of all ages. Given at 9 months of age, concomitantly with measles vaccine, Vi-CRM197 shows a promise for potential inclusion in EPI schedules of countries endemic for typhoid. An apparent absence of booster response and a reduction in antibody titres 6 months after immunisation should be further investigated, but data show that an immunogenic typhoid vaccine can be safely delivered to infants during EPI visits recommended by WHO. FUNDING: Sclavo Vaccines Association and Regione Toscana.

Tumorigenicity of IL-1alpha- and IL-1beta-deficient fibrosarcoma cells.

Analyzing the growth of fibrosarcoma lines derived from IL-1alpha-, IL-1beta- , or IL-1alphabeta-knockout (-/-) mice in the immunocompetent host revealed that tumor-derived IL-1alpha and IL-1beta exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1-deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1-deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1alpha(-/-)beta-competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1beta(comp) lines, IL-1alpha strengthens anchorage-independent growth, but both IL-1alpha and IL-1beta support drug resistance. Corresponding to the aggressive growth, IL-1beta(comp) cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated alpha(v)/beta3 and matrix metalloroteinase expression. Recruitment of myeloid cells requires IL-1beta but is regulated by IL-1alpha, because inflammatory chemokine and cytokine expression is stronger in IL-1alpha(-/-)beta(comp) than in IL-1(wt) lines. This regulatory effect of tumor-derived IL-1alpha is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1beta. Both sarcoma cell-derived IL-1alpha and IL-1beta promote tumor growth. However, IL-1alpha exerts regulatory activity on the tumor cell-matrix cross-talk, and only IL-1beta initiates systemic inflammation.

The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a chronic contact eczema.

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4(+)CD25(high) lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4(+)CD25(+) T cells. Instead, a population of Gr-1(+)CD11b(+) cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1(+)CD11b(+) spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-gamma receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1(+) cells expressed several chemokines and CCR8 at high levels. Gr-1(+)CD11b(+) cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4(+) or CD8(+) cells from AA mice, the Gr-1(+)CD11b(+) cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, zeta-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via zeta-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.

In vivo CD44-CD49d complex formation in autoimmune disease has consequences on T cell activation and apoptosis resistance.

CD44 is involved in leukocyte migration and activation and has recently been reported to contribute to leukocyte extravasation by associating with CD49d. We explored whether similar changes in CD44 activity are seen in vivo using murine alopecia areata (AA) as a chronic, organ-related autoimmune disease model system. Expression of the activated, hyaluronan-binding form of CD44, and of CD49d, was elevated in draining lymph node cells (LNC) of AA-affected mice as compared to control mice. LNC of AA mice displayed increased motility, proliferative activity and apoptosis resistance, which were equally well inhibited by anti-CD44 and anti-CD49d. The latter is the sequelae of the association between CD44 and CD49d that is seen in activated lymphocytes. Significantly, due to CD44-CD49d complex formation, CD44 gains access to focal adhesion kinase and CD49d gains access to CD44-associated lck and ezrin, such that downstream kinases become activated via CD44 or CD49d engagement. Thus, by their association, CD44 and CD49d mutually avail themselves of the partner's signaling pathways and the ligand binding of each one triggers signaling pathways of both. This strongly influences the lymphocytes' activation state and function.