Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin’s mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim^−/−) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim^−/−mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin’s enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim^−/−mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim^−/−mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim^−/−mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young’s moduli of normal and vim^−/−mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.     

Viscoelastic Parameters for Quantifying Liver Fibrosis: Three-Dimensional Multifrequency MR Elastography Study on Thin Liver Rat Slices

Objective

To assess in a high-resolution model of thin liver rat slices which viscoelastic parameter at three-dimensional multifrequency MR elastography has the best diagnostic performance for quantifying liver fibrosis.

Materials and Methods

The study was approved by the ethics committee for animal care of our institution. Eight normal rats and 42 rats with carbon tetrachloride induced liver fibrosis were used in the study. The rats were sacrificed, their livers were resected and three-dimensional MR elastography of 5±2 mm liver slices was performed at 7T with mechanical frequencies of 500, 600 and 700 Hz. The complex shear, storage and loss moduli, and the coefficient of the frequency power law were calculated. At histopathology, fibrosis and inflammation were assessed with METAVIR score, fibrosis was further quantified with morphometry. The diagnostic value of the viscoelastic parameters for assessing fibrosis severity was evaluated with simple and multiple linear regressions, receiver operating characteristic analysis and Obuchowski measures.

Results

At simple regression, the shear, storage and loss moduli were associated with the severity of fibrosis. At multiple regression, the storage modulus at 600 Hz was the only parameter associated with fibrosis severity (r = 0.86, p<0.0001). This parameter had an Obuchowski measure of 0.89+/−0.03. This measure was significantly larger than that of the loss modulus (0.78+/−0.04, p = 0.028), but not than that of the complex shear modulus (0.88+/−0.03, p = 0.84).

Conclusion

Our high resolution, three-dimensional multifrequency MR elastography study of thin liver slices shows that the storage modulus is the viscoelastic parameter that has the best association with the severity of liver fibrosis. However, its diagnostic performance does not differ significantly from that of the complex shear modulus.     

Cellular Response to Substrate Rigidity Is Governed by Either Stress or Strain

Cells sense the rigidity of their substrate; however, little is known about the physical variables that determine their response to this rigidity. Here, we report traction stress measurements carried out using fibroblasts on polyacrylamide gels with Young’s moduli ranging from 6 to 110 kPa. We prepared the substrates by employing a modified method that involves N-acryloyl-6-aminocaproic acid (ACA). ACA allows for covalent binding between proteins and elastomers and thus introduces a more stable immobilization of collagen onto the substrate when compared to the conventional method of using sulfo-succinimidyl-6-(4-azido-2-nitrophenyl-amino) hexanoate (sulfo-SANPAH). Cells remove extracellular matrix proteins off the surface of gels coated using sulfo-SANPAH, which corresponds to lower values of traction stress and substrate deformation compared to gels coated using ACA. On soft ACA gels (Young’s modulus <20 kPa), cell-exerted substrate deformation remains constant, independent of the substrate Young’s modulus. In contrast, on stiff substrates (Young’s modulus >20 kPa), traction stress plateaus at a limiting value and the substrate deformation decreases with increasing substrate rigidity. Sustained substrate strain on soft substrates and sustained traction stress on stiff substrates suggest these may be factors governing cellular responses to substrate rigidity.     

Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth (~1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4–37°C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37–4°C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G′, from 23 ± 3 Pa to 0.28 ± 0.16 Pa, respectively, mean ± SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 ± 3.5 Pa before to 138 ± 40 Pa after cross-linking, mean ± SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.     

Influenza-Infected Neutrophils within the Infected Lungs Act as Antigen Presenting Cells for Anti-Viral CD8+ T Cells

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8⁺ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8⁺ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8⁺ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8⁺ T cells within the infected lung interstitium.     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

Characterizing the Lymphopoietic Kinetics and Features of Hematopoietic Progenitors Contained in the Adult Murine Liver In Vivo

The appearance of donor-derived lymphocytes in liver transplant patients suggests that adult livers may contain cells capable of lymphopoiesis. However, only a few published studies have addressed the lymphopoietic capacity of adult liver cells, and its kinetics and features remain unclear. Herein, we investigated the lymphopoietic capacity of adult liver mononuclear cells (MNCs) and purified liver hematopoietic progenitor cells (HPCs) in vivo. Similar to bone-marrow transplantation (BMT), transplantation of liver MNCs alone was able to rescue survival of lethally irradiated mice. In terms of kinetics, liver MNC-derived myeloid lineage cells reconstituted more slowly than those from BMT. Liver MNC-derived lymphocyte lineage cells in the blood, spleen and BM also reconstituted more slowly than BMT, but lymphocytes in the liver recovered at a similar rate. Interestingly, liver MNCs predominantly gave rise to CD3⁺CD19⁻ T cells in both irradiated WT and non-irradiated lymphocyte-deficient Rag-1^−/−Il2rg^−/− recipients. To define the lymphopoietic potential of various cell populations within liver MNCs, we transplanted purified lineage-negative (Lin⁻) liver HPCs into recipient mice. Unlike total liver MNCs, liver HPCs reconstituted T and B cells in similar frequencies to BMT. We further determined that the predominance of T cells observed after transplanting total liver MNCs likely originated from mature T cells, as purified donor liver T cells proliferated in the recipients and gave rise to CD8⁺ T cells. Thus, the capacity of donor adult liver cells to reconstitute lymphocytes in recipients derives from both HPCs and mature T cells contained in the liver MNC population.     

TRAF3 Regulates Homeostasis of CD8+ Central Memory T Cells

Our laboratory reported previously that TNF receptor associated factor 3 (TRAF3) is a positive regulator of TCR signaling and T cell function. In the current study, we present new findings that reveal differential roles for TRAF3 in the regulation of CD4⁺ and CD8⁺ T cells. In response to TCR stimulation in vitro, TRAF3 has greater impact in CD4⁺ T cells than in CD8⁺ T cells. However, T cell-specific TRAF3 deficient mice (CD4^Cre TRAF3^fl°^x/fl°^x; T-TRAF3^−/−) have a greater number of CD4⁺CD44^hi effector/memory T cells than littermate control (LMC) mice, possibly due to an inefficient suppressive effect of TRAF3 deficient Foxp3⁺ regulatory T cells. In contrast, CD8⁺CD44^hiCD62L^hi central memory (Tcm) cells are markedly reduced in T-TRAF3^−/− mice in comparison to LMC mice, although CD8⁺CD44^hiCD62L^l°^w effector memory T (Tem) cells and naïve T cells (CD8⁺CD44^l°^wCD62L^hi) do not show significant differences in number. Importantly, TRAF3-deficient Tcm cells exhibit defective homeostasis due to impaired IL-15 signaling. These results indicate that the involvement of TRAF3 in IL-15 mediated signaling to T cells plays a previously unappreciated and critical role in CD8⁺ Tcm cell regulation and maintenance.     

An Inverse Power-Law Distribution of Molecular Bond Lifetimes Predicts Fractional Derivative Viscoelasticity in Biological Tissue

Viscoelastic characteristics of many materials falling under the category of soft glassy substances, including biological tissue, often exhibit a mechanical complex modulus Y(ω) well described by a fractional derivative model: Y(ω) = E(iω/ϕ)^k, where E = a generalized viscoelastic stiffness; i = (−1)^1/2; ω = angular frequency; ϕ = scaling factor; and k = an exponent valued between 0 and 1. The term “fractional derivative” refers to the value of k: when k = 0 the viscoelastic response is purely elastic, and when k = 1 the response is purely viscous. We provide an analytical derivation of the fractional derivative complex modulus based on the hypothesis that the viscoelastic response arises from many intermittent molecular crosslinks, whose lifetimes longer than a critical threshold lifetime, t_crit, are distributed with an inverse power law proportional to t^-(k+2). We demonstrate that E is proportional to the number and stiffness of crosslinks formed at any moment; the scaling factor ϕ is equivalent to reciprocal of t_crit; and the relative mean lifetime of the attached crosslinks is inversely proportional to the parameter k. To test whether electrostatic molecular bonds could be responsible for the fractional derivative viscoelasticity, we used chemically skinned human skeletal muscle as a one-dimensional model of a soft glassy substance. A reduction in ionic strength from 175 to 110 mEq resulted in a larger E with no change in k, consistent with a higher probability of interfilament molecular interactions. Thick to thin filament spacing was reduced by applying 4% w/v of the osmolyte Dextran T500, which also resulted in a larger E, indicating a greater probability of crosslink formation in proportion to proximity. A 10°C increase in temperature resulted in an increase in k, which corresponded to a decrease in cross-bridge attachment lifetime expected with higher temperatures. These theoretical and experimental results suggest that the fractional derivative viscoelasticity observed in some biological tissue arises as a mechanical consequence of electrostatic interactions, whose longest lifetimes are distributed with an inverse power law.     

p38α Protein Negatively Regulates T Helper Type 2 Responses by Orchestrating Multiple T Cell Receptor-associated Signals

Mitogen-activated protein kinase p38α is a critical regulator of certain inflammatory diseases. However, its role in T helper type 2 (Th2) responses and allergic inflammation remains unknown. Here we show an increase in the production of interleukin-4 (IL-4) in p38α^−/− CD4⁺ T cells in response to antigen stimulation. p38α-deficient naïve CD4⁺ T cells preferentially differentiate into Th2 cells through increased endogenous production of IL-4. Consistent with those results, we also observed decreased expression of p38α during T helper cell differentiation. Furthermore, deficiency of p38α alters the balance in the expression of NFATc1 and NFATc2 under steady-state conditions and enhances the expression and nuclear translocation of NFATc1 in CD4⁺ T cells upon antigen stimulation. Knockdown of NFATc1 significantly inhibits Th2 differentiation in p38α^−/− T cells but not in p38α^+/− T cells. p38α deficiency also inhibits the activation of Akt but enhances the activation of ERK in response to T cell receptor engagement without impacting IL-2/Stat5 signaling. In a model of ovalbumin-induced acute allergic airway inflammation, mice with induced deletion of p38α show elevated serum ovalbumin-specific IgE level, increased infiltration of eosinophils, and higher concentrations of Th2 cytokines including IL-4 and IL-5 in the bronchoalveolar lavage fluid relative to control mice. Taken together, p38α regulates multiple T cell receptor-associated signals and negatively influences Th2 differentiation and allergic inflammation.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^−/− mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10^−/− mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b⁻CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^−/− mice and IL-10^−/− mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.     

T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm⁻². T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Contrasted TCRβ Diversity of CD8+ and CD8? T Cells in Rainbow Trout

Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8⁺ T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8⁺ and CD8⁻ T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8⁺ T cells of naïve fish, it appeared very different in CD8⁻ lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8⁺ T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8⁻ fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8⁺ and CD8⁻ αβ T cells may be subjected to different regulatory patterns in fish and in mammals.     

Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar ^−/− mice completely lacking type I IFN signaling. In Mavs^−/−×Ifnar^−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar ^−/− and CD11c Cre⁺ Ifnar ^f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.     

Skewed Distribution of IL-7 Receptor-α-Expressing Effector Memory CD8+ T Cells with Distinct Functional Characteristics in Oral Squamous Cell Carcinoma

CD8⁺ T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8⁺ T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻ CD45RA⁺CD8⁺ T cells negatively correlated with each other. A significantly higher frequency of CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells among total CD8⁺ T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127^hi CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells maintained significantly higher IFN-γ, IL-2 productivity and ex vivo proliferative capacity, while the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7⁻CD45RA⁺CD8⁺ T cells to CCR7⁻CD45RA⁻CD8⁺ T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻CD45RA⁺CD8⁺ T cells are functionally similar CD8⁺ T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8⁺ effector memory balance toward CCR7⁻CD45RA⁺CD8⁺ T cells is associated with OSCC progression.     

Colorectal Cancer with Residual Polyp of Origin: A Model of Malignant Transformation

The majority of colorectal cancers (CRCs) arise from adenomatous polyps. In this study, we sought to present the underrecognized CRC with the residual polyp of origin (CRC RPO +) as an entity to be utilized as a model to study colorectal carcinogenesis. We identified all subjects with biopsy-proven CRC RPO + that were evaluated over 10 years at Mayo Clinic, Rochester, MN, and compared their clinical and pathologic characteristics to CRC without remnant polyps (CRC RPO −). Overall survival and disease-free survival overlap with an equivalent hazard ratio between CRC RPO + and RPO − cases when age, stage, and grade are adjusted. The somatic genomic profile obtained by whole genome sequencing and the gene expression profiles by RNA-seq for CRC RPO + tumors were compared with that of age -and gender-matched CRC RPO − evaluated by The Cancer Genome Atlas. CRC RPO + cases were more commonly found with lower-grade, earlier-stage disease than CRC RPO −. However, within the same disease stage and grade, their clinical course is very similar to that of CRC RPO −. The mutation frequencies of commonly mutated genes in CRC are similar between CRC RPO + and RPO − cases. Likewise, gene expression patterns are indistinguishable between the RPO + and RPO − cases. We have confirmed that CRC RPO + is clinically and biologically similar to CRC RPO − and may be utilized as a model of the adenoma to carcinoma transition.     

Spontaneous Proliferation of H2M-/- CD4 T Cells Results in Unusual Acute Hepatocellular Necrosis

Naïve CD4 T cells are triggered to undergo spontaneous proliferation, a proliferative response induced in response to homeostatic stimulation, when exposed to severe lymphopenic environments. They spontaneously acquire proinflammatory effector phenotypes, playing a major role in inducing chronic inflammation in the intestine that is believed to be induced by T cell recognition of commensal antigens. While the antigens inducing the T cell responses and inflammation are being extensively investigated, the role of clonality of T cells involved in this process remains poorly understood. In this study, we utilized naïve CD4 T cells isolated from B6 H2M−/− mice, in which MHCII molecules are complexed with a single CLIP molecule, and examined spontaneous proliferation and intestinal inflammation of CD4 T cells expressing limited T cell receptor repertoire diversity. We found that H2M−/− CD4 T cells undergo robust spontaneous proliferation, differentiate into IFNγ-producing Th1 type effector cells, and, most unexpectedly, induce severe acute hepatocellular necrosis. T cell interaction with MHCII molecule on cells of hematopoietic origin was essential to induce the pathology. Interestingly, B cells are fully capable of preventing necrotic inflammation via IL-10-independent and B7-H1-dependent mechanism. This could be a useful animal model to examine T cell-mediated liver inflammation and B cell-mediated immune regulation.