T cell therapy against cancer: A predictive diffuse-interface mathematical model informed by pre-clinical studies.

T cell therapy has become a new therapeutic opportunity against solid cancers. Predicting T cell behaviour and efficacy would help therapy optimization and clinical implementation. In this work, we model responsiveness of mouse prostate adenocarcinoma to T cell-based therapies. The mathematical model is based on a Cahn-Hilliard diffuse interface description of the tumour, coupled with Keller-Segel type equations describing immune components dynamics. The model is fed by pre-clinical magnetic resonance imaging data describing anatomical features of prostate adenocarcinoma developed in the context of the Transgenic Adenocarcinoma of the Mouse Prostate model. We perform computational simulations based on the finite element method to describe tumor growth dynamics in relation to local T cells concentrations. We report that when we include in the model the possibility to activate tumor-associated vessels and by that increase the number of T cells within the tumor mass, the model predicts higher therapeutic effects (tumor regression) shortly after therapy administration. The simulated results are found in agreement with reported experimental data. Thus, this diffuse-interface mathematical model well predicts T cell behavior in vivo and represents a proof-of-concept for the role such predictive strategies may play in optimization of immunotherapy against cancer.

Development and validation of a sleep questionnaire, SNoRE 3.0, to evaluate sleep in companion dogs.

Disturbances in the sleep-wake cycle are a debilitating, yet rather common condition not only in humans, but also in family dogs. While there is an emerging need for easy-to-use tools to document sleep alterations (in order to ultimately treat and/or prevent them), the veterinary tools which yield objective data (e.g. polysomnography, activity monitors) are both labor intensive and expensive. In this study, we developed a modified version of a previously used sleep questionnaire (SNoRE) and determined criterion validity in companion dogs against polysomnography and physical activity monitors (PAMs). Since a negative correlation between sleep time and cognitive performance in senior dogs has been demonstrated, we evaluated the correlation between the SNoRE scores and the Canine Dementia Scale (CADES, which includes a factor concerning sleep). There was a significant correlation between SNoRE 3.0 questionnaire scores and polysomnography data (latency to NREM sleep, ρ = 0.507, p < 0.001) as well as PAMs' data (activity between 1:00 and 3:00 AM, p < 0.05). There was a moderate positive correlation between the SNoRE 3.0 scores and the CADES scores (ρ = 0.625, p < 0.001). Additionally, the questionnaire structure was validated by a confirmatory factor analysis, and it also showed an adequate test-retest reliability. In conclusion the present paper describes a valid and reliable questionnaire tool, that can be used as a cost-effective way to monitor dog sleep in clinical settings.

In vivo detection of dendritic cell antigen presentation to CD4(+) T cells.

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4(+) T cells specific for an OVA peptide-I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism.

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.

Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells.

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.

Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor).

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Surface proteins involved in T cell costimulation.

The activation and eventual clonal expansion of individual antigen-specific CD4+ T cell clones are dependent on the production of autocrine growth factors such as interleukin-2 (IL-2). The specificity of CD4+ T cell activation is imparted by T cell antigen receptor (TCR) recognition of peptide antigens bound to class II major histocompatibility complex (MHC)-encoded molecules on the surface of antigen-presenting cells (APCs), for example B cells, macrophages, and dendritic cells. To induce maximal IL-2 production by T cells, however, APCs must also provide non-antigen-specific costimulatory signals. Recent work indicates that APC-derived costimulatory signals play a critical role in determining whether lymphokine production, apoptotic cell death, or functional anergy is induced by TCR engagement. This information has allowed immunologists to manipulate costimulatory molecules to prevent allograft rejection and enhance tumor immunity. Here we review current information on the biologic effects of, and signal transduction pathways engaged by, several known receptor-ligand pairs that transduce costimulatory signals in T cells. Special emphasis will be placed on the interaction of CD28 on the T cell with its ligands, B7-1, B7-2, and B7-3 on the APC.

Structure and function of the urokinase receptor.

The binding of the urokinase plasminogen activator (uPA) to its receptor (uPAR) regulates cell adhesion, surface proteolysis, chemotaxis and cell extravasation in a number of experimental systems. Recent evidences have suggested that uPAR can by itself mediate chemotaxis of human monocytes and cause profound changes in cytoskeletal organization indicating that this receptor has the properties of a cell-surface regulated chemokine. Indeed, it is likely that upon binding to uPA, uPAR undergoes a conformational change that uncovers a new epitope located in the linker region between domain 1 and 2 of the receptor and is endowed with a potent chemotactic activity. This conformational change can be mimicked in vitro by enzymatic processing of a recombinant receptor. We have shown that chymotrypsin cleaves uPAR between domain 1 and 2 in an area that can be also cleaved by uPA at high efficiency and generate a receptor that can mediate monocytes migration independently of uPA binding. This mechanism is pertussis-toxin sensitive and involves activation of tyrosine kinases and cytoskeletal reorganization events in vitro. These studies indicate that in addition to its receptor function, upon binding to uPA, uPAR becomes a pleiotropic ligand for other still to be identified surface molecules.

Antigen-specific CD4+ T cells that survive after the induction of peripheral tolerance possess an intrinsic lymphokine production defect.

Injection of soluble foreign antigen without an adjuvant induces a state of antigen-specific immunological unresponsiveness. We investigated the cellular mechanisms that underlie this form of peripheral tolerance by physically tracking a small population of ovalbumin (OVA) peptide/I-Ad-specific, CD4+ T cell receptor (TCR) transgenic T cells following adoptive transfer into normal recipients. Injection of OVA peptide in the absence of adjuvant caused the antigen-specific T cells to proliferate for a brief period after which most of the T cells disappeared. The remaining OVA-specific T cells had converted to a memory phenotype but were poorly responsive in vivo as evidenced by a failure to accumulate in the draining lymph nodes following immunization with OVA peptide in adjuvant. These surviving T cells possessed a long-lasting, but reversible, defect in Il-2 and TNF-alpha production and in vivo proliferation, but did not gain capacity to produce Th2-type cytokines or suppress the clonal expansion of T cells specific for another antigen. Therefore, some antigen-specific T cells survive this peripheral tolerance protocol but are functionally unresponsive due to an intrinsic activation defect.

A tyrosine protein kinase activated by bombesin in normal fibroblasts and small cell carcinomas.

In Swiss 3T3 fibroblasts, antibodies which recognize a phosphotyrosine residue (P-Tyr antibodies) identify a 115-kDa cell surface protein (p115) that becomes phosphorylated on tyrosine as a response to bombesin stimulation of quiescent cells. The extent of phosphorylation is dose-dependent and correlates with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 is detectable minutes after addition of bombesin and precedes the activation of c-fos and c-myc gene transcription. Immunocomplexes of phosphorylated p115 with P-Tyr antibodies bind 125I-labeled [Tyr4]bombesin in a specific and saturable manner and display an associated tyrosine protein kinase activity enhanced by bombesin. P-Tyr antibodies also recognize a protein of 115 kDa, phosphorylated at tyrosine, in four human SCLC lines producing bombesin but not in a non-producer "variant" line. Phosphorylation of SCLC p115 does not require the addition of exogenous bombesin. As in the case of the p115 immunoprecipitated from mouse fibroblasts, the SCLC p115 is phosphorylated in an immunocomplex kinase assay. These observations are in agreement with the hypothesis of autocrine activation of bombesin receptors in human small cell lung carcinoma cells.

Longitudinal changes of brain amino acid content occurring before, during and after epileptic activity.

In cats affected with cortical epileptogenic foci induced by penicillin application to and cobalt implantation into the pericruciate area, the brain amino acids contents were determined in the focus as well as in extrafocal areas. In different groups of animals, brain removal for biochemical determinations was performed at different times before, during and after epilepsy and the values compared to controls. The only amino acid to show a significant change before appearance of spikes in both types of epilepsy was taurine, which decreased. Cobalt epilepsy was accompanied by changes in a larger number of amino acids than penicillin epilepsy: in the former the brain content of taurine, GABA, aspartate, glutamate, serine, threonine, glycine and alanine was altered. The changes were proportional to the severity of epilepsy and more prominent in the focus area. After disappearance of spikes the levels of most amino acids returned to normal except for some amino acids, previously unaffected by penicillin epilepsy, which were decreased. It is proposed that the decrease in brain taurine, occurring before the appearance of penicillin and cobalt epilepsy, could increase the excitability of a certain neuronal population and thus, by potentiating the effects on neurons of penicillin and cobalt, contribute to the initiation of epilepsy.