Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

Teleseismic array analysis of upper mantle compressional velocity structure

Large quantities of teleseismic short-period seismograms recorded at SCARLET provide travel time, apparent velocity and waveform data for study of upper mantle compressional velocity structure. Relative array analysis of arrival times from distant (30° < Δ < 95°) earthquakes at all azimuths constrains lateral velocity variations beneath southern California. We compare dT/dΔ back azimuth and averaged arrival time estimates from the entire network for 154 events to the same parameters derived from small subsets of SCARLET. Patterns of mislocation vectors for over 100 overlapping subarrays delimit the spatial extent of an east-west striking, high-velocity anomaly beneath the Transverse Ranges. Thin lens analysis of the averaged arrival time differences, called 'net delay' data, requires the mean depth of the corresponding lens to be more than 100 km. Our results are consistent with the PKP-delay times of Hadley and Kanamori (1977), who first proposed the high-velocity feature, but we place the anomalous material at substantially greater depths than their 40-100 km estimate.

Detailed analysis of travel time, ray parameter and waveform data from 29 events occurring in the distance range 9° to 40° reveals the upper mantle structure beneath an oceanic ridge to depths of over 900 km. More than 1400 digital seismograms from earthquakes in Mexico and Central America yield 1753 travel times and 58 dT/dΔ measurements as well as high-quality, stable waveforms for investigation of the deep structure of the Gulf of California. The result of a travel time inversion with the tau method (Bessonova et al., 1976) is adjusted to fit the p(Δ) data, then further refined by incorporation of relative amplitude information through synthetic seismogram modeling. The application of a modified wave field continuation method (Clayton and McMechan, 1981) to the data with the final model confirms that GCA is consistent with the entire data set and also provides an estimate of the data resolution in velocity-depth space. We discover that the upper mantle under this spreading center has anomalously slow velocities to depths of 350 km, and place new constraints on the shape of the 660 km discontinuity.

Seismograms from 22 earthquakes along the northeast Pacific rim recorded in southern California form the data set for a comparative investigation of the upper mantle beneath the Cascade Ranges-Juan de Fuca region, an ocean-continent transit ion. These data consist of 853 seismograms (6° < Δ < 42°) which produce 1068 travel times and 40 ray parameter estimates. We use the spreading center model initially in synthetic seismogram modeling, and perturb GCA until the Cascade Ranges data are matched. Wave field continuation of both data sets with a common reference model confirms that real differences exist between the two suites of seismograms, implying lateral variation in the upper mantle. The ocean-continent transition model, CJF, features velocities from 200 and 350 km that are intermediate between GCA and T7 (Burdick and Helmberger, 1978), a model for the inland western United States. Models of continental shield regions (e.g., King and Calcagnile, 1976) have higher velocities in this depth range, but all four model types are similar below 400 km. This variation in rate of velocity increase with tectonic regime suggests an inverse relationship between velocity gradient and lithospheric age above 400 km depth.

Tunable heparan sulfate glycomimetics for modulating chemokine activity

Heparan sulfate (HS) glycosaminoglycans participate in critical biological processes by modulating the activity of a diverse set of protein binding partners. Such proteins include all known members of the chemokine superfamily, which are thought to guide the migration of distinct subsets of immune cells through their interactions with HS proteoglycans on endothelial cell surfaces. Animal-derived heparin polysaccharides have been shown to reduce inflammation levels through the inhibition of HS-chemokine interactions; however, the clinical usage of heparin as an anti-inflammatory drug is hampered by its anticoagulant activity and potential risk for side effects, such as heparin-induced thrombocytopenia (HIT).

Here, we describe an expedient, divergent synthesis to prepare defined glycomimetics of HS that recapitulate the macromolecular structure and biological activity of natural HS glycosaminoglycans. Our synthetic approach uses a core disaccharide precursor to generate a library of four differentially sulfated polymers. We show that a trisulfated glycopolymer antagonizes the chemotactic activities of pro-inflammatory chemokine RANTES with similar potency as heparin polysaccharide, without potentiating the anticoagulant activities of antithrombin III. The same glycopolymer also inhibited the homeostatic chemokine SDF-1 with significantly more efficacy than heparin. Our work offers a general strategy for modulating chemokines and dissecting the pleiotropic functions of HS/heparin through the presentation of defined sulfation motifs within multivalent polymeric scaffolds.

Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

Molecular genetic studies on voltage-gated ion channels

Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K⁺) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant para^ts. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.

In Drosophila, diverse voltage-gated K⁺ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K⁺-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K⁺-channel gene family probably diversified prior to the mammalian radiation. Functional K⁺ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K⁺ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K⁺-channel genes may efficiently co-assemble into heterooligomeric K⁺ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K⁺-channel diversity.

Paranormal operator on a Hilbert space

In this thesis an extensive study is made of the set P of all paranormal operators in B(H), the set of all bounded endomorphisms on the complex Hilbert space H. T ϵ B(H) is paranormal if for each z contained in the resolvent set of T, d(z, σ(T))//(T-zI)^-1 = 1 where d(z, σ(T)) is the distance from z to σ(T), the spectrum of T. P contains the set N of normal operators and P contains the set of hyponormal operators. However, P is contained in L, the set of all T ϵ B(H) such that the convex hull of the spectrum of T is equal to the closure of the numerical range of T. Thus, N≤P≤L.

If the uniform operator (norm) topology is placed on B(H), then the relative topological properties of N, P, L can be discussed. In Section IV, it is shown that: 1) N P and L are arc-wise connected and closed, 2) N, P, and L are nowhere dense subsets of B(H) when dim H ≥ 2, 3) N = P when dimH ˂ ∞ , 4) N is a nowhere dense subset of P when dimH ˂ ∞ , 5) P is not a nowhere dense subset of L when dimH ˂ ∞ , and 6) it is not known if P is a nowhere dense subset of L when dimH ˂ ∞.

The spectral properties of paranormal operators are of current interest in the literature. Putnam [22, 23] has shown that certain points on the boundary of the spectrum of a paranormal operator are either normal eigenvalues or normal approximate eigenvalues. Stampfli [26] has shown that a hyponormal operator with countable spectrum is normal. However, in Theorem 3.3, it is shown that a paranormal operator T with countable spectrum can be written as the direct sum, N ⊕ A, of a normal operator N with σ(N) = σ(T) and of an operator A with σ(A) a subset of the derived set of σ(T). It is then shown that A need not be normal. If we restrict the countable spectrum of T ϵ P to lie on a C²-smooth rectifiable Jordan curve G_o, then T must be normal [see Theorem 3.5 and its Corollary]. If T is a scalar paranormal operator with countable spectrum, then in order to conclude that T is normal the condition of σ(T) ≤ G_o can be relaxed [see Theorem 3.6]. In Theorem 3.7 it is then shown that the above result is not true when T is not assumed to be scalar. It was then conjectured that if T ϵ P with σ(T) ≤ G_o, then T is normal. The proof of Theorem 3.5 relies heavily on the assumption that T has countable spectrum and cannot be generalized. However, the corollary to Theorem 3.9 states that if T ϵ P with σ(T) ≤ G_o, then T has a non-trivial lattice of invariant subspaces. After the completion of most of the work on this thesis, Stampfli [30, 31] published a proof that a paranormal operator T with σ(T) ≤ G_o is normal. His proof uses some rather deep results concerning numerical ranges whereas the proof of Theorem 3.5 uses relatively elementary methods.

Locally convex Riesz spaces and Archimedean quotient spaces

A Riesz space with a Hausdorff, locally convex topology determined by Riesz seminorms is called a locally convex Riesz space. A sequence {x_n} in a locally convex Riesz space L is said to converge locally to x ϵ L if for some topologically bounded set B and every real r ˃ 0 there exists N (r) and n ≥ N (r) implies x – x_n ϵ r^b. Local Cauchy sequences are defined analogously, and L is said to be locally complete if every local Cauchy sequence converges locally. Then L is locally complete if and only if every monotone local Cauchy sequence has a least upper bound. This is a somewhat more general form of the completeness criterion for Riesz – normed Riesz spaces given by Luxemburg and Zaanen. Locally complete, bound, locally convex Riesz spaces are barrelled. If the space is metrizable, local completeness and topological completeness are equivalent.

Two measures of the non-archimedean character of a non-archimedean Riesz space L are the smallest ideal A_o (L) such that quotient space is Archimedean and the ideal I (L) = { x ϵ L: for some 0 ≤ v ϵ L, n |x| ≤ v for n = 1, 2, …}. In general A_o (L) ᴝ I (L). If L is itself a quotient space, a necessary and sufficient condition that A_o (L) = I (L) is given. There is an example where A_o (L) ≠ I (L).

A necessary and sufficient condition that a Riesz space L have every quotient space Archimedean is that for every 0 ≤ u, v ϵ L there exist u₁ = sup (inf (n v, u): n = 1, 2, …), and real numbers m₁ and m₂ such that m₁ u₁ ≥ v₁ and m₂ v₁ ≥ u₁. If, in addition, L is Dedekind σ – complete, then L may be represented as the space of all functions which vanish off finite subsets of some non-empty set.

λ-designs and related combinatorial configurations

This thesis deals with two problems. The first is the determination of λ-designs, combinatorial configurations which are essentially symmetric block designs with the condition that each subset be of the same cardinality negated. We construct an infinite family of such designs from symmetric block designs and obtain some basic results about their structure. These results enable us to solve the problem for λ = 3 and λ = 4. The second problem deals with configurations related to both λ -designs and (ѵ, k, λ)-configurations. We have (n-1) k-subsets of {1, 2, ..., n}, S₁, ..., S_n-1 such that S_i ∩ S_j is a λ-set for i ≠ j. We obtain specifically the replication numbers of such a design in terms of n, k, and λ with one exceptional class which we determine explicitly. In certain special cases we settle the problem entirely.

Distributed Optimal Control of Cyber-Physical Systems: Controller Synthesis, Architecture Design and System Identification

The centralized paradigm of a single controller and a single plant upon which modern control theory is built is no longer applicable to modern cyber-physical systems of interest, such as the power-grid, software defined networks or automated highways systems, as these are all large-scale and spatially distributed. Both the scale and the distributed nature of these systems has motivated the decentralization of control schemes into local sub-controllers that measure, exchange and act on locally available subsets of the globally available system information. This decentralization of control logic leads to different decision makers acting on asymmetric information sets, introduces the need for coordination between them, and perhaps not surprisingly makes the resulting optimal control problem much harder to solve. In fact, shortly after such questions were posed, it was realized that seemingly simple decentralized optimal control problems are computationally intractable to solve, with the Wistenhausen counterexample being a famous instance of this phenomenon. Spurred on by this perhaps discouraging result, a concerted 40 year effort to identify tractable classes of distributed optimal control problems culminated in the notion of quadratic invariance, which loosely states that if sub-controllers can exchange information with each other at least as quickly as the effect of their control actions propagates through the plant, then the resulting distributed optimal control problem admits a convex formulation.

The identification of quadratic invariance as an appropriate means of "convexifying" distributed optimal control problems led to a renewed enthusiasm in the controller synthesis community, resulting in a rich set of results over the past decade. The contributions of this thesis can be seen as being a part of this broader family of results, with a particular focus on closing the gap between theory and practice by relaxing or removing assumptions made in the traditional distributed optimal control framework. Our contributions are to the foundational theory of distributed optimal control, and fall under three broad categories, namely controller synthesis, architecture design and system identification.

We begin by providing two novel controller synthesis algorithms. The first is a solution to the distributed H-infinity optimal control problem subject to delay constraints, and provides the only known exact characterization of delay-constrained distributed controllers satisfying an H-infinity norm bound. The second is an explicit dynamic programming solution to a two player LQR state-feedback problem with varying delays. Accommodating varying delays represents an important first step in combining distributed optimal control theory with the area of Networked Control Systems that considers lossy channels in the feedback loop. Our next set of results are concerned with controller architecture design. When designing controllers for large-scale systems, the architectural aspects of the controller such as the placement of actuators, sensors, and the communication links between them can no longer be taken as given -- indeed the task of designing this architecture is now as important as the design of the control laws themselves. To address this task, we formulate the Regularization for Design (RFD) framework, which is a unifying computationally tractable approach, based on the model matching framework and atomic norm regularization, for the simultaneous co-design of a structured optimal controller and the architecture needed to implement it. Our final result is a contribution to distributed system identification. Traditional system identification techniques such as subspace identification are not computationally scalable, and destroy rather than leverage any a priori information about the system's interconnection structure. We argue that in the context of system identification, an essential building block of any scalable algorithm is the ability to estimate local dynamics within a large interconnected system. To that end we propose a promising heuristic for identifying the dynamics of a subsystem that is still connected to a large system. We exploit the fact that the transfer function of the local dynamics is low-order, but full-rank, while the transfer function of the global dynamics is high-order, but low-rank, to formulate this separation task as a nuclear norm minimization problem. Finally, we conclude with a brief discussion of future research directions, with a particular emphasis on how to incorporate the results of this thesis, and those of optimal control theory in general, into a broader theory of dynamics, control and optimization in layered architectures.