The immune system produces antibodies to recognize and provide protection against infection. The immunoglobulin G (IgG) antibody isotype is present at high serum concentrations and has a longer half-life than other isotypes due to the interaction between its fragment crystallizable (Fc) region with the neonatal Fc receptor (FcRn). This Fc-FcRn interaction, which takes place in many cell types throughout the cardiovascular system, mediates pH-dependent formation of the IgG-FcRn complex and leads to the rescue of IgG from eventual degradation via transport from the low-pH early endosome back to the cell surface for release into serum at pH 7.4. Because this process is the primary determinant of IgG antibody half-life, and because the Fc region is common to all antibodies of the same subtype, the Fc-FcRn system has been a target of numerous antibody design and engineering studies. Indeed, several engineered Fcs have been reported with extended serum half-lives. These novel Fc variants, however, have generally been the result of extensive experimental screening and combinations of individual Fc mutations with known biophysical properties; there are few reports of predominantly structure-based rational Fc design. Notably, simply increasing Fc binding affinity for FcRn at low pH does not appear to be sufficient to achieve the largest increases in half-life (and in some cases, very high affinity results in reduced serum half-life). Most of these engineered Fcs have increased affinity not only at low pH (~6.0), but also at pH 7.4. The longest-lived Fc variant known to date, however, with mutations L309D/Q311H/N434S (“DHS”), has only a modest 5-fold increase in binding affinity compared to wt Fc at low pH, but also exhibits negligible binding to the receptor at pH 7.4 (Lee et al., 2019). This is consistent with previous reports that identify efficient release at physiologic serum pH to be critical to FcRn-mediated half-life extension. Thus, while engineering for affinity at low pH, it is also important to optimize the pH dependence of binding for optimal release at serum pH. The rational design process requires a detailed understanding of the structural and functional details of the interaction, which for a pH-dependent complex like Fc-FcRn must also include an accurate model of the pH-sensing mechanism. Unfortunately, the only publicly available crystal structure of a human Fc-FcRn complex is of the M252Y/S254T/T256E (“YTE”) variant, and was determined only to a relatively low 3.8 Å resolution, leaving the atomic positions of many sidechains, and even regions of the protein backbone, subject to substantial uncertainty. Furthermore, the widely accepted conventional mechanism of pH sensing, involving protonation of key histidine residues on Fc at low pH due to the assumed histidine pKa of 6.5 being within the range of interest (pH 6.0-7.4), is thermodynamically impossible. In this thesis I present an extensive analysis of the Fc-FcRn system, including the generation of all-atom models of human wild-type (wt) and variant complexes and the rat wt complex, and assignment of dominant protonation states at pH 6.0, at which most binding experiments are performed. I validate these models using retrospective molecular dynamics (MD)-based free-energy perturbation (FEP) calculations to compare to a large dataset of wt and mutant binding affinities. During this validation process I identify a residue on FcRn, glutamic acid 133, which adopts a highly unusual configuration in the complex and, due to quantum mechanical electronic polarization effects, is not described well by the fixed-charge molecular mechanics force field used by the FEP calculations, resulting in systematic errors for mutations that affect its hydrogen bonding network. I also identify a new variant, with a V308P mutation in a YTE background (“YTEP”), which induces a previously unreported conformational change that accounts for its high binding affinity compared to YTE and wt. To address the problem of the pH-sensing mechanism, I describe a general method for calculating the pH-sensing free energy of binding for any complex, based on a study of the pH dependence of protein unfolding free energies (Yang and Honig, 1993). The key observation underlying this method is that pH-dependent complex formation must be accompanied by a change in the pKa of one or more titratable groups between the unbound and bound states. Furthermore, the change in binding energy between two pHs can be directly calculated based on those pKas alone. As there are no experimental pKa measurements available for the Fc-FcRn interface residues, I perform these pH-sensing free energy calculations using FEP-based calculated pKas to quantitatively assess which residues at the interface are involved in sensing pH over the physiologically relevant pH range, and present a residue-level model for pH sensing in the Fc-FcRn system. Finally, I present some preliminary work toward the rational design of modified Fc regions with both increased affinity at low pH, and increased pH dependence of binding, using FEP calculations to guide experiment. This type of approach, of computational screening of a large number of different variants, followed by more limited experimental testing of promising leads, has the potential to streamline Fc design efforts and provide further insight into the structural basis of function for the Fc-FcRn system.

Knowledge of human T cell responses and the pathways for their differentiation and maintenance from development into adulthood remains largely sparse. Much of what is known concerning the adaptive immune response in humans derives from analysis of peripheral blood, even though the majority of T cells within the body reside in tissue sites. We have established a protocol with LiveOnNY, the organ procurement organization of the New York metropolitan area allowing us access to healthy tissues from individual organ donors of a diverse background. Through novel analysis of lymphoid and mucosal tissues from infant and adult organ donors, we reveal how naïve, regulatory, and memory T cells dynamically populate and are maintained in tissues and circulation over the human lifespan. An initial multidimensional, quantitative analysis of human T cell compartmentalization involving 56 organ donors of a broad age range revealed that distribution of naïve, effector, and memory T cell subsets is largely dependent on tissue localization and differentiation state. Furthermore, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+or CD8+ T cell subset. Examining whether T cell subset distribution was set at birth, we compared T cell populations from a cohort of pediatric organ donors in the first two years of life to tissues from young adult donors aged 15-25 years. Results show a dynamic compartmentalization of naïve and regulatory T cells in all tissues early in life that is rapidly replaced with effector memory T cells (TEM) especially in mucosal sites further revealing the importance of a local adaptive immune response. Interestingly, the proportion and distribution of these T cell populations in tissue sites show varying levels of reliance on thymic output. Further evidence for the compartmentalization of the adaptive immune response and mechanisms for T cell maintenance throughout life can be shown through the analysis of T cell receptor sequences. We examined naïve and TEM populations in lymph nodes and spleen as a function of thymic output revealing low sharing of the naïve T cell repertoire between tissue sites with higher amounts of overlapping clones seen in TEM populations, especially with CD8+ T cells. These differences highlight potentially different roles for CD4+ and CD8+ T cells in the response to pathogen and their maintenance with age.

In conclusion, the studies reported in this thesis suggested that the CHO amount, whether provided by a pre-exercise CHO meal or short-time, i.e., 3-day, CHO loading, plays a pivotal role in regulating the immune responses before, during, and after endurance exercise. Although GI and GL independently affect the exercise performance and immune responses, the amount of CHO consumed remains a determining factor. The potential benefits on immune system and endurance performance after the low GI and low GL diet (L-L) should be noted and warrant further investigation. Although the HGI and LGI meals demonstrated similar effects on endurance performance when large amount of CHO-electrolyte solution consumed during the exercise, pre-exercise LGI meal can hasten the IL-6 responses during the recovery. (Abstract shortened by UMI.) / The aim of this thesis was to investigate the influence of pre-exercise carbohydrate (CHO) meal(s) with different glycemic index (GI) and glycemic load (GL) on endurance running performance, physiological, and immune responses. / The first study (Chapter 4) examined the influence of a pre-exercise meal with different GI and GL on subsequent endurance running performance, physiological, and immune responses. Eight endurance-trained male runners completed three trials in a randomized order, separated by at least seven days. These responses were characterized by a lower CHO oxidation with a concomitant higher glycerol and FFA in the H-L trial. Consumption of a pre-exercise high CHO meal, i.e., H-H and L-L, resulted in less perturbation of circulating numbers of leukocytes, neutrophils, and T lymphocyte subsets, decreased elevation of plasma IL-6 concentrations immediately after exercise and during the 2 h recovery period when compared to the H-L trial. These responses were accompanied by an attenuated increase in plasma IL-10 concentrations and plasma cortisol concentrations at the end of 2 h recovery. It was concluded that the amount of CHO consumed 2 h before endurance exercise appears to be the main influencing factor on immune responses irrespective of its GI and GL value. / The second study (Chapter 5) examined the influence of a 3-day CHO loading with different GI and GL meals on the supercompensation status, running performance, physiological and immune responses. Nine endurance-trained male runners were recruited in this study. The procedures basically involved a 3-day CHO loading with different GI and GL meal [CHO intake (% of energy intake), GI, and GL per day are 73%, 80, and 553 for the high-GI and high-GL (H-H); 73%, 42, and 249 for the low-GI and low-GL (L-L); 31%, 78.5, and 227 for the high-GI and low-GL (H-L) respectively] after a glycogen-lowering exercise. Two hours after the breakfast on day 4, participants performed the running protocol as described in the first study. There was no difference in time to complete the 10-km TT between the two trials with high-CHO loading, i.e., H-H and L-L (51.3 +/- 5.3 min vs 48.6 +/- 1.3 min, NS). These results suggested that 3-day CHO loading with low GI and low GL (L-L) is more effective in improving endurance performance when compared to a high GI but low GL diet (H-L). It appears that the amount of CHO consumed during the 3-day CHO loading remains the key influencing factor on immune responses despite of the differences in the GI and GL value. / The third study (Chapter 6) investigated the influence of pre-exercise meal with different GI on subsequent endurance running performance and immune responses when CHO-electrolyte solution was consumed during exercise. Pre-exercise LGI meal attenuated the increases of cortisol when compared with CON and hastened the recovery of the IL-6 value to baseline when compared to that in HGI and CON trials during the first hour of the recovery. The results suggested that beside of CHO quantity (CHO content), the role of CHO quality (GI) in the diet consumed 2 h before exercise should be considered when investigating the influence of CHO supplementation on the exercise-induced transitory immunosuppressive effects. / Chen Yajun. / "August 2006." / Adviser: Stephen Wong Heung-Sang. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1597. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 199-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Apoptosis (programmed cell death) and exercise immunology have been the focus of research for the past five years. Trained athletes are particularly susceptible to a wide variety of viral and bacterial infections and this has been related to oxidative damage which is a mediator of apoptosis. Apoptosis, a normal physiological mechanism has also been implicated in the pathogenesis of a wide-variety of diseases. To date, the link between apoptosis and exercise has not been shown by established methods or ultrastructurally. The objective of the study was t.o determine the effects of a single bout of high intensity exercise on lymphocyte DNA and antioxidant status in trained athletes. The study was carried out in two phases. In the first phase, 11 trained athletes were subjected to a treadmill run to exhaustion using a ramp protocol to determine their maximum oxygen uptake (V02 max). Fifteen millimetres of blood was collected before exercise, immediately after exercise, 24 hours and 48 hours after exercise. Whole blood (4 ul) was used in the determination of DNA damage in lymphocytes using the single cell gel electrophoresis (SCGE) assay. The remaining blood was centrifuged and used for the following: Vitamin C concentration was determined by the 2,4 dinitrophenylhydrazine method, vitamin E concentration was determined by the High Pressure Liquid Chromatography (HPLC) method and lipid peroxides were determined by the measurement ofhydroperoxides. In the second phase, 3 trained athletes who had participated in phase 1, were subjected to a V02 max. test. Blood samples (10 ml) were collected before and immediately after exercise, 24 hours and 48 hours later. Lymphocytes were isolated using Histopaque 1077. An in situ cell death detection kit, Fluorescein was used for the detection and quantification of apoptosis in lymphocytes at a single cell level, based on labelling of DNA strand breaks. Analysis was carried out using flow cytometry. Lymphocytes were also prepared for Transmission Electron Microscopy (TEM) using conventional techniques. The results showed that immediately after exercise there was a non-significant decrease in vitamin C concentrations (p=o, 16), and a non-significant increase in vitamin E (p=0,82) and lipid peroxide concentrations (p=0,21). There was no significant difference in all 3 levels over the 48 hour period, when compared to the pre-exercise values. The SCGE assay revealed that the immediate post exercise samples showed DNA damage in lymphocytes of all subjects as evidenced by fluorescent strands of DNA outside the cell while DNA damage was observed in only one subsequent sample. In the pre-exercise samples, DNA was visualised as a central core, whereas in all samples taken after exercise, DNA was located at the periphery or confined to one pole of the cell. The pattern of DNA distribution seen in the SCGE assay over the 48 hour period were characteristic features of apoptosis. Flow cytometric analysis showed an increase in apoptosis in lymphocytes immediately after exercise with a further increase after 24 hours. After 48 hours the numbers decreased to control levels. TEM showed that majority of cells were normal before exercise while other lymphocytes were smaller with indented nuclei. Immediately after exercise the lymphocytes displayed features of indented nuclei and microsegregation, cell shrinkage, swelling of the endoplasmic reticulum, mitochondria and Golgi. These changes persisted after 24 hours but were not observed after 48 hours when most of the cells showed normal morphology. The ultrastructural changes observed were also characteristic features of apoptosis. These results suggest that high intensity exercise may cause an increase in apoptosis as evidenced by DNA damage in the SCGE assay and fully supported by the results achieved during flow cytometry and by the ultrastructural changes observed. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Indiana University-Purdue University Indianapolis (IUPUI) / Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.