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Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft-versus-host disease (GVHD). CD4+ T cells that produce granulocyte-macrophage colony stimulating factor (GM-CSF) have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T-cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if a unique CD4+ GM-CSF+ subset exists that differs from other defined T helper subtypes. Using single-cell RNA sequencing analysis (scRNAseq), we identified two CD4+ GM-CSF+ T-cell populations that arose during GVHD and were distinguishable according to the presence or absence of interferon-γ (IFN-γ) coexpression. CD4+ GM-CSF+ IFN-γ− T cells, which emerged preferentially in the colon, had a distinct transcriptional profile, used unique gene regulatory networks, and possessed a nonoverlapping T-cell receptor repertoire compared with CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T-cell populations in the colon. Functionally, this CD4+ GM-CSF+ T-cell population contributed to pathologic damage in the GI tract that was critically dependent on signaling through the interleukin-17 (IL-7) receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic T helper subtype GM-CSF+ subset that mediates immunopathology.

Vaccines against seasonal infections like influenza offer a recurring testbed, encompassing challenges in design, implementation, and uptake to combat a both familiar and ever-shifting threat. One of the pervading mysteries of influenza epidemiology is what causes the distinctive seasonal outbreak pattern. Proposed theories each suggest different paths forward in being able to tailor precision vaccines and/or deploy them most effectively. One of the greatest challenges in contrasting and supporting these theories is, of course, that there is no means by which to actually test them. In this communication we revisit theories and explore how the ongoing coronavirus disease 2019 (COVID-19) pandemic might provide a unique opportunity to better understand the global circulation of respiratory infections. We discuss how vaccine strategies may be targeted and improved by both isolating drivers and understanding the immunological consequences of seasonality, and how these insights about influenza vaccines may generalize to vaccines for other seasonal respiratory infections.

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell–mediated destruction of pancreatic β cells. We have previously shown that diabetogenic CD8 T cells in the islets of non-obese diabetic mice are phenotypically heterogeneous, but clonal heterogeneity remains relatively unexplored. Here, we use paired single-cell RNA and T-cell receptor sequencing (scRNA-seq and scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of non-obese diabetic mice. scTCR-seq demonstrates that CD8 T cells targeting the immunodominant β-cell epitope IGRP206-214exhibit restricted TCR gene usage. scRNA-seq identifies six clusters of autoreactive CD8 T cells in the islets and six in the spleen, including memory and exhausted cells. Clonal overlap between IGRP206-214–reactive CD8 T cells in the islets and spleen suggests these cells may circulate between the islets and periphery. Finally, we identify correlations between TCR genes and T-cell clonal expansion and effector fate. Collectively, our work demonstrates that IGRP206-214–specific CD8 T cells are phenotypically heterogeneous but clonally restricted, raising the possibility of selectively targeting these TCR structures for therapeutic benefit.

Objective In the presented study, we examined the impact of school holidays (Autumn, Winter, Summer, and Spring Breaks) and social events (Super Bowl, NBA Finals, World Series, and Black Friday) for five age groups ( 65 years) on four health outcomes of influenza (total tested, all influenza positives, positives for influenza A, and B) in Milwaukee, WI, in 2004-2009 using routine surveillance. Introduction Influenza viral infection is contentious, has a short incubation period, yet preventable if multiple barriers are employed. At some extend school holidays and travel restrictions serve as a socially accepted control measure 1,2 . A study of a spatiotemporal spread of influenza among school-aged children in Belgium illustrated that changes in mixing patterns are responsible for altering disease seasonality 3 . Stochastic numerical simulations suggested that weekends and holidays can delay disease seasonal peaks, mitigate the spread of infection, and slow down the epidemic by periodically dampening transmission. While Christmas holidays had the largest impact on transmission, other school breaks may also help in reducing an epidemic size. Contrary to events reducing social mixing, sporting events and mass gatherings facilitate the spread of infections 4 . A study on county-level vital statistics of the US from 1974-2009 showed that Super Bowl social mixing affects influenza dissemination by decreasing mortality rates in older adults in Bowl-participating counties. The effect is most pronounced for highly virulent influenza strains and when the Super Bowl occurs closer to the influenza seasonal peak. Simulation studies exploring how social mixing affects influenza spread 5 demonstrated that impact of the public gathering on prevalence of influenza depends on time proximity to epidemic peak. While the effects of holidays and social events on seasonal influenza have been explored in surveillance time series and agent-based modeling studies, the understanding of the differential effects across age groups is incomplete. Methods The City of Milwaukee Health Department Laboratory (MHDL), Wisconsin routinely collect tests from residents of metropolitan areas and vicinities of the Marquette University (MU). We obtained weekly counts of total tested, all influenza positives, positives for influenza A and B, from MHDL between 5/16/04-3/7/09 (before the surge of tests associated with “swine flu”). Cases for

Social outings can trigger influenza transmission, especially in children and elderly. In contrast, school closures are associated with reduced influenza incidence in school-aged children. While influenza surveillance modelling studies typically account for holidays and mass gatherings, age-specific effects of school breaks, sporting events and commonly celebrated observances are not fully explored. We examined the impact of school holidays, social events and religious observances for six age groups (all ages, ⩽4, 5–24, 25–44, 45–64, ⩾65 years) on four influenza outcomes (tests, positives, influenza A and influenza B) as reported by the City of Milwaukee Health Department Laboratory, Milwaukee, Wisconsin from 2004 to 2009. We characterised holiday effects by analysing average weekly counts in negative binomial regression models controlling for weather and seasonal incidence fluctuations. We estimated age-specific annual peak timing and compared influenza outcomes before, during and after school breaks. During the 118 university holiday weeks, average weekly tests were lower than in 140 school term weeks (5.93 vs. 11.99 cases/week, P < 0.005). The dampening of tests during Winter Break was evident in all ages and in those 5–24 years (RR = 0.31; 95% CI 0.22–0.41 vs. RR = 0.14; 95% CI 0.09–0.22, respectively). A significant increase in tests was observed during Spring Break in 45–64 years old adults (RR = 2.12; 95% CI 1.14–3.96). Milwaukee Public Schools holiday breaks showed similar amplification and dampening effects. Overall, calendar effects depend on the proximity and alignment of an individual holiday to age-specific and influenza outcome-specific peak timing. Better quantification of individual holiday effects, tailored to specific age groups, should improve influenza prevention measures.

The amino acids at the V - J rearrangement junction of TCR are encoded by the D region, and by N or P nucleotides. Together they comprise the NDN region, the specific pMHC selection surface of the TCR β-chain. As an extension of our earlier work on the recall response to influenza M158-66 in HLA-A2 individuals, we have been analyzing the circulating BV19 CD8 T cell repertoires. We observed that NDN regions of the CDR3 often start at positions that are V-region encoded. Here we examine NDN encoded amino acid motifs of BV19 rearrangements in circulating CD8 T cells based on the CDR3 length, the CDR3 start position of the NDN, and the motif length. Motifs that start at V region-encoded positions could be expected to be CDR3 length independent as indeed is the case. Motifs that included sequential proline and glycine showed a CDR3 length independent distribution and examining codon usage indicates that a large proportion of these can be explained by P-nucleotide addition from the 5’ end of the D region. Other examples of skewed codon usage were observed indicating possible additional rearrangement mechanisms. Another pattern of motif distributions was a shift of position along the CDR3 as a function of the CDR3 length. As these data were collected from an older healthy individual they can be used to model successful repertoire selection and to further define characteristics associated with a positive history of responses to pathogen exposures.

Generating a detailed description of human T cell repertoire diversity is an important goal in the study of human immunology. The circulation is the source of most T cells used for studies in humans. Here we use high throughput sequencing of TCR BV19 transcripts from CD8 T cells derived from unmanipulated PBMC from an older HLA-A2 individual to provide a quantitative and qualitative description of the clonotypic CDR3 nucleotide and amino acid composition of the TCR β-chain from this subset of circulating CD8 T cells. Aggregated samples from six time points spanning ∼1.5 years were analyzed to smooth possible temporal fluctuation. BV19 encompasses the well studied RS-encoding clonotypes involved in recognition of the M1(58-66) epitope from influenza A in HLA-A2 individuals. The clonotype distribution was diverse, complex and self-similar. The amino acid composition was generally skewed in favor of glycines and there were specific amino acids observed at higher frequency at the NDN start position. The motif repertoire distribution was also diverse, complex and self-similar with respect to CDR3 length, NDN start and length.

Background Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. Methods In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. Results There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. Conclusions Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

The CD8 memory T cell repertoire to the influenza A derived M1(58-66) epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8(+)CD107(+) repertoires are complex in the same manner as previously reported. The clonotype composition of CD8(+)CD107(+) repertoires is also very similar to CD8 only repertoires, and to CD8(+)HLA-A2-M1(58-66) pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

In temperate regions, influenza typically arrives with the onset of colder weather. Seasonal waves travel over large spaces covering many climatic zones in a relatively short period of time. The precise mechanism for this striking seasonal pattern is still not well understood, and the interplay of factors that influence the spread of infection and the emergence of new strains is largely unknown. The study of influenza seasonality has been fraught with problems. One of these is the ever-shifting description of illness resulting from influenza and the use of both the historical definitions and new definitions based on actual isolation of the virus. The compilation of records describing influenza oscillations on a local and global scale is massive, but the value of these data is a function of the definitions used. In this review, we argue that observations of both seasonality and deviation from the expected pattern stem from the nature of this disease. Heterogeneity in seasonal patterns may arise from differences in the behaviour of specific strains, the emergence of a novel strain, or cross-protection from previously observed strains. Most likely, the seasonal patterns emerge from interactions of individual factors behaving as coupled resonators. We emphasize that both seasonality and deviations from it may merely be reflections of our inability to disentangle signal from noise, because of ambiguity in measurement and/or terminology. We conclude the review with suggestions for new promising and realistic directions with tangible consequences for the modelling of complex influenza dynamics in order to effectively control infection.

Memory T-cell repertoires are populated by clonotypes selected by an individual's history of antigen exposures. Our previous analysis of middle-age CD8+ T-cell memory repertoires to the influenza-derived epitope M158–66, described a network of highly cross-reactive BV19 clonotypes responding to M158–66 and at least one peptide with a conservative amino acid substitution at either of two TCR contact positions. Here, we report that some substitutions abrogate BV19 responses and favor responses with different BV. Cross-reactive T cells using seven other BV families responded to 12 of 13 peptides tested. BV12 clonotypes define the most extensive cross-reactive network that encompasses seven peptides. We generated 3D networks based on the peptides recognized and BV family used and observed a cluster of five peptides that includes M158–66 and another cluster of five peptides that does not include M158–66. The first cluster represents peptides structurally similar to M158–66, and the second represents peptides with more considerable changes in epitope recognition surface. We hypothesize that the second cluster represents the cross-reactive network around another unknown epitope or epitopes. This data supports a model of stable CD8+ T-cell memory networks that include a substantial contribution from cross-reactive T cells.

Ligand binding is a thermodynamically cooperative process in many biochemical systems characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to cooperativity of ligation needs to be elucidated. Here, we perform kinetic and thermodynamic analyses on a panel of cycle-mutated peptides, derived from influenza H3 HA(306-319), interacting with wild type and a mutant HLA-DR. We observe that, within a certain range of peptide affinity, this system shows isothermal entropy-enthalpy compensation (iEEC). The incremental increases in conformational entropy measured as disruptive mutations are added in the ligand or receptor are more than sufficient in magnitude to account for the experimentally observed lack of free-energy decrease cooperativity. Beyond this affinity range, compensation is not observed, and therefore, the ability of the residual interactions to form a stable complex decreases in an exponential fashion. Taken together, our results indicate that cooperativity and iEEC constitute the thermodynamic epiphenomena of the structural fluctuation that accompanies ligand-receptor complex formation in flexible systems. Therefore, ligand binding affinity prediction needs to consider how each source of binding energy contributes synergistically to the folding and kinetic stability of the complex in a process based on the trade-off between structural tightening and restraint of conformational mobility.

T-cell receptors recognize peptides presented by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APC). The ability of the T-cell receptor (TCR) to recognize more than one peptide-MHC structure defines cross-reactivity. Cross-reactivity is a documented phenomenon of the immune system whose importance is still under investigation. There are a number of rational arguments for cross-reactivity. These include the discrepancy between the theoretical high number of pathogen-derived peptides and the lower diversity of the T-cell repertoire, the need for recognition of escape variants, and the intrinsic low affinity of this receptor-ligand pair. However, quantifying the phenomenon has been difficult, and its immunological importance remains unknown. In this review, we examined the cases for and against an important role for cross reactivity. We argue that it may be an essential feature of the immune system from the point of view of biological robustness.

SummaryAlthough both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

The aging of T cell memory is often considered in terms of senescence, a process viewed as decay and loss of memory T cells. How senescence would affect memory is a function of the initial structure of the memory repertoire and whether the clonotypes that make up the repertoire decay at random. We examine this issue using the T cell memory generated to the conserved influenza A epitope M158–66, which induces a strong, focused, but polyclonal CD8 T cell response in HLA-A2 individuals. We analyzed the CD8 T cell memory repertoires in eight healthy middle-aged and eight healthy older blood donors representing an average age difference of ∼27 y. Although the repertoires show broadly similar clonotype distributions, the number of observable clonotypes decreases significantly. This decrease disproportionally affects low-frequency clonotypes. Rank frequency analysis shows the same two-component clonotype distribution described earlier for these repertoires. The first component includes lower frequency clonotypes for which distribution can be described by a power law. The slope of this first component is significantly steeper in the older cohort. Generating a representative repertoire for each healthy cohort allowed agent-based modeling of the aging process. Interestingly, simple senescence of middle-aged repertoires is insufficient to describe the older clonotype distribution. Rather, a selective clonotype expansion must be included to achieve the best fit. We propose that responses to periodic virus exposure may drive such expansion, ensuring that the remaining clonotypes are optimized for continued protection.

Cross-reactivity of T cells is defined as recognition of two or more peptide–MHC complexes by the same T cell. Although examples of cross-reactivity have been reported, a detailed examination of cross-reactivity has not been performed. In this study, we took advantage of the high degree of polyclonality in the BV19 T cell repertoire responding to influenza M158–66 in HLA-A2 individuals to obtain a measure of simple cross-reactivity. We used substitutions that incrementally change the structure of the M158–66 peptide to measure how the HLA-A2–restricted response adapts to these changes. In three HLA-A2 adult subjects, we identified the BV19 clonotypes in the recall response to the influenza epitope M158–66 and 12 M1 peptides substituted at TCR contact position 63 or 65. The fraction of cross-reactive clonotypes in the M158–66 repertoire varied from 45–58% in the three donors. The extent of cross-reactivity, which is the additional number of peptides recognized by a single clonotype, is as high as six. We summarized the data using graph theory, with the cross-reactive clonotypes connecting the different HLA-A2 peptides recognized. The cross-reactive clonotypes form a well-connected network that could provide protection from virus-escape variants. We predict that any new pathogen with an epitope whose shape corresponds to that of the peptides that we studied would find a pre-existing repertoire ready to respond to it. We propose that in adult memory repertoires, previously encountered epitopes may have generated similar cross-reactive repertoires.

Please cite this paper as: Lofgren et al. (2010) Disproportional effects in populations of concern for pandemic influenza: insights from seasonal epidemics in Wisconsin, 1967–2004. Influenza and Other Respiratory Viruses 4(4), 205–212. Background Influenza infections pose a serious burden of illness in the United States. We explored age, influenza strains, and seasonal epidemic curves in relation to influenza-associated mortality. Methods The state of Wisconsin death records for the years 1967–2004 were analyzed for three distinct populations: children, general population, and elderly. Yearly parameters of duration, intensity, and peak timing were obtained from Annual Harmonic Regression coefficients. Results Overall, elderly had the highest rate and intensity of influenza mortality. The children and infant subpopulations showed an earlier and wider range in duration of peak timing than elderly. During A/Hong Kong/1/68 pandemic years, the elderly subpopulation showed no change in mortality rates while a sharp increase was observed for the children and infant subpopulations. In epidemic years such as 1966–1969, children and infants showed a dramatic decrease in the severity of influenza outbreaks over time. The elderly had increased baseline mortality in years (1986–1987) where predominant strain was characterized as A/Singapore/6/86. Conclusions Our findings indicate that the younger populations may have benefited from the lack of a major shift in viral strains for a number of decades. Furthermore, we demonstrate considerable heterogeneity in the spread of seasonal influenza across age categories, with implications both for the modeling of influenza seasonality, risk assessment, and effective distribution and timing of vaccine and prophylactic interventions.

Classical and nonclassical MHC class II (MHCII) genes are coregulated by the transcription factor RFX (regulatory factor X) and the transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein “enhanceosome” complex on MHCII promoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome in recruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome was revealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA was found to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was associated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNA methylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of the absence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQB promoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-type fibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA-independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retaining MHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors of APC and nonprofessional APC before induction with IFN-γ.

The time-dependent changes of human memory T cell repertoires are still poorly understood. We define a T cell memory repertoire as the pool of clonotypic lineages participating in a recall response to the influenza M158–66 epitope. In HLA-A2 individuals, this response predominantly uses BV19 chains with Arg-Ser (RS) in the CDR3 loop. We previously showed that the repertoire is polyclonal with a large fraction of clonotype that are only observed once. In this study, we perform longitudinal analyses of memory repertoires in three middle-aged individuals at times that spanned from 7 to 10 years. In these individuals, who are well into thymic involution, a substantial number of clonotypes were stable, e.g., detected at two times. The shape of the repertoire was stable over time as reflected by a number of repertoire characteristics, including singletons, i.e., the fraction of clonotypes observed only once, and repertoire diversity. However, the RS-clonotype subset showed a significant decline in the fraction of singletons and in clonotypic diversity. Thus, repertoire structure is maintained over time by a recruitment of non-RS-clonotypes and a shift of existing RS-clonotypes into higher frequencies. The recruitment of new clonotypes into the low-frequency component of the repertoire implies a role for these clonotypes.

The loss of Gimap5 (GTPase of the immune-associated protein 5) gene function is the underlying cause of lymphopenia and autoimmune diabetes in the BioBreeding (BB) rat. The in vivo function of murine gimap5 is largely unknown. We show that selective gene ablation of the mouse gimap5 gene impairs the final intrathymic maturation of CD8 and CD4 T cells and compromises the survival of postthymic CD4 and CD8 cells, replicating findings in the BB rat model. In addition, gimap5 deficiency imposes a block of natural killer (NK)- and NKT-cell differentiation. Development of NK/NKT cells is restored on transfer of gimap5−/− bone marrow into a wild-type environment. Mice lacking gimap5 have a median survival of 15 weeks, exhibit chronic hepatic hematopoiesis, and in later stages show pronounced hepatocyte apoptosis, leading to liver failure. This pathology persists in a Rag2-deficient background in the absence of mature B, T, or NK cells and cannot be adoptively transferred by transplanting gimap5−/− bone marrow into wild-type recipients. We conclude that mouse gimap5 is necessary for the survival of peripheral T cells, NK/NKT-cell development, and the maintenance of normal liver function. These functions involve cell-intrinsic as well as cell-extrinsic mechanisms.

This communication provides an illustration for the use of computer simulations in human immunology. When traditional experiments are impossible, unethical, or unfeasible, in silico modeling procedures may help to fill the gaps in our knowledge of an immune system response to a pathogen. In our study, we define terms and properties of modeled entities: "a clonotype", its distribution, and rank-frequency summaries, and describe properties associated with each of these three clonotype-related entities. We simulate a multistage dynamic process of an immune memory response to influenza. We believe that illustrated properties of fractality and self-similarity might arise due to the following process. The memory T cells operate in a complex environment of shifting pathogen concentrations, increasing and then decreasing inflammatory signals, and multiple interactions with other immune cells and their infected targets. Therefore, a fractal structure to such a population would represent an optimization in terms of percolation into immune/inflammatory space.

Background Therapeutic options for DiGeorge syndrome (DGS) with profound T-cell deficiency are very limited. Thymic transplantation has shown promising results but is not easily available. Hematopoietic cell transplantation (HCT) has been successful in restoring immune competence in the short term. Objective Present the long-term follow-up of 2 patients with complete DGS who received bone marrow transplants in the neonatal period from HLA-matched siblings, and perform a multicenter survey to document the status of other patients with DGS who have undergone HCT. Methods Immune function assessment by immunophenotyping, lymphocyte proliferation, T-cell receptor excision circles, single nucleotide polymorphism mapping arrays, spectratyping, cytogenetics, and fluorescence in situ hybridization were used. Results Among reported patients with DGS receiving HCT, survival is greater than 75%. Our patients are in their 20s and in good health. Their hematopoietic compartment shows continuous engraftment with mixed chimerism, normal T-cell function, and humoral immunity. Circulating T cells exhibit a memory phenotype with a restricted repertoire and are devoid of T-cell receptor excision circles. Conclusion These features suggest that T-cell reconstitution has occurred predominantly through expansion of the donors' mature T-cell pool. Although restricted, their immune systems are capable of providing substantial protection to infection and respond to vaccines. We conclude that bone marrow transplant achieves long-lived reconstitution of immune function in complete DGS and is a good alternative to thymic transplantation in patients with a suitable donor. Clinical implications Bone marrow transplant in complete DGS using an HLA-matched sibling donor provides long-lasting immunity and is a suitable and more available alternative to thymic transplantation.

We describe structural studies of the human leukocyte antigen DR52a, HLA-DRA/DRB3*0101, in complex with an N-terminal human platelet integrin alphaII(B)betaIII glycoprotein peptide which contains a Leu/Pro dimorphism. The 33:Leu dimorphism is the epitope for the T cell directed response in neonatal alloimmune thrombocytopenia and post-transfusion purpura in individuals with the alphaII(B)betaIII 33:Pro allele, and defines the unidirectional alloimmune response. This condition is always associated with DR52a. The crystallographic structure has been refined to 2.25 A. There are two alphabeta heterodimers to the asymmetric unit in space group P4(1)2(1)2. The molecule is characterized by two prominent hydrophobic pockets at either end of the peptide binding cleft and a deep, narrower and highly charged P4 opening underneath the beta 1 chain. Further, the peptide in the second molecule displays a sharp upward turn after pocket P9. The structure reveals the role of pockets and the distinctive basic P4 pocket, shared by DR52a and DR3, in selecting their respective binding peptide repertoire. We observe an interesting switch in a residue from the canonically assigned pocket 6 seen in prior class II structures to pocket 4. This occludes the P6 pocket helping to explain the distinctive "1-4-9" peptide binding motif. A beta57 Asp--Val substitution abrogates the salt-bridge to alpha76 Arg and along with a hydrophobic beta37 is important in shaping the P9 pocket. DRB3*0101 and DRB1*0301 belong to an ancestral haplotype and are associated with many autoimmune diseases linked to antigen presentation, but whereas DR3 is susceptible to type 1 diabetes DR52a is not. This dichotomy is explored for clues to the disease.

Peptide binding to MHC class II (MHCII) molecules is stabilized by hydrophobic anchoring and hydrogen bond formation. We view peptide binding as a process in which the peptide folds into the binding groove and to some extent the groove folds around the peptide. Our previous observation of cooperativity when analyzing binding properties of peptides modified at side chains with medium to high solvent accessibility is compatible with such a view. However, a large component of peptide binding is mediated by residues with strong hydrophobic interactions that bind to their respective pockets. If these reflect initial nucleation events they may be upstream of the folding process and not show cooperativity. To test whether the folding hypothesis extends to these anchor interactions, we measured dissociation and affinity to HLA-DR1 of an influenza hemagglutinin-derived peptide with multiple substitutions at major anchor residues. Our results show both negative and positive cooperative effects between hydrophobic pocket interactions. Cooperativity was also observed between hydrophobic pockets and positions with intermediate solvent accessibility, indicating that hydrophobic interactions participate in the overall folding process. These findings point out that predicting the binding potential of epitopes cannot assume additive and independent contributions of the interactions between major MHCII pockets and corresponding peptide side chains.

Influenza (or “flu”) leads to the hospitalization of more than 200,000 people yearly and results in 36,000 deaths from flu or flu-related complications in the United States ([15][1]), striking both the elderly and infant populations particularly hard ([24][2]). Two members of the

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host-reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2b] → AKR/J [H-2k]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell-depleted bone marrow plus thymidine kinase-positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline-treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell-depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline-treated animals had persistent skewing in most Vβ families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4+ T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.

Peptides presented via the class II MHC (MHCII) pathway are selected based on affinity for MHCII and stability in the presence of HLA-DM. Currently, epitope selection is thought to be controlled by the ability of peptide to sequester “anchor” residues into pockets in the MHCII. Residues exhibiting higher levels of solvent accessibility have been shown to contact TCR, but their roles in affinity and complex stability have not been directly studied. Using the HLA-DR1-binding influenza peptide, hemagglutinin (306–318), as a model, we show that side chain substitutions at these positions influence affinity and HLA-DM stability. Multiple substitutions reduce affinity to a greater extent than the loss of the major P1 anchor residue. We propose that these effects may be mediated through the H-bond network. These results demonstrate the importance of solvent-exposed residues in epitope selection and blur the distinctions between anchor and TCR contact residues.

Donor T cells are able to effect a graft-vs-leukemia (GVL) response but also induce graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation. We used an AKR leukemia murine transplant model, analogous to human acute lymphoblastic leukemia, in which donor T cells expressed a thymidine kinase suicide gene, to test whether separation of GVL and graft-vs-host (GVH) responses was feasible by selectively eliminating alloactivated donor T cells at defined time points posttransplant. Under experimental conditions where untreated mice could not be cured of disease without dying from GVHD, mice transplanted with thymidine kinase-positive T cells and subsequently administered ganciclovir (GCV) could eliminate leukemia without lethal GVHD. Timing of GCV administration, donor T cell dose, and preexisting leukemia burden were observed to be critical variables. Eradication of leukemia without lethal GVHD in GCV-treated mice implied that the kinetics of GVL and GVH responses were asynchronous and could therefore be temporally dissociated by timely GCV administration. That this strategy was feasible in a murine leukemia model in which GVHD and GVL reactivity are tightly linked suggests that this approach may be relevant to the treatment of selected human leukemias where similar constraints exist. This strategy represents an alternative approach to separating GVL and GVH reactivity and challenges the current paradigm that separation of these responses is dependent upon the administration of donor T cells with restricted specificity for leukemia as opposed to host Ags.

The rate of dissociation of recombinant, purified human estrogen receptor α (ERα) from a fluorescein-labeled DNA containing the consensus vitellogenin ERE sequence (F-vitERE) was determined in real time using fluorescence anisotropy. The complex of estradiol-occupied ERα with F-vitERE had an apparent dissociation rate of 1.48±0.06×10 −2 s −1 and a half-life of 46.6 s at room temperature. The dissociation rate was characterized by a single exponential decay, suggesting that ER dissociates from the DNA as a preformed dimer, rather than as two individual monomers. The association rate of estradiol-occupied ERα for the F-vitERE was calculated as 7×10 6 M −1 s −1 based on the dissociation rate measured and previous determinations of the equilibrium dissociation constant ( K d ) in similar assay conditions ( Ozers et al., 1997 ). In buffer containing various concentrations of salt, the rate of dissociation of estradiol-occupied ERα from F-vitERE was accelerated by increasing salt concentrations. Compared to estradiol-occupied ERα, the rate of dissociation of unoccupied ERα from the F-vitERE was very similar, indicating that estradiol occupancy does not affect the dissociation rate of ERα from the ERE.

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.

Estrogen treatment to rats of the Fischer 344 (F344) strain induces growth of pituitary tumors that exhibit accelerated cell proliferation, breakdown of basement membrane, and formation of hemorrhagic lakes. Estrogen-dependent pituitary growth is due to variation in a group of quantitative trait loci (QTL), called Edpm for estrogen-dependent pituitary mass, that we previously identified in an F(2) intercross of F344 and the tumor-resistant Brown Norway strain. We previously identified 5 QTL, and microsatellite markers developed since our earlier work have allowed us to scan new chromosomal regions, resulting in two new QTL for estrogen-dependent pituitary mass: Edpm9-2 and a possible QTL on the X Chromosome (Chr). Here we report evidence that these QTL differ from each other in how they affect growth. To examine the effect of the Edpm QTL on biochemical components of tumor growth, we tested their effects in 138 progeny of a backcross to the F344 strain which were given a 10-week chronic estrogen treatment. Hemoglobin/DNA ratio (a measure of blood volume relative to cell number) and total pituitary DNA (a measure of cell number) correlated only weakly, and very large pituitaries were observed which had a low hemoglobin/DNA ratio resembling a normal gland. Through QTL mapping, we found that Edpm2-1, Edpm3, Edpm5, and Edpm9-2 all had significant effects on pituitary mass, but Edpm2-1 and Edpm9-2 primarily affected DNA content, Edpm5 primarily affected hemoglobin/DNA ratio, and Edpm3 affected all traits equally.

Data from animal models have shown that hepatic graft-versus-host disease (GVHD) may be mediated by donor T cells interacting with liver adhesion molecules, other minor histocompatibility antigens, or both. We hypothesized that T-cell infiltrates within a liver biopsy during clinical GVHD would show a restricted T-cell response because the T cells would be responding to a limited number of antigens. We studied the peripheral T-cell repertoire and the liver-infiltrating T-cell repertoire of a patient who developed skin GVHD and subsequent liver GVHD after a matched sibling bone marrow transplantation for acute myeloid leukemia. Spectratype analysis of peripheral blood at the time of liver GVHD revealed that the patient had reconstituted a complex peripheral T-cell repertoire as evidenced by the presence of complementarity-determining region 3 (CDR3) length heterogeneity in most of the T-cell families. The repertoire complexity was skewed in variable gene beta (VB) 5.3, VB4, VB7, VB8, and VB15. Spectratype analysis on the liver biopsy sample revealed a limited infiltrate with an oligoclonal expansion in VBs 4, 7, and 8. We evaluated the T-cell infiltrate in more detail by sequencing the relevant expansions noted by spectratype and developing probes for the predominant CDR3 sequences. These clonotype probes were hybridized to peripheral blood and liver samples from the patient, a T-cell line developed from the patient's peripheral blood at the time of the initial skin GVHD, the donor's blood and marrow, and control samples. The results showed that the T-cell infiltrate during liver GVHD is mediated by a limited number of T cells, and that those cells are mostly different from the ones expanded from the peripheral blood during an acute skin GVHD reaction. These data support the concept that liver GVHD is a response to tissue-specific minor histocompatibility antigens. Biol Blood Marrow Transplant 2000;6(4):408-15.

Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene.

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous β-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.

A series of metabolic changes within the immature rat uterus begins minutes after the administration of microgram quantities of estradiol (E2). One of the earliest effects that has been measured is an increase in the rate at which the uterus takes up glucose. To characterize the effect of E2 on glucose transport stimulation, whole protein preparations were examined for the presence of mammalian glucose transport proteins Glut1 through Glut5. E2-stimulated changes in the steady state levels of messenger RNA (mRNA) and protein were measured for Glut1 and Glut4 by quantitative competitive RT-PCR and Western blots. Both Glut1 mRNA and protein increased approximately 3- to 4-fold within 4-8 h. This increase in Glut1 mRNA and protein agrees with the maximal stimulation of the glucose transport rate that was observed. No translocation of either Glut1 or Glut4 was observed 2 h after E2 injection, indicating that translocation is not the mechanism responsible for the initial E2-stimulated increase in glucose transport observed in immature rat uterus. These data support the conclusion that the prolonged increase in glucose transport rate is due to either the transcriptional activation of Glut1 and/or the increased Glut1 mRNA half-life.

Chronic treatment of rats with the estrogens 17 β -estradiol or diethylstilbestrol (DES) induces pituitary tumors in Fischer 344 but not Brown–Norway or Sprague–Dawley rats. Functional loss of retinoblastoma susceptibility gene product (pRb), a major regulatory protein for the G1 to S transition of the cell cycle, has been shown in several tumors. Here we report a decreased level of pRb in pituitary tumors of the Fischer 344 rat as compared with resistant Sprague–Dawley and Brown–Norway strains. pRb protein levels decreased 70% in Fischer 344 rats that were treated with diethylstilbestrol for 10 weeks as compared with tumor resistant control animals. Interestingly, the F1 hybrid (Fischer 344×Norway) showed an intermediate range of pRb protein expression as compared with those of the parental strains. pRb expression levels in nonhemmorhagic F2 (F1×F1) rats correlated with the size of the tumors. One week withdrawal of DES increased pRb levels as compared with continuously treated rats. Also, there was a decreased association of cyclin D and cyclin dependent kinase in susceptible tumors, supporting the hypothesis of a physical and possibly functional loss of pRb in the diethylstilbestrol-induced pituitary tumor. These results suggest that the difference in pRb regulation, whether it is a direct or indirect effect of estrogen, is related to tumor resistance or susceptibility in these two rat strains.

In this study, we analyze the recall response to influenza A matrix peptide M1(58-66) restricted by HLA-A2 in one individual and find a strict CDR3 selection as well as a high degree of polyclonality. The TCR β-chain repertoire of memory T cells specific for this Ag system has been shown previously to be constrained by the use of the BV17 family and the I/sRSA/S amino acid motif in the CDR3 region. Our sequence analysis of BV17 TCR from a CTL line showed the repertoire to be highly polyclonal, as 95 distinct CDR3 sequences (clonotypes) were identified expressing this CDR3 motif. The clonotype frequencies showed a power law distribution with an extensive low-frequency tail. The clonotypes present in the high-frequency component of the distribution could be measured directly in the PBMC. This measurement showed that the relative frequencies of these clonotypes before stimulation were similar to their frequencies after culturing. Analysis of short-term cultures showed that the responding clonotypes have a similar ability to proliferate, which is independent of TCR β-chain CDR3 sequence or precursor frequency. These data indicate that the memory T cell repertoire is composed of a surprisingly diverse set of T cell clonotypes with a limited potential for expansion. We propose that the high-frequency component represents T cells that have existed the longest. In keeping with this hypothesis, these clonotypes were measured over a 2-year period, during which their precursor frequency did not change.

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17β-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [≈0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction (≈50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The K d of ER binding in these cells is about 3 × 10 −11 M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.

We examined noncooperativity of estrogen action by studying the transcription of estrogen receptor (ER) down-regulation in an ER stably transfected cell line (Rat1+ER). The time-course of the ER transcription rate following 17 β -estradiol (E2) (10 nM) treatment was measured by nuclear run-on assays. ER transcription rates decreased to 40±3% within 1 h as compared with no treatment and stayed suppressed to 24 h of E2 treatment. The ER transcription rate decrease was noncooperative when measured at 24 h of E2 treatment. This suggested that initial E2 binding to the ER is also a noncooperative process. The Hill coefficients of E2 binding to ER were near unity with an equilibrium dissociation constant of 1–3 nM when measured after a 1 h incubation. These results show that E2 noncooperatively binds ER and down-regulates transcription of ER noncooperatively in intact Rat1+ER cells.

The first hydrophobic pocket, P1, of class II MHC has been shown to be an important site of peptide anchoring. Two polymorphisms occur in this pocket in the human class II MHC beta chain at position 85 and 86. beta 85 is usually Val, occasionally Ala, whereas beta 86 can be Gly or Val. However, Ala85 is found only in conjunction with Val86. The independent effect of the polymorphism at these two positions on the binding of normal and substituted antigenic peptides has never been examined. To do so, three soluble HLA-DR1 variants that contain the naturally occurring combinations of these side chains at these two positions were generated and tested with a panel of influenza matrix peptides varying at anchor P1. DR1 alleles differing only at position 86 are very similar in the binding of a panel of antigenic peptide, indicating that beta 86 does not substantially influence the peptide binding of DR1. In contrast, DR1 varying only at position beta 85 differ in their binding of substituted peptides containing Ala, Tyr or Trp at the P1 anchor position. Thus, beta 85 shows the predominant effect on the P1 anchor side chain preference of the P1 pocket in DR1. This is in contrast to other HLA-DR alleles where beta 86 has been shown to control the nature of the P1 anchor. These previous data together with our own imply that the role of polymorphism in P1 may be influenced by the contextual framework of the remaining allelic polymorphism.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.