The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

HIV-1 gp41 envelope IgA is frequently elicited after transmission but has an initial short response half-life.

Prevention of HIV-1 transmission at mucosal surfaces will likely require durable pre-existing mucosal anti-HIV-1 antibodies (Abs). Defining the ontogeny, specificities and potentially protective nature of the initial mucosal virus-specific B-cell response will be critical for understanding how to induce protective Ab responses by vaccination. Genital fluids from patients within the earliest stages of acute HIV-1 infection (Fiebig I-VI) were examined for multiple anti-HIV specificities. Gp41 (but not gp120) Env immunoglobulin (Ig)A Abs were frequently elicited in both plasma and mucosal fluids within the first weeks of transmission. However, shortly after induction, these initial mucosal gp41 Env IgA Abs rapidly declined with a t(½) of ∼2.7 days. B-cell-activating factor belonging to the TNF family (BAFF) was elevated immediately preceding the appearance of gp41 Abs, likely contributing to an initial T-independent Ab response. HIV-1 transmission frequently elicits mucosal HIV-1 envelope-specific IgA responses targeted to gp41 that have a short half-life.

Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-poisson distribution of transmitted variants.

Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood.

Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope +/- cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1alpha plus IL-18, IL-1alpha plus IL-12, and IL-1alpha plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60-71% peptide-specific (51)Cr release). Peak H-2D(d)-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8(+) PBMC. Nasal immunization with HIV immunogen and IL-1alpha, IL-12, and GM-CSF also induced Ag-specific IFN-gamma-secreting cells in the draining cervical lymph node and the lung. The use of IL-1alpha, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1alpha, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.

T-Cell recovery in adults and children following umbilical cord blood transplantation.

T-cell reconstitution following allogeneic stem cell transplantation may involve thymic education of donor-derived precursors or peripheral expansion of mature T cells transferred in the graft. T cell-receptor excision circles (sjTRECs) are generated within the thymus and identify new thymic emigrants and those that have not divided. We measured quantitative and qualitative immunologic reconstitution and sjTREC levels in adult and pediatric recipients of umbilical cord blood transplants (UCBTs). sjTRECs were detected at normal levels in all children, starting 12 months after transplantation. sjTRECs were not detected until 18 months after transplantation in adults, and then only at a 3-fold lower level than expected for age. We used complementarity-determining region 3 (CDR3) spectratyping to measure changes in T cell-receptor diversity occurring with restoration of thymic function. T-cell repertoires were skewed in adults and children at 12 to 18 months after transplantation but recovered to near-normal diversity at 2 to 3 years post-UCBT. T-cell repertoires appeared more diverse earlier in children (at 1 to 2 years post-UCBT) than in adults (at 3 to 4 years post-UCBT). We conclude that early T-cell recovery after UCBT occurs primarily through peripheral expansion of adoptively transferred donor T cells and results in skewing of the T-cell repertoire. The reappearance of sjTREC-containing cells after UCBT is associated with increasing numbers of phenotypicaly naive T cells, improved mitogen and recall antigen responses, and diversification of the T-cell repertoire. The delay in central T-cell recovery in adults relative to children may be due to differences in thymic function resulting from age-related atrophy, graft-versus-host disease, or the pharmacologic effects of prophylaxis and treatment of graft-versus-host disease.

The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans.

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.

Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope.

Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens.

Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice.

CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-gamma and tumor necrosis factor (TNF)-alpha production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P < or = 0.01 and P < or = 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-gamma and TNF-alpha production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P < or = 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.

Thymopoiesis in HIV-infected adults after highly active antiretroviral therapy.

The thymus of HIV-seropositive patients can enlarge as CD4+ T cell counts increase on highly active anti-retroviral therapy (HAART). This may indicate development of new T cells or represent mature peripheral T cells recirculating to the thymus. To define the etiology of the enlargement, the thymuses of two HIV-infected individuals on HAART were biopsied. For more than 3 years before initiation of HAART, both patients (38 and 41 years of age) had documented CD4+ T lymphopenia. Peripheral blood samples were obtained to assess circulating CD4+ CD45RA+ CD62L+ T cells, which were thought to have recently developed in the thymus. Peripheral blood T cells from both patients and thymocytes from the second patient were also tested for levels of DNA episomes formed during T cell receptor gene rearrangement (T cell receptor rearrangement excision circles, TRECs). With HAART, peripheral blood CD4+ T cell counts increased from approximately 60/mm(3) to 552/mm(3) and 750/mm(3) for patients 1 and 2, respectively. Thymic biopsies from both patients showed normal thymus histology with active thymopoiesis. Percentages of peripheral blood CD4+ CD45RA+ CD62L+ T cells and quantitation of T cell TRECs also reflected active thymopoiesis in both patients. Thus, in these two HIV-seropositive adults examined after initiation of HAART, thymic enlargement represented active thymopoiesis. Thymopoiesis in adult AIDS patients may contribute to immune reconstitution even after prolonged CD4+ T lymphopenia.

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma.

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.

Cytokines and adhesion molecules in the pathogenesis of vasculitis.

Upregulated adhesion molecule and cytokine expression on endothelial cells and infiltrating inflammatory cells occur in most vasculitis syndromes. These observations suggest that vasculitis is associated with pathologic activation of normal immune cell cytokine cascades. Abnormal expression of adhesion molecules and cytokines in vascular endothelium is a manifestation of endothelial dysfunction that can be triggered by a variety of stimuli, including infectious agents, immune complexes, and antiendothelial cell antibodies. Dysregulated adhesion molecule expression, uncontrolled inflammation, thrombosis, and vessel occlusion lead to the clinical manifestations of vasculitis.

The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection.

The human thymus is a complex chimeric organ comprised of central (thymic epithelial space) and peripheral (perivascular space) components that functions well into adult life to produce naive T lymphocytes. Recent advances in identifying thymic emigrants and development of safe methods to study thymic function in vivo in adults have provided new opportunities to understand the role that the human thymus plays in immune reconstitution in aging, in bone marrow transplantation, and in HIV-1 infection. The emerging concept is that there are age-dependent contributions of thymic emigrants and proliferation of postthymic T cells to maintain the peripheral T cell pool and to contribute to T cell regeneration, with the thymus contributing more at younger ages and peripheral T cell expansion contributing more in older subjects. New studies have revealed a dynamic interplay between postnatal thymus output and peripheral T cell pool proliferation, which play important roles in determining the nature of immune reconstitution in congenital immunodeficiency diseases, in bone marrow transplantation, and in HIV-1 infection. In this paper, we review recent data on human postnatal thymus function that, taken together, support the notion that the human thymus is functional well into the sixth decade and plays a role throughout life to optimize human immune system function.

Linkage of the CCR5 Delta 32 mutation with a functional polymorphism of CD45RA.

A 32-bp deletion in CCR5 (CCR5 Delta 32) confers to PBMC resistance to HIV-1 isolates that use CCR5 as a coreceptor. To study this mutation in T cell development, we have screened 571 human thymus tissues for the mutation. We identified 72 thymuses (12.6%) that were heterozygous and 2 (0.35%) that were homozygous for the CCR5 Delta 32 mutation. We found that thymocyte development was normal in both CCR5 Delta 32 heterozygous and homozygous thymuses. In 3% of thymuses we identified a functional polymorphism of CD45RA, in which cortical and medullary thymocytes failed to down-regulate the 200- and 220-kDa CD45RA isoforms during T cell development. Moreover, we found an association of this CD45 functional polymorphism in thymuses with the CCR5 Delta 32 mutation (p = 0.00258). In vitro HIV-1 infection assays with CCR5-using primary isolates demonstrated that thymocytes with the heterozygous CCR5 Delta 32 mutation produced less p24 than did CCR5 wild-type thymocytes. However, the functional CD45RA polymorphism did not alter the susceptibility of thymocytes to HIV-1 infection. Taken together, these data demonstrate association of the CCR5 Delta 32 mutation with a polymorphism in an as yet unknown gene that is responsible for the ability to down-regulate the expression of high m.w. CD45RA isoforms. Although the presence of the CCR5 Delta 32 mutation down-regulates HIV-1 infection of thymocytes, the functional CD45RA polymorphism does not alter the susceptibility of thymocytes to HIV-1 infection in vitro.

Thymic function after hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency.

BACKGROUND: Immune function can be restored in infants with severe combined immunodeficiency by transplantation of unfractionated bone marrow from HLA-identical donors or T-cell-depleted marrow stem cells from haploidentical donors, with whom there is a single haplotype mismatch, without the need for chemotherapy before transplantation or prophylaxis against graft-versus-host disease. The role of the thymus in this process is unknown. METHODS: We analyzed the phenotypes of circulating T cells and the proliferative responses of peripheral-blood mononuclear cells to phytohemagglutinin in 83 patients with severe combined immunodeficiency who received allogeneic marrow transplants without T-cell ablation from related donors over an 18-year period. We also tested for the presence of episomes of T-cell antigen receptors (extrachromosomal DNA circles formed during intrathymic T-cell development) to assess thymus-dependent T-cell reconstitution. RESULTS: Before and early after transplantation, the numbers of circulating T cells were low, with a predominance of mature CD45RO+ T cells (primarily resulting from the transplacental transfer of maternal cells); T-cell antigen-receptor episomes were undetectable in peripheral-blood mononuclear cells. In 73 of the infants, thymus-derived T cells expressing CD45RA and T-cell antigen-receptor episomes were detected within three to six weeks after transplantation. The mean (+/-SD) value for thymus-dependent T-cell antigen-receptor episomes peaked (at 7311+/-8652 per microgram of peripheral-blood mononuclear-cell DNA) 1 to 2 years after transplantation and declined to low levels (less than 100 episomes per microgram of DNA) within 14 years, as compared with a gradual decline from birth to the age of about 80 years in normal subjects. CONCLUSIONS: The vestigial thymus in infants with severe combined immunodeficiency is functional and can produce enough T cells after bone marrow transplantation to provide normal immune function.

Effect of highly active antiretroviral therapy and thymic transplantation on immunoreconstitution in HIV infection.

The purpose of this study was to determine whether thymic transplantation in addition to highly active antiretroviral therapy (HAART) will restore T cell function in HIV infection. Eight treatment-naive HIV-infected patients with CD4+ T cell counts of 200-500/mm3 were randomized into thymic transplantation and control arms. All patients received HAART (zidovudine, lamivudine, and ritonavir) for 6 weeks prior to transplantation. Thymic transplantation was done without immunosuppression, using postnatal HLA-unmatched cultured allogeneic thymus tissue. Patients were immunized every 6 months with the neoantigen keyhole limpet hemocyanin (KLH) and the recall antigen tetanus toxoid (TT). T cell phenotype and function and T cell receptor rearrangement excision circles (TRECs) were assessed. Thymic allografts were biopsied at 2 months. Six HIV-infected patients completed the study. Four patients received cultured allogeneic postnatal thymic grafts, two others were controls. CD4+ T cell counts increased and T cell-proliferative responses to Candida antigen and TT normalized in all patients. Proliferative responses to KLH developed in three of four transplant recipients and one of two controls. Patients responding to KLH after secondary immunization had greater TREC increases compared with the patients who did not respond. All thymic allografts were rejected within 2 months. In summary, four of six patients developed T cell-proliferative responses to the neoantigen KLH over the first 2 years of HAART. The transplanted thymus tissue, however, was rejected. There was no clear difference in restoration of T cell function in the transplant recipients compared with the controls. Increases in TRECs after initiation of HAART may correlate with improved immune function.

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy.

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.