HIV Malaria Co-Infection Is Associated with Atypical Memory B Cell Expansion and a Reduced Antibody Response to a Broad Array of Plasmodium falciparum Antigens in Rwandan Adults

HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4⁺ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.     

BILL-Cadherin/Cadherin-17 Contributes to the Survival of Memory B Cells

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) are responsible for immunological “memory”, which can last for many years. The long-term survival niche for LLPCs in the bone marrow is well characterized; however, the corresponding niche for MBCs is unclear. BILL-cadherin/cadherin-17 (CDH17) is the only member of the cadherin superfamily that is expressed on mouse B lymphocytes in a spatiotemporally regulated manner. Here, we show that half of all MBCs regain expression of CDH17 during the later stage of development. The maintenance of high affinity antigen-specific serum antibodies was impaired in CDH17^-/- mice and the number of antigen-specific MBCs was reduced as compared to wild-type mice (WT). Also, specific responses to secondary antigens were ablated in CDH17^-/- mice, whereas primary antibody responses were the same as those in WT mice. Cell cycle analysis revealed a decline in the proliferation of CDH17^- MBCs as compared to CDH17⁺ MBCs. In addition, we identified a subpopulation of splenic stromal cells, MAdCAM-1⁺ blood endothelial cells (BEC), which was CDH17⁺. Taken together, these results suggest that CDH17 plays a role in the long-term survival of MBCs, presumably via an “MBC niche” comprising, at least in part, BEC in the spleen.     

Infants' Peripheral Blood Lymphocyte Composition Reflects Both Maternal and Post-Natal Infection with Plasmodium falciparum

Maternal parasitoses modulate fetal immune development, manifesting as altered cellular immunological activity in cord blood that may be linked to enhanced susceptibility to infections in early life. Plasmodium falciparum typifies such infections, with distinct placental infection-related changes in cord blood exemplified by expanded populations of parasite antigen-specific regulatory T cells. Here we addressed whether such early-onset cellular immunological alterations persist through infancy. Specifically, in order to assess the potential impacts of P. falciparum infections either during pregnancy or during infancy, we quantified lymphocyte subsets in cord blood and in infants'' peripheral blood during the first year of life. The principal age-related changes observed, independent of infection status, concerned decreases in the frequencies of CD4⁺, NK^dim and NKT cells, whilst CD8⁺, Treg and Teff cells'' frequencies increased from birth to 12 months of age. P. falciparum infections present at delivery, but not those earlier in gestation, were associated with increased frequencies of Treg and CD8⁺ T cells but fewer CD4⁺ and NKT cells during infancy, thus accentuating the observed age-related patterns. Overall, P. falciparum infections arising during infancy were associated with a reversal of the trends associated with maternal infection i.e. with more CD4⁺ cells, with fewer Treg and CD8⁺ cells. We conclude that maternal P. falciparum infection at delivery has significant and, in some cases, year-long effects on the composition of infants'' peripheral blood lymphocyte populations. Those effects are superimposed on separate and independent age- as well as infant infection-related alterations that, respectively, either match or run counter to them.     

Parasite Specific Antibody Increase Induced by an Episode of Acute P. falciparum Uncomplicated Malaria

Introduction

There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated.

Methods

This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4⁺ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured.

Results

On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4⁺ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients.

Conclusion

In the majority (70%) of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status.     

Human “Orchestrator” CD11b+ B1 Cells Spontaneously Secrete Interleukin-10 and Regulate T-Cell Activity

Immune regulation produced by B cells has been attributed to production and secretion of interleukin (IL)-10, which is a characteristic of mouse B1 cells. In view of the widespread clinical use of B-cell depletion therapies in autoimmune and malignant diseases, it is important to monitor the function and fate of regulatory B cells. However, there is no consensus regarding the phenotypic identity of human IL-10⁺ B cells. Here we show that human CD11b⁺ B1 cells, one of two recently described subpopulations of B1 cells, spontaneously produce IL-10 and suppress T-cell activation. In view of the capacity of these B cells to either stimulate T-cell proliferation or suppress T-cell activation, CD11b⁺ B1 cells are considered to be capable of orchestrating elements of immune responsiveness and thus are termed “orchestrator B1 cells,” or “B1orc,” whereas CD11b⁻ B1 cells that primarily secrete antibody are termed “secretor B1 cells,” or “B1sec.”     

Involvement of CD244 in Regulating CD4+ T Cell Immunity in Patients with Active Tuberculosis

CD244 (2B4) is a member of the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors and it plays an important role in modulating NK cell and CD8⁺ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4⁺ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4⁺ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4⁺ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4⁺ T cells, CD244/2B4-bright CD4⁺ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4⁺ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4⁺ T cell function.     

T Cell Responses Induced by Adenoviral Vectored Vaccines Can Be Adjuvanted by Fusion of Antigen to the Oligomerization Domain of C4b-Binding Protein

Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell “molecular adjuvant” when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4⁺ and CD8⁺ T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP1₄₂) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation.     

Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Marginal zone (MZ) B cells, identified as surface (s)IgM^highsIgD^lowCD23^low/−CD21⁺CD38⁻ B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27⁺ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.     

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background

The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8⁺ and CD4⁺ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.

Methodology/Principal Findings

To establish the basis for a SAPN-based vaccine, B- and CD8⁺ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ⁺, IL-2⁺) long-lived central memory CD8⁺ T-cells. Furthermore, we demonstrated that these Ab or CD8⁺ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.

Conclusion

The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.     

Babesia argentina, Plasmodium vivax and P. falciparum: antigenic cross-reactions

An indirect fluorescent antibody test was used to analyze the antigenic relationships between Babesia argentina, a parasite of cattle, and two human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Elevated antibody titers to P. falciparum were found in cattle infected with B. argentina. Some persons infected with P. falciparum or P. vivax were found to produce antibodies to B. argentina. Explanations for the occurrence of these cross reactions are considered.     

Decreased Frequency of Intestinal Regulatory CD5+ B Cells in Colonic Inflammation

Background

CD5⁺ B cells are a type of regulatory immune cells, though the involvement of this B cell subset in intestinal inflammation and immune regulation is not fully understood.

Methods

We examined the distribution of CD5⁺ B cells in various mouse organs. Expression levels of CD11b, IgM, and toll-like receptor (TLR)-4 and -9 in B cells were evaluated. In vitro, TLR-stimulated IL-10 production by colonic lamina propria (LP) CD5⁺ and CD5^- B cells was measured. In vivo, mice with acute or chronic dextran sulfate sodium (DSS)-induced colonic injury were examined, and the frequency of colonic LP CD5⁺ B cells in those was assessed by flow cytometry.

Results

The expression level of TLR9 was higher in colonic LP CD5⁺ B cells as compared to CD5^- B cells. Colonic LP CD5⁺ B cells produced greater amounts of IL-10 following stimulation with TLR ligands, especially TLR9, as compared with the LP CD5^- B cells. Acute intestinal inflammation transiently decreased the frequency of colonic LP CD5⁺ B cells, while chronic inflammation induced a persistent decrease in colonic LP CD5⁺ B cells and led to a CD5^- B cell-dominant condition.

Conclusion

A persistent altered mucosal B cell population caused by chronic gut inflammation may be involved in the pathogenesis of inflammatory bowel diseases.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

TIGIT Marks Exhausted T Cells,Correlates with Disease Progression,and Serves as a Target for Immune Restoration in HIV and SIV Infection

HIV infection induces phenotypic and functional changes to CD8⁺ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8⁺ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT⁺ and TIGIT⁺ PD-1⁺ CD8⁺ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8⁺ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8⁺ T cell effector responses. The frequency of TIGIT⁺ CD4⁺ T cells correlated with the CD4⁺ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.     

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P⁺) and nonpromotable (P⁻) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P⁺ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO₄ precipitated and used to transfect the P⁻ cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10⁻⁸ M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P⁻ JB6 Cl 30 cells yielded 40 to 50 colonies per 10⁵ cells. In contrast, transfection of Cl 30 cells with “P⁺ DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10⁵ cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P⁺ DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P⁺ lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P⁺ DNA, no evidence for acquisition of promotability was obtained. P⁻ JB6 Cl 25, like Cl 30, also permitted expression of transfected P⁺ DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P⁺ DNA and requires recipient cells of the appropriate phenotype for expression.     

Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1⁺ICOS⁺, PD1⁺ ICOS^-, or PD1^-ICOS^-. We used these markers to identify different subsets of CXCR5⁺CD4⁺ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD^-CD38^HiCD27⁺ memory B cells by flow cytometry. PD1⁺ICOS⁺ CXCR3⁺CCR6^-CXCR5⁺CD4⁺ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1⁺ICOS⁺ CXCR3^-CCR6^-CXCR5⁺CD4⁺ (Tfh2-like) cells for both vaccines, and PD1⁺ICOS⁺ CXCR3^-CCR6⁺CXCR5⁺CD4⁺ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1⁺ICOS⁺Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5⁺CD4⁺ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.     

Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL⁺ killer B cells, showing they are enriched in the IgM^highCD5⁺CD1d^high B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL⁺ B cells, but most FasL⁺ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL⁺ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL⁺ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4⁺ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL⁺ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.     

Differential susceptibility of peripheral blood CD5 and CD5 B cells to apoptosis in chronic hepatitis C patients

A body of evidence has suggested a close link between chronic hepatitis C virus (HCV) infection and B cell abnormalities, including mixed cryoglobulinemia, rheumatoid factor (RF) production, and lymphoproliferative disorders that may develop into non-Hodgkin’s lymphoma. Recent studies have demonstrated the expansion of CD5⁺ B cells in the peripheral blood of chronic hepatitis C patients (CHC). As CD5⁺ B cells, which are capable of producing autoantibodies and RF, are apparently crucial for the development of HCV-associated pathogenesis, the fate of both the CD5⁺ and CD5⁻ B cell subsets upon chronic HCV infection is of interest. In this study, the degree to which chronic HCV infection induces apoptosis in each B cell subset was investigated. Our results demonstrated that peripheral CD5⁻ B cells were more susceptible to apoptosis than CD5⁺ B cells in CHC. Furthermore, plasma levels of IL-4, IL-10, and IL-12 were significantly elevated in CHC, thus suggesting that these interleukins protect CD5⁺ B cells from apoptosis. The rationale for the differential susceptibility of distinct B cell subsets in CHC is also discussed with regard to extrahepatic manifestations associated with chronic HCV infection.