Biomedical subjects

Michael R Betts

Publications and source records attributed to Michael R Betts.

24 records · Page 2Linked to original sources

PubMed2003

Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation.

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).

Antigens, CD↗

PubMed2003

Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses.

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.

AIDS Vaccines↗

PubMed2002

Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells.

Virus-specific CD8(+) T-cell responses play a pivotal role in limiting viral replication. Alterations in these responses, such as decreased cytolytic function, inappropriate maturation, and limited proliferative ability could reduce their ability to control viral replication. Here, we report on the capacity of HIV-specific CD8(+) T cells to secrete cytokines and proliferate in response to HIV antigen stimulation. We find that a large proportion of HIV-specific CD8(+) T cells that produce cytokines in response to cognate antigen are unable to divide and die during a 48-hour in vitro culture. This lack of proliferative ability of HIV-specific CD8(+) T cells is defined by surface expression of CD57 but not by absence of CD28 or CCR7. This inability to proliferate in response to antigen cannot be overcome by exogenous interleukin-2 (IL-2) or IL-15. Furthermore, CD57 expression on CD8(+) T cells, CD4(+) T cells, and NK cells is a general marker of proliferative inability, a history of more cell divisions, and short telomeres. We suggest, therefore, that the increase in CD57(+) HIV-specific CD8(+) T cells results from chronic antigen stimulation that is a hallmark of HIV infection. Thus, our studies define a phenotype associated with replicative senescence in HIV-specific CD8(+) T cells, which may have broad implications to other conditions associated with chronic antigenic stimulation.

Apoptosis↗

PubMed2002

HIV preferentially infects HIV-specific CD4+ T cells.

HIV infection is associated with the progressive loss of CD4(+) T cells through their destruction or decreased production. A central, yet unresolved issue of HIV disease is the mechanism for this loss, and in particular whether HIV-specific CD4(+) T cells are preferentially affected. Here we show that HIV-specific memory CD4(+) T cells in infected individuals contain more HIV viral DNA than other memory CD4(+) T cells, at all stages of HIV disease. Additionally, following viral rebound during interruption of antiretroviral therapy, the frequency of HIV viral DNA in the HIV-specific pool of memory CD4(+) T cells increases to a greater extent than in memory CD4(+) T cells of other specificities. These findings show that HIV-specific CD4(+) T cells are preferentially infected by HIV in vivo. This provides a potential mechanism to explain the loss of HIV-specific CD4(+) T-cell responses, and consequently the loss of immunological control of HIV replication. Furthermore, the phenomenon of HIV specifically infecting the very cells that respond to it adds a cautionary note to the practice of structured therapy interruption.

Anti-HIV Agents↗

PubMed2002

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape.

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.

Adult↗

PubMed2002

Optimal antigens for HIV vaccines based on CD8+ T response, protein length, and sequence variability.

The identification of optimal antigens to include in an HIV vaccine designed to elicit a cellular immune response requires careful consideration of protein length, variability, and immunogenicity. Here, we have examined the relationship of these parameters in a cohort of HIV-infected subjects. We find that HIV Gag and Nef represent optimal antigens for the CD8+ T cell response, based on size, variability, and immunogenicity. Although the Env and Pol proteins have a poorer response to length (Pol) or variability (Env) ratio than Gag or Nef, they are still strong candidates for inclusion into an HIV vaccine, based on overall response frequency in the cohort. The accessory proteins Tat, Rev, Vif, Vpr, and Vpu in general all elicit very low CD8+ T cell responses, and this, in combination with their high variability, makes them less attractive as vaccine antigen.

AIDS Vaccines↗