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Chapter 97 : Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection
Authors:
Richard L. Hengel,
Tomozumi Imamichi,
Stephen A. Migueles
Content Type: Reference
Publication Year:
2006
Category: Immunology
Book DOI:
10.1128/9781555815905
Chapter DOI:
10.1128/9781555815905.ch97
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Abstract:
While the ultimate mechanism of human immunodeficiency virus (HIV) pathogenesis remains elusive, one fortunate consequence of two decades of HIV research has been the development and optimization of new tools to investigate the human immune response, both in health and in disease. A T-cell receptor (TCR) that binds with too much or too little avidity for a self-peptide presented within a self-major histocompatibility complex molecule will cause the death of that T cell. This chapter outlines both old and new tools used in evaluating patients with HIV disease. RNA extractions should be performed in a PCR hood, making sure to run the UV light for sufficient time before beginning in order to avoid any cross-contamination. Two methods, chemical (phenol) and nonchemical (column), are available to isolate RNA from cells. A section of the chapter outlines two popular methods to detect cytokine-secreting antigen-specific T cells using multiparameter flow cytometry. The ability of T cells to rapidly secrete cytokines following stimulation by their cognate antigen is an important hallmark of the memory T-cell response. HLA tetramer technology revolutionized the investigation of antigen-specific T-cell clones. Unlike other assays and technologies, HLA tetramers can enumerate T-cell clones regardless of their functional behavior, such as proliferation, killing, or cytokine production. The ability of lymphocytes to expand exponentially after encountering their cognate antigen is a critical attribute that allows the acquired immune response to contain otherwise lethal pathogenic challenges.
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
Figures
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection
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Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97fig01
FIGURE 1
As recently reported by Gea-Banacloche et al., PBMCs were separated into CD4+ T-cell fractions and then compared at two different time points for the same HIV-infected individual starting treatment for HIV infection. Such an analysis is typical of how the CDR3 sizing assay is applied to HIV-infected patients. The Vβ family is indicated along the y axis, while CDR3 sizing analysis is indicated along the x axis (each peak corresponding to the relative frequency of PCR products of the same size). Abnormal patterns are identified by bolded boxes around the frequency distribution graphs. Comparisons between the early and late time periods indicate an improvement in the Vβ repertoire (i.e., less skewing), presumably because of this individual’s HIV treatment regimen (
5
).
10.1128/9781555815905/f0879-01_thmb.gif
10.1128/9781555815905/f0879-01.gif
FIGURE 1
As recently reported by Gea-Banacloche et al., PBMCs were separated into CD4+ T-cell fractions and then compared at two different time points for the same HIV-infected individual starting treatment for HIV infection. Such an analysis is typical of how the CDR3 sizing assay is applied to HIV-infected patients. The Vβ family is indicated along the y axis, while CDR3 sizing analysis is indicated along the x axis (each peak corresponding to the relative frequency of PCR products of the same size). Abnormal patterns are identified by bolded boxes around the frequency distribution graphs. Comparisons between the early and late time periods indicate an improvement in the Vβ repertoire (i.e., less skewing), presumably because of this individual’s HIV treatment regimen ( 5 ).
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97fig02
FIGURE 2
PBMCs from an HIV-seronegative individual were separated into CD4+ CD45RA- and then into CD62L+ and CD62L- fractions using magnetic beads to determine whether T-cell repertoire differences observed in later-stage HIV-seropositive individuals might be because these individuals have decreased ratios of central to effector memory (as well as naive to memory) CD4+ T cells accompanying their advancing HIV infection. CDR3 gene length analysis was performed on the separated subsets, and while abnormal patterns were found among both subsets, further exaggeration was found among CD62L- effector memory subsets (as indicated with adjacent stars), suggesting some support for the hypothesis that a decreased Vβ repertoire in late-stage HIV infection may be due to a combination of a relative decrease in naive and central memory and a reciprocal increase in effector memory CD4+ T cells.
10.1128/9781555815905/f0880-01_thmb.gif
10.1128/9781555815905/f0880-01.gif
FIGURE 2
PBMCs from an HIV-seronegative individual were separated into CD4+ CD45RA- and then into CD62L+ and CD62L- fractions using magnetic beads to determine whether T-cell repertoire differences observed in later-stage HIV-seropositive individuals might be because these individuals have decreased ratios of central to effector memory (as well as naive to memory) CD4+ T cells accompanying their advancing HIV infection. CDR3 gene length analysis was performed on the separated subsets, and while abnormal patterns were found among both subsets, further exaggeration was found among CD62L- effector memory subsets (as indicated with adjacent stars), suggesting some support for the hypothesis that a decreased Vβ repertoire in late-stage HIV infection may be due to a combination of a relative decrease in naive and central memory and a reciprocal increase in effector memory CD4+ T cells.
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97fig03
FIGURE 3
PBMCs were separated into CD4+ T cells using magnetic beads and then labeled with CFSE to track cell division over 5 days following stimulation with either medium control or phytohemagglutinin (PHA), as indicated in the two dot plots. Arbitrary cell number is indicated on the y axis, while CFSE fluorescence intensity (measured in FL-1 on the flow cytometer) is shown on the x axis. A single CFSE bright peak (0) is shown under the medium control conditions, indicating that no cell division has occurred (the dim peak is from non-CFSE-labeled irradiated PBMCs that were added into these and parallel tubes for presentation of other antigens in the experiment). Conversely, the PHA-stimulated condition shows four cell (1 to 4) divisions beyond the peak of undivided cells (0), in addition to the dim peak of unlabeled irradiated PBMCs.
10.1128/9781555815905/f0887-01_thmb.gif
10.1128/9781555815905/f0887-01.gif
FIGURE 3
PBMCs were separated into CD4+ T cells using magnetic beads and then labeled with CFSE to track cell division over 5 days following stimulation with either medium control or phytohemagglutinin (PHA), as indicated in the two dot plots. Arbitrary cell number is indicated on the y axis, while CFSE fluorescence intensity (measured in FL-1 on the flow cytometer) is shown on the x axis. A single CFSE bright peak (0) is shown under the medium control conditions, indicating that no cell division has occurred (the dim peak is from non-CFSE-labeled irradiated PBMCs that were added into these and parallel tubes for presentation of other antigens in the experiment). Conversely, the PHA-stimulated condition shows four cell (1 to 4) divisions beyond the peak of undivided cells (0), in addition to the dim peak of unlabeled irradiated PBMCs.
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97fig04
FIGURE 4
PBMCs were either set aside, irradiated, or separated into CD4+ CD62L+ or CD4+ CD62L- T cells. PBMCs (unirradiated) and separated CD4+ T-cell subsets were then stimulated with medium control, tetanus toxoid, CMV, or phytohemagglutinin (PHA) (as shown across the top of all dot plots) for 2 days in the presence of irradiated PBMCs as APC. Cells were then harvested and labeled with CFSE and placed back into medium for an additional 6 days. Cells were harvested again and then labeled with MAb for surface markers (CD3 and CD4) and analyzed on a flow cytometer to track cell division. The arrowheads identify tetanus toxoid-specific cell division (bright for CD4) found in PBMCs that corresponded to division found in CD4+ CD62L+ T cells but not CD4+ CD62L- T cells, while the arrows identify homeostatic cell division (dim for CD4) that is mostly confined to CD4+ CD62L+ T cells. Proliferation measured by CFSE cell division was confirmed with standard tritiated-thymidine LPA results at 5 days (results not shown). Separated PHA stimulations do not show proliferation, likely as these cells lost CFSE signal (death or >8 divisions) by day 8 of stimulation.
10.1128/9781555815905/f0888-01_thmb.gif
10.1128/9781555815905/f0888-01.gif
FIGURE 4
PBMCs were either set aside, irradiated, or separated into CD4+ CD62L+ or CD4+ CD62L- T cells. PBMCs (unirradiated) and separated CD4+ T-cell subsets were then stimulated with medium control, tetanus toxoid, CMV, or phytohemagglutinin (PHA) (as shown across the top of all dot plots) for 2 days in the presence of irradiated PBMCs as APC. Cells were then harvested and labeled with CFSE and placed back into medium for an additional 6 days. Cells were harvested again and then labeled with MAb for surface markers (CD3 and CD4) and analyzed on a flow cytometer to track cell division. The arrowheads identify tetanus toxoid-specific cell division (bright for CD4) found in PBMCs that corresponded to division found in CD4+ CD62L+ T cells but not CD4+ CD62L- T cells, while the arrows identify homeostatic cell division (dim for CD4) that is mostly confined to CD4+ CD62L+ T cells. Proliferation measured by CFSE cell division was confirmed with standard tritiated-thymidine LPA results at 5 days (results not shown). Separated PHA stimulations do not show proliferation, likely as these cells lost CFSE signal (death or >8 divisions) by day 8 of stimulation.
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
References
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Tables
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection
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Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97tab01
TABLE 1
PCR primers for Vβ CDR3 sizing analysis
TABLE 1
PCR primers for Vβ CDR3 sizing analysis
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97utab01
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97utab02
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97utab03
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97utab04
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97
/content/10.1128/9781555815905.ch97.ch97utab05
Citation:
Hengel R, Imamichi T, Migueles S.
2006.
Measurement of T-Cell-Specific Immunity in Patients with Human Immunodeficiency Virus Infection,
p 878-890.
In
Detrick B, Hamilton R, Folds J (ed),
Manual of Molecular and Clinical Laboratory Immunology, 7th Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815905.ch97