New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

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CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

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Angin, M., King, M., Addo, M. M. New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals. J. Vis. Exp. (75), e50244, doi:10.3791/50244 (2013).


CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.

Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.

Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.


With more than 34 million individuals living with HIV/AIDS worldwide and an estimated 2.5 million people newly infected in 2011, the need for an effective HIV vaccine to curb the worldwide HIV epidemic remains paramount. However, despite three decades of intense research efforts, the HIV-1 vaccine efficacy trials to date have resulted in only modest protection 1-3 and the correlates of protective immunity remain poorly understood. Elucidating the nature of the immune response needed for protection is essential for the strategic design of an effective HIV-1 vaccine and other immunotherapeutic strategies targeting HIV-1 infection.

Natural CD4+ regulatory T cells (Tregs) are critical to the maintenance of immune cell homeostasis by controlling excessive immune activation, thus limiting immune-mediated tissue damage. However, they can also suppress immune responses against pathogens and prevent their clearance. Cancer and Hepatitis B vaccine studies have demonstrated that decreasing the activity of Tregs can enhance vaccine response and antigen-specific immunity against viruses 4-7. However, in the context of HIV-1 infection, the exact impact of regulatory T cells remains incompletely understood. Tregs were shown to decrease virus replication in activated T cells 8 and possibly impact immune activation 9. They were also shown to suppress HIV-1-specific immune responses, which could have negative outcomes for disease progression 10,11. Thus, before being able to modulate Treg activity to enhance the efficacy of an HIV-1 vaccine, it is important to gain further insight into their function in this disease context.

Human CD4+ regulatory T cells are a relatively scarce cell population, representing about 5% of CD4+ T cells in the peripheral blood, and their absolute numbers further decrease with HIV-associated CD4+ T cell depletion 12. Current assays to assess Treg function, such as T cell proliferation assays with Treg co-culture, use relatively large cell numbers 12. Therefore, characterizing function and specificity of regulatory T cells in individuals with advanced HIV-1 disease has been challenging, despite their importance for HIV pathogenesis.

The ex vivo isolation and expansion of Tregs from HIV-1 patients could represent a solution to overcome some of these limitations. Here we describe an easy and robust protocol to expand functional Tregs derived from HIV-1 infected individuals in vitro; we further explain how to phenotype them and test their suppressive function using flow cytometric assays. We believe this protocol will facilitate access to Tregs and help understanding their role in HIV-1 disease progression.

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1. Regulatory T cell isolation from HIV-1 Positive Blood

  1. Carefully transfer blood, collected in ACD tubes, into a 50 ml conical tube for a final volume of 15 ml blood per tube.
  2. Add 25 μl/ml of blood of RosetteSep Human CD4+ T Cell Enrichment Cocktail, mix carefully and incubate 20 min at room temperature.
  3. Add 15 ml of PBS/2% FBS to the blood and mix carefully. Layer the diluted blood sample on top of 15 ml of Histopaque at room temperature in a 50 ml conical tube. Spin the conical tube for 20 min at 1,200 x g with a slow start and no brakes.
  4. Transfer the CD4+ T cell enriched PBMC layer in a new 50 ml conical tube, wash the cells by adding PBS/2% FBS and spin them down for 10 min at 1,200 x g. Then count the cells, wash again and resuspend the cells at about 20 x 106/200 μl.
  5. Add the following antibodies (concentration):
    anti-CD3-Phycoerythrin-Cyanine 7 (PE-Cy7) (1/100)
    anti-CD4-Fluorescein Isothiocyanate (FITC) (1/40)
    anti-CD25-Allophycocyanin (APC) (1/40)
    anti-CD127-Phycoerythrin (PE) (1/20)
    Incubate 30 min in the dark at 4 °C
  6. Wash the cells with PBS/2% FBS. Resuspend the cells at 20 x 106/ml in PBS/2% FBS and filter them on a 35 μm nylon mesh.
  7. Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the CD3+CD4+CD25+CD127low Treg in X-VIVO 15 media (see gating strategy in Figure 1). Conventional T cells (CD3+CD4+CD25-CD127+) can be isolated and expanded as negative controls.

2. Cell Culture

  1. After isolation, wash the Treg with X-VIVO 15 media.
  2. Resuspend the cells at 250 x 103/ml in X-VIVO 15 media complemented with 10% Human Serum and Penicillin-Streptomycin (50 U/ml).
  3. Wash Human T-Activator CD3/CD28 beads according to manufacturer's protocol. Add beads to isolated Tregs at a ratio of 1:1 bead per cell.
  4. After two days of culture, double the media volume and add IL-2 (300 U/ml).
  5. Culture the Tregs for 2 weeks. Change media (X-VIVO 15/Human serum/P/S/IL-2) at days 5, 7, 9, 12. Add beads at a 1:1 ratio at day 9. When changing media, keep cells at 250 x 103/ml.

3. Phenotyping

At the end of the expansion culture, expanded CD3+CD4+CD25+CD127low Treg can be phenotyped by flow cytometry and compared to expanded CD3+CD4+CD25-CD127+ conventional T cells as a control.

  1. Harvest expanded Tregs/Tconvs and wash them in PBS. Label dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to manufacturer's protocol. Wash the cells in PBS/2% FBS.
  2. Add the following antibodies (concentration):
    anti-CD3-PECy7 (1/100)
    anti-CD4-Qdot-655 (1/200)
    anti-CD25-PECy5 (1/100)
    Incubate 30 min in the dark at 4 °C
  3. Wash the cells and perform the intracellular staining using the Foxp3/ Transcription Factor Staining Buffer Set according to manufacturer's protocol and the following antibodies:
    anti-FOXP3-PE (1/50)
    anti-HELIOS-FITC (1/40)
    anti-CTLA4-APC (1/20)
    Acquire the data on a flow cytometer.

4. Suppression Assay

At the end of the expansion culture, the suppressive function i.e. the capacity of the expanded Treg isolated from HIV-1 positive individuals to suppress the proliferation of activated T cells can be assessed in vitro.

  1. Thaw autologous cryopreserved ex vivo PBMCs. Leave them for about 3 hr in a 37 °C incubator in RPMI 1640 medium containing penicillin/streptomycin, L-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (=R+ media), and 10% FBS (=R10 media).
  2. Label the dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to the manufacturer's protocol. Wash the cells in PBS/2% FBS.
  3. Incubate the cells with anti-CD3-PECy7 for 30 min in the dark at 4 °C. Wash the cells with PBS/2% FBS. Resuspend the cells in PBS/2% FBS and filter them on a 35 μm nylon mesh.
  4. Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the viable CD3+ T cells in R10 media.
  5. Label the T cells with a cell tracing reagent such as CellTrace Violet or Vybrant CFDA SE Cell Tracer at 5 μM diluted in PBS for 7 min at 37 °C according to the manufacturer's protocol. Resuspend cells in R+ media supplemented with 10% human serum (=hR10 media) at 1 x 106/ml.
  6. Harvest the expanded Tregs, resuspend the cells at 0.5 x 106/ml in hR10 and prepare dilutions at 0.25 x 106/ml and 0.125 x 106/ml.
  7. Prepare anti-CD2/anti-CD3/anti-CD28 microbeads according to the manufacturer's protocol, resuspend the microbeads at 0.75 x 106/ml and prepare dilutions at 0.625, 0.562, 0.5 x 106/ml in hR10 media.
  8. In a 96 wells round bottom plate, transfer cells and beads according to the following plan:
T cells:Treg ratio 1:0 1:1/2 1:1/4 1:1/8
T cells (50 μl) 1 x 106/ml 1 x 106/ml 1 x 106/ml 1 x 106/ml
Tregs (50 μl) no 0.5 x 106/ml 0.25 x 106/ml 0.125 x 106/ml
Beads (100 μl) 0.5 x 106/ml 0.75 x 106/ml 0.625 x 106/ml 0.562 x 106/ml
hR10 (50 μl) yes no no no
T cells 50 x 103 50 x 103 50 x 103 50 x 103
Tregs 0 25 x 103 12.5 x 103 6.25 x 103
Beads 50 x 103 75 x 103 62.5 x 103 56.25 x 103
  1. After 4 days of culture, wash the cells and incubate them for 30 min at 4 °C with the following antibodies:
    anti-CD3-PECy7 (1/100)
    anti-CD4-APC (1/100)
    anti-CD8-AF700 (1/100)

Acquire the data on a flow cytometer. Use the FlowJo proliferation platform to calculate the percentage of divided cells.

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Representative Results

The expression of interleukin 2 receptor (CD25) and the interleukin 7 receptor (CD127) have been described as reliable surface markers to identify functional Treg populations 13 and have been shown to correlate with CD4+CD25+FOXP3+ Tregs 9,12. Figure 1 represents the gating strategy used to flow-sort single CD3+CD4+CD25+CD127low Tregs from PBMC isolated from an HIV-1-positive individual. The CD25/CD127 antibody clones and their conjugate are crucial to reach a good separation of the Treg population; we found the CD25 APC (eBioscience)/ CD127 PE (BD Pharmingen) combination to work well in our hands.

In a representative example (Figure 2), the Treg expansion fold change reached 765 after 14 days of culture. The expansion fold change can vary from one individual to another and can be as low as ca. 100 or reach ca. 2500. The human serum lot used in the media can also impact the expansion fold change; it is suggested to screen different lots of human serum to achieve optimal expansion capacity.

Examples of histograms staining for selected markers of Tregs (CTLA4, FOXP3 and HELIOS) are represented in Figure 3. Since most of the Treg markers are also expressed by activated conventional T cells, displaying the mean fluorescence intensity (MFI) may be more informative than the frequency of the positive population in this context.

A fundamental characteristic of regulatory T cells is their capacity to suppress cellular immune responses. Using a standardized flow-based T cell proliferation assay (Figure 4) expanded Tregs can suppress the proliferation of activated autologous T cells. Proliferation of T cells are assayed by a membrane-permeant reactive tracer (CellTrace Violet) after 4 days of co-culture with different anti-CD2/anti-CD3/antiCD28 microbeads activated T cells to Tregs ratios. The numbers of divided cells were calculated using the FlowJo Proliferation platform. CD2 stimulation was shown to up-regulate FOXP3 expression and lead to enhanced Treg function 14. In our hands the use of microbeads gives a stronger and more consistent proliferation than plate-bound anti-CD3 with or without the addition of anti-CD28. For the suppression assay, media is complemented with human serum, we suggest to screen different serum lots to achieve optimal and constant suppressive capacity.

Figure 1
Figure 1. Flow cytometry gating strategy to sort singles CD3+CD4+CD25+CD127low Tregs from PBMC isolated from an HIV-1-positive individual. Click here to view larger figure.

Figure 2
Figure 2. Representative example of the expansion fold change of Tregs isolated from an HIV-1-infected individual cultured during 14 days.

Figure 3
Figure 3. Examples of flow cytometry histograms staining of expanded Tregs isolated from an HIV-1-infected Treg using different Tregs markers. Expanded Tregs are figured in grey and expanded Tconvs in white. Click here to view larger figure.

Figure 4
Figure 4. Example of flow-based T cell proliferation assay using a membrane-permeant reactive tracer and activated T cells at different responder T cell to Treg ratios. CD4+ T cells are figured in grey and CD8+ T cells in black. The numbers of divided cells are figured in the histograms.

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Using the protocol described above, Tregs can be successfully isolated and expanded from HIV-1-infected individuals in vitro. Expanded Tregs express high levels of FOXP3, CTLA4 and HELIOS, are highly suppressive and display a highly demethylated Treg-Specific Demethylation Region (TSDR) locus of the FOXP3 gene (data not shown) 15, suggesting true origin from the regulatory T cell lineage, as opposed to activation-induced transient FOXP3 upregulation. Deep sequencing demonstrated that the TCR repertoire of the Tregs is not skewed during the expansion (data not shown).

Regulatory T cells are naturally anergic and need strong stimuli to proliferate; here we used beads coated with anti-CD3 and anti-CD28 16 antibodies in the presence of exogenous IL-2 17. Other groups have successfully used similar methods to expand polyclonal Tregs from healthy donors 18, diabetic patients 19, transplant recipients 20 or antigen-specific Tregs from HCV patients 21 but, to our knowledge, this is the first description of a successful protocol to expand functional Tregs from HIV-1 positive donors.

We chose to use flow-cytometry over immunomagnetic cell sorting in order to isolate the cell population with the best purity possible and to avoid contamination with CD3+CD4+CD25-CD127+ conventional T cells (Tconvs) during expansion. Avoiding contamination with Tconvs is crucial since we found that under the same conditions Tconvs proliferate more rapidly than regulatory T cells, and thus could overgrow Tregs during a prolonged expansion culture. Rapamycin was previously used to inhibit Tconvs growth while favoring expansion and survival of Tregs 22. However rapamycin was found to downregulate CCR5 expression in vitro, an important co-factor of R5 HIV virus entry, which would have made expanded cells less usable to study HIV-1 infection of regulatory T cells by this HIV-1 strain 23.

In the present protocol, Tregs are expanded for 14 days. Although we found expanded Tregs to be still actively suppressive after 21 days, we chose not to prolong our cell culture and preserve the suppressive activity of our cells. Indeed, the group of M. Edinger reported a decrease in FOXP3 when Tregs were expanded more than two weeks and this was associated with moderate Treg suppression 24,25.

Most markers used to characterize regulatory T cells are shared by activated T cells (e.g. FOXP3 26, CTLA4 27). In our hands, even though expanded Tregs expressed higher levels of FOXP3 and CTLA4 compared to expanded conventional T cells (Tconvs), HELIOS was the only marker that was almost exclusively expressed by the expanded Tregs. This protein is mostly expressed by thymic-derived but not by peripherally induced FOXP3+ regulatory T cells 28. Thus, the Tregs isolated and expanded by our protocol could be used to understand the role of thymic-derived Tregs in HIV-immunopathogenesis. The expansion of other Tregs subsets, such as induced antigen-specific Tr1 or Th3 suppressor cells from HIV-1 positive individuals would need alternative adapted protocols 29,30.

In summary, using the described protocol we successfully expanded Tregs from frozen adult and pediatric PBMC as well as GALT (gut associated lymphoid tissue), the major site of HIV-1 replication. Easier access to large numbers of Tregs will help to address important questions concerning their role in HIV-1 immunopathogenesis and to better understandthe role of this T cell subpopulation in HIV-1 immune evasion. We believe that answering these questions may have a strong impact in the development of an effective HIV-1 vaccine and potential new strategies for immunotherapeutic intervention in HIV-1 infection.

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The authors declare that they have no competing financial interests.


This work was supported in part by research funding from the Elisabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00 to MMA), MGH/ECOR (Physician Scientist Development Award to MMA), NIH NIAID (KO8219 AI074405 and AI074405-03S1 to MMA), and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. These studies were furthermore supported by the Bill & Melinda Gates Foundation and the Terry and Susan Ragon Foundation.


Name Company Catalog Number Comments
RosetteSep Human CD4+ T Cell Enrichment Cocktail Stemcells technologies 15062
PBS Sigma D8537
FBS Sigma F4135
Histopaque Sigma H8889
Anti-CD3-PECy7 BD Pharmingen 557851
Anti-CD4-FITC eBioscience 11-0049-42
Anti-CD25-APC eBioscience 17-0259-42
Anti-CD127-PE BD Pharmingen 557938
Round-Bottom tube with 35 μm a nylon mesh BD Falcon 352235
X-VIVO 15 Lonza 04-418Q
Penicillin/Streptomycin Mediatech 30-001-Cl
Human Serum Gemini Bio-Products 100-512
Human T-activator CD3/CD28 Life Technologies 111.31D
IL-2 NIH Aids Research Reference Reagent Program 136
LIVE/DEAD Fixable Violet Dead Cell Stain Kit Life technologies L34955
Anti-CD4-qdot-655 Life Technologies Q10007
Anti-CD25-PECy5 eBiosciences 15-0259-42
Foxp3 / Transcription Factor Staining Buffer Set eBiosciences 00-5523-00
Anti-FOXP3-PE eBiosciences 12-4776-42
Anti-HELIOS-FITC Biolegend 137204
Anti-CTLA4-APC BD Pharmingen 555855
CellTrace Violet Cell Proliferation Kit Life Technologies C34557
Vybrant CFDA SE Cell Tracer Kit Life Technologies V12883
HEPES Mediatech 25-060-Cl
Treg Suppression inspector Miltenyi Biotec 130-092-909
Anti-CD4-APC BD Pharmingen 340443
Anti-CD8-AF700 BD Pharmingen 557945
RPMI 1640 Sigma R0883
Glutamine Mediatech 25-002-Cl
BD Vacutainer Blood Collection Tube w/ ACID CITRATE DEXTROSE (ACD) Becton, Dickinson and Company (BD) 364606
FACSAria IIu Cell Sorter BD Biosciences -
LSR II Flow Cytometer BD Biosciences -
FlowJo Tree Star v887



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