# Double-negative T cells are increased in HIV-infected patients under antiretroviral therapy

Fatma Korbi, Imen Zamali, Raja Rekik, Ahlem Ben Hmid, Mouldi Hidri, Wafa Kammoun Rebai, Zeineb Jelili, Senda Masmoudi, Souleiman Karim Rahal, Azza Ben Ayed, et al.

► To cite this version:

Fatma Korbi, Imen Zamali, Raja Rekik, Ahlem Ben Hmid, Mouldi Hidri, et al. Double-negative T cells are increased in HIV-infected patients under antiretroviral therapy. Medicine, 2022, 101 (36), pp.e30182. 10.1097/MD.00000000000030182. pasteur-05018233

HAL Id: pasteur-05018233

https://riip.hal.science/pasteur-05018233v1

Submitted on 3 Apr 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Double-negative T cells are increased in HIV-infected patients under antiretroviral therapy

Fatma Korbi, MDª, Imen Zamali, MDª,ᵇ, Raja Rekik, PhDª, Ahlem Ben Hmid, MDª,ᵇ, Mouldi Hidri, MScª, Wafa Kammoun Rebai, PhDᶜ, Zeineb Jelili, PharmDᵈ, Senda Masmoudi, PharmDᵈ, Souleiman Karim Rahal, PharmDᵈ, Azza Ben Ayed, PharmDᵈ, Mélika Ben Ahmed, MD, PhDª,ᵇ ID

**Abstract**

Double-negative T (DNT) cells are a T-cell subset with a CD4⁺CD8⁻ phenotype. They represent 1% to 5% of circulating lymphocytes, but an increase in this proportion can be found during lymphoproliferative and autoimmune diseases. This increase has also been reported in persons with HIV (PWH). The aim of this work was to better describe the proportion of DNT cell subset in PWH. We retrospectively collected 984 samples from PWH referred for lymphocyte immunophenotyping over a 7.5-year period. Quantification of DNT cells was performed by flow cytometry. DNT cell proportion was calculated by subtracting the CD4⁺ and CD8⁺ subsets proportions from the total of T cells. A total of 984 blood samples from PWH were collected. Mean CD4 T-cell count was decreased in such patients while DNT cell frequency was increased with a mean of 6.7%. More than half of the patients had a DNT cell proportion >5%. Patients with DNT cell proportion over 5% exhibited significantly reduced CD3⁺ and CD4⁺ T-cell counts, while CD8⁺ T-cell count was unchanged compared to patients with normal DNT cell rates. Interestingly, DNT cell percentage was negatively correlated with CD4 and CD3 T-cell counts in all included patients. Moreover, the DNT cell proportion was significantly increased in subjects with CD4⁺ T cells <200/mm³ compared to those with CD4⁺ T cells >200/mm³. Interestingly, DNT cell proportions were significantly higher in patients with high viral load compared with those presenting undetectable viral load. HIV infection is associated with an increase in DNT cell proportion. This increase is more frequent as the CD4 count is decreased and the viral load is increased. DNT cell subset should not be omitted when interpreting immunophenotyping in PWH as it appears to be associated with disease progression in patients under antiretroviral therapy.

**Abbreviations:** ART = antiretroviral therapy, DN = double negative, HIV = human immunodeficiency virus, PWH = persons with human immunodeficiency virus, SIV = Simian immunodeficiency virus.

**Keywords:** double-negative T cells, HIV, immunophenotyping, viral load

## 1. Introduction

Double-negative (DN) T cells express a CD3⁺CD4⁻CD8⁻ phe-
notype. They are present in peripheral blood, lymph nodes,
and mucosa-associated lymphoid tissue.[1] Their functions and
ontogeny are still poorly established. The variety of surface
markers as well as the different cytokine and chemokine expres-
sion profiles of such cells point out the presence of several dif-
ferentiation pathways.[1]

DNT cells exhibit a duality of function as they appear to have
effector as well as regulatory functions. DNT cell effector functions
have been suggested by rapid expansion of such cells following par-
asitic, viral and intracellular bacterial infection, as well as the expres-
sion of an array of proinflammatory effector cytokines.[2-4] On the
other hand, DNT cells seem to be involved in the maintenance of
immune homeostasis as they are able to regulate autoimmunity and
contribute to tolerance induction after transplantation.[1,5]

DNT cells represent 1% to 5% of circulating T cells.[1,6] An increase in this rate is associated with several pathological conditions, mainly lymphoproliferative and autoimmune disorders.[7] A high rate has also been reported during infections, mainly with the human immunodeficiency virus (HIV) and its precursor in nonhuman primates, Simian immunodeficiency virus (SIV).[6]

In this study, we proposed to evaluate DNT cell levels in per-
sons with HIV (PWH) under antiretroviral therapy (ART) and
to look for a possible correlation with other lymphocyte popu-
lations and the viral load.

## 2. Material and Methods

### 2.1. Study population

This retrospective study included 984 PWH referred to the clinical immunology laboratory of Pasteur institute of Tunis for

<footnote>The authors have no funding and conflicts of interest to disclose.</footnote>

<footnote>All data generated or analyzed during this study are included in this published
article [and its supplementary information files].</footnote>

<footnote>Ethical approval: The study was approved by the Bio-Medical Ethics Committee of Pasteur Institute of Tunis (reference 2022/7/I/LR16IPT).</footnote>

<footnote>ª Department of Clinical Immunology, Pasteur Institute of Tunis, Tunis, Tunisia,</footnote>

<footnote>ᵇ Faculty of Medicine of Tunis, University of Tunis El Manar, 1068, Tunis, Tunisia,</footnote>

<footnote>ᶜ Laboratory of Biomedical Genomics and Oncogenetics, Pasteur Institute of Tunis, Tunis, Tunisia, ᵈ Faculty of Pharmacy, Monastir, Tunisia.</footnote>

<footnote>*Correspondence: Mélika BEN AHMED, Laboratory of Clinical Immunology, Pasteur Institute of Tunis, 13, Place Pasteur, 1002, Le Belvédère, Tunis, Tunisia (e-mail: Melika.BenAhmed@pasteur.tn).</footnote>

<footnote>Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.
This is an open access article distributed under the Creative Commons
Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly
cited.</footnote>

<footnote>How to cite this article: Korbi F, Zamali I, Rekik R, Ben Hmid A, Hidri M, Kammoun Rebai W, Jelili Z, Masmoudi S, Rahal SK, Ben Ayed A, Ben Ahmed M. Double-negative T cells are increased in HIV-infected patients under antiretroviral therapy. Medicine 2022;101:36(e30182).</footnote>

<footnote>Received: 16 March 2022 / Received in final form: 7 June 2022 / Accepted: 7 July 2022</footnote>

<footnote>http://dx.doi.org/10.1097/MD.00000000000030182</footnote>
lymphocyte immunophenotyping from January 2014 to June 2021. At the time of blood collection, patients were already on ART. The Bio-Medical Ethics Committee of Pasteur Institute of Tunis approved the study and informed consent was obtained from all participants.

## 2.2. Immunophenotyping

Freshly collected EDTA-anticoagulated whole blood was processed within 2 to 4 hours after sampling. After lysis of red blood cells and washings with PBS containing 1% BSA, cells were incubated at room temperature for 20 minutes with the following conjugated monoclonal antibodies from BD Biosciences: anti-CD45-allophycocyanin-H7, CD4-fluorescein isothiocyanate, anti-CD8-phycioerythrin, and CD3-phycioerythrin-cyanin-5. Cells were fixed with Cell Fix (BD Biosciences) and kept at 4°C until analysis with a BD FACSCanto™ II flow cytometer. DNT cell proportions were determined by subtracting the CD4+ and CD8+ T-cell subsets proportions from the total of T cells: CD3+ – (CD3+CD4+ + CD3+CD8+). For each sample, a complete blood count was performed as well to assess total lymphocyte count and calculate the different T-cell subset counts.

## 2.3. Viral load measurement

Plasma HIV-1 viral load was quantified by real-time PCR. The sensitivity of viral RNA detection was 40 copies/mL of plasma.

## 2.4. Statistical analysis

Statistical analyses were performed using SPSS (Version 22, IBM®, USA). The difference between the groups was determined using the nonparametric Mann-Whitney U test. Correlation between different T-cell subset proportions or counts were calculated with the Spearman Rank correlation coefficient. P values <.05 were considered statistically significant.

# 3. Results

A total of 984 blood samples were collected from HIV-infected patients with a sex ratio of 1.16 and an average age of 36 ± 14.9 years. The different mean T-cell subset counts and proportions are shown in Table 1. Fifty-one percent of patients (500/984) had a CD4 T-cell count <500/mm³ and 45% (443/984) had a

CD8 T-cell count >900/mm³. Half PWH (51.7%; 509/984) had a DNT cell proportion >5% and 15.8% (155/984) of them had a proportion >10%.

When comparing different T-cell subset levels in patients presenting a DNT cell proportion >5% to those with DNT cell proportions within normal range, we noted significantly reduced CD3⁺ (1521 vs 1660.5/mm³, *P* = .016) and CD4⁺ (363 vs 601/mm³, *P* < .001) T-cell counts while CD8⁺ T-cell count was similar (836 vs 837.9/mm³, *P* = .49; Fig. 1). Subsequently, CD4/CD8 ratio was statistically decreased in the former group of patients (0.4 vs 0.69; *P* < .001). Besides, lymphocyte count was also lower in these patients (1859 vs 2001/mm³; *P* = .017).

We analyzed the relationship between T-cell subset counts and DNT cell proportion and observed a negative correlation with CD3 and CD4 T-cell counts (Fig. 2A, B) and no significant correlation with CD8 T-cell count (*P* = .16). Interestingly, the DNT cell proportion was significantly increased in subjects with CD4⁺ T cells < 200/mm³ compared to those with CD4⁺ T cells >200/mm³ (8.3% vs 5.7%, *P* < .001; Fig. 2C).

The viral load was available at the time of lymphocyte immunophenotyping in 221 cases. Among these, 117 (52.94%) patients had a DNT cell proportion above 5%, and 37 (16.74%) patients above 10%. Ninety-six patients had undetectable viral load with a median CD4 T-cell count of 603/mm³ and median DNT cell proportion of 4.85%. In patients with detectable viral load (n = 125, median viral load = 33884), the median CD4 T-cell count was 271/mm³ and their median DNT cell proportion was 5.7%, which was higher than that in patients with undetectable viral load (*P* = .049; Fig. 3A). DNT cell proportion was positively correlated with the viral load (*P* = .008; Fig. 3B). Furthermore, the viral load was significantly higher in patients with DNT cell proportion >5% compared to those within the normal range (*P* = .015; data not shown).

# 4. Discussion

Our study showed a CD4 T-cell number near the lower limit, and a slightly increased CD8 T-cell count in PWH with a clearly inverted CD4/CD8 ratio. These variations are frequent in the early stages of infection or in the AIDS stage. Less commonly reported, DNT cell proportion was increased in nearly half of the patients and was negatively correlated with CD3 and CD4 T-cell counts. Interestingly, increase in DNT cell proportion

<table><caption>**Table 1**
Mean counts and proportions of the different T-cell subsets.</caption><thead><tr><td>Parameter</td><td>Mean</td><td>Reference range</td></tr></thead><tbody><tr><td>White blood cells</td><td>5900/mm³ (840–16,300)</td><td>4000–10,000/mm³</td></tr><tr><td>Lymphocytes</td><td>1920/mm³ (66–8890)</td><td>1500–4000/mm³</td></tr><tr><td>T cells %</td><td>83.1% (14–97)</td><td>60%–85%</td></tr><tr><td>T-cell count</td><td>1593/mm³ (24–8287)</td><td>850–2200/mm³</td></tr><tr><td>CD4 T cells %</td><td>25.3% (0–85)</td><td>35–65%</td></tr><tr><td>CD4 T-cell count</td><td>487.1/mm³ (0–4672)</td><td>500–1400/mm³</td></tr><tr><td>CD8 T cells %</td><td>47.6% (2.2–95.2)</td><td>13%–40%</td></tr><tr><td>CD8 T-cell count</td><td>836/mm³ (0–3850)</td><td>200–900/mm³</td></tr><tr><td>CD4/CD8 ratio</td><td>0.53 (0–8.95)</td><td>1.0–3.0</td></tr><tr><td>DNT cell %</td><td>6.7% (0–27)</td><td>1%–5%</td></tr><tr><td>DNT cell count</td><td>98.67/mm³ (0–1041)</td><td></td></tr></tbody></table>

DN = double negative.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Mean of CD3, CD4, and CD8 T-cell subset counts in patients with DNT cell proportion within normal range (<5%) and in patients with increased DNT cell proportion (>5%). DNT = double negative T.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** (A) Correlation of DNT cell proportion with CD3 T-cell counts. (B) Correlation of DNT cell proportion with CD3 T-cell counts. The Spearman rank test was used for correlation analysis. (C) Mean of DNT cell proportion according to CD4 T-cell count. ns: nonsignificant. DNT = double negative T.</figcaption></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3.** (A) Mean of DNT cell proportions in patients with undetectable and detectable viral load. (B) Correlation of DNT cell proportion with the viral load. The Spearman rank test was used for correlation analysis. DNT = double negative T.</figcaption></figure>
was more pronounced in subjects with CD4+ T cells <200/mm³. Although Liang et al[8] showed that DNT cell levels decrease as the disease progressed in ART-naive PWH, ART was reported to be able to restore DNT cell levels in PWH whether they revealed to be responders or not.[9] That may explain the increased DNT cell frequency in our patients, all of which were under ART.

The significant positive correlation between DNT cells and the viral load observed in our cohort was consistent with the negative correlation found between DNT cells and CD4 T-cell count. Indeed, patients with an undetectable viral load and a restored CD4 T-cell count under ART had a lower proportion of DNT cells than patients with a detectable viral load and a low CD4 T-cell count. Previous studies have reported different results from ours. The correlation between DNT cells and viral load was either negative or absent in untreated patients.[8-10] No study has yet examined this association in treated patients. Nevertheless, considering the correlation between CD4 and DNT cells, our results align with those of Singleterry et al.[11] Their study demonstrated an increase of DNT cells in 31 PWH who had advanced to the AIDS stage of infection, and this expansion increased as the CD4 count lowered.[11] Our results confirm these data on a larger cohort.

The increase in DNT cells in PWH may reflect a systemic immune response to the CD4 T-cell depletion and thus would be of central thymic origin. A second hypothesis could be that DNT cells have a peripheral origin due to the loss of CD4 expression. HIV-1 gene products such as Nef, Env, and Vpu have been involved in CD4 down-modulation.[12,13] Further studies suggest that it can also occur independently of viral proteins, implicating rather an atypical regulation of the DNA methylation machinery leading to epigenetic silencing of CD4 expression.[14]

DNT cells seem essential in compensating CD4 T-cell depletion as they are associated to the nonpathogenic nature of the infection in SIV-infected natural hosts.[15] Natural hosts, such as sooty mangabeys and African green monkeys, do not develop AIDS and are able to maintain preserved T-cell populations and low levels of systemic immune activation despite high levels of virus replication.[16,17] These primates exhibit up to 40% of DNT cells that are capable of proliferating and producing T helper cytokines following antigen stimulation.[6,15]

Transforming growth factor-beta 1 and interleukin-10-producing DNT cells are associated with the control of chronic immune activation, which plays an essential role in the pathogenesis of HIV infection and is a key predictor of progression to AIDS.[9,10] Furthermore, DNT cells seem to be involved in the immune reconstitution occurring during ART and to predict future immune activation.[9] This immunoregulatory function might explain the persistence of high levels of immune activation in PWH nonresponding to long-term ART, whose DNT cell levels were shown to be only partially restored[9]

This study presents some limitations. Its retrospective nature leads to a lack of clinical data on the stage of the disease that would allow a better analysis of the correlation with the progression of the infection. Another limitation is that we only included PWH under ART and this does not allow us to generalize our results to all PWH. On the other hand, the strong point of this study is a large number of patients included.

## 5. Conclusion

Altogether, data from the present study show that, in addition to a decrease in CD4 and an increase in CD8 T cells, an increase in DNT cell proportion is frequently found in HIV-infected patients. It is important for the biologist and the clinician to keep in mind the possible DNT cell level variations when interpreting

immunophenotyping results in PWH. The significant associations with CD4 T-cell count and the viral load strongly support that DNT cells are correlated with disease progression in treated PWH. Therefore, this marker should be considered for use in clinical practice.

## Author contributions

Conceptualization: IZ and MBA; Acquisition of data: FK, IZ, RR, ABH, ZJ, ABA, SKR, WKR and MH; Analysis and interpretation of data: FK, IZ and MBA; Writing - Original draft preparation: FK and IZ; Final approval: MBA.

## References

[1] D'Acquisto F, Crompton T. CD3+CD4-CD8- (double negative) T cells: savours or villains of the immune response? Biochem Pharmacol. 2011;82:333-40.

[2] Amaral Villani FN, Da Costa Rocha MO, Pereira Nunes MC, et al. Trypanosoma Cruzi-induced activation of functionally distinct αβ and γδ CD4- CD8- T cells in individuals with polar forms of Chagas' disease. Infect Immun. 2010;78:4421-30.

[3] Neyt K, GeurtsvanKessel CH, Lambrecht BN. Double-negative T resident memory cells of the lung react to influenza virus infection via CD11c hi dendritic cells. Mucosal Immunol. 2016;9:999-1014.

[4] Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8+ double-negative T cells are prominent producers of IL-17A and IFN-gamma during primary respiratory murine infection with Francisella tularensis live vaccine strain. J Immunol. 2010;184:5791-801.

[5] Juvet SC, Zhang L. Double negative regulatory T cells in transplantation and autoimmunity: recent progress and future directions. J Mol Cell Biol. 2012;4:48-58.

[6] Sundaravaradan V, Mir KD, Sodora DL. Double-negative T cells during HIV/SIV infections: potential pinch hitters in the T-cell lineup. Curr Opin HIV AIDS. 2012;7:164-71.

[7] Brandt D, Hedrich CM. TCRαβ + CD3 + CD4 - CD8 - (double negative) T cells in autoimmunity. Autoimmun Rev. 2018;17:422-30.

[8] Liang Q, Jiao Y, Zhang T, et al. Double negative (DN) [CD3(+)-CD4(-) CD8(-)] T cells correlate with disease progression during HIV infection. Immunol Invest. 2013;42:431-7.

[9] Lu X, Su B, Xia H, et al. Low double-negative CD3+CD4-CD8- T cells are associated with incomplete restoration of CD4+ T cells and higher immune activation in HIV-1 immunological non-responders. Front Immunol. 2016;7:579.

[10] Petitjean G, Chevalier MF, Tibaoui F, et al. Level of double negative T cells, which produce TGF-β and IL-10, predicts CD8 T-cell activation in primary HIV-1 infection. AIDS. 2012;26:139-48.

[11] Singleterry WL, Henderson H, Cruse JM. Depletion of pro-inflammatory CD161+ double negative (CD3+CD4-CD8-) T cells in AIDS patients is ameliorated by expansion of the γδ T cell population. Exp Mol Pathol. 2012;92:155-9.

[12] Wildum S, Schindler M, Münch J, et al. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection. J Virol. 2006;80:8047-59.

[13] DeMaster LK, Liu X, VanBelzen DJ, et al. A subset of CD4/CD8 double-negative T cells expresses HIV proteins in patients on antiretroviral therapy. J Virol. 2016;90:2165-79.

[14] Mudd JC, Lai S, Shah S, et al. Epigenetic silencing of CD4 expression in nonpathogenic SIV infection in African green monkeys. JCI Insight. 2020;5:139043.

[15] Milush JM, Mir KD, Sundaravaradan V, et al. Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells. J Clin Invest. 2011;121:1102-10.

[16] Silvestri G, Sodora DL, Koup RA, et al. Nonpathogenic SIV infection of sooty mangabeys is characterized by limited bystander immunopathology despite chronic high-level viremia. Immunity. 2003;18:441-52.

[17] Milush JM, Reeves JD, Gordon SN, et al. Virally induced CD4+ T cell depletion is not sufficient to induce AIDS in a natural host. J Immunol. 2007;179:3047-56.