CD154 and IL-2 Signaling of CD4+ T Cells Play a Critical Role in Multiple Phases of CD8+ CTL Responses Following Adenovirus Vaccination

Adenoviral (AdV) vectors represent most commonly utilized viral vaccines in clinical studies. While the role of CD8⁺ cytotoxic T lymphocyte (CTL) responses in mediating AdV-induced protection is well understood, the involvement of CD4⁺ T cell-provided signals in the development of functional CD8⁺ CTL responses remain unclear. To explore CD4⁺ T helper signals required for AdVova-stimulated CTL responses, we established an adoptive transfer system by transferring CD4⁺ T cells derived from various knock out and transgenic mice into wild-type and/or CD4-deficient animals, followed by immunizing with recombinant ovalbumin (OVA)-expressing AdVova vector. Without CD4⁺ T help, both primary and memory CTL responses were greatly reduced in this model, and were associated with increased PD-1 expression. The provision of OVA-specific CD4⁺ T help in CD4⁺ T cell-deficient mice restored AdVova-induced primary CTL responses, and supported survival and recall responses of AdVova-stimulated memory CTLs. These effects were specifically mediated by CD4⁺ T cell-produced IL-2 and CD154 signals. Adoptive transfer of “helped” or “unhelped” effector and memory CTLs into naïve CD4⁺ T cell-deficient or -sufficient mice also revealed an additional role for polyclonal CD4⁺ T cell environment in the survival of AdVova-stimulated CTLs, partially explaining the extension of CTL contraction phase. Finally, during recall responses, CD4⁺ T cell environment, particularly involving memory CD4⁺ T cells, greatly enhanced expansion of memory CTLs. Collectively, our data strongly suggest a critical role for CD4⁺ T help in multiple phases of AdV-stimulated CTL responses, and could partially explain certain failures in AdV-based immunization trials targeting malignant tumors and chronic diseases that are often associated with compromised CD4⁺ T cell population and function.     

Influenza-Infected Neutrophils within the Infected Lungs Act as Antigen Presenting Cells for Anti-Viral CD8+ T Cells

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8⁺ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8⁺ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8⁺ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8⁺ T cells within the infected lung interstitium.     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

TLR2 Directing PD-L2 Expression Inhibit T Cells Response in Schistosoma japonicum Infection

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2^−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2^−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4⁺T cells was down-regulated in TLR2^−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4⁺T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4⁺T cells, to help inhibit T cells response in Schistosoma japonicum infection.     

Mechanism of γδ T-Cell-Mediated Inhibition of Stem Cell Differentiationin Vitro:Possible Relevance for Myelosuppression in HIV-Infected Individuals

We investigated whether γδ T cells contribute to the suppression of myelopoiesis in HIV infection. Freshly isolated γδ T cells from HIV seropositive patients suppressed CFU–GM growthin vitro.Preactivation of γδ T cells with IL-2 and/or IL-15 further reduced the number of CFU–GM. Natural killer cells and to a lower extent CD4⁺and CD8⁺cells also inhibited CFU–GM growth. In contrast to γδ T cells, this effect was not dependent on IL-15 or IL-2 preactivation. Moreover, no enhanced inhibitory effect of CD56⁺and CD4⁺cells was observed in HIV⁺subjects compared to HIV⁻donors. The myelosuppressive effect of supernatants of γδ T cells could be inhibited by antibodies against IFN-γ or TNF-α. Accordingly, we found increased numbers of TNF-α- or IFN-γ-secreting CD8⁺γδ T cells in HIV⁺patients. We conclude that the increased fraction of activated γδ T cells producing myelosuppressive cytokines might contribute to the dyshematopoiesis frequently observed in HIV-infected individuals.     

Epitope-Specific CD8+ T Cells Play a Differential Pathogenic Role in the Development of a Viral Disease Model for Multiple Sclerosis

Theiler''s virus-induced demyelinating disease has been extensively investigated as a model for persistent viral infection and multiple sclerosis (MS). However, the role of CD8⁺ T cells in the development of disease remains unclear. To assess the role of virus-specific CD8⁺ T cells in the pathogenesis of demyelinating disease, a single amino acid substitution was introduced into the predominant viral epitope (VP3 from residues 159 to 166 [VP3_159-166]) and/or a subdominant viral epitope (VP3_173-181) of susceptible SJL/J mice by site-directed mutagenesis. The resulting variant viruses (N160V, P179A, and N160V/P179A) failed to induce CD8⁺ T cell responses to the respective epitopes. Surprisingly, mice infected with N160V or N160V/P179A virus, which lacks CD8⁺ T cells against VP3_159-166, did not develop demyelinating disease, in contrast to wild-type virus or P179A virus lacking VP3_173-181-specific CD8⁺ T cells. Our findings clearly show that the presence of VP3_159-166-specific CD8⁺ T cells, rather than viral persistence itself, is strongly correlated with disease development. VP3_173-181-specific CD8⁺ T cells in the central nervous system (CNS) of these virus-infected mice expressed higher levels of transforming growth factor β, forkhead box P3, interleukin-22 (IL-22), and IL-17 mRNA but caused minimal cytotoxicity compared to that caused by VP3_159-166-specific CD8⁺ T cells. VP3_159-166-specific CD8⁺ T cells exhibited high functional avidity for gamma interferon production, whereas VP3_173-181-specific CD8⁺ T cells showed low avidity. To our knowledge, this is the first report indicating that the induction of the IL-17-producing CD8⁺ T cell type is largely epitope specific and that this specificity apparently plays a differential role in the pathogenicity of virus-induced demyelinating disease. These results strongly advocate for the careful consideration of CD8⁺ T cell-mediated intervention of virus-induced inflammatory diseases.     

Human Langerhans Cells Control Th Cells via Programmed Death-Ligand 1 in Response to Bacterial Stimuli and Nickel-Induced Contact Allergy

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4⁺T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6⁺/CCR4⁺ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6⁺ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6⁺/CCR4⁺ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.     

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during Brucella abortus infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.     

HIV-specific cytotoxic T lymphocytes against alveolar macrophages: specificities and downregulation

To analyse the evolution of alveolar-lymphocyte-mediated cytotoxic activity directed against autologous alveolar macrophages (AM), cytotoxic assays against various HIV⁺ target cells were performed in a cohort of 75 patients with HIV-associated lymphoid interstitial pneumonitis (LIP) studied at distinct stages of HIV infection. Our data confirm that alveolar HIV-specific cytotoxic T lymphocytes (CTL) against AM were detectable before AIDS in patients with CD8⁺ LIP. Mild CD8⁺ lymphocytic alveolitis occurs silently in 62% of stage II and III patients with no respiratory symptoms. In these cases, the lack of spontaneous alveolar-lymphocyte-mediated cytotoxic activity against autologous AM may contrast with the detection of primary alveolar CTL specific for HIV proteins such as nef. In AIDS patients, the alveolar CTL lytic efficiency against both AM- and HIV-antigen-expressing cells can be inhibited by a suppressore factor produced by alveolar CD8⁺ CD57⁺ cells. Therefore, spontaneous CTL lysis of AM may be (1) limited to a subgroup of patients with active LIP and (2) controlled by distinct mechanisms, including suppressor phenomenons, and HIV replication levels in AM.     

Enhanced and prolonged efficacy of superantigen-induced cytotoxic T lymphocyte activity by interleukin-2 in vivo

The bacterial superantigen, staphylococcal enterotoxin A (SEA) activates T cells with high frequency and directs them to lyse MHC-class-II-expressing cells in superantigen-dependent cell-mediated cytotoxicity (SDCC). Treatment of mice with SEA induced strong CD8⁺ T-cell(CTL)-mediated SDCC, as well as abundant cytokine production from CD4⁺ and CD8⁺ T cells. However, both cytotoxicity and cytokine release were transient. In contrast, combined treatment with SEA and recombinant interleukin-2 (rIL-2) increased peak levels and maintained CTL activity. These effects were concomitant with an increased number of SEA-reactive V11⁺ T cells. Both the CD4⁺ and CD8⁺ populations contained higher frequencies of cells expressing IL-2 receptor (IL-2R) , which suggests that continuous IL-2R signaling preserves its high expression and subsequently prevents loss of growth factor signal necessary for expansion of T cells. Although IL-2R expression was increased among both CD4⁺ and CD8⁺ cells, only the cytotoxic function of CTL, but not cytokine production from either CD4 or CD8, was augmented. These findings demonstrate that treatment with rIL-2 potentiates superantigen-induced cytotoxicity and maintains high CTL activity. rIL-2 might therefore be useful in improving superantigenbased tumor therapy.     

Development of Skewed Functionality of HIV-1-Specific Cytotoxic CD8+ T Cells from Primary to Early Chronic Phase of HIV Infection

In recent years, the prevalence of HIV-1 infection has been rapidly increasing among men who have sex with men (MSM). However, it remains unknown how the host immune system responds to the infection in this population. We assessed the quantity of HIV-specific CD8⁺ T-cell responses by using Elispot assay and their functionalities by measuring 5 CD8⁺ T-cell evaluations (IL-2, MIP-1β, CD107a, TNF-α, IFN-γ) with flow cytometry assays among 18 primarily and 37 early chronically HIV-infected MSM. Our results demonstrated that subjects at early chronic phase developed HIV-specific CD8⁺ T-cell responses with higher magnitudes and more diversified functionalities in comparison with those at primary infection. However, populations with IL-2⁺ CD107a⁺ or in combination with other functionality failed to develop in parallel. The multifunctional but not monofunctional HIV-specific CD8⁺ T cells were associated with higher CD4⁺ T -cell counts and lower viral loads. These data revealed that prolonged infection from primary to early chronic infection could selectively increase the functionalities of HIV-specific CD8⁺ T cells in HIV-infected MSM population, the failure to develop IL-2 and cytotoxic functionalities in parallel may explain why the increased HIV-specific CD8⁺ T cells were unable to enhance the containment of HIV-1 replication at the early chronic stage.     

Myeloid-derived suppressor cells regulate the immunosuppressive functions of PD-1−PD-L1+ Bregs through PD-L1/PI3K/AKT/NF-κB axis in breast cancer

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells that are closely related to tumor immune escape, but the mechanism by which MDSCs regulate B cells has not been elucidated. Our previous studies revealed that breast cancer-derived MDSCs could induce a group of PD-1⁻PD-L1⁺ Bregs with immunosuppressive functions. Here, we reported that blocking PD-1/PD-L1 interaction between MDSCs and B cells could reverse the immunosuppressive functions of PD-1⁻PD-L1⁺ Bregs. The activation of PI3K/AKT/NF-κB signaling pathway is essential for PD-1⁻PD-L1⁺ Bregs to exert immunosuppressive effects. MDSCs activated the PI3K/AKT/NF-κB pathway in B cells via the PD-1/PD-L1 axis. Furthermore, inhibition of PD-1/PD-L1 or PI3K/AKT signaling suppressed both tumor growth and the immunosuppressive functions of PD-1⁻PD-L1⁺ Bregs. Dual suppression of PD-1/PD-L1 and PI3K/AKT exerted better antitumor effect. Finally, MDSCs and PD-1⁻PD-L1⁺ Bregs were colocalized in breast cancer tissues and PD-1⁻PD-L1⁺ Bregs were positively correlated with poor prognosis. Thus, MDSC-educated PD-1⁻PD-L1⁺ Bregs and their regulatory mechanisms could contribute to the immunosuppressive tumor microenvironment. Our study proposes a novel mechanism for MDSC-mediated regulation of B cell immunity, which might shed new light on tumor immunotherapy.⁺Subject terms: Breast cancer, Cancer microenvironment     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Vaccination Expands Antigen-Specific CD4+ Memory T Cells and Mobilizes Bystander Central Memory T Cells

CD4⁺ T helper memory (Th_mem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Th_mem cells as expected, but was accompanied by parallel increase of Th_mem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Th_mem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Th_mem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Th_mem cells were activated (CD38^High HLA-DR⁺), cycling or recently divided (Ki-67⁺), and apparently vulnerable to death (IL-7Rα^Low and Bcl-2 ^Low). In contrast, bystander Th_mem cells were resting (CD38^Low HLA-DR^- Ki-67^-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Th_mem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines.     

4-1BB Signaling Enhances Primary and Secondary Population Expansion of CD8+ T Cells by Maximizing Autocrine IL-2/IL-2 Receptor Signaling

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is primarily expressed on activated T cells and is known to enhance proliferation of T cells, prevent activation-induced cell death, and promote memory formation of CD8⁺ T cells. In particular, it is well acknowledged that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells rather than CD4⁺ T cells, but the underlying mechanism remains unclear. Here we found that 4-1BB triggering markedly increased IL-2Rα (CD25) and IL-2 expressions of CD8+ T cells but minimally for CD4⁺ T cells. Proliferation of CD8⁺ T cells was moderately enhanced by direct 4-1BB triggering in the absence of signaling through IL-2Rα/IL-2 interactions, but further promoted in the presence of IL-2Rα/IL-2 interactions. Among the TNFRSF members including OX40, GITR, CD30, and CD27, 4-1BB was superior in the ability to induce IL-2Rα expression on CD8+ T cells. When the primary and secondary expansions of CD8⁺ T cells in vivo were examined by adoptively transferring OVA-specific CD8⁺ T cells along with the treatment with agonistic anti-4-1BB and/or antagonistic anti-CD25 F(ab’)₂ mAb, 4-1BB triggering enhanced both primary and secondary expansion of CD8⁺ T cells in vivo, and the 4-1BB effects were moderately suppressed in primary expansion while completely abolished in secondary expansion of OVA-specific CD8⁺ T cells by blocking IL-2Rα. These results suggest that 4-1BB-mediated increases of IL-2Rα and IL-2 prolong the effects of transient TCR- and 4-1BB-mediated signaling in CD8⁺ T cells, and that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells through the amplification of autocrine IL-2/IL-2R signaling loop.     

Poor Immune Reconstitution in HIV-Infected Patients Associates with High Percentage of Regulatory CD4+ T Cells

CD4⁺ regulatory T cells (Tregs) are essential for the maintenance of the immune system''s equilibrium, by dampening the activation of potential auto-reactive T cells and avoiding excessive immune activation. To correctly perform their function, Tregs must be maintained at the right proportion with respect to effector T cells. Since this equilibrium is frequently disrupted in individuals infected with the human immunodeficiency virus (HIV), we hypothesize that its deregulation could hamper immune reconstitution in patients with poor CD4⁺ T cell recovery under highly active antiretroviral therapy (HAART). We analysed Tregs percentages amongst CD4⁺ T cells in 53 HIV-infected patients under HAART, with suppression of viral replication and distinct levels of immune reconstitution. As controls, 51 healthy individuals were also analysed. We observed that amongst the patients with Nadir values (the lowest CD4⁺ T cell counts achieved) <200 cells/µL, the individuals with high Tregs percentages (≥10% of total CD4⁺ T cells) had the worse CD4⁺ T cell reconstitution. In accordance, the well-described direct correlation between the Nadir value and CD4⁺ T cell reconstitution is clearly more evident in individuals with high Tregs proportions. Furthermore, we observed a strong negative correlation between Tregs percentages and CD4⁺ T cell recovery among immunological non-responder HIV⁺ individuals. All together, this work shows that high Tregs frequency is an important factor associated with sub-optimal CD4⁺ T cell recovery. This is particularly relevant for immunological non-responders with low Nadir values. Our results suggest that the Tregs proportion might be of clinical relevance to define cut-offs for HAART initiation.