Immediate major dynamic changes in the T- and NK-cell subset composition after cardiac transplantation

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3 wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4⁺ or CD8⁺ T cells and CD56^dimCD16⁺ NK cells immediately after HTx linked to a decrease in naïve CD8⁺ and CM CD4⁺ T as well as CD56^brightCD16⁻ NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69⁺CD25⁻ and diminished CD69⁻CD25⁻CD4⁺ or CD8⁺ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.     

Enhancement of cytokine‐driven NK cell IFN‐γ production after vaccination of HCMV infected Africans

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine‐induced responses, we studied NK‐cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK‐cell responses (IFN‐γ, CD107a, CD25) occurred after in vitro re‐stimulation of post‐vaccination NK cells with TIV or DTPiP antigens compared to pre‐vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN‐γ in response to exogenous IL‐12 with IL‐18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN‐γ⁺ cells observed within CD56^brightCD57^?, CD56^dimCD57^?NKG2C^? and CD56^dimCD57^?NKG2C⁺ NK‐cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN‐γ function in HCMV infected donors and raise the potential for further exploitation of NK cell “pre‐activation” to improve vaccine effectiveness.     

Type 2 diabetes mellitus is associated with altered CD8+ T and natural killer cell function in pulmonary tuberculosis

Type 2 diabetes mellitus (DM) is associated with expanded frequencies of mycobacterial antigen-specific CD4⁺ T helper type 1 (Th1) and Th17 cells in individuals with active pulmonary tuberculosis (TB). No data are available on the role of CD8⁺ T and natural killer (NK) cells in TB with coincident DM. To identify the role of CD8⁺ T and NK cells in pulmonary TB with diabetes, we examined mycobacteria-specific immune responses in the whole blood of individuals with TB and DM (TB-DM) and compared them with those without DM (TB-NDM). We found that TB-DM is characterized by elevated frequencies of mycobacterial antigen-stimulated CD8⁺ T cells expressing type 1 [interferon-γ and interleukin-2 (IL-2)] and type 17 (IL-17F) cytokines. We also found that TB-DM is characterized by expanded frequencies of TB antigen-stimulated NK cells expressing type 1 (tumour necrosis factor-α) and type 17 (IL-17A and IL-17F) cytokines. In contrast, CD8⁺ T cells were associated with significantly diminished expression of the cytotoxic markers perforin, granzyme B and CD107a both at baseline and following antigen or anti-CD3 stimulation, while NK cells were associated with significantly decreased antigen-stimulated expression of CD107a only. This was not associated with alterations in CD8⁺ T-cell or NK cell numbers or subset distribution. Therefore, our data suggest that pulmonary TB complicated with type 2 DM is associated with an altered repertoire of cytokine-producing and cytotoxic molecule-expressing CD8⁺ T and NK cells, possibly contributing to increased pathology.     

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

Increased frequency of CD56Bright NK‐cells,CD3−CD16+CD56− NK‐cells and activated CD4+T‐cells or B‐cells in parallel with CD4+CDC25High T‐cells control potentially viremia in blood donors with HCV

A detailed phenotypic analysis of major and minor circulating lymphocyte subsets is described in potential blood donors with markers of hepatitis C virus (HCV), including non‐viremic and viremic groups. Although there were no changes in the hematological profile of either group, increased the levels of pre‐NK cells (CD3^?CD16⁺CD56^?) and a lower frequency of mature NK cells (CD3^?CD16⁺CD56⁺) characterized innate immunity in the non‐viremic group. Both non‐viremic and viremic groups displayed significantly increased levels of CD56^Bright NK cells. Furthermore, this subset was significantly elevated in the viremic subgroup with a low viral load. In addition, an increase in the NKT2 subset was observed only in this subgroup. An enhanced frequency of activated CD4⁺ T‐cells (CD4⁺HLA‐DR⁺) was a characteristic feature of the non‐viremic group, whereas elevated CD19⁺ B‐cells and CD19⁺CD86⁺ cell populations were the major phenotypic features of the viremic group, particularly in individuals with a low viral load. Although CD4⁺CD25^High T‐cells were significantly elevated in both the viremic and non‐viremic groups, it was particularly evident in the viremic low viral load subgroup. A parallel increase in CD4⁺CD25^High T‐cells, pre‐NK, and activated CD4⁺ T‐cells was observed in the non‐viremic group, whereas a parallel increase in CD4⁺CD25^High T‐cells and CD19⁺ B‐cells was characteristic of the low viral load subgroup. These findings suggest that CD56^Bright NK cells, together with pre‐NK cells and activated CD4⁺ T‐cells in combination with CD4⁺CD25^High T‐cells, might play an important role in controlling viremia. Elevated CD56^Bright NK cells, B‐cell responses and a T‐regulated immunological profile appeared to be associated with a low viral load. J. Med. Virol. 81:49–59, 2009. © 2008 Wiley‐Liss, Inc.     

Immune regulation by non‐lymphoid cells in transplantation

Regulatory cells play a crucial role in the induction and maintenance of tolerance by controlling T cell as well as B and natural killer (NK) cell‐mediated immunity. In transplantation, CD4⁺CD25⁺forkhead box P3⁺ T regulatory cells are instrumental in the maintenance of immunological tolerance, as are several other T cell subsets such as NK T cells, double negative CD3⁺ T cells, γδ T cells, interleukin‐10‐producing regulatory type 1 cells, transforming growth factor‐β‐producing T helper type 3 cells and CD8⁺CD28^‐ cells. However, not only T cells have immunosuppressive properties, as it is becoming increasingly clear that both T and non‐T regulatory cells co‐operate and form a network of cellular interactions controlling immune responses. Non‐T regulatory cells include tolerogenic dendritic cells, plasmacytoid dendritic cells, mesenchymal stem cells, different types of stem cells, various types of alternatively activated macrophages and myeloid‐derived suppressor cells. Here, we review the mechanism of action of these non‐lymphoid regulatory cells as they relate to the induction or maintenance of tolerance in organ transplantation.     

Glatiramer acetate attenuates the pro‐migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell‐bound and soluble AM in the peripheral blood of patients with relapsing–remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)‐1, ICAM‐3, lymphocyte function‐associated antigen (LFA)‐1 and very late activation antigen (VLA)‐4 were assessed in T cells (CD3⁺CD8⁺, CD3⁺CD4⁺), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five‐colour flow cytometry. Soluble E‐selectin, ICAM‐1, ICAM‐3, platelet endothelial cell adhesion molecule (PECAM)‐1, P‐selectin and vascular cell adhesion molecule (VCAM)‐1 were determined with a fluorescent bead‐based immunoassay. The pro‐migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up‐regulation of LFA‐1 (CD3⁺CD4⁺ T cells, B cells), VLA‐4 (CD3⁺CD8⁺ T cells, NK cells), ICAM‐1 (B cells) and ICAM‐3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA‐1 (CD3⁺CD4⁺) and VLA‐4 (CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NK T, monocytes). Further effects included lowering of ICAM‐1 and ICAM‐3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro‐migratory expression profile of cell‐bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.     

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c-kit⁺ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3^? IL-2Rβ⁺) and extrathymic T cells (CD3^int IL-2Rβ⁺) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8⁺ CD3^int IL-2Rβ⁺ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1⁺ and NK1.1^? subsets of CD3^int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1⁺ subset of CD3^int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1^? subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.     

Excessive CD4+ T cells co-expressing interleukin-17 and interferon-γ in patients with Behçet's disease

Excessive T helper type 1 (Th1) cell activity has been reported in Behçet's disease (BD). Recently, association of Th17 cells with certain autoimmune diseases was reported, and we thus investigated circulating Th17 cells in BD. CD4⁺CD45RO^– (naive) T cells were cultured with Th0‐, Th1‐, Th2‐ and Th17‐related cytokines and antibodies, and their mRNA was studied by real‐time polymerase chain reaction (PCR). When naive CD4⁺ T cells were cultured with Th1‐ and Th17‐related cytokines, interferon (IFN)‐γ mRNA and interleukin (IL)‐17 mRNA were up‐regulated, respectively, in BD patients. Naive CD4⁺ T cells cultured in a Th17 cell‐inducing condition expressed IL‐23 receptor (IL‐23R) mRNA excessively. IL‐17 mRNA expression was induced only when naive CD4⁺T cells were cultured in the presence of IL‐23. CD4⁺ T cells cultured with Th17 cytokines expressed excessive RAR‐related orphan receptor C (RORC) mRNA. Using intracellular cytokine staining, we found that CD45RO⁺(memory) CD4⁺ T cells producing IL‐17 and IFN‐γ simultaneously were increased significantly. Memory CD4⁺ T cells producing IFN‐γ but not IL‐17 decreased profoundly in BD patients. CD4⁺ T cells producing IL‐17 and IFN‐γ simultaneously were found in BD skin lesions. Collectively, we found excessive CD4⁺ T cells producing IL‐17 and IFN‐γ (Th1/Th17) cells in patients with BD, and possible involvement of IL‐23/IL‐23R pathway for the appearance of excessive Th1/Th17 cells.     

The differential impact of natural killer (NK) cell education via KIR2DL3 and KIR3DL1 on CCL4 secretion in the context of in‐vitro HIV infection

Carriage of certain inhibitory natural killer (NK) cell receptor (iNKR)/HLA ligand pairs is associated with protection from infection and slow time to AIDS implicating NK cells in HIV control. NK cells acquire functional potential through education, which requires the engagement of iNKRs by their human leucocyte antigen (HLA) ligands. HIV infection down‐regulates cell surface HLA‐A/B, but not HLA‐C/E. We investigated how NK cell populations expressing combinations of the iNKRs NKG2A, KIR2DL3 (2DL3) and KIR3DL1 (3DL1) responded to autologous HIV infected CD4 (iCD4) cells. Purified NK cells from HIV‐uninfected individuals were stimulated with autologous HIV iCD4 or uninfected CD4 T cells. Using flow cytometry we gated on each of the 8 NKG2A^+/–2DL3^+/–3DL1^+/‐ populations and analysed all possible combinations of interferon (IFN)‐γ, CCL4 and CD107a functional subsets responding to iCD4 cells. Infected CD4 cells induced differential frequencies of NKG2A^+/–2DL3^+/–3DL1^+/– populations with total IFN‐γ⁺, CCL4⁺ and CD107a⁺ functional profiles. 2DL3⁺NKG2A⁺ NK cells had a higher frequency of responses to iCD4 than other populations studied. A higher frequency of 2DL3⁺ NK cells responded to iCD4 from individuals that were not HLA‐C1 homozygotes. These results show that 2DL3⁺ NK cells are mediators of HIV‐specific responses. Furthermore, responses of NK cell populations to iCD4 are influenced not only by NK cell education through specific KIR/HLA pairs, but also by differential HIV‐mediated changes in HLA expression.     

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4⁺CD25⁺ regulatory T cells (T_reg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T_reg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4⁺CD25⁺ T_reg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4⁺CD25⁺ T_reg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4⁺CD25⁺ T_reg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4⁺CD25⁺ T_reg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.     

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV‐1) associated neurological disease

Human T lymphotropic virus type 1 (HTLV‐1) infects 10–20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV‐1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4⁺ and fewer CD8⁺ cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4⁺ NK T subset are associated with HTLV‐1 disease progression.     

Variable immune cell frequencies in peripheral blood of LEW.1AR1‐iddm rats over time compared to other congenic LEW strains

The LEW.1AR1‐iddm rat is an animal model of human type 1 diabetes (T1D), which arose through a spontaneous mutation within the major histocompatibility complex (MHC)‐congenic background strain LEW.1AR1. The LEW.1AR1‐iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1AR1‐iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1AR1 and the LEW rat strain as well as the LEW.1WR1 rat strain. The LEW.1AR1‐iddm rats are characterized by a high variability of CD3⁺, CD4⁺ and CD8⁺ T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4⁺ : CD8⁺ T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain‐specific T cell frequency resulting in a specific CD4⁺ : CD8⁺ T cell ratio. The frequency of natural killer (NK) cells and B cells in LEW.1AR1‐iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1AR1‐iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development.     

Murine peripheral NK‐cell populations originate from site‐specific immature NK cells more than from BM‐derived NK cells

Murine NK cells can be divided by the expression of two cell surface markers, CD27 and Mac‐1 (a.k.a. CD11b), into four separate subsets. These subsets suggest a linear development model: CD27^– Mac‐1^– → CD27⁺ Mac‐1^– → CD27⁺ Mac‐1⁺ → CD27^– Mac‐1⁺. Here, we used a combination of BrdU labeling experiments and mathematical modeling to gain insights regarding NK‐cell development in mouse bone marrow (BM), spleen and liver. The modeling results that best fit the experimental data show that the majority of NK cells already express CD27 upon entering the NK‐cell developmental pathway. Additionally, only a small fraction of NK cells exit the BM to other sites, suggesting that peripheral NK‐cell populations originate from site‐specific immature NK cells more than from BM‐derived mature NK cells.     

Effect of standard tuberculosis treatment on naive,memory and regulatory T‐cell homeostasis in tuberculosis–diabetes co‐morbidity

Perturbations in CD4⁺ and CD8⁺ T‐cell phenotype and function are hallmarks of tuberculosis–diabetes co‐morbidity. However, their contribution to the pathogenesis of this co‐morbidity and the effect of anti‐tuberculosis treatment on the phenotype of the T‐cell subsets is poorly understood. In this study, we examined the frequency of different T‐cell subsets in individuals with pulmonary tuberculosis (PTB) with diabetes mellitus (DM) or without coincident diabetes mellitus (NDM) before, during and after completion of anti‐tuberculosis chemotherapy. PTB‐DM is characterized by heightened frequencies of central memory CD4⁺ and CD8⁺ T cells and diminished frequencies of naive, effector memory and/or effector CD4⁺ and CD8⁺ T cells at baseline and after 2 months of treatment but not following treatment completion in comparison with PTB‐NDM. Central memory CD4⁺ and CD8⁺ T‐cell frequencies exhibited a positive correlation with fasting blood glucose and glycated haemoglobin A1c levels, whereas the frequencies of naive and effector memory or effector CD4⁺ and CD8⁺ T cells exhibited a negative correlation. However, the frequencies of CD4⁺ and CD8⁺ T‐cell subsets in individuals with PTB exhibited no significant relationship with bacterial burdens. Finally, although minor alterations in the T‐cell subset compartment were observed at 2 months of treatment, significantly decreased frequencies of central memory and significantly enhanced frequencies of naive CD4⁺ and CD8⁺ T cells were observed at the completion of treatment. Our data reveal a profound effect of coexistent diabetes on the altered frequencies of central memory, effector memory and naive T cells and its normalization following therapy.     

Proteomic definition of human mucosal‐associated invariant T cells determines their unique molecular effector phenotype

Mucosal‐associated invariant T cells (MAIT) constitute the most abundant anti‐bacterial CD8⁺ T‐cell population in humans. MR1/TCR‐activated MAIT cells were reported to organize cytotoxic and innate‐like responses but knowledge about their molecular effector phenotype is still fragmentary. Here, we have examined the functional inventory of human MAIT cells (CD3⁺Vα7.2⁺CD161⁺) in comparison with those from conventional non‐MAIT CD8⁺ T cells (cCD8⁺) and NK cells. Quantitative mass spectrometry characterized 5500 proteins of primary MAIT cells and identified 160 and 135 proteins that discriminate them from cCD8⁺ T cells and NK cells donor‐independently. Most notably, MAIT cells showed a unique exocytosis machinery in parallel to a proinflammatory granzyme profile with high levels of the granzymes A, K, and M. Furthermore, 24 proteins were identified with highest abundances in MAIT cells, including CD26, CD98, and L‐amino‐oxidase (LAAO). Among those, expression of granzyme K and CD98 were validated as MAIT‐specific with respect to non‐MAIT CD8⁺ effector subsets and LAAO was found to be recruited together with granzymes, perforin, and CD107a at the immunological synapse of activated MAIT cells. In conclusion, this study complements knowledge on the molecular effector phenotype of MAIT cells and suggest novel immune regulatory functions as part of their cytotoxic responses.     

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34⁺ cells are enriched in lymphoid‐committed precursors, while PB CD34⁺ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56⁺CD161⁺ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56⁺CD161⁺ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34⁺ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.