Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity

Natural killer T (NKT) cells are important regulators of the immune system, but their trafficking machinery, including expression of chemokine receptors, has been poorly defined. Unlike other conventional T-cell populations, we show that most NKT cells express receptors for extralymphoid tissue or inflammation-related chemokines (CCR2, CCR5, and CXCR3), while few NKT cells express lymphoid tissue-homing chemokine receptors (CCR7 and CXCR5). A population with homing potential for lymph nodes (L selectin(+) CCR7(+)) exists only within a small subset of CD4 NKT cells. We show differential expression of chemokine receptors among NKT cell subsets: CCR4 is mainly expressed by a high cytokine (interleukin-4/interleukin-2)-producing (CD4) NKT subset, while CCR1, CCR6, and CXCR6 are preferentially expressed by the low cytokine-producing CD8 and CD4(-)CD8(-) subsets. In line with this, TARC/CCL17 (a CCR4 ligand) induces preferential chemotaxis of the CD4 NKT subset, while chemotactic activities of LARC/CCL20 (a CCR6 ligand) and MIP-1 alpha/CCL3 (a CCR1 ligand) are focused on the CD8 and CD4(-)CD8(-) NKT cells. We conclude that, unlike conventional naive, memory, or effector T cells, the entire NKT cell population expresses nonlymphoid tissue homing chemokine receptors, yet NKT cell subsets differ considerably from each other by displaying distinct and reciprocal expression patterns of some chemokine receptors. Our results identify chemokine receptors that are potentially important for trafficking of human blood NKT cell subsets and reveal their function (cytokine production capacity)-dependent differential trafficking potentials.     

High-scatter T cells: a reliable biomarker for malignant T cells in cutaneous T-cell lymphoma

In early-stage cutaneous T-cell lymphoma (CTCL), malignant T cells are confined to skin and are difficult to isolate and discriminate from benign reactive cells. We found that T cells from CTCL skin lesions contained a population of large, high-scatter, activated skin homing T cells not observed in other inflammatory skin diseases. High-scatter T (T(HS)) cells were CD4(+) in CD4(+) mycosis fungoides (MF), CD8(+) in CD8(+) MF, and contained only clonal T cells in patients with identifiable malignant Vβ clones. T(HS) cells were present in the blood of patients with leukemic CTCL, absent in patients without blood involvement, and contained only clonal malignant T cells. The presence of clonal T(HS) cells correlated with skin disease in patients followed longitudinally. Clonal T(HS) cells underwent apoptosis in patients clearing on extracorporeal photopheresis but persisted in nonresponsive patients. Benign clonal T-cell proliferations mapped to the normal low-scatter T-cell population. Thus, the malignant T cells in both MF and leukemic CTCL can be conclusively identified by a unique scatter profile. This observation will allow selective study of malignant T cells, can be used to discriminate patients with MF from patients with other inflammatory skin diseases, to detect peripheral blood involvement, and to monitor responses to therapy.     

CCR6/CCR10-mediated plasmacytoid dendritic cell recruitment to inflamed epithelia after instruction in lymphoid tissues

Absent in peripheral tissues during homeostasis, human plasmacytoid dendritic cells (pDCs) are described in inflamed skin or mucosa. Here, we report that, unlike blood pDCs, a subset of tonsil pDCs express functional CCR6 and CCR10, and their respective ligands CCL20 and CCL27are detected in inflamed epithelia contacting blood dendritic cell antigen 2(+) pDCs. Moreover, pDCs are recruited to imiquimod-treated skin tumors in WT but not CCR6-deficient mice, and competitive adoptive transfers reveal that CCR6-deficient pDCs are impaired in homing to inflamed skin tumors after intravenous transfer. On IL-3 culture, CCR6 and CCR10 expression is induced on human blood pDCs that become responsive to CCL20 and CCL27/CCL28, respectively. Interestingly, unlike myeloid DC, blood pDCs initially up-regulate CCR7 expression and CCL19 responsiveness on IL-3 ± CpG-B and then acquire functional CCR6 and CCR10. Finally, IL-3-differentiated CCR6(+) CCR10(+) pDCs secrete high levels of IFN-α in response to virus. Overall, we propose an unexpected pDCs migratory model that may best apply for mucosal-associated lymphoid tissues. After CCR7-mediated extravasation into lymphoid tissues draining inflamed epithelia, blood pDCs may be instructed to up-regulate CCR6 and/or CCR10 allowing their homing into inflamed epithelia (in mucosae or skin). At this site, pDCs can then produce IFN-α contributing to pathogen clearance and/or local inflammation.     

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.     

CCR2 expressing CD4+ T lymphocytes are preferentially recruited to the ileum in Crohn's disease

BACKGROUND AND AIMS: Chemokine receptors are key determinants of leucocyte trafficking. While the chemokine receptor CCR9 and its chemokine ligand CCL25 (TECK) mediate lymphocyte homing to the healthy small intestine, the chemokine receptors important for recruitment during intestinal inflammation are undefined. Animal studies have suggested potential roles for CCR2 and CCR5 in inflammatory bowel disease (IBD). The aim of this study was to understand the role of CCR2 in human IBD. METHODS: Resections of ileum or colon were obtained from patients undergoing surgery for small bowel Crohn's disease (SBCD; n = 10), Crohn's colitis (n = 5), ulcerative colitis (n = 6), and non-IBD related conditions (control ileum n = 11; control colon n = 11). Expression of CCR2 by lamina propria lymphocytes (LPLs) was determined by both flow cytometry and immunohistochemistry. As a functional correlate, chemotaxis assays using the CCR2 ligand, CCL2 (MCP-1), were performed. Expression of CCR2 by peripheral blood lymphocytes was determined by flow cytometry. RESULTS: There were greater than 30-fold more CCR2(+) LPLs in SBCD than in control ileum (29.3% (19.9-55.1) v 0.9% (0.4-11.5); p = 0.0007). Specifically, CCR2(+)CD4(+) LPLs were increased (p = 0.002) whereas CCR2(+)CD8(+) LPLs were not. Increased expression included both memory (CD45RO(+); p = 0.005) and na?ve (CD45RO(-); p = 0.01) CCR2(+) populations. The increase in CCR2(+) LPLs in SBCD was confirmed by both immunohistochemistry (p = 0.0002) and enhanced chemotactic responses to CCL2. CCR2 expression was not increased in the peripheral blood of patients with SBCD, suggesting ongoing recruitment of the CCR2(+) population to the ileum. In contrast with SBCD, there was no significant increase in CCR2(+) LPLs in Crohn's colitis or ulcerative colitis samples. CONCLUSIONS: The chemokine receptor CCR2 appears to be an important contributor to accumulation of CD4(+) T lymphocytes in the ileum in small bowel Crohn's disease. Blockade of CCR2 may provide a novel therapeutic alternative.     

CD30-CD30 ligand interaction in primary cutaneous CD30(+) T-cell lymphomas: A clue to the pathophysiology of clinical regression.

Primary CD30(+) cutaneous T-cell lymphomas (CTCLs) represent a spectrum of non-Hodgkin's lymphomas (NHLs) that have been well defined at the clinical, histologic, and immunologic level. This group, which includes 2 main entities (large cell lymphoma and lymphomatoid papulosis [LyP]) and borderline cases, is characterized by the expression of CD30 antigen by neoplastic large cells at presentation, possible spontaneous regression of the skin lesions, and generally favorable clinical course. Although the functional relevance of CD30 and its natural ligand (CD30L) expression in most cases of NHL is presently undefined, previous studies indicate that CD30L is likely to mediate reduction of proliferation in CD30(+) anaplastic large-cell NHL. No information is currently available concerning the expression of CD30L in primary CD30(+) CTCLs. In this study, we investigated the immunophenotypic and genotypic expression of CD30 and CD30L in different developmental phases of skin lesions (growing v spontaneously regressing). By immunohistochemistry, CD30L expression was detected in regressing lesions only; by molecular analysis, the expression of CD30L was clearly higher in regressing lesions than in growing ones. CD30L, while expressed by some small lymphocytes, was most often coexpressed by CD30(+) neoplastic large cells, as demonstrated by 2-color immunofluorescence and by immunohistochemistry on paraffin sections. Taken together, these data suggest that CD30-CD30L interaction may play a role in the pathobiology of primary cutaneous CD30(+) lymphoproliferative disorders. In particular, CD30L (over)expression might have a major role in the mechanism of self-regression of skin lesions, the most distinctive clinical feature of this cutaneous lymphoma subtype.     

Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma

Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4(+)CD25(+) T cells with high levels of CTLA-4 and Foxp3 (intratumoral T(reg) cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4(+)CD25(+) T cells suppressed the proliferation and cytokine (IFN-gamma and IL-4) production of infiltrating CD4(+)CD25(-) T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4(+)CD25(-) T cells, and B7-H1 could be induced on intratumoral CD4(+)CD25(+) T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4(+)CD25(-) T cells when cocultured with intratumoral T(reg) cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral T(reg) cells that express CCR4, but not CCR8. Taken together, our results suggest that T(reg) cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.     

Loss of receptors for transforming growth factor beta in human T-cell malignancies.

Ki-1 (CD30)+ cutaneous T-cell lymphomas CTCLs) are slowly progressive lymphomas in which initial spontaneous regression is often observed. To better understand the mechanisms of spontaneous regression and eventual tumor progression in Ki-1+ CTCLs, type beta transforming growth factor (TGF-beta)-mediated growth inhibition of clonally related cell lines derived from two time points, before and after tumor progression, was studied. TGF-beta 1 inhibited colony-forming efficiency (CFE) of a cell line (Mac-1) derived from clinically indolent Ki-1+ CTCLs but failed to inhibit CFE of Mac-2A and -2B cell lines from advanced CTCLs. To determine the basis for TGF-beta 1 resistance in advanced CTCL cells, we looked for possible defects in the expression of cell surface TGF-beta receptors. Mac-1 cells were found to express TGF-beta receptors I and II, which mediate growth inhibition, and the TGF-beta-binding proteoglycan betaglycan. In contrast, receptors I and II were not detected in CTCL lines Mac-2A and -2B even though these cell lines did express betaglycan. Various treatments that unmask or induce TGF-beta receptors in other cells failed to show evidence for these receptors in advanced CTCL cells. Loss of TGF-beta receptor expression in these cells correlated with a marked decrease in TGF-beta receptor II mRNA levels. Loss of cell surface TGF-beta receptors was also found in two of five other patients with T-cell lymphomas including the Sezary syndrome and a noncutaneous T-cell lymphoma, suggesting that loss of TGF-beta receptor expression may be a recurrent feature of human T-cell malignancies.     

Peripheral T-cell lymphomas unspecified presenting in the skin: analysis of prognostic factors in a group of 82 patients

In the present study the clinicopathologic and immunophenotypic features of 82 patients with a CD30- peripheral T-cell lymphoma, unspecified, presenting in the skin were evaluated. The purpose of this study was to find out whether subdivision of these lymphomas on the basis of cell size, phenotype, or presentation with only skin lesions is clinically relevant. The study group included 46 primary cutaneous CD30- large cell lymphomas and 17 small/medium-sized T-cell lymphomas as well as 17 peripheral T-cell lymphomas with both skin and extracutaneous disease at the time of diagnosis. Patients with primary cutaneous small- or medium-sized T-cell lymphomas had a significantly better prognosis (5-year-overall survival, 45%) than patients with primary cutaneous CD30- large T-cell lymphomas (12%) and patients presenting with concurrent extracutaneous disease (12%). The favorable prognosis in this group with primary cutaneous small- or medium-sized T-cell lymphomas was particularly found in patients presenting with localized skin lesions expressing a CD3+CD4+CD8- phenotype. In the primary cutaneous T-cell lymphoma (CTCL) group and in the concurrent group, neither extent of skin lesions nor phenotype had any effect on survival. Our results indicate that peripheral T-cell lymphomas, unspecified, presenting in the skin have an unfavorable prognosis, irrespective of the presence or absence of extracutaneous disease at the time of diagnosis, cell size, and expression of a CD4+ or CD8+ phenotype. The only exception was a group of primary cutaneous small- or medium-sized pleomorphic CTCLs with a CD3+CD4+CD8- phenotype and presenting with localized skin lesions.     

A possible role for CCL27/CTACK-CCR10 interaction in recruiting CD4 T cells to skin in human graft-versus-host disease

Graft-versus-host disease (GvHD) is a serious complication of allogeneic stem cell transplantation (SCT) affecting the skin, gut and liver. The involvement of distinct organs suggests a role for tissue-specific chemokines and their receptors in directing activated donor T cells to these sites. In this study the potential involvement of the skin-specific CCL27/CTACK-CCR10 interaction was investigated in 15 paediatric SCT patients with skin GvHD. During the course of skin GvHD, peripheral blood T cells from these patients contained a high proportion of CD4+ CCR10+ T cells that disappeared after the GvHD was resolved. These cells were CD45RO+, expressed additional skin homing markers (cutaneous lymphocyte-associated antigen and CCR4), and produced the T-cell helper type 1-cytokines tumour necrosis factor-alpha and interleukin-2. The increase in CD4+ CCR10+ T cells was absent in SCT patients without GvHD. Immunohistochemical investigations showed CD4+ CCR10+ T cells in the GvHD skin biopsies of the same patients, but not in the gut biopsies of patients also suffering from gut GvHD. The infiltration of CD4+ CCR10+ T cells in the GvHD-affected skin correlated with an enhanced epidermal expression of CCL27/CTACK, the ligand for CCR10. These findings support the involvement of CCL27/CTACK-CCR10 interaction in recruiting CD4+ T cells to the skin, thus contributing to the pathogenesis of acute GvHD.     

CCR4 versus CCR10 in human cutaneous TH lymphocyte trafficking

The chemokine receptors (CCRs) CCR4 and CCR10, and the cutaneous lymphocyte antigen (CLA), have each been proposed as critical mediators of skin-specific TH lymphocyte homing in mice and humans. CLA initiates skin homing by mediating E-selectin-dependent tethering and rolling within cutaneous venules, but the specific roles of CCR4 and CCR10 are unclear. We have generated an antihuman CCR10 monoclonal antibody (mAb; 1B5) to illuminate the individual contributions of these molecules. This mAb allows us to compare CCR10, CCR4, and CLA expression within human TH populations. The mAb 1B5 recognizes functional CCR10 expression, as chemotactic responsiveness to cutaneous T-cell-attracting chemokine (CTACK)/CCL27 (a CCR10 ligand) parallels the staining of TH subsets. We find CCR10 expressed by only a minority (approximately 30%) of blood-borne, skin-homing (CLA+/CCR4+) TH cells. However, essentially all members of the relatively small "effector" (CLA+/CCR4+/CD27-/CCR7-) skin-homing TH population express CCR10. Most skin-infiltrating lymphocytes in allergic delayed-type hypersensitivity (DTH) and bacterial chancroid skin lesions express both CCR4 and CLA, but only about 10% express CCR10. This suggests for the 2 models of TH skin homing studied here that CCR10+ TH cells have no advantage over other CLA+/CCR4+ TH cells in homing to cutaneous sites. We conclude that the skin-homing TH compartment is itself divided into distinct subpopulations, the smaller of which expresses both CCR4 and CCR10, and the larger of which expresses only CCR4. Thus, CCR10 is unlikely to be necessary for cutaneous homing of TH cells in the models studied here. CCR10 may instead play a role in the movement of specialized "effector" cutaneous TH cells to and/or within epidermal microenvironments.     

Pancreatic islet expression of chemokine CCL2 suppresses autoimmune diabetes via tolerogenic CD11c+ CD11b+ dendritic cells

Development of type 1 diabetes in the nonobese diabetic (NOD) mouse is preceded by an immune cell infiltrate in the pancreatic islets. The exact role of the attracted cells is still poorly understood. Chemokine CCL2/MCP-1 is known to attract CCR2(+) monocytes and dendritic cells (DCs). We have previously shown that transgenic expression of CCL2 in pancreatic islets via the rat insulin promoter induces nondestructive insulitis on a nonautoimmune background. We report here an unexpected reduction of diabetes development on the NOD background despite an increased islet cell infiltrate with markedly increased numbers of CD11c(+) CD11b(+) DCs. These DCs exhibited a hypoactive phenotype with low CD40, MHC II, CD80/CD86 expression, and reduced TNF-α but elevated IL-10 secretions. They failed to induce proliferation of diabetogenic CD4(+) T cells in vitro. Pancreatic lymph node CD4(+) T cells were down-regulated ex vivo and expressed the anergy marker Grail. By using an in vivo transfer system, we show that CD11c(+) CD11b(+) DCs from rat insulin promoter-CCL2 transgenic NOD mice were the most potent cells suppressing diabetes development. These findings support an unexpected beneficial role for CCL2 in type 1 diabetes with implications for current strategies interfering with the CCL2/CCR2 axis in humans, and for dendritic cell biology in autoimmunity.     

Skin homing of Sézary cells involves SDF-1-CXCR4 signaling and down-regulation of CD26/dipeptidylpeptidase IV

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma (CTCL) characterized by a distinct metastatic pattern mainly involving blood and skin. Chemokines and their receptors play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors including non-Hodgkin T-cell lymphomas (NHLs). Here we report that SS cells express a functionally active CXCR4 and that its ligand SDF-1 is abundantly produced in the skin, which represents the main destination of SS cell spreading. SDF-1 is normally inactivated by proteolytic cleavage by the CD26/dipeptidylpeptidase IV (DPPIV). The lack of CD26 from the cell surface is a hallmark of circulating SS cells. We also show that the CD26(-) phenotype is maintained also in skin-infiltrating neoplastic T lymphocytes and that SS-affected individuals exhibit a reduced activity of plasma soluble CD26. Finally, we observe that the addition of soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26(+) CTCL cell line, enhances the SDF-1-induced migration of these cells. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.     

Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis

BACKGROUND: Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue. OBJECTIVE: To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention. METHODS: Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non-parametric tests were used for statistical analysis. RESULTS: Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen. DISCUSSION: A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1(+) and CCR5(+) cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.     

Cutaneous T-cell lymphoma: malignant proliferation of T-regulatory cells

Studies in an in vitro model of cutaneous T-cell lymphoma (CTCL) demonstrated that CTCL cell proliferation is stimulated by direct contact with autologous, immature dendritic cells (DCs), suggesting that CD4(+) CTCL cell division is driven by antigens presented by DC major histocompatibility complex (MHC) class 2. We now report that the T-cell receptor (TCR) of the CD4(+) CTCL cells is triggered after interaction with DCs loaded with apoptotic CTCL cells, as shown by reduced membrane expression of CD3 and the TCR, up-regulation of cytotoxic T lymphocyte antigen-4 (CTLA-4), and calcium mobilization. CTCL cells adopt a T-regulatory (Treg) phenotype expressing CD25/CTLA-4 and FoxP3 and secreting interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Treg CTCL cells suppress normal T-cell antigen-driven secretion of IL-2 and interferon-gamma (IFN-gamma). Blocking DC MHC class 2 expression or transport inhibited CTCL cell adoption of a Treg phenotype. Allogeneic CTCL cells or normal CD4 T cells served as sources of apoptotic material for CTCL cell conversion to a Treg phenotype. Conversion of CTCL cells to Treg cells may explain the anergic, immunosuppressive nature of the malignancy.     

Comparative analysis of naïve and memory CD4+ and CD8+ T-cell subsets in bone marrow and G-CSF-mobilized peripheral blood stem cell allografts: impact of donor characteristics

OBJECTIVE: Donor T cells expressing lymph node homing receptors are the foremost initiators of acute graft-vs-host disease (aGVHD), and a high proportion of CD4(+)CCR7(+) T cells in human leukocyte antigen-matched allografts has been shown to confer a high risk of aGVHD without interfering in other outcomes. METHODS: Na?ve, central memory (T(CM)), effector memory (T(EM)), and terminally differentiated effector memory (T(TD)) subsets, further subdivided by CD28 expression, were compared in 52 bone marrow and 37 granulocyte colony-stimulating factor-mobilized peripheral blood harvests. RESULTS: CCR7(+) cells (na?ve and T(CM)) predominated in the CD4(+) population, whereas CD8(+) memory cells were chiefly CCR7(neg) in the grafts. Donor age, antecedent of chronic infections, and graft type were independent factors influencing graft composition. CD8(+) na?ve cells negatively correlated and CD8(+) T(EM) positively correlated with age. Cytomegalovirus seropositivity was associated with more CD8(+) T(TD) and diminished CD28 expression. Toxoplasmosis seropositivity was associated with more CD4(+) T(CM) (p = 0.021). Marrow grafts comprised more CD28(+) cells within CD8(+) T(TD), but the percentage of CD4(+)CCR7(+) cells did not differ significantly between the two graft sources. Each of the four CD4(+) subsets and the percentage of CD4(+)CCR7(+) cells (p < 0.001) were correlated between graft and venous blood analyzed in 42 donors before harvest procedures. CONCLUSION: This study provides reference values for CD4(+) and CD8(+) na?ve and memory subsets within allografts applicable to the healthy donor population and indicates that beforehand analysis of a whole-blood sample can help evaluating the risk of aGVHD conferred by each donor and, when possible, choosing the one conferring the lowest risk.