Cytokines, particularly those of the common gamma chain receptor family, provide extrinsic signals that regulate naive CD4 cell survival. Whether these cytokines are required for the maintenance of memory CD4 cells has not been rigorously assessed. In this paper, we examined the contribution of interleukin (IL) 7, a constitutively produced common gamma chain receptor cytokine, to the survival of resting T cell receptor transgenic memory CD4 cells that were generated in vivo. IL-7 mediated the survival and up-regulation of Bcl-2 by resting memory CD4 cells in vitro in the absence of proliferation. Memory CD4 cells persisted for extended periods upon adoptive transfer into intact or lymphopenic recipients, but not in IL-7- mice or in recipients that were rendered deficient in IL-7 by antibody blocking. Both central (CD62L+) and effector (CD62L-) memory phenotype CD4 cells required IL-7 for survival and, in vivo, memory cells were comparable to naive CD4 cells in this regard. Although the generation of primary effector cells from naive CD4 cells and their dissemination to nonlymphoid tissues were not affected by IL-7 deficiency, memory cells failed to subsequently develop in either the lymphoid or nonlymphoid compartments. The results demonstrate that IL-7 can have previously unrecognized roles in the maintenance of memory in the CD4 cell population and in the survival of CD4 cells with a capacity to become memory cells.

Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin(+) cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin(+) DCs in the skin DLNs. Our results show that in contrast to the current view, langerin(+)CD8(-) DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin(+) DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin(+) DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin(+) DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.

Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8(+) T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127(high) and CD62L(high)) and peripheral effector memory T cells (CD127(high) and CD62L(low)). Using this new tool of memory T cell analysis, we demonstrate that CD8(+) T cell priming in the absence of T cell help or CD40L specifically alters the generation of the effector memory T cell subset, which appears to be crucial for immediate memory responses and long-term maintenance of effective protective immunity. Our data reveal a unique strategy to obtain information about the quality of long-term protective immunity early during an immune response, a finding that may be applied in a variety of clinical settings, including the rapid monitoring of vaccination success.

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However, it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells, designated tumor-evoked Bregs (tBreg), phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-β-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs, 4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications, as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis.

Immune activation during chronic HIV infection is a strong clinical predictor of death and may mediate CD4(+) T cell depletion. Regulatory T cells (Tregs) are CD4(+)CD25(bright)CD62L(high) cells that actively down-regulate immune responses. We asked whether loss of Tregs during HIV infection mediates immune activation in a cross-sectional study of 81 HIV-positive Ugandan volunteers. We found that Treg number is strongly correlated with both CD4(+) and CD8(+) T cell activation. In multivariate modeling, this relationship between Treg depletion and CD4(+) T cell activation was stronger than any other clinical factor examined, including viral load and absolute CD4 count. Tregs appear to decline at different rates compared with other CD4(+) T cells, resulting in an increased regulator to helper ratio in many patients with advanced disease. We hypothesize that this skewing may contribute to T cell effector dysfunction. Our findings suggest Tregs are a major contributor to the immune activation observed during chronic HIV infection.

In the current study, we address the underlying mechanism for the selective generation of gut-homing T cells in the gut-associated lymphoid tissues (GALT). We demonstrate that DCs in the GALT are unique in their capacity to establish T cell gut tropism but in vivo only confer this property to T cells in the presence of DC maturational stimuli, including toll-like receptor-dependent and -independent adjuvants. Thus, DCs from mesenteric LNs (MLNs), but not from spleen, supported expression of the chemokine receptor CCR9 and integrin alpha4beta7 by activated CD8+ T cells. While DCs were also required for an efficient down-regulation of CD62L, this function was not restricted to MLN DCs. In an adoptive CD8+ T cell transfer model, antigen-specific T cells entering the small intestinal epithelium were homogeneously CCR9+alpha4beta7+CD62Llow, and this phenotype was only generated in GALT and in the presence of adjuvant. Consistent with the CCR9+ phenotype of the gut-homing T cells, CCR9 was found to play a critical role in the localization of T cells to the small intestinal epithelium. Together, these results demonstrate that GALT DCs and T cell expression of CCR9 play critical and integrated roles during T cell homing to the gut.

The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector- and central-memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Effector cells derived from central memory CD8(+) T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1(-) phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8(+) T cells may allow superior efficacy upon adoptive transfer.

OBJECTIVE

Immunosuppression, including that mediated by CD4(+)CD25(high)Foxp3(+) regulatory T cells (Treg), is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Tregs with a distinct phenotype in tumor-infiltrating lymphocytes (TIL) contribute to local immune suppression.

METHODS

The frequency and phenotype of Treg in TIL and/or peripheral blood mononuclear cells (PBMC) in 15 HNSCC patients and PBMC in 15 normal controls were compared. Single-cell sorted CD4(+)CD25(high) T cells were tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidyl ester-labeled and activated autologous CD4(+)CD25(-) responder T cells. Transwell inserts separating Treg from responders and neutralizing interleukin-10 (IL-10) or transforming growth factor-beta1 (TGF-beta1) antibodies were used to evaluate the mechanisms used by Treg to suppress responder cell proliferation.

RESULTS

In TIL, CD25(+) cells were enriched in the CD3(+)CD4(+) subset (13 +/- 3%) relative to circulating CD3(+)CD4(+) T cells (3 +/- 0.7%) in HNSCC patients (P < or = 0.01) or normal controls (2 +/- 1.5%; P < or = 0.001). Among the CD3(+)CD4(+) subset, CD25(high) Treg represented 3 +/- 0.5% in TIL, 1 +/- 0.3% in PBMC, and 0.4 +/- 0.2% in normal controls. Tregs in TIL were GITR(+), IL-10(+), and TGF-beta1(+), although circulating Treg up-regulated CD62L and CCR7 but not GITR, IL-10, or TGF-beta1. Treg in TIL mediated stronger suppression (P < or = 0.001) than Treg in PBMC of HNSCC patients. The addition of neutralizing IL-10 and TGF-beta antibodies almost completely abrogated suppression (5 +/- 2.51%). Transwell inserts partly prevented suppression (60 +/- 5% versus 95 +/- 5%).

CONCLUSIONS

Suppression in the tumor microenvironment is mediated by a unique subset of Treg, which produce IL-10 and TGF-beta1 and do not require cell-to-cell contact between Treg and responder cells for inhibition.

Mature myeloid cells (macrophages and CD11b(+) dendritic cells) form a prominent component of neuroinflammatory infiltrates in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). The mechanism by which these cells are replenished during relapsing and chronic neuroinflammation is poorly understood. Here we demonstrate that CD11b(+)CD62L(+)Ly6C(hi) monocytes with colony-forming potential are mobilized into the bloodstream by a granulocyte-macrophage colony-stimulating factor-dependent pathway immediately before EAE relapses. Circulating Ly6C(hi) monocytes traffic across the blood-brain barrier, up-regulate proinflammatory molecules, and differentiate into central nervous system dendritic cells and macrophages. Enrichment of Ly6C(hi) monocytes in the circulating pool is associated with an earlier onset and increased severity of clinical EAE. Our studies indicate that granulocyte-macrophage colony-stimulating factor-driven release of Ly6C(hi) precursors from the bone marrow prevents exhaustion of central nervous system myeloid populations during relapsing or chronic autoimmune demyelination, suggesting a novel pathway for therapeutic targeting.

Globally suppressed T-cell function has been described in many patients with cancer to be a major hurdle for the development of clinically efficient cancer immunotherapy. Inhibition of antitumor immune responses has been mainly linked to inhibitory factors present in cancer patients. More recently, increased frequencies of CD4+CD25hi regulatory T cells (Treg cells) have been described as an additional mechanism reducing immunity. We assessed 73 patients with B-cell chronic lymphocytic leukemia (CLL) and 42 healthy controls and demonstrated significantly increased frequencies of cytotoxic T lymphocyte-associated protein 4 (CTLA4+)-, Forkhead box P3 (FOXP3+)-, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR+)-, CD62L+-, transforming growth factor beta1 (TGF-beta1+)-, interleukin 10 (IL-10+)-Treg cells in patients with CLL, with highest frequencies in untreated or progressing patients presenting with extended disease. Most surprisingly, in the majority of patients with CLL treated with fludarabine-containing therapy regimens the inhibitory function of Treg cells was decreased or even abrogated. In addition, frequencies of Treg cells were significantly decreased after therapy with fludarabine. In light of similar findings for cyclophosphamide the combination of fludarabine and cyclophosphamide might be further exploited in strategies reducing immunosuppression prior to cancer immunotherapy.

Most treatments that prevent autoimmune diabetes in nonobese diabetic (NOD) mice require intervention at early pathogenic stages, when insulitis is first developing. We tested whether dendritic cell (DC)-expanded, islet antigen-specific CD4+ CD25+ suppressor T cells could treat diabetes at later stages of disease, when most of the insulin-producing islet beta cells had been destroyed by infiltrating lymphocytes. CD4+ CD25+ CD62L+ regulatory T cells (T reg cells) from BDC2.5 T cell receptor transgenic mice were expanded with antigen-pulsed DCs and IL-2, and were then injected into NOD mice. A single dose of as few as 5x10(4) of these islet-specific T reg cells blocked diabetes development in prediabetic 13-wk-old NOD mice. The T reg cells also induced long-lasting reversal of hyperglycemia in 50% of mice in which overt diabetes had developed. Successfully treated diabetic mice had similar responses to glucose challenge compared with nondiabetic NOD mice. The successfully treated mice retained diabetogenic T cells, but also had substantially increased Foxp3+ cells in draining pancreatic lymph nodes. However, these Foxp3+ cells were derived from the recipient mice and not the injected T reg cells, suggesting a role for endogenous T reg cells in maintaining tolerance after treatment. Therefore, inoculation of DC-expanded, antigen-specific suppressor T cells has considerable efficacy in ameliorating ongoing diabetes in NOD mice.

CD4(+) T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4(+) T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation-a process involving integrin alpha2. Antigen-specific memory CD4(+) T lymphocytes highly expressed Ly-6C, unlike most splenic CD44(hi)CD62L(-) CD4(+) T lymphocytes. In adult mice, more than 80% of Ly-6C(hi)CD44(hi)CD62L(-) memory CD4(+) T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1(+) stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper T cells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.

CD4+CD25+ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4+CD25+ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4+CD25- T cells. Here we examined the differential effect of CD62L+ and CD62L- subsets of CD4+CD25+ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4+CD25+ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4+CD25- T cells, only the CD62L+ subset of CD4+CD25+ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4+CD25+CD62L+ Treg cells were transferred compared with the CD62L- subset. Subsequently, CD4+CD25+CD62L+ Treg cells showed a significantly higher capacity than their CD62L- counterpart to inhibit the expansion of donor CD4+CD25- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.

Graft-versus-host disease (GVHD) is caused by alloreactive donor T cells that trigger host tissue injury. GVHD develops over weeks or months, but how this immune response is maintained over time is unknown. In mouse models of human GVHD, we identify a new subset of postmitotic CD44(lo)CD62L(hi)CD8(+) T cells that generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8(+) T cells, while self-renewing. These cells express Sca-1, CD122 and Bcl-2, and induce GVHD upon transfer into secondary recipients. The postmitotic CD44(lo)CD62L(hi)CD8(+) T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigens and dendritic cells and require interleukin-15. Thus, their long life, ability to self-renew and multipotentiality define these cells as candidate memory stem cells. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including GVHD.

The induction of regulatory T (T reg) cells holds considerable potential as a treatment for autoimmune diseases. We have previously shown that CD4+CD25hi T reg cells isolated from patients with active rheumatoid arthritis (RA) have a defect in their ability to suppress proinflammatory cytokine production by CD4+CD25- [corrected] T cells. This defect, however, was overcome after anti-tumor necrosis factor (TNF)-alpha antibody (infliximab) therapy. Here, we demonstrate that infliximab therapy gives rise to a CD4+CD25hiFoxP3+ T reg cell population, which mediates suppression via transforming growth factor (TGF)-beta and interleukin 10, and lacks CD62L expression, thereby distinguishing this T reg cell subset from natural T reg cells present in healthy individuals and patients with active RA. In vitro, infliximab induced the differentiation of CD62L- T reg cells from CD4+CD25- T cells isolated from active RA patients, a process dependent on TGF-beta. In spite of the potent suppressor capacity displayed by this CD62L- T reg cell population, the natural CD62L+ T reg cells remained defective in infliximab-treated patients. These results suggest that anti-TNF-alpha therapy in RA patients generates a newly differentiated population of T reg cells, which compensates for the defective natural T reg cells. Therefore, manipulation of a proinflammatory environment could represent a therapeutic strategy for the induction of T reg cells and the restoration of tolerance.

T-cell depletion facilitates reduced immunosuppression following organ transplantation and has been suggested to be pro-tolerant. However, the characteristics of post-depletional T cells have not been evaluated as they relate to tolerance induction. We therefore studied patients undergoing profound T-cell depletion with alemtuzumab or rabbit anti-thymocyte globulin following renal transplantation, evaluating the phenotype and functional characteristics of their residual cells. Naive T cells and T cells with potential regulatory function (CD4+CD25+) were not prevalent following aggressive depletion. Rather, post-depletion T cells were of a single phenotype (CD3+CD4+CD45RA-CD62L-CCR7-) consistent with depletion-resistant effector memory T cells that expanded in the first month and were uniquely prevalent at the time of rejection. These cells were resistant to steroids, deoxyspergualin or sirolimus in vitro, but were calcineurin-inhibitor sensitive. These data demonstrate that therapeutic depletion begets a limited population of functional memory-like T cells that are easily suppressed with certain immunosuppressants, but cannot be considered uniquely pro-tolerant.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). Donor T cells that accompany stem cell grafts cause GVHD by attacking recipient tissues; therefore, all patients receive GVHD prophylaxis by depletion of T cells from the allograft or through immunosuppressant drugs. In addition to providing a graft-versus-leukemia effect, donor T cells are critical for reconstituting T cell-mediated immunity. Ideally, immunity to infectious agents would be transferred from donor to host without GVHD. Most donors have been exposed to common pathogens and have an increased precursor frequency of memory T cells against pathogenic antigens. We therefore asked whether memory CD62L-CD44+ CD4+ T cells would induce less GVHD than unfractionated or naive CD4+ T cells. Strikingly, we found that memory CD4 cells induced neither clinical nor histologic GVHD. This effect was not due to the increased number of CD4+CD25+ regulatory T cells found in the CD62L-CD44+ fraction because memory T cells depletion of these cells did not cause GVHD. Memory CD4 cells engrafted and responded to antigen both in vivo and in vitro. If these murine results are applicable to human alloSCT, selective administration of memory T cells could greatly improve post-transplant immune reconstitution.

The immune response of naive CD4 T cells to influenza virus is initiated in the draining lymph nodes and spleen, and only after effectors are generated do antigen-specific cells migrate to the lung which is the site of infection. The effector cells generated in secondary organs appear as multiple subsets which are a heterogeneous continuum of cells in terms of number of cell divisions, phenotype and function. The effector cells that migrate to the lung constitute the more differentiated of the total responding population, characterized by many cell divisions, loss of CD62L, down-regulation of CCR7, stable expression of CD44 and CD49d, and transient expression of CCR5 and CD25. These cells also secrete high levels of interferon gamma and reduced levels of interleukin 2 relative to those in the secondary lymphoid organs. The response declines rapidly in parallel with viral clearance, but a spectrum of resting cell subsets reflecting the pattern at the peak of response is retained, suggesting that heterogeneous effector populations may give rise to corresponding memory populations. These results reveal a complex response, not an all-or-none one, which results in multiple effector phenotypes and implies that effector cells and the memory cells derived from them can display a broad spectrum of functional potentials.

Effective cell-mediated antitumor immunity requires the activation of tumor-reactive T cells and the trafficking of activated T cells to tumor sites. These processes involve the extravasation of lymphocytes from the blood and lymphatics, and their homing to lymph nodes and tumors. L-selectin (CD62L) is an important molecule in these processes. It directs naive lymphocytes to peripheral lymph nodes where they become activated and it traffics naive lymphocytes to inflammatory environments, such as tumors. Individuals with advanced cancer are immune suppressed due to myeloid-derived suppressor cells (MDSC), a population of immature myeloid cells that accumulate to high levels in response to tumor-secreted and proinflammatory factors. We now demonstrate that the reduction in T cell levels of L-selectin that is commonly seen in individuals with cancer inversely correlates with MDSC levels. Three lines of evidence demonstrate that MDSC directly down-regulate L-selectin on naive T cells: 1) naive T cells cocultured with tumor-induced MDSC have reduced L-selectin; 2) T cells in tumor-free aged mice with elevated levels of MDSC have reduced L-selectin, and 3) peritoneal exudate T cells of tumor-free mice treated with plasminogen activator urokinase to elevate MDSC have reduced levels of L-selectin. MDSC are likely to down-regulate L-selectin through their plasma membrane expression of ADAM17 (a disintegrin and metalloproteinase domain 17), an enzyme that cleaves the ectodomain of L-selectin. Therefore, MDSC down-regulate L-selectin levels on naive T cells, decreasing their ability to home to sites where they would be activated. This is another mechanism by which MDSC inhibit antitumor immunity.

Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4(+) T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA(+)CD62L(+) and signal-joint TCR rearrangement excision circle-bearing CD4(+) populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4(+) T cell populations. This recovery encompassed the recovery of normal CD4(+) T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vbeta diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.

Naturally occurring CD4(+)CD25(+) regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4(+)CD25(+) non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4(+)CD25(high) T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4(+)CD25(low) or CD4(+)CD25(-) T cells did not. The expanding pure populations of CD4(+)CD25(high) T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4(+)CD25(-) T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4(+)CD25(high) T cell populations retained a broad TCR repertoire and, like CD4(+) CD25(+) T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4(+) and CD8(+) T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.