Inhibition of accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonged allograft survival by arsenic trioxide

The aim of this study was to evaluate and determine the potential mechanisms of As₂O₃ in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As₂O₃ was administered to recipient mice from Day 0 to 10. As a result, As₂O₃ could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As₂O₃ group was prolonged from 5.8?±?0.7 to 14.2?±?2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As₂O₃ treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As₂O₃ as a novel immunosuppressant targeting CD4⁺ memory T cells.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8⁺ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8⁺ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8⁺ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8⁺T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8⁺ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8⁺ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8⁺ T-cell survival. mTOR may be a possible target to reverse CD8⁺ T-cell immune dysfunction in lethal fungal sepsis.     

In vitro selective depletion of CD4+CD25+ regulatory T-cells from PBMC using anti-tac-SAP

It has been shown that naturally occurring regulatory T-cells (CD4⁺CD25⁺ Foxp3⁺ T-cells) have critical roles in tumor invasion and down-regulation of immune response against established tumors. High expression of CD25 (IL-2Rα) by regulatory T (T_reg) cells may cause an inefficient response when using IL-2-based cancer vaccines. It seems that selective elimination of T_reg cells before treatment of tumor-bearing T-cells can strongly increase the efficacy of a vaccine. The aim of this study was to set up an efficient cost-effective protocol to eliminate CD4⁺CD25⁺ T-cells—using the immunotoxin anti-tac-SAP. Peripheral blood mononuclear cells (PBMC) taken from colon cancer patients were treated with different concentrations (i.e., 0–100 µg/dl) of the immunotoxin. Flow cytometric analyses were then preformed to analyze expression of CD4, CD25, CD3, CD8, and CD45 surface markers; semi-quantitative fluorescent-PCR was used for the detection of Foxp3 expression before and after anti-tac-SAP treatment. The results indicated that anti-tac-SAP effectively eliminated CD4⁺CD25⁺ T_reg cells and that 25 µg/dl was the optimal concentration of anti-tac-SAP for selective depletion of these cells. These outcomes were verified by analyses of Foxp3 expression. The results also indicated that this immunotoxin had no non-specific effects on other T-cells, including CD4⁺CD25^? and CD8⁺CD45⁺ T-cells. Building on the work here, ongoing/future studies with the anti-tac-SAP will focus on functional assessments of the remaining (i.e., non-eliminated) T-cells (i.e., CD8, CD4; using proliferation and peptide sensitization assays) to ascertain if the immunotoxin inadvertently alters the functions of these cells—an untoward outcome.     

IL-4 synthesis byin vivo-primed memory CD4+ T Cells: II. Presence of IL-4 is not required for IL-4 synthesis in primed CD4+ T Cells

Previous studies have shown that the presence of IL-4 is required for the development of IL-4 synthesis in naive CD4⁺ T cells. The purpose of our current studies was to investigate the role of IL-4 in the development of IL-4 synthesis in primed memory T cells. We therefore examined CD4⁺ T cells taken from lymph nodes of BALB/c mice immunized with keyhole limpet hemocyanin (KLH) and restimulatedin vitro with KLH. Our results with such primed resting CD4⁺ T cells programmed to produce IL-4 indicated that the production of IL-4 did not require the presence of IL-4 (although the presence of IL-2 was absolutely necessary), and was only slightly limited by the presence of anti-IL-4 MAb. These results with resting memory T cells were not biased by the presence of activated T cells already producing substantial quantities of IL-4, since we demonstrated that high-density memory T cells could produce IL-4 in the absence of IL-4, and because T cells that actively produce IL-4 do not persistin vivo very long after antigen exposure. These results indicate that IL-4 synthesis in T cells committed to IL-4 production can indeed occur in the absence of IL-4 when culture conditions have been optimized and suggest that therapies with anti-IL-4 MAb or with soluble IL-4 receptors designed to control the development of IL-4 synthesis in memory T cells from individuals exhibiting excessive IL-4 synthesis will be unsuccessful. Therefore, other therapies, for example, utilizing IL-12, will be required to modulate the relatively fixed programs in memory T cells that direct the development of cytokine synthesis.     

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice

CD40‐CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T‐cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8⁺ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus‐specific CD8⁺ T‐cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L^?/? mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV‐specific effector and memory T cells consists of CD40L⁺ CD8⁺ T cells. However, deficiency of CD40L in CD8⁺ T cells did influence neither the quantity nor the quality of primary T‐cell responses in LCMV infection. Virus‐specific CD8⁺ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8⁺ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8⁺ T‐cell responses also under inflammatory conditions, CD40L expression by CD8⁺ T cells themselves is dispensable in acute LCMV infection.     

Characteristics of Expanded CD4+CD28null T Cells in Patients with Chronic Hepatitis B

Expanded CD4⁺CD28^null T cells have not been described in the circulation of patients with chronic hepatitis B (CHB). The aim of the present was to detect and characterize the surface phenotype and functional capacity of these cells in CHB patients. Expanded CD4⁺CD28^null T cells were detected in the circulation of CHB patients with high viral load and elevated aminotransferase levels. Most CD4⁺CD28^null T cells showed a CD27?CD45RA^?CD45RO⁺ surface phenotype. The markers CD56, CD57 and killer immunoglobulin-like receptor (KIR) were detected on CD4⁺CD28^null T cells, but the majority were positive for CD57. Functionally, CD4⁺CD28^null T cells were found to be potent cytotoxic T lymphocytes with perforin and granzyme B secretion profiles. These findings indicate that the expanded CD4⁺CD28^null T cells are cytotoxic memory T cells and display a distinct functional phenotype in comparison with CD4⁺CD28⁺ T cells. The presence of these cells appears to be associated with inflammatory conditions, suggesting that these elevated CD4⁺CD28^null T cells might be involved in the pathogenesis of CHB.     

Neonatal Autoimmune Disease: Influence of CD4+ CD25+ Regulatory T Cells

Although previous studies have emphasized the tolerogenic property of murine neonatal immune system, recent studies indicate that neonatal mice are prone to autoimmune disease. This chapter will summarize the evidence for neonatal propensity to autoimmune ovarian disease (AOD) and describe the new finding that autoantibody can trigger a T cell–dependent autoimmune disease in neonatal but not adult mice. Based on depletion or addition of the CD4⁺CD25⁺ T cells, disease resistance of older mice is explicable by the emergence of CD4⁺CD25⁺ regulatory T-cell function after day 5, whereas disease susceptibility is associated with resistance to regulation by CD4⁺CD25⁺ T cells.     

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet β-cell ovalbumin antigen leading to diabetes

CD4⁺ helper T (Th) cells play crucial role in priming, expansion and survival of CD8⁺ cytotoxic T lymphocytes (CTLs). However, how CD4⁺ Th cell's help is delivered to CD8⁺ T cells in vivo is still unclear. We previously demonstrated that CD4⁺ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC_OVA) stimulation, and then stimulate OVA-specific CD8⁺ CTL responses in C57BL/6 mice. In this study, we further investigated CD4⁺ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4⁺ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC_OVA-activated CD4⁺ Th cells (3 × 10⁶ cells/mouse) can directly stimulate OVA-specific CD8⁺ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4⁺ T cells. A large amount of CD4⁺ Th cells (12 × 10⁶ cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8⁺ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4⁺ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8⁺ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4⁺ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.     

Expression of CD62L on Donor CD4<Superscript>+</Superscript> T Cells in Allografts: Correlation with Graft-Versus-Host Disease after Unmanipulated Allogeneic Blood and Marrow Transplantation

Introduction  The aim of this study was to investigate the association of donor CD4⁺ T cells expressing CD62L with transplant outcomes. Materials and Methods  We report a prospective analysis of 31 patients who were treated with a Bu/Cy regimen, followed by unmanipulated blood and marrow transplantation. Results  Median number (range) of CD4⁺CD62L⁺, CD4⁺CD45RA⁺CD62L⁺, and CD4⁺CD45RO⁺CD62L⁺ cells infused were 0.31(0.05–1.10)×10⁸/kg, 0.22(0.03–0.95)× 10⁸/kg, and 0.17(0.01–0.81)×10⁸/kg, respectively. The incidence of grades II to IV aGVHD was 36%. In a multivariate analysis, infusion of >0.22 × 10⁸ CD4⁺CD45RA⁺CD62L⁺ cells infused/kg increased the risk of grades II to IV aGVHD (HR = 4.741, 95% CI = 1.037–21.662, P = 0.045). Thirteen of 31 patients experienced cGVHD, the risk of cGVHD was increased in patients receiving >0.45 × 10⁸ CD4⁺CD45RA⁺ cells infused/kg (HR = 4.614, 95% CI = 1.265–16.829, P = 0.021). Conclusion  Our results suggest that a high cell dose of CD4⁺CD45RA⁺CD62L⁺ cells increase the incidence of grades II–IV aGVHD. A high number of CD4⁺CD45RA⁺ cells infused were associated with increased risk of cGVHD in our transplant settings. Ying-Jun Chang: performed research, analysis and interpretation of data, and drafting of the article, and gave final approval of the version to be published; Xiang-Yu Zhao: performed research, analysis and interpretation of data, and drafting of the article and gave final approval of the version to be published; Ming-Rui Huo: performed research, analysis and interpretation of data, and drafting of the article and gave final approval of the version to be published; Xiao-Jun Huang: involved in conception and design, revising the article critically, and final approval of the version to be published.     

Memory CD8+ T cells exhibit tissue imprinting and non‐stable exposure‐dependent reactivation characteristics following blood‐stage Plasmodium berghei ANKA infections

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8⁺ T‐cell accumulation within the brain. Whilst the dynamics of CD8⁺ T‐cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite‐specific CD8⁺ T cells upon resolution of ECM is not understood. In this study, we show that memory OT‐I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA‐OVA infection. Whereas memory OT‐I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT‐I cells within the brain post‐PbA‐OVA infection displayed an enriched CD69⁺CD103⁻ profile and expressed low levels of T‐bet. OT‐I cells within the brain were excluded from short‐term intravascular antibody labelling but were targeted effectively by longer‐term systemically administered antibodies. Thus, the memory OT‐I cells were extravascular within the brain post‐ECM but were potentially not resident memory cells. Importantly, whilst memory OT‐I cells exhibited strong reactivation during secondary PbA‐OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA‐OVA infection. Overall, our results demonstrate that memory CD8⁺ T cells are systemically distributed but exhibit a unique phenotype within the brain post‐ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8⁺ T‐cell populations within the brain and other tissues during repeat Plasmodium infections.     

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8⁺ CD28⁻ T cells with a corresponding reduction in the percentage of CD8⁺ CD28⁺ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4⁺ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8⁺ CD28⁺ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8⁺ CD28⁻ T cells in peripheral blood of HLA-A3⁺ but not HLA-A3⁻ HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR⁺ but not CD45RO⁺ cells was also found within the peripheral CD8⁺ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8⁺ CD28⁺ T cells both in controls and HH were able to expand in vitro; (ii) that CD8⁺ CD28⁻ T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8⁺ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard ⁵¹Cr-release assays when compared with CD8⁺ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8⁺ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.     

Human Purified CD8+ T Cells: Ex vivo Expansion Model to Generate a Maximum Yield of Functional Cytotoxic Cells

CD8⁺ T cells are a critical component of the cellular immune response. They play an important role in the control of viral infection and eliminating cells with malignant potential. However, attempts to generate and expand human CD8⁺ T cells in vitro for an adoptive immunotherapy have been conducted with limitation of the very low frequency of CD8⁺ T cells in blood. Therefore, several expansion protocols have been developed to obtain large and efficient numbers of human CD8⁺ T cells for use in adoptive immunotherapies. In this study various common culture conditions using different cytokines IL-2, IL-4, IL-7, IL-10, IL-12 and IL-15 and autologous feeders and sera were investigated to expand human purified CD8⁺ T cells. The importance and the influence of these factors on the growth and phenotype of CD8⁺ T cell were assessed by serially sampling cultures using flow cytometry. We demonstrated that combination of IL-2 (50U/ml) and autologous feeders induced maximal CD8⁺ T cell proliferation (40–50 folds) compared to other cytokines. Immunophenotypic analysis of cultured cells showed that expanded CD8⁺ T cells were activated and differentiated. Furthermore our expansion model also demonstrated that expanded CD8⁺ T cells are functionally cytotoxic active by killing Allogeneic LCLs cells. In conclusion, we have developed a reliable, simple method that uses minimal cell numbers to generate a high yield of functional cytotoxic CD8⁺ T cells, which can be used for the development of cellular immunotherapies.     

Features of peripheral CD8+CD57+ lymphocytes in patients with autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is an acquired condition characterized by the presence of autoantibodies recognizing erythrocyte-related antigens. Several components of the immune system are involved in disease pathogenesis. Among them, as for other autoimmune disorders, a role for specific CD8⁺CD57⁺ regulatory cells subset could be hypothesized. We evaluated this lymphocyte subset by flow cytometry in 18 AIHA patients randomly selected in a retrospective population of 29 cases. Secondary forms were observed in 65.5% of cases, whereas frequencies of warm, cold, mixed, and atypical forms were similar. Cold agglutinins and cryoglobulins tested positive in 44.8% and 10.3% of cases, respectively. These patients exhibited a higher frequency of peripheral vascular symptoms (odds ratio?=?8.2, p?=?.04) and complement consumption (odds ratio?=?7.2, p?=?.02). Frequency of CD8⁺CD57⁺ cells resulted significantly higher in AIHA patients than in control group (17.0?±?15.8% vs 8.2?±?5.0%, p?=?.04). Regardless of therapeutic schedule, patients with partial or no response to therapy (8/18) showed higher frequencies of CD8⁺CD57⁺ cells as compared with controls (23.6?±?21.3% vs 8.9?±?4.9%, p?=?.01), whereas 10/18 complete responders (CR) showed lower levels of CD8⁺CD57⁺ cells (11.7?±?6.9%, p?=?.11). CR and controls showed similar values (p?=?.24). This study suggests that monitoring this lymphocyte subset before and after treatment administration might have a prognostic value. Moreover, CD8⁺CD57⁺ cells may represent a possible therapeutic target to restore the normal balance between lymphocyte populations.     

Involvement of Foxp3-expressing CD4+ CD25+ regulatory T cells in the development of tolerance induced by transforming growth factor-beta2-treated antigen-presenting cells

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-β (TGF-β) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-β-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-β₂ induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4⁺ T cells from mice receiving an intravenous injection of TGF-β₂-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4⁺ CD25⁺ T cells was observed in mice with tolerance. CD4⁺CD25⁺ T cells from TGF-β₂-treated APC-injected mice produced a large amount of TGF-β₁ and exhibited an in vitro antigen-specific suppressive activity. CD4⁺ CD25⁺ T cells from TGF-β₂-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4⁺ CD25⁺ T cells might be actively involved in the development of tolerance induced by TGF-β₂-treated APC.     

Chemokines,chemokine receptors and CD4+CD25+ regulatory T cells

The fields of regulatory T (Treg) cells and chemokines/chemokine receptors have progressed rapidly in the last few years. Treg cells, especially CD4⁺CD25⁺ Treg cells, play a critical role in maintaining self-tolerance and immune homeostasis. Chemokines and chemokine receptors are crucial for lymphoid development, homing and immunological regulation. This review will discuss the biological effects of chemokines and chemokine receptors on regulating the migration and development of CD4⁺CD25⁺ Treg cells, and the potential clinical implications of these findings when considering chemokine receptors as therapeutic targets.     

Failure of tumor-reactive lymph node cells to kill tumor in the presence of immune-suppressive CD34+ cells can be overcome with vitamin D3 treatment to diminish CD34+ cell levels

Growth of Lewis lung carcinoma (LLC-LN7) tumors results in an increase in CD34⁺ granulocyte-macrophage progenitor cells having natural suppressor (NS) activity. These CD34⁺ NS cells were capable of inhibiting the cytotoxic activity of tumor-reactive lymph node cells. In vivo studies showed that adoptive treatment of LLC-LN7 tumor-bearing mice with tumor-reactive lymph node cells plus IL-2 failed to reduce the development of metastases. Studies were conducted to determine if diminishing the levels of CD34⁺ NS cells would allow for improved anti-tumor effectiveness of the adoptively transferred cells. The suppressive activity of CD34⁺ cells toward the cytolytic activity of tumor-reactive lymph node cells could be blocked by in vitro culture of CD34⁺ cells with the differentiation-inducing hormone 1a,25-dihydroxyvitamin D₃. Similarly, treatment of LLC-LN7-bearing mice with vitamin D₃ alone diminished the levels of CD34⁺ NS cells within regional lymph nodes, spleens and tu mors. This treatment resulted in an increased immune reactivity to autologous tumor, as shown by the production of IFN-g by lymph node cells in response to the presence of LLC-LN7 cells. The extent of tumor metastasis in mice receiving vitamin D₃ treatment was also reduced. When tumor-reactive lymph node cells were adoptively transferred into these LLC-LN7-bearing mice that were receiving vitamin D₃ treatment, there resulted a pronounced synergistic reduction in tumor metastasis. The results of this study show that treatment of tumor bearers with vitamin D₃ to eliminate CD34⁺ NS cells improves the anti-tumor effectiveness of adoptively transferred tumor-reactive lymph node cells. © Rapid Science 1998