Autologous designer antigen-presenting cells by gene modification of T lymphocyte blasts with IL-7 and IL-12

An effective immune response to antigen requires professional antigen-presenting cell (APC), which not only present antigen, but also provide costimulation and cytokines (eg, IL-12) that drive T cell differentiation down the appropriate effector pathway (Tc1/TH1). For T cell-based immunotherapy protocols, the availability of large numbers of autologous professional APC is a major limitation because professional APC do not proliferate in vitro. T cells themselves can proliferate exponentially in vitro and have the ability to present antigen. They can also express costimulatory molecules after activation. Therefore, we hypothesized that if activated T cells were genetically modified to express proinflammatory cytokines required to polarize T cells toward a Tc1 response, they could fulfill the requirements for an abundant, autologous APC. To test this potential, T cells were activated by CD3/CD28 antibodies and pulsed with model HLA-A2+ peptides derived from CMVpp65, MAGE-3, and MART-1. Activated T-APC readily reactivated CD8 pp65 memory T cells from healthy CMV seropositive donors; however, the activation of MAGE-3 and MART-1-specific CD8 T cells required both IL-7 and IL-12, which could be provided either exogenously or by genetic modification of the T-APC. Responder T cells could be expanded to large numbers with subsequent stimulations using activated, peptide-pulsed T-APC and IL-2. Tumor antigen-specific T cell lines killed both peptide-pulsed target cells and tumor cell lines. Thus, T cells provide a platform for the generation of autologous APC that can be customized to express both antigens and therapeutic molecules for the induction of antigen-specific T cell immunity.     

Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis.

The T cell proliferative response to autologous non-T cells is termed the autologous mixed lymphocyte reaction (AMLR). Recent studies have suggested that the AMLR represents an inducer circuit for the activation of T8+ suppressor/cytotoxic effector cells. Since atopic dermatitis (AD) patients are deficient in T8+ cytolytic T cell function, we investigated the AMLR in AD. When sheep erythrocytes were used to separate T cells from non-T cells, the AMLR was found to be significantly decreased (P less than 0.001) in AD patients (n = 11; delta cpm = 1,550 +/- 393) when compared with normal control subjects (n = 13; delta cpm = 25,819 +/- 4,609). To exclude the possibility that these results were an artifact of the sheep erythrocyte separation, T cells were also separated on a fluorescence-activated cell sorter after treatment of peripheral blood lymphocytes with the OKT3 monoclonal antibody. AD T cells separated by the latter method were also found to have a significantly reduced AMLR response when compared with similarly treated normal T cells. Co-culture studies using cells from AD patients and their HLA identical siblings indicated that the defect resided at the responder T cell level rather than at the stimulator non-T cell level. Co-culture studies revealed no evidence for excessive suppressor cell activity resulting in the decreased AMLR. However, enumeration of T cells reactive with the monoclonal antibody T29, which recognizes a subset of T cells proliferating in the AMLR, demonstrated that AD patients (n = 8; % T29 = 2.5 +/- 0.7) had a significantly decreased (P less than 0.001) number of circulating T29+ T cells when compared with normal controls (n = 8; % T29 = 10.4 +/- 0.8). These studies suggest that a deficiency of T4+ T29+ cells contributes to the deficient AMLR in AD and possibly underlies the abnormalities of T8+ effector cells present in this disease.     

IL-6-dependent spontaneous proliferation is required for the induction of colitogenic IL-17-producing CD8+ T cells

We propose a novel role for interleukin (IL) 6 in inducing rapid spontaneous proliferation (SP) of naive CD8(+) T cells, which is a crucial step in the differentiation of colitogenic CD8(+) T cells. Homeostasis of T cells is regulated by two distinct modes of cell proliferation: major histocompatibility complex/antigen-driven rapid SP and IL-7/IL-15-dependent slow homeostatic proliferation. Using our novel model of CD8(+) T cell-dependent colitis, we found that SP of naive CD8(+) T cells is essential for inducing pathogenic cytokine-producing effector T cells. The rapid SP was predominantly induced in mesenteric lymph nodes (LNs) but not in peripheral LNs under the influence of intestinal flora and IL-6. Indeed, this SP was markedly inhibited by treatment with anti-IL-6 receptor monoclonal antibody (IL-6R mAb) or antibiotic-induced flora depletion, but not by anti-IL-7R mAb and/or in IL-15-deficient conditions. Concomitantly with the inhibition of SP, anti-IL-6R mAb significantly inhibited the induction of CD8(+) T cell-dependent autoimmune colitis. Notably, the transfer of naive CD8(+) T cells derived from IL-17(-/-) mice did not induce autoimmune colitis. Thus, we conclude that IL-6 signaling is crucial for SP under lymphopenic conditions, which subsequently caused severe IL-17-producing CD8(+) T cell-mediated autoimmune colitis. We suggest that anti-IL-6R mAb may become a promising strategy for the therapy of colitis.     

Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent

Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide-specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti-IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene-deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell-driven response to allergen in vivo by an IL-10-dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.     

Responder cells in the human autologous mixed lymphocyte reaction.

Isolated human T4+ cells proliferate in the autologous mixed lymphocyte reaction (AMLR), whereas isolated T8+ cells do not. However, in the presence of Interleukin 2 or T4+ cells, the T8+ cells demonstrated substantial proliferation. These studies suggest that T8+ cells recognize signals from autologous non-T cells, but require an additional factor for the subsequent proliferative response. Since this stimulus can be provided by T4+ cells, the AMLR appears to constitute an inducer circuit. Different defects in this circuit may be responsible for the common abnormality of the AMLR in different diseases.     

Enhanced susceptibility to apoptosis in T cells recovering after autologous peripheral blood progenitor cell transplantation: reversal by interleukin-15

T-cell number and competence are profoundly impaired after transplantation of autologous cytokine-mobilized peripheral blood progenitor cells (PBPC). The objective of the present study was to evaluate the occurrence of T-cell spontaneous apoptosis (Aspont) and its modulation in vitro by the interleukin-2 receptor (IL-2R) gamma-chain (gammac)-signaling cytokine interleukin-15 (IL-15) in the peripheral blood of patients transplanted with autologous PBPC for hematological malignancies. An average 45%+/-6% of CD4+ and 55%+/-6% of CD8+ T cells cultured in the absence of exogenous cytokines underwent Aspont; of interest, IL-15 and, to a lesser extent, its structural cousin IL-2 counteracted T-cell Aspont and upregulated Bcl-2 levels. IL-15 did not rescue T cells from Aspont by promoting proliferation, but rather it acted as a genuine survival factor. Furthermore, T-cell preincubation with a gammac-blocking antibody was capable of abrogating both apoptosis inhibition and Bcl-2 induction by IL-15. These in vitro findings suggest that IL-15 might represent a promising immunomodulating agent to improve T-cell function after autologous PBPC transplantation.     

Generation of antigen receptor-specific suppressor T cell clones in man

We have shown previously that CD8+ T cells proliferate upon exposure to autologous, antigen primed CD4+ T cells, and suppress the response of fresh T cells to the priming antigen but not irrelevant antigens. The stimulus and target of suppression in this system appears to be the antigen receptor on the surface of CD4+ cells, rather than the nominal antigen. In the current study, alloantigen primed CD4+ inducer cells and IL-2-containing medium were used to generate clones of suppressor cells from several individuals. The clones inhibited the response of fresh autologous T cells only to the original allogeneic stimulator cell and to stimulator cells that shared HLA-DR antigens with the priming cell. The clones were also genetically restricted, since they inhibited the response of HLA-A,B-compatible but not HLA-A,B-incompatible individuals. The availability of a method for reproducibly generating antigen receptor-specific suppressor T cell clones in vitro should make it possible to clarify the mechanism, whereby such cells are activated and exert their suppressive effect.     

Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis

CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.     

Defective regulatory and effector T cell functions in patients with FOXP3 mutations

The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+ CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+ CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+ CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by "weak" TCR stimuli. In contrast, the suppressive function of CD4+ CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+ CD25high T cells from either FOXP3+ or FOXP3- IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-gamma production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+ CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.     

Increased prevalence of regulatory T cells (Treg) is induced by pancreas adenocarcinoma

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.     

Redirecting human CD4+ T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR alphabeta gene transfer requires CD8alpha

Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.     

Anti-TNF-alpha therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-beta

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.     

Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells

Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor alpha+ (CD11b+IL-4Ralpha+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b+IL-4Ralpha+ cells produced IL-13 and IFN-gamma and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.     

Idiotype-like determinants on human T lymphocytes alloactivated in mixed lymphocyte culture

T cells alloactivated in 5-d MLC with an HLA-DR-different stimulator acquire the capacity of stimulating the autologous mixed lymphocyte response (AMLR). We have demonstrated that activation of AMLR by allosensitized T cells is determined by the expression of the idiotype receptor for the stimulating HLA-DR alloantigen. This has been shown in experiments in which purified, OKT-3-positive T cell suspensions were first primed for 9 d with AMLR-activated T lymphoblasts, then tested in secondary AMLR with autologous lymphoblasts sensitized to various HLA- DR alloantigens. Accelerated memory responses were induced only by autologous lymphoblasts that had been sensitized against the same HLA- DR specificity as the primary AMLR stimulators. This response was not inhibited by a mouse monoclonal antibody recognizing Ia-like determinants, and was not triggered by human allogeneic resting peripheral blood lymphocytes. Thus, recognition of alloactivated T lymphoblasts in secondary AMLR seems to be specific for the idiotype- like determinants expressed by the autologous stimulators.     

CD8+ T cell subsets of cytotoxic T lymphocytes induced by Epstein-Barr virus infection in infectious mononucleosis

Mononuclear peripheral blood lymphocytes (PBL) from patient with infectious mononucleosis (IM) were tested in a 51Cr-release assay for cytotoxicity against autologous and allogeneic lymphoblastoid cell line (LCL), or Epstein-Barr virus (EBV)-genome positive and negative cell line. In acute phase, PBL lyse an autologous LCL as well as allogeneic LCL (Wa cells). High levels of cytotoxicity were observed in the combinations between effector and target cells sharing HLA-Class 1 product. EBV-genome positive Daudi and Raji cells which lack HLA-Class 1 antigen and have mismactched HLA-Class 1 antigen, respectively showed resistance to killing. EBV-genome negative tumor cells except NK sensitive K562 cells were not killed by IM lymphocytes. However, the IM lymphocytes without atypical form in convalescent phase failed to show killing activity against autologous and allogeneic LCL. These findings suggest that cell surface membrane antigen structure on EBV-infected LCL may be able to explain the recognition and triggering of lysis of target cells by HLA-Class 1 restricted cytotoxic T cells (CTL) from acute IM. Phenotypic analysis of PBL with atypical form from IM was made by two-color flow cytometry. The data demonstrate that CD8+ T cells quantitatively represent the major population of lymphocytes expanded during acute IM. Furthermore, approximately 70% of these CD8+ T cells express HLA-DR on these surface, suggesting that they have undergone activation. However, IL 2R (CD25 antigen) expression was not significantly elevated on activated T cells. The salient profile on cytofluorographs of an acute IM was the increased number of CD3+CD19-, CD8+CD11b-, CD8+CD28+ and CD8+S6F1+ cells. However, CD3-CD19+, CD8+CD11b+, CD8+S6F1-, CD4+Leu8- and CD25+HLA-DR+ antigens were little expressed. Increased number of CD8+CD11b-, CD8+CD28+ and CD8+S6F1+ cells, which are regarded as CTL were reduced according to the improvement of the clinical symptoms and laboratory findings. These results together with HLA typing analysis suggested a possibility HLA-Class 1 restriction of the CTL with surface phenotype of CD8+CD11b-, CD8+CD28+, and CD8+S6F1+.     

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.     

Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma

T cell-mediated immune response against autologous melanoma cells was analyzed, at population and clonal levels, in 31 patients with recurrent and/or metastatic disease. Fresh PBL and lymph node lymphocytes (LNL) from melanoma-involved nodes were not cytotoxic against the respective melanoma cells. When activated in in vitro coculture (IVC) against the autologous melanoma cells in the presence of IL-2, a majority of the activated PBL and LNL became cytotoxic against the autologous targets. The activated effector cells were cloned in limiting dilution microcultures, and growing clones were phenotypically defined and were functionally characterized for cytotoxicity and for potential regulatory function. Functional T cell clones were obtained from 15 of 31 cases. Of these, CTL responses exhibiting cytotoxicity restricted against the autologous melanoma were seen in four cases. All four CTL clones were CD3+, CD8+, and CD4-. Three of these four CTL clones were studied extensively. All three of these CTL clones expressed MHC class I-restricted cytotoxicity. mAb anti-CD3 blocked cytotoxicity in two and enhanced cytotoxicity in the other. Neither autologous sera nor autologous nonactivated fresh PBL modulated the cytotoxic functions of the CTL clones at the effector phase. T cell lines exhibiting regulatory function were obtained in 11 cases. The regulatory T cell lines were CD3+, CD4+, and CD8-. In three cases CD4+ clones amplified the cytotoxic response in the PBL in coculture, while in eight other cases the T cell lines downregulated the cytotoxic responses. Such T cell-mediated down-regulations were either restricted to the autologous system, induced by D/DR antigens expressed by the autologous or allogeneic melanoma cells, or induced by stimulus other than D/DR antigens. Taken together, these findings clearly demonstrate the existence of T cell-mediated cytotoxic and regulatory responses against human melanoma.     

The role of transforming growth factor beta in the generation of suppression: an interaction between CD8+ T and NK cells

CD8+ T cells have suppressor effector functions, but the mechanisms involved in the generation of this activity are poorly understood. We report that natural killer (NK) cells have an important role in the acquisition of this function. CD8+ cells induce NK cells to produce transforming growth factor-beta (TGF-beta) which, in turn, stimulates CD8+ T cells to become suppressors of antibody production. Using a monocyte-dependent and -independent method to induce antibody production, we first observed that the addition of NK cells to CD8+ cells was required for optimal suppression. Next, we determined that the interaction of CD8+ T cells with NK cells resulted in a striking increase NK cell TGF-beta mRNA and its production. This cytokine appeared to be involved in the induction of T suppressor cell activity since: (a) anti-TGF-beta 1 completely abrogated the suppression of immunoglobulin G synthesis; (b) TGF-beta 1 could substitute for NK cells in inducing CD8+ T cells to develop suppressor activity; and (c) a short exposure of T cells to TGF-beta 1 in the absence of B cells was sufficient for the generation of suppressor activity by CD8+ T cells. Interferon gamma did not have this property. These studies provide strong evidence that in addition to its suppressive properties, TGF- beta is involved in the generation of CD8+ T suppressor effector cells. Because NK cell function is decreased in many autoimmune diseases, these cells may fail to interact properly with these individuals' CD8+ cells in generating suppressors of aggressive anti-self responses.     

The effect of in vivo IL-7 deprivation on T cell maturation

A number of previous studies have suggested a key role for interleukin 7 (IL-7) in the maturation of T lymphocytes. To better assess the function of IL-7 in lymphopoiesis, we have deprived mice of IL-7 in vivo by long-term administration of a neutralizing anti-IL-7 antibody. In a previous report (Grabstein, K. H., T. J. Waldschmidt, F. D. Finkelman, B. W. Hess, A. R. Alpert, N. E. Boiani, A. E. Namen, and P. J. Morrissey. 1993. J. Exp. Med. 178:257-264), we used this system to demonstrate the critical role of IL-7 in B cell maturation. After a brief period of anti-IL-7 treatment, most of the pro-B cells and all of the pre-B and immature B cells were depleted from the bone marrow. In the present report, we have injected anti-IL-7 antibody for periods of up to 12 wk to determine the effect of in vivo IL-7 deprivation on the thymus. The results demonstrate a > 99% reduction in thymic cellularity after extended periods of antibody administration. Examination of thymic CD4- and CD8- defined subsets revealed that, on a proportional basis, the CD4+, CD8+ subset was most depleted, the CD4 and CD8 single positive cells remained essentially unchanged, and the CD4-, CD8- compartment actually increased to approximately 50% of the thymus. Further examination of the double negative thymocytes demonstrated that IL-7 deprivation did, indeed, deplete the CD3-, CD4-, CD8- precursors, with expansion of this subset being interupted at the CD44+, CD25+ stage. The proportional increase in the CD4-, CD8- compartment was found to be due to an accumulation of CD3+, T cell receptor alpha, beta + double negative T cells. Additional analysis revealed that anti-IL-7 treatment suppressed the audition/selection process of T cells, as shown by a significant reduction of single positive cells expressing CD69 and heat stable antigen. Finally, the effects of IL-7 deprivation on the thymus were found to be reversible, with a normal pattern of thymic subsets returning 4 wk after cessation of treatment. The present results thus indicate a central role for IL-7 in the maturation of thymic-derived T cells.