Interleukin 2-activated human killer cells are derived from phenotypically heterogeneous precursors

When cultured with native or recombinant human interleukin 2 (IL 2), human peripheral blood non-adherent mononuclear cells (NAMNC) acquire the ability to lyse both NK-sensitive and NK-resistant tumor target cells. The development of these IL 2-activated killer (IAK) cells, also known as LAK, is observed in the absence of exogenous antigen or mitogen. This study describes the ability of various subpopulations of human peripheral blood NAMNC with defined surface phenotype to generate the IAK activity. Human NAMNC were separated into various subpopulations on the basis of the ability to bind monoclonal antibodies, activated with IL 2, and were examined for the cytolytic effect on various tumor target cells. Although CD16+ (Leu-11+) NK cells from NAMNC could become IAK cells when cultured with IL 2, removal of these cells from NAMNC had no effect on the latter's ability to generate the IAK effect. When CD16- NAMNC were separated into CD2+ E rosette-forming T cells (ERFC) and CD2- non-T (non-ERFC) subpopulations, both subpopulations generated the IAK activity. The ability of monoclonal antibody-defined subpopulations of T and non-T cells to generate IAK cells was then examined. Both CD4+ and CD8+ subsets isolated by either positive or negative selection generated the IAK activity. Similarly, CD20+ (B1+) B cells and CD20- non-T (null) cells developed into IAK cells when cultured with IL 2. In contrast, Leu-7+ T cells failed to generate the IAK activity. CD4+ and CD8+ subsets were additionally separated into narrower subpopulations by using monoclonal antibodies anti-Leu-8 and 9.3 respectively, and were examined for their ability to generate IAK cells. Precursors of IAK cells were derived from each of the four: CD4+, Leu-8+ (inducer), CD4+, Leu-8- (helper/amplifier), CD8+, 9.3+ (cytolytic), and CD8+, 9.3- (suppressor) subpopulations of T cells. Thus, the IAK activity appears to be derived from phenotypically heterogeneous and otherwise functionally diverse human lymphoid cells and is not confined to any single subpopulation.     

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.     

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.     

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.     

Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells

Acute viral infections induce extensive proliferation and differentiation of virus-specific CD8+ T cells. One mechanism reported to regulate the proliferative capacity of activated lymphocytes is mediated by the effect of telomerase in maintaining the length of telomeres in proliferating cells. We examined the regulation of telomerase activity and telomere length in naive CD8+ T cells and in virus-specific CD8+ T cells isolated from mice infected with lymphocytic choriomeningitis virus. These studies reveal that, compared with naive CD8+ T cells, which express little or no telomerase activity, Ag-specific effector and long-lived memory CD8+ T cells express high levels of telomerase activity. Despite the extensive clonal expansion that occurs during acute lymphocytic choriomeningitis virus infection, telomere length is maintained in both effector and memory CD8+ T cells. These results suggest that induction of telomerase activity in Ag-specific effector and memory CD8+ T cells is important for the extensive clonal expansion of both primary and secondary effector cells and for the maintenance and longevity of the memory CD8+ T cell population.     

The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28

CD28 plays crucial costimulatory roles in T cell proliferation, cytokine production, and germinal center response. Mice that are deficient in the inducible costimulator (ICOS) also have defects in cytokine production and germinal center response. Because the full induction of ICOS in activated T cells depends on CD28 signal, the T cell costimulatory capacity of ICOS in the absence of CD28 has remained unclear. We have clarified this issue by comparing humoral immune responses in wild-type, CD28 knockout (CD28 KO), and CD28-ICOS double-knockout (DKO) mice. DKO mice had profound defects in Ab responses against environmental Ags, T-dependent protein Ags, and vesicular stomatitis virus that extended far beyond those observed in CD28 KO mice. However, DKO mice mounted normal Ab responses against a T-independent Ag, indicating that B cell function itself was normal. Restimulated CD4(+) DKO T cells that had been primed in vivo showed decreased proliferation and reduced IL-4 and IL-10 production compared with restimulated CD4(+) T cells from CD28 KO mice. Thus, in the absence of CD28, ICOS assumes the major T cell costimulatory role for humoral immune responses. Importantly, CD28-mediated ICOS up-regulation is not essential for ICOS function in vivo.     

Different signals for stimulation of proliferation and lymphokine secretion by a CD3+ WT31- cloned cytotoxic lymphocyte

CD3+ WT31- T cells were sorted from peripheral blood of a normal healthy donor by a FACS IV and cloned by limiting dilution in the presence of a phorbol ester (tetradecanoyl phorbol acetate, TPA), calcium ionophore (ionomycin, Io), interleukin-2 (IL-2), allogeneic cells, and phytohemagglutinin (PHA). One of the derived clones, 290-2, was investigated in detail. 290-2 mediated strong natural killer (NK) but not lymphokine-activated killer (LAK) activity. It proliferated in the presence of IL-2 but not IL-4. It carried the surface phenotype CD3+ WT31- CD4weak+ CD8-, CD16-, and Leu 19+. Expression of CD4 was heterogeneous within the clone, since two of three subclones were also CD4weak+ but one was CD4-. NK activity was blocked by monoclonal antibody (moAb) to CD1 1a (LFA1), but not by monoclonal antibody (moAb) to either CD3 or CD4. Northern blotting revealed T-cell receptor (TCR-gamma) but not alpha- or full-length beta-chain mRNA. 290-2 proliferated autonomously when stimulated with a combination of TPA +Io, with PHA or CD3 moAb and autologous B-cell lines (B-LCL) (and this was inhibited by an anti-IL-2 receptor moAb), but not to allogeneic B-LCL or any of the other stimulating agents alone. Unexpectedly, the TPA + Io stimulus which resulted in maximal proliferative responses did not trigger interferon-gamma or granulocyte/macrophage colony-stimulating factor production, although both lymphokines were secreted in the presence of B-LCL + TPA + Io. Proliferative responses were not enhanced by the presence of B-LCL. Thus, activation signals sufficient for autocrine proliferative responses were insufficient for secretion of other lymphokines. Such clones will provide valuable reagents for investigating the biology of the TCR-gamma+ T cell.     

High IFN-gamma production of individual CD8 T lymphocytes is controlled by CD152 (CTLA-4)

CD8 T cell expansion and cytokine production is needed to generate an effective defense against viral invasion of the host. These features of CD8 T lymphocytes are regulated, especially during primary responses, by positive and negative costimulation. We show in this study that surface expression of CD152 is highly up-regulated on activated CD8 T lymphocytes during primary immune responses, suggesting a prominent regulatory role. Indeed, production of the proinflammatory cytokine IFN-gamma, but not TNF-alpha, by CD8 T cells was inhibited by CD152 engagement. The inhibition was regulated independent of proliferation and IL-2 production, but dependent on the quality of the TCR signaling. We show that signals induced by CD152 on activated CD8 T lymphocytes reduce the frequency of IFN-gamma(high)-expressing cells. Our data also show that in activated CD8 T cells, the CD152-mediated inhibition of cytokine production is more pronounced than inhibition of their proliferation.     

Promotion of human T lymphocyte proliferation by IL-4

The capacity of human rIL-4 to support the proliferation of mitogen-stimulated T cells directly as well as by increasing IL-2 production or enhancing IL-2 responsiveness was investigated. IL-4 augmented proliferation of T cells stimulated with PHA, Con A, immobilized mAb to the CD3 molecular complex (OKT3), or PMA. IL-4 increased the number of mitogen-stimulated cells entering the cell cycle as well as enhancing ongoing proliferation of mitogen-activated lymphoblasts. Facilitation of initial activation by IL-4 was not inhibited by mAb to the p55 component of the IL-2R, anti-Tac, and, therefore, was not dependent on endogenous IL-2 activity. However, IL-4-mediated enhancement of ongoing T cell proliferation stimulated by PHA or OKT3 was partially but not completely blocked by anti-Tac. Analysis of the supernatants from PHA-stimulated T cell cultures indicated that IL-4 increased the production of IL-2 by mitogen-activated cells. Moreover, IL-4 increased the amount of IL-2 mRNA that accumulated in mitogen-stimulated T cells. In addition, IL-4 markedly augmented IL-2R expression by PHA-stimulated T cells. Although IL-4 promoted ongoing DNA synthesis of mitogen-stimulated T cells in an IL-2-dependent manner, it was also able to sustain their proliferation directly. Thus, IL-4 supported proliferation of PMA-activated T cells in a manner that was not inhibited by anti-Tac. Furthermore, IL-4 could augment proliferation and IL-2R expression of T cells stimulated with PHA in the presence of cyclosporin A, which blocks endogenous cytokine production or anti-Tac. Finally, IL-4 was noted to enhance proliferation of both CD4+ and CD8+ T cell subsets. The results indicate that IL-4 enhances proliferation of mitogen-activated human T cells by a number of mechanisms, including the direct promotion of cell cycle entry and subsequent DNA synthesis, enhanced production of IL-2, and increased responsiveness to IL-2 in part by up-regulation of IL-2R expression.     

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.     

HIV-induced immunodeficiency. Relatively preserved phytohemagglutinin as opposed to decreased pokeweed mitogen responses may be due to possibly preserved responses via CD2/phytohemagglutinin pathway

We studied the proliferative response of PBL to the mitogens PHA and PWM and Candida albicans Ag in 301 HIV seropositive homosexual men, of whom 55 had AIDS. The responses to PHA were reduced only in the clinically ill HIV seropositive subjects. In contrast, the responses to PWM were profoundly reduced in most HIV seropositive subjects including the asymptomatic group. Further analysis of 16 HIV seropositive subjects showed that the proliferative responses were reduced in both CD4 and CD8 T cell subsets. A total of 15 HIV seropositive individuals with low responses to PWM, of whom seven had AIDS and eight controls were chosen for the following studies. Expression of T3, Ti, delta receptors, and CD2 was investigated and showed an increased percentage of CD2 receptors positive cells in HIV seropositive subjects without AIDS. The proliferative responses of PBL to stimulation with PHA, PWM, antibodies to CD3, or antibodies to CD2 were investigated and showed significant correlation in controls, whereas in contrast, only the responses to PHA and CD2ab correlated in patients with AIDS. The proliferative responses to CD2ab and CD3ab in controls were larger than the responses to both PHA and PWM. In patients, these responses were less suppressed than the responses to PWM indicating that stimulation with mitogens is more complex than a simple stimulation of Ti/T3 and CD2 receptors. Further investigations were done on resting T cells, i.e., lymphocytes depleted of macrophages and pre-activated cells. Addition of PHA to these cells resulted in preactivation with expression of IL-2R (CD25) but not in proliferation. In contrast, addition of PHA plus SRBC, which bind to the CD2 receptors caused IL-2R expression, IL-2 production, and proliferation. Addition of PWM + SRBC did not result in proliferation. A comparison of the responses to PHA + SRBC of resting T cells from 26 HIV seropositive individuals, of whom seven had AIDS and 12 seronegative controls, showed that these responses were normal or only slightly decreased in the 19 seropositive men without AIDS whereas it was decreased in AIDS patients. Nevertheless, all AIDS patients showed clear-cut responses in this assay. Thus, the discrepancy between responses to PHA and PWM may be explained by an at least partially preserved function of the PHA/CD2-dependent pathway. We suggest that the defect induced by the HIV infection primarily concerns T3/Ti-induced responses.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

NTB-A, a new activating receptor in T cells that regulates autoimmune disease

The CD28 co-stimulatory pathway is well established for T cell activation; however, results from CD28 -/- mice suggest the existence of additional co-stimulatory pathways. Here we report the further characterization of a new member of the CD2 superfamily, NTB-A, important in T cell co-stimulation. NTB-A is expressed on T cells, and its expression is up-regulated on activated cells. Triggering of NTB-A with monoclonal antibodies in the absence of CD28 signals leads to T cell proliferation and interferon-gamma secretion but not interleukin-4. Cross-linking of NTB-A also induces phosphorylation of NTB-A and the association of SAP (SLAM-associated protein), the protein absent in X-linked lymphoproliferative disease. T helper cells differentiated by cross-linking NTB-A and CD3 developed predominantly into Th1 cells not Th2 cells. In vivo blocking of NTB-A interactions with its ligands by using soluble NTB-A-Fc fusion protein inhibits B cell isotype switching to IgG2a and IgG3, commonly induced by Th1-type cytokines. Most important, treatment of mice with NTB-A-Fc delays the onset of antigen-induced experimental allergic encephalomyelitis in myelin basic protein-T cell receptor transgenic mice, suggesting a role in T cell-mediated autoimmune disease. Regulation of interferon-gamma secretion, and not interleukin-4 in vitro, as well as inhibition of Th1 cell-induced isotype switching and attenuation of experimental allergic encephalomyelitis indicate that NTB-A is important for Th1 responses. The observation that cross-linking of NTB-A induces T cell activation, expansion, and Th1-type cytokine production suggests NTB-A is a novel co-stimulatory receptor. The identification of NTB-A as a regulator of T cell response paves the way to provide novel therapeutic approaches for modulation of the immune response.