DAP10 contributes to CD8(+) T cell-mediated cytotoxic effector mechanisms during Mycobacterium tuberculosis infection

The activating C-type lectin-like receptor NKG2D, which is expressed by mouse NK cells and activated CD8 T cells, was previously demonstrated to be involved in tumor rejection and as a defense mechanism against viral and bacterial infections. Because CD8 T cells are important for protective immune responses during chronic Mycobacterium tuberculosis (Mtb) infection and represent a promising target for new vaccine strategies to prevent human pulmonary tuberculosis (TB), we studied the immune response in mice deficient for the NKG2D adapter molecule DAP10 during experimental TB. After aerosol infection, DAP10-defcient mice displayed an unimpaired recruitment, activation and development of antigen-specific CD8 T cells. Whereas the frequency of interferon-gamma-producing CD8 T cells from Mtb-infected DAP10-defcient mice was not affected, CD8 T cell-mediated cytotoxicity was significantly reduced in the absence of DAP10. The loss of cytotoxic activity in DAP10-deficient CD8 T cells was associated with an impaired release of cytotoxic granules. Together, our results suggest that during Mtb infection DAP10 is required for maximal cytolytic activity of CD8 T cells.     

Induction of Mycobacterium tuberculosis-specific primary and secondary T-cell responses in interleukin-15-deficient mice

Several studies have provided evidence that interleukin-15 (IL-15) can enhance protective immune responses against Mycobacterium tuberculosis infection. However, the effects of IL-15 deficiency on the functionality of M. tuberculosis-specific CD4 and CD8 T cells are unknown. In this study, we investigated the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection of IL-15(-/-) mice. IL-15(-/-) mice had slightly higher bacterial numbers during chronic infection, which were accompanied by an increase in gamma interferon (IFN-gamma)-producing CD4 and CD8 T cells. There was no evidence of increased apoptosis or a defect in proliferation of CD8 effector T cells following M. tuberculosis infection. The induction of cytotoxic and IFN-gamma CD8 T-cell responses was normal in the absence of IL-15 signaling. The infiltration of CD4 and CD8 T cells into the lungs of "immune" IL-15(-/-) mice was delayed in response to M. tuberculosis challenge. These findings demonstrate that efficient effector CD4 and CD8 T cells can be developed following M. tuberculosis infection in the absence of IL-15 but that recall T-cell responses may be impaired.     

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A

The contribution of CD8+ and CD4+ T cell-mediated effector functions against Mycobacterium tuberculosis infection elicited by i.m. vaccination with plasmid DNA encoding the immunodominant Ag85A antigen of M. tuberculosis was studied. Ag85A DNA-vaccinated beta2-microglobulin gene-deficient (beta2m-/-) mice, which lack CD8+ T cells, produced Ag85-specific antibodies and Th1 type cytokines similar to wild-type mice. Although beta2m-/- mice were more susceptible to M. tuberculosis infection, following vaccination they efficiently controlled bacterial replication in spleen and lungs 4 weeks post-infection. In contrast, mice lacking CD4+ T cells were neither sensitized by the Ag85A DNA vaccine to produce Ag85-specific antibodies or Th1 type cytokines nor did they contain a M. tuberculosis challenge infection. In addition, Ag85A DNA-vaccinated IFN-gamma gene knockout mice produced Ag85-specific antibodies and IL-2 but died rapidly following a M. tuberculosis challenge infection. Collectively, these data support the view that IFN-gamma-producing CD4+ T cells, independently of CD8+ T cells, may mediate the protective effect of the Ag85A DNA vaccine.     

Expansion of circulating NKG2D+ effector memory T-cells and expression of NKG2D-ligand MIC in granulomaous lesions in Wegener's granulomatosis

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.     

NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation

NKG2D is a receptor on natural killer (NK) cells and cytotoxic T lymphocytes that binds major histocompatibility complex (MHC) class I-like ligands expressed primarily on virally infected and neoplastic cells. In vitro studies indicate that NKG2D provides costimulation through an associated adapter, DAP10, which recruits phosphatidylinositol-3 kinase. Here we show that in DAP10-deficient mice, CD8+ T cells lack NKG2D expression and are incapable of mounting tumor-specific responses. However, DAP10-deficient NK cells express a functional NKG2D receptor due to the association of NKG2D with another adapter molecule, DAP12 (also known as KARAP), which recruits protein tyrosine kinases. Thus, NKG2D is a versatile receptor that, depending on the availability of adapter partners, mediates costimulation in T cells and/or activation in NK cells.     

Efficacy of IL-2- versus IL-15-stimulated CD8 T cells in adoptive immunotherapy

We determined the efficacy of in vitro expanded P14 TCR transgenic CD8 T cells to mediate tumor cell elimination and to protect against viral infection in mice. Contrary to previous studies, an adoptive transfer model without lymphodepletion, vaccination or cytokine treatment was used. Antigen-activated P14 T cells cultured in IL-2-containing medium for 7 days (P14IL-2) exhibited potent effector cell functions in vitro but did not confer protection against melanoma growth or viral infection. In contrast, P14 T cells cultured in IL-15 (P14IL-15) were highly effective in vivo although they displayed only moderate effector functions in vitro. Therapeutic efficacy correlated with the survival of the transferred T cells in the recipients: P14IL-2 cells disappeared rapidly whereas P14IL-15 cells persisted for prolonged time. Decreasing the IL-2 concentration in the culture media improved in vivo survival and efficacy but also lowered the cell yield of the cultures. Finally, we could extend the findings with monoclonal P14 T cells to polyclonal CD8 T cells. Thus, in vitro expansion of antigen-specific CD8 T cells in IL-15 allowed the generation of substantial numbers of T cells without inducing terminally differentiated effector cells that turned out to be unfavorable in the transfer model examined here.     

IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D

目的 观察大剂量IL-2活化的人外周血单个核细胞（PBMC）中，NKG2D在NK细胞、T细胞和NKT细胞表面的表达规律。方法 使用三重免疫荧光标记的流式细胞术检测NKG2D的表达情况。使用sMICA蛋白与人PBMC共同培养，之后使用流式细胞术分析NKG2D在NK细胞中的表达情况。使用半定量RT-PCR方法检测大剂量IL-2活化的人PBMC中NKG2D及其锚定蛋白DAP10 mRNA的表达变化。结果 使用大剂量IL-2活化人PBMC细胞后，NKG2D在NK细胞、CD^＋T细胞和NKT细胞表面的表达均增加，但是在CD4^＋T细胞表面始终不表达。同时IL-2可以拮抗sMICA对NKG2D的下调作用。半定量RT-PCR结果显示，使用大剂量IL-2活化人PBMC之后，NKG2D及其锚定蛋白DAP10的mRNA水平并不发生明显变化。结论 大剂量IL-2培养人PBMC之后，NKG2D在NK细胞、CD8^＋T细胞和NKT细胞表面的表达均增加，可能是PBMC活化并获得广谱抗肿瘤效应的机制之一．     

IL-15 induces CD8+ T cells to acquire functional NK receptors capable of modulating cytotoxicity and cytokine secretion

During the last years several authors have described a small population of CD8+ T cells expressing NK receptors (NKRs). Although their origin remains largely unknown, we have recently demonstrated that IL-15 is capable of inducing NKR expression in purified human CD8+CD56− T cells. In this study we show that IL-15-driven NKR induction in CD8+ T cells was linked with CD56 de novo acquisition, consistent with an effector-memory phenotype, increased anti-apoptotic levels, high granzyme B/perforin expression and with the ability of displaying in vitro NK-like cytotoxicity. Interestingly, dissection of NKR functional outcome in IL-15-cultured CD8+ T cells revealed: (i) that NKG2D cross-linking was able per se to upregulate degranulation levels and (ii) that KIR and NKG2A cross-linking upregulated secretion of cytokines such as IFN-γ, TNF-α, IL-1β and IL-10. These results suggest that IL-15 is capable of differentiating CD8+ T cells into NK-like T cells displaying a regulatory phenotype.     

Protective heterologous immunity against fatal ehrlichiosis and lack of protection following homologous challenge

The roles of antibodies and memory T cells in protection against virulent Ehrlichia have not been completely investigated. In this study, we addressed these issues by using murine models of mild and fatal ehrlichiosis caused by related monocytotropic Ehrlichia strains. Mice were primed with either Ehrlichia muris or closely related virulent ehrlichiae transmitted by Ixodes ovatus (IOE) ticks given intraperitoneally or intradermally. All groups were reinfected intraperitoneally, 30 days later, with a lethal high dose of IOE. Priming with E. muris, but not IOE, induced strong CD4+ and CD8+ memory type 1 T-cell responses, Ehrlichia-specific immunoglobulin G (IgG) antibodies, and persistent infection. Compared to IOE-primed mice, subsequent lethal IOE challenge of E. muris-primed mice, resulted in (i) 100% protection against lethal infection, (ii) strong Ehrlichia-specific secondary gamma interferon (IFN-gamma)-producing effector/effector memory CD4+ and CD8+ T-cell responses, (iii) enhanced secondary anti-ehrlichial antibody response, (iv) accelerated bacterial clearance, and (v) the formation of granulomas in the liver and lung. E. muris-primed mice challenged with IOE had lower levels of serum interleukin-1alpha (IL-1alpha), IL-6, and IL-10 compared to unprimed mice challenged with IOE. Interestingly, the fatal secondary response in IOE-primed mice correlated with (i) decline in the Ehrlichia-specific CD4+ and CD8+ type 1 responses, (ii) marked hepatic apoptosis and necrosis, and (iii) substantial bacterial clearance, suggesting that fatal secondary response is due to immune-mediated tissue damage. In conclusion, protection against fatal ehrlichial infection correlates with strong expansion of IFN-gamma-producing CD4+ and CD8+ effector memory type 1 T cells, which appear to be maintained in the presence of IgG antibodies and persistent infection.     

Decreased IL-10 and IL-13 production and increased CD44hi T cell recruitment contribute to Leishmania major immunity induced by non-persistent parasites

Leishmaniasis is currently classified as category 1 disease, i.e. emerging and uncontrolled. Since the importance of persistent infection for maintaining an effective long-lasting protective response is controversial, the present study asks whether immunisation with non-persistent parasites leads to protection against Leishmania infection and to the recruitment of T cells of a specific phenotype. Our study shows that vaccination of susceptible BALB/c mice with live Leishmania major phosphomannomutase-deficient parasites, which are avirulent and non-persistent in vivo, leads to protection against infection. Immunisation with phosphomannomutase-deficient parasites neither leads to differences in IFN-gamma, IL-12, IL-4 production nor alters the expression of effector and memory markers, including CD62L, IL-7Ralpha and IL-2Ralpha, when compared with unvaccinated controls. Observed protection is due to the ability of vaccinated animals to suppress early IL-10 and IL-13 production and to recruit a higher number of antigen-experienced CD44hiCD4+ and CD44hiCD8+ T cells into draining LN following infection. Thus, expansion of T-cell numbers and their rapid recruitment to LN upon infection as well as the restriction of IL-13 and IL-10 production leading to high IFN-gamma/IL-10 ratio play an important role in protection against Leishmania affecting the outcome of the disease in favour of the host.     

NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.     

TCR-independent cytokine stimulation induces non-MHC-restricted T cell activity and is negatively regulated by HLA class I

Recent evidence suggests that the functional status of T cells activated independently from their TCR differs substantially from classical MHC-restricted T cells. Here, we show that TCR-independent, short-term stimulation via the common gamma-chain of the IL-2/IL-15 receptor induces non-MHC-restricted cytotoxicity and sustained cytokine secretion in purified CD4+ or CD8+ T cells. NK-like cytotoxicity is directed against MHC class I-negative targets and can be inhibited by classical and non-classical HLA class I molecules. Known inhibitory receptors, such as CD85j (ILT2) and leukocyte-associated Ig-like receptor-1, are not responsible for this HLA-mediated inhibition. NK-like cytotoxicity can be costimulated by NKG2D (CD314) triggering, but 2B4 (CD244) and DNAM-1 (CD226) are not involved. NK-like T cells display an activated phenotype and secrete various cytokines, including IFN-gamma, TNF-alpha, IL-5, IL-13 and MIP-1beta. Under normal conditions, HLA class I-mediated inhibition may function as a safety mechanism to prevent unbalanced cytokine production and effector killing mechanisms by T cells that were activated independently from their TCR. Non-MHC-restricted activity represents a functional status rather than a property of distinct T cell subpopulations. Thus, cytokine-induced, non-MHC-restricted T cells may be relevant in immune responses against tumors showing aberrant MHC expression through their capacities of cytokine production and direct tumor cell eradication.     

Interleukin-12 primes CD4+ T cells for interferon-γ production and protective immunity during Mycobacterium avium infection

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.     

The CD94/NKG2C killer lectin-like receptor constitutes an alternative activation pathway for a subset of CD8+ T cells

The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.     

NKG2D receptor activation of NF-κB enhances inflammatory cytokine production in murine effector CD8 T cells

To induce strong immune responses, naïve CD8⁺ T cells require stimulation through the TCR and costimulatory receptors. However, the biological effect of activating costimulatory receptors on effector T cells is still unclear. One costimulatory receptor that is likely to be engaged at the target site is NKG2D. This activating receptor is expressed on human and murine CD8⁺ T cells with its ligands expressed on the majority of tumor cells and during some infections. In order to determine how activation of costimulatory receptors alters effector CD8⁺ T cell functions, this study compared the activation of the NF-κB signaling pathway by two costimulatory receptors, CD28 and NKG2D. Compared to CD28 costimulation, activation of murine effector CD8⁺ T cells through CD3 and NKG2D receptors enhanced activation of NF-κB as shown by increased phosphorylation of IKKα, IκBα, and NF-κB and IκBα degradation. NKG2D costimulation also increased activation, nuclear translocation, and DNA binding of NF-κB p65/p50 dimers. Activation of the NF-κB pathway also lead to increased gene expression and secretion of pro-inflammatory cytokines, including IFNα and IFNγ, and decreased gene expression and secretion of anti-inflammatory cytokines, including IL-10 and CCL2. Altered NF-κB activation also increased expression of the effector molecules TNFα, lymphotoxins α and β, and Fas ligand, and increased tumor cell killing through FasL. These data show that compared to CD28 costimulation, activation through the NKG2D receptor leads to the differential activation of the NF-κB signaling pathway and potentially enhances the anti-tumor and anti-viral functions of effector CD8⁺ T cells.     

NKG2D stimulation of CD8+ T cells during priming promotes their capacity to produce cytokines in response to viral infection in mice

Natural killer group 2 member D (NKG2D) is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ, and CD8⁺ T cells. It is still a matter of debate whether and how NKG2D mediates priming of CD8⁺ T cells in vivo, due to a lack of studies where NKG2D is eliminated exclusively in these cells. Here, we studied the impact of NKG2D on effector CD8⁺ T‐cell formation. NKG2D deficiency that is restricted to murine CD8⁺ T cells did not impair antigen‐specific T‐cell expansion following mouse CMV and lymphocytic choriomeningitis virus infection, but reduced their capacity to produce cytokines. Upon infection, conventional dendritic cells induce NKG2D ligands, which drive cytokine production on CD8⁺ T cells via the Dap10 signaling pathway. T‐cell development, homing, and proliferation were not affected by NKG2D deficiency and cytotoxicity was only impaired when strong T‐cell receptor (TCR) stimuli were used. Transfer of antigen‐specific CD8⁺ T cells demonstrated that NKG2D deficiency attenuated their capacity to reduce viral loads. The inability of NKG2D‐deficient cells to produce cytokines could be overcome with injection of IL‐15 superagonist during priming. In summary, our data show that NKG2D has a nonredundant role in priming of CD8⁺ T cells to produce antiviral cytokines.     

Partial reconstitution of the CD4+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA vaccines

Reactivation tuberculosis (TB) is a serious problem in immunocompromised individuals, especially those with human immunodeficiency virus (HIV) coinfection. The adaptive immune response mediated by CD4+ and CD8+ T cells is known to confer protection against TB. Hence, vaccines against TB are designed to activate these two components of the immune system. Anti-TB DNA vaccines encoding the immunodominant proteins Ag85A, Ag85B, and PstS-3 from Mycobacterium tuberculosis are ineffective in mice lacking CD4+ T cells (CD4-/- mice). In this study, we demonstrate that reconstitution of the T-cell compartment in CD4-/- mice restores vaccine-specific antibody and gamma interferon (IFN-gamma) responses to these DNA vaccines. The magnitude of the immune responses correlated with the extent of reconstitution of the CD4+-T-cell compartment. Reconstituted mice vaccinated with DNA encoding PstS-3, known to encode a dominant D(b)-restricted CD8+-T-cell epitope, displayed CD8+-T-cell responses not observed in CD4-/- mice. M. tuberculosis challenge in reconstituted mice led to the extravasation of IFN-gamma-producing CD4+ and CD8+ T cells into lungs, the primary site of bacterial replication. Importantly, a reconstitution of 12 to 15% of the CD4+-T-cell compartment resulted in Ag85B plasmid DNA-mediated protection against a challenge M. tuberculosis infection. Our findings provide evidence that anti-TB DNA vaccines could be effective in immunodeficient individuals after CD4+-T-lymphocyte reconstitution, as may occur following antiretroviral therapy in HIV+ patients.     

IL-15-independent antiviral function of primary and memory CD8+ T cells

Memory CD8+ T cells are comprised of CD122hi IL-15-dependent and CD122lo IL-15-independent subsets. Induction and retention of IL-15-independent memory CD8+ T cells was assessed in IL-15-/- and wild-type (wt) mice immunized with recombinant vaccinia virus (rVV) or Sindbis virus (rSIN) vectors expressing the identical foreign epitope. Both vectors induced epitope-specific CD8+ T cell expansion and function, independent of IL-15. Similar kinetics of rVV clearance confirmed effective CD8+ T cell function in IL-15-/- mice. CD44hi CD122hi CD8+ T cells, mainly of the CD62L-/lo phenotype, increased more dramatically and declined more rapidly in IL-15-/- mice, independent of the vector. Rapid IL-15-independent memory CD8+ T cell expansion following challenge of immune mice compensated for the limited memory CD8+ populations in IL-15-/- mice. However, despite expansion and expression of potent effector function, viral clearance was delayed in the absence of IL-15, coinciding with a rapid loss in cytolytic function.     

Bcl6 is essential for the generation of long-term memory CD4+ T cells

Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells. We analyzed here a role for Bcl6 in the generation of long-term memory CD4(+) T cells. Naive CD45RB(+) CD4(+) T cells from Bcl6-deficient DO11.10 (KJ1.26(+)) transgenic mice were transferred into BALB/c mice and immunized with ovalbumin peptide and LPS. Long-term memory KJ1.26(+) CD4(+) T cells from wild-type mice were detected in the spleen, lungs and liver during 10 weeks after immunization; however, Bcl6-deficient KJ1.26(+) CD4(+) T cells were vanished completely in those organs 4 weeks after immunization. Since memory CD4(+) T cells can be generated from effector CD4(+) T cells, properties of Bcl6-deficient effector CD4(+) T cells were compared with those wild-type effector CD4(+) T cells 10 days after immunization. Numbers of IFN-gamma-non-producing CD45RB(-), CD62L(+) or IL-7Ralpha(+) effector CD4(+) T cells in the spleen, lungs and liver were similar between Bcl6-deficient and wild-type CD4(+) T cells. However, the percentage of apoptotic cells in Bcl6-deficient effector CD4(+) T cells was higher than that in wild-type effector CD4(+) T cells. At the late effector phase, the number of IFN-gamma-non-producing cells and the percentage of apoptotic cells in Bcl6-deficient CD4(+) T cells were smaller and higher than those in wild-type CD4(+) T cells, respectively. These data suggest that Bcl6 in CD4(+) T cells plays a role in protection of memory precursor CD4(+) T cells from apoptosis and may involve in survivability of long-term memory CD4(+) T cells.