T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8⁺ and CD4⁺ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8⁺ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4⁺ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4⁺ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4⁺ T cells, supernatants from (i) CD4⁺ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4⁺ T cells from GM-CSF −/− mice.     

A critical role for neutralizing-antibody-producing B cells, CD4+ T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: Implications for adoptive immunotherapy of virus carriers

This study demonstrates that neutralizing-antibody-producing B cells, CD4⁺ T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4⁺ T cells but not necessarily CD8⁺ T cells. (ii) At the doses examined, CD8⁺ T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-γ, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-γ, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8⁺ T cells were needed for virus clearance in addition to B and CD4⁺ T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8⁺ T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4⁺ T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.     

Ly-6C regulates endothelial adhesion and homing of CD8+ T cells by activating integrin-dependent adhesion pathways

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8⁺ T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8⁺ T cells (but not of lymphocytes depleted of CD8⁺ T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8⁺ T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7.G10 (whole antibody) or with its fragments [F(ab) or F(ab)₂] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8⁺ T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C⁺ CD8⁺ T cells in vivo.     

An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4⁺, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Rα^−/− mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Rα^−/−, β₂-microglobulin^−/− double homozygous mice produced less IL-4 than did either IL-4Rα^−/− or wild-type mice. These results indicate that an IL-4-independent, β₂-microglobulin-dependent pathway exists through which the CD62L-low CD4⁺ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.     

Development and function of T cells in T cell antigen receptor/CD3 ζ knockout mice reconstituted with FcɛRI γ

Engagement of α–β T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and ζ–ζ or ζ–η dimers. Previous experiments using knockout (KO) mice that lacked ζ (ζKO) showed that ζ is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in ζKO mice, a subset of T cells in the gut of both ζKO and normal mice bore nearly normal levels of TCR on its surface. This was because ζ was replaced by the FcRI γ (FcRγ). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that ζ replacement by the FcRγ chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of ζ substitution by FcRγ for T cell development and function in vivo, we produced ζKO mice expressing FcRγ in all of their T cells (FcRγTG ζKO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcRγ⁺ lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcRγTG ζKO mice were lower than those of controls. This was particularly true for the CD4⁺ T cells. We conclude that FcRγ can replace the functions of ζ in T cell development in vivo but that TCR/CD3 complexes associated with FcRγ rather than ζ are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4⁺ and CD8⁺ T cells.     

A role for B cells in the development of T cell helper function in a malaria infection in mice

B cell knockout mice are unable to clear a primary erythrocytic infection of Plasmodium chabaudi chabaudi. However, the early acute infection is controlled to some extent, giving rise to a chronic relapsing parasitemia that can be reduced either by drug treatment or by adoptive transfer of B cells. Similar to mice rendered B-cell deficient by lifelong treatment with anti-μ antibodies, B cell knockout mice (μMT) retain a predominant CD4⁺ Th1-like response to malarial antigens throughout a primary infection. This contrasts with the response seen in control C57BL/6 mice in which the CD4⁺ T-cell response has switched to that characteristic of Th2 cells at the later stages of infection, manifesting efficient help for specific antibodies in vitro and interleukin 4 production. Both chloroquine and adoptive transfer of immune B cells reduced parasite load. However, the adoptive transfer of B cells resulted in a Th2 response in recipient μMT mice, as indicated by a relative increase in the precursor frequency of helper cells for antibody production. These data support the idea that B cells play a role in the regulation of CD4⁺ T subset responses.     

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4⁺ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8⁺ regulatory T cells that are specific for pathogenic CD4⁺ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8⁺ T cells emerge that preferentially lyse and regulate activated autologous CD4⁺ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β₂-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8⁺ T cells are also induced by TCV with activated CD4⁺ Vβ8⁺ T cells. These CD8⁺ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8⁺ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4⁺Vβ8⁺ T cell clone specifically recognize other CD4⁺ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1^a but not the allogeneic Qa-1^b molecule. Thus, Vβ-specific Qa-1-restricted CD8⁺ T cells are induced by activated CD4⁺ T cells. We suggest that these CD8⁺ T cells may function to specifically regulate activated CD4⁺ T cells during immune responses.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4⁺ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4⁺ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4⁺ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4⁺ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.     

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8⁺ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8⁺ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Thymocyte development is normal in CTLA-4-deficient mice

Recent studies indicate that CTLA-4 interaction with B7 ligands transduces an inhibitory signal to T lymphocytes. Mice homozygous for a null mutation in CTLA-4 have provided the most dramatic example of the functional importance of CTLA-4 in vivo. These animals develop a fatal lymphoproliferative disorder and were reported to have an increase in CD4⁺ and CD8⁺ thymocytes and CD4⁻CD8⁻ thymocytes, and a decrease in CD4⁺CD8⁺ thymocytes. Based on these observations, it was proposed that CTLA-4 is necessary for normal thymocyte development. In this study, CTLA-4-deficient mice carrying an insertional mutation into exon 3 of the ctla-4 gene were generated. Although these mice display a lymphoproliferative disorder similar to previous reports, there was no alteration in the thymocyte profiles when the parathymic lymph nodes were excluded from the thymi. Further, thymocyte development was normal throughout ontogeny and in neonates, and there was no increase in thymocyte production. Finally, T cell antigen receptor signaling, as assessed by proximal and distal events, was not altered in thymocytes from CTLA-4^−/− animals. Collectively, these results clearly demonstrate that the abnormal T cell expansion in the CTLA-4-deficient mice is not due to altered thymocyte development and suggest that the apparent altered thymic phenotype previously described was due to the inclusion of parathymic lymph nodes and, in visibly ill animals, to the infiltration of the thymus by activated peripheral T cells. Thus it appears that CTLA-4 is primarily involved in the regulation of peripheral T cell activation.     

Independent signals regulate development of primary and secondary follicle structure in spleen and mesenteric lymph node

Lymphotoxin-α-deficient (LT-α^−/−) mice manifest congenital absence of lymph nodes (LNs) and Peyer’s patches and disturbed spleen follicle structure. The splenic white pulp areas show loss of discrete T and B lymphocyte zones, of follicular dendritic cell (FDC) clusters, and of germinal centers (GCs). Tumor necrosis factor receptor I-deficient (TNFR-I^−/−) mice show similar absence of FDC clusters and GCs but retain segregation of T and B cell zones. Rarely are mesenteric LNs found in LT-α^−/− mice. These mesenteric LNs show segregation of T and B cell zones similar to wild-type mice. In contrast, mesenteric LNs in TNFR-I^−/− mice manifest grossly disturbed organization of T and B cells. Both LT-α^−/− and TNFR-I^−/− mice lacked FDC clusters in LNs and spleen. Interestingly, although both LT-α^−/− and TNFR-I^−/− mice that had been immunized with sheep red blood cells failed to form GCs in the spleen, they both developed GC-like clusters of peanut agglutinin-positive (PNA⁺) cells in their LNs. Furthermore, when lethally irradiated recombination activating gene (RAG)-1-deficient (RAG-1^−/−) mice that had received spleen cells from LT-α^−/− mice were immunized with sheep red blood cells, they failed to generate PNA⁺ clusters in the reconstituted spleen but showed robust PNA⁺ clusters in the reconstituted LNs. These data demonstrate that the signals that regulate the development of distinct T and B cell zones as well as the signals that regulate B cell activation to produce clusters of PNA⁺ cells differ between the spleen and LNs.     

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4⁻CD8⁻ double negative (DN) cells to CD4⁺CD8⁺ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.     

β cell apoptosis in T cell-mediated autoimmune diabetes

Insulin-dependent diabetes mellitus results from T cell-mediated destruction of insulin-producing, pancreatic islet β cells. How this destruction takes place has remained elusive—largely due to the slow kinetics of disease progression. By crossing a transgenic mouse carrying a β cell-specific T cell receptor onto the NOD.scid background, we produced a simplified but robust and accelerated model of diabetes. This mouse produces CD4⁺ T cells bearing transgenic T cell receptor but is devoid of CD8⁺ T cells and B cells. More importantly, this mouse develops a rapid diabetes, which has allowed us to record and quantify β cell death. We have determined that β cells within the inflamed islets die by apoptosis.     

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.     

Virus-specific immunity after gene therapy in a murine model of severe combined immunodeficiency

Human severe combined immunodeficiency (SCID) can be caused by defects in Janus kinase 3 (JAK3)-dependent cytokine signaling pathways. As a result, patients are at high risk of life-threatening infection. A JAK3 −/− SCID mouse model for the human disease has been used to test whether transplant with retrovirally transduced bone marrow (BM) cells (JAK3 BMT) could restore immunity to an influenza A virus. The immune responses also were compared directly with those for mice transplanted with wild-type BM (+/+ BMT). After infection, approximately 90% of the JAK3 BMT or +/+ BMT mice survived, whereas all of the JAK3 −/− mice died within 29 days. Normal levels of influenza-specific IgG were present in plasma from JAK3 BMT mice at 14 days after respiratory challenge, indicating restoration of B cell function. Influenza-specific CD4⁺ and CD8⁺ T cells were detected in the spleen and lymph nodes, and virus-specific CD8⁺ effectors localized to the lungs of the JAK3 BMT mice. The kinetics of the specific host response correlated with complete clearance of the virus within 2 weeks of the initial exposure. By contrast, the JAK3 −/− mice did not show any evidence of viral immunity and were unable to control this viral pneumonia. Retroviral-mediated JAK3 gene transfer thus restores diverse aspects of cellular and humoral immunity and has obvious potential for human autologous BMT.     

Point mutations at the purine nucleoside phosphorylase locus impair thymocyte differentiation in the mouse

Three point mutations on the Np^b allele of the purine nucleoside phosphorylase locus in the mouse have been recovered by male germ cell mutagenesis. The mutants were backcrossed, 12–14 generations, and are designated in increasing order of severity of enzyme deficiency and phenotype: B6-NPE, Met-87 → Lys; B6-NPF, Ala-228 → Thr; and B6-NPG, Trp-16 → Arg. A marked decline in total cell numbers per thymus occurs between 2 and 3 months for the more severe B6-NPF and B6-NPG mutants (35% and 52%, respectively) and by 8 months for the less severe B6-NPE mutation. The thymocyte population is thereafter characterized by a 3- or 8-fold expanded precursor, CD4⁻CD8⁻ double-negative population and 15% or 55% reduced CD4⁺CD8⁺ double-positive cells for the B6-NPF and B6-NPG strains, respectively. Spleen lymphocyte Thy-1⁺ cells are reduced by 50% and spleen lymphocyte response to T cell mitogen and interleukin 2 is reduced by 80%. Increases of thymocyte dGTP pools of 5- and 2.5-fold for B6-NPF and B6-NPG mutants, respectively, are observed. The purine nucleoside phosphorylase-deficient mouse exhibits age-dependent progressive perturbations in thymocyte differentiation, reduced numbers of thymocytes, and reduced splenic T cell numbers and response. The progressive T cell deficit is similar to the human disorder.     

Primary CD8+ cells from HIV-infected individuals can suppress productive infection of macrophages independent of β-chemokines

The productive infection of human monocyte-derived macrophages (M) by HIV was suppressed by primary CD8⁺ cells from asymptomatic HIV-infected individuals. This anti-HIV response was noncytotoxic; removal of the CD8⁺ cells from the infected M leads to virus production. CD8⁺ cells inhibited HIV replication when separated from the infected M by a transwell filter insert, indicating a diffusible factor made by the CD8⁺ cells suppressed productive infection of M. Three β-chemokines, which can be secreted by activated CD8⁺ cells, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α and MIP-1β prevented HIV replication in the M cultures. In addition, incubation of acutely infected M with a mixture of neutralizing antibodies to RANTES, MIP-1α, and MIP-1β enhanced virus replication. Nevertheless, neutralization of β-chemokines with specific antibodies did not abolish the suppression by CD8⁺ cells of HIV replication in M. Thus, even though β-chemokines decrease HIV replication in M, these cytokines are not responsible for the ability of CD8⁺ cells to inhibit HIV production in these cells.     

The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes

The chemokine receptors CXCR4 and CCR5 function as coreceptors for HIV-1 entry into CD4⁺ cells. During the early stages of HIV infection, viral isolates tend to use CCR5 for viral entry, while later isolates tend to use CXCR4. The pattern of expression of these chemokine receptors on T cell subsets and their regulation has important implications for AIDS pathogenesis and lymphocyte recirculation. A mAb to CXCR4, 12G5, showed partial inhibition of chemotaxis and calcium influx induced by SDF-1, the natural ligand of CXCR4. 12G5 stained predominantly the naive, unactivated CD26^low CD45RA⁺ CD45R0⁻ T lymphocyte subset of peripheral blood lymphocytes. In contrast, a mAb specific for CCR5, 5C7, stained CD26^high CD45RA^low CD45R0⁺ T lymphocytes, a subset thought to represent previously activated/memory cells. CXCR4 expression was rapidly up-regulated on peripheral blood mononuclear cells during phytohemagglutinin stimulation and interleukin 2 priming, and responsiveness to SDF-1 increased simultaneously. CCR5 expression, however, showed only a gradual increase over 12 days of culture with interleukin 2, while T cell activation with phytohemagglutinin was ineffective. Taken together, the data suggest distinct functions for the two receptors and their ligands in the migration of lymphocyte subsets through lymphoid and nonlymphoid tissues. Furthermore, the largely reciprocal expression of CXCR4 and CCR5 among peripheral blood T cells implies distinct susceptibility of T cell subsets to viral entry by T cell line-tropic versus macrophage-tropic strains during the course of HIV infection.     

A single peripheral CD8+ T cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions

The lineage relationships between murine CD8⁺ T cells with different cytokine profiles were investigated by paired-daughter analysis in the presence and absence of the type 2 cytokine-inducing stimulus, interleukin 4 (IL-4). Single CD8⁺ CD44^low lymph node T cells were activated to divide at high frequency with IL-2 and immobilized antibodies to CD3, CD8, and LFA-1. When these parent cells were subcloned by transferring their daughter or granddaughter cells into secondary cultures with or without IL-4, the subclones expressed diverse combinations of the mRNAs for the type 1 cytokines, interferon γ (IFN-γ), and IL-2, and the type 2 cytokines, IL-4, IL-5, IL-6, and IL-10. Frequencies of subclones that expressed IL-4, IL-6, and, to a lesser extent, IL-2, IL-5, and IL-10 were higher among those grown with IL-4, but a significant proportion of those grown without exogenous IL-4 also expressed one or more type 2 cytokines. Subclones within 89% of families displayed different cytokine profiles, indicating that their parent cells were multipotential for this function. Because 98% of parent cells yielded subclones that produced type 1 cytokines and 77% yielded type 2 cytokine producers, we conclude that type 1 and type 2 cytokine-producing CD8⁺ T cells can be derived from a common precursor. Similar analyses performed by subcloning after ≥7 or ≥13 cell divisions without IL-4 showed that many CD8⁺ T cells retained the potential to shift toward a type 2 cytokine profile in response to IL-4, even after prolonged expansion under conditions that favored type 1 cytokine expression. CD8⁺ T cells that express type 1 and/or type 2 cytokines therefore are derived from the same peripheral T cell lineage whose multipotentiality can persist through many cell divisions.