Distinct CD4+ helper T cells involved in primary and secondary responses to infection

Helper T cells are critical for protective immunity, CD8(+) T-cell memory, and CD4(+) recall responses, but whether the same or distinct CD4(+) T cells are involved in these responses has not been established. Here we describe two CD4(+) T cells, LLO118 and LLO56, specific for an immunodominant Listeria monocytogenes epitope, with dramatically different responses to primary and secondary infection. Comparing in vivo responses, LLO118 T cells proliferate more strongly to primary infection, whereas surprisingly, LLO56 has a superior CD4(+) recall response to secondary infection. LLO118 T cells provide more robust help for CD8(+) T-cell responses to secondary infection than LLO56. We found no detectable differences in antigen sensitivity, but naive LLO118 T cells have much lower levels of CD5 and their T-cell receptor levels are dramatically down-regulated after their strong primary response. Thus, distinct CD4(+) helper T cells are specialized to help either in primary or secondary responses to infection.     

Enhancement of T-cell-depleted bone marrow allografts in the absence of graft-versus-host disease is mediated by CD8+ CD4- and not by CD8- CD4+ thymocytes.

Transplantation of T-cell-depleted C57BL/6-Nu/Nu ("nude") bone marrow (BM) into C3H/HeJ recipients, conditioned with 8 Gy total body irradiation plus chemotherapy with the myeloablative drug dimethyl myleran, resulted in poor hematopoietic reconstitution 14 days posttransplant, compared with transplantation with T-cell-depleted BM from normal C57BL/6 donors. Hematopoietic reconstitution of "nude" BM could be improved by the addition of (C57BL/6xC3H/HeJ)F1 thymocytes void of graft-versus-host activity. Enhancement of BM allografting by thymocytes is sensitive to low radiation doses (> or = 5.0 Gy) and can be achieved by transplanting the BM 24 hours before the administration of thymocytes. Fractionation of F1 thymocytes by differential agglutination with peanut agglutinin (PNA) and by fluorescence activated cell sorting showed that this hematopoietic enhancing activity is enriched in the unagglutinated (PNA-) thymocyte fraction and is mediated by PNA- CD8+ and not by PNA- CD4+ thymocytes.     

Both CD4+ and CD8+ T cells can mediate vaccine-induced protection against Coccidioides immitis infection in mice

To determine which lymphocytes are required for vaccine-induced immunity to coccidioidomycosis, we used a temperature-sensitive mutant of Coccidioides immitis to immunize mice lacking subsets of lymphocytes or specific cytokines and infected the mice 4 weeks later with virulent C. immitis. After 2 weeks, we determined the number of fungi in their lungs and spleens. Vaccine-induced immunity required alpha beta T lymphocytes. beta -2 microglobulin knockout (KO) mice were protected by immunization, and we transferred protection using CD4+ T cells from immunized mice. However, vaccination also protected CD4+ KO mice, which suggests that CD8+ T cells played a role in vaccine-induced immunity, even though they were not required. We adaptively transferred protection using spleen cells from immunized CD4+ KO mice to nonimmune B6 mice, but CD8+ -depleted spleen cells did not protect against infection. Recipients of spleen cells from immunized CD4+ KO mice had 6 times more tumor necrosis factor (TNF)- alpha mRNA in their lungs than did mice that received nonimmune spleen cells, and TNF receptor-1 KO mice were not fully protected by immunization. These results show that both CD4+ and CD8+ T cells can protect against coccidioidomycosis and that TNF- alpha is a necessary component of the acquired immune response.     

Type 1 and type 2 cytokine-producing CD4+ and CD8+ T cells in primary antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by thrombosis and/or recurrent fetal loss as well as the presence of autoantibodies against epitopes present on phospholipid-binding proteins. The role of cellular immunity in the pathogenesis of the syndrome remains unclear. We studied the cellular phenotype and the production of type 1 [interferon (IFN)-, interleukin (IL)-2] and type 2 (IL-4, IL-10) cytokines by CD4+ and CD8+ T-lymphocyte subsets in 13 patients with untreated primary APS (PAPS) and in 32 healthy controls. The production of cytokines was determined in T cells after a 5-h culture with or without mitogenic stimulation using a flow cytometric method of intracellular cytokine staining. In six of the patients these studies were repeated 6 months later. In PAPS patients we found a reduced percentage of circulating CD4+CD45RA+ and an increased percentage and absolute number of CD8+HLA-DR+ cells. A type 1 response was observed in the patients unstimulated cells, indicated by an increase in IFN--producing CD8+, IL-2-producing CD4+ T cells, and a decrease in IL-4-producing CD4+ and CD8+ T cells. Similar results were obtained in the patients at follow-up. Taken together, these results suggest a chronic in vivo stimulation of CD4+ and CD8+ T cells in PAPS patients exhibiting a type 1 polarization. Changes of cellular immunity may contribute to the pathogenesis of the clinical manifestations of the syndrome and might be proven to be useful targets for therapeutic interventions in the future.     

Human cytomegalovirus-specific memory CD8+ and CD4+ T cell differentiation after primary infection

BACKGROUND: The development of human cytomegalovirus (HCMV)-specific T cell immunity after primary infection and its correlation with virus transmission to the fetus were investigated. METHODS: The membrane phenotype (CCR7 and CD45RA expression) of and intracellular cytokine (interferon [IFN]-gamma and interleukin-2) production by HCMV-specific T cells (stimulated with HCMV-infected dendritic cells) were investigated in 21 immunocompetent pregnant women (12 transmitters and 9 nontransmitters) and in 5 nonpregnant subjects during the first year after infection. RESULTS: IFN-gamma-producing CD4+ and CD8+ T cells were readily detected during the first month, and their levels did not significantly change with time. CCR7 expression was negligible during both the early and the late stage of infection. Among CCR7- cells, those reexpressing CD45RA progressively increased until they reached median levels of 33% (range, 7%-51%) and 51% (range, 22%-76%) for HCMV-specific CD4+ and CD8+ T cells, respectively, similar to those observed in subjects with remote infection. CD45RA reexpression correlated with HCMV disappearance from blood. The level of HCMV-specific CD45RA+ T cells during the first months after infection was significantly lower in mothers who were transmitters than in those who were nontransmitters. CONCLUSIONS: After primary infection, circulating HCMV-specific effector T cells revert to the CD45RA+ phenotype, which appears to be associated with control of viremia and vertical transmission. Thus, these cells may represent long-lived true memory lymphocytes in the HCMV-specific pool.     

CD4+ T cell regulation of CD25 expression controls development of short-lived effector CD8+ T cells in primary and secondary responses

Both CD4⁺ T cell help and IL-2 have been postulated to “program” activated CD8⁺ T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8⁺ T cell differentiation following infection with three pathogens (Listeria monocytogenes, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4⁺ T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8⁺ T cells peaked 3–4 days after initial priming and was dependent on CD4⁺ T cell help, likely through a CD28:CD80/86 mediated pathway. CD4⁺ T cell or CD25-deficiency led to normal early effector CD8⁺ T cell differentiation, but a subsequent lack of accumulation of CD8⁺ T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1^high CD127^low short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8⁺ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4⁺ T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8⁺ T cells, rather than “programming” memory cell traits.     

Acquisition of HIV type 1 resistance by beta-chemokine-producing CD4+ T cells.

The CD4+ T cell is a major target cell type for human immunodeficiency virus type 1 (HIV-1) infection. In this study, we provide evidence that the susceptibility to HIV-1 infection is variable in individual CD4+ T cells. Five CD4+ T cell clones were isolated from an HIV-1-seronegative donor and were investigated for their susceptibility to HIV-1 infection. Four CD4+ T cell clones were resistant to infection by a macrophage-tropic (R5) HIV-1 isolate whereas one clone was fully permissive. The level of susceptibility to HIV-1 correlated inversely with beta-chemokine production, including RANTES (regulated on activation, normally T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. Resistance to HIV-1 infection was abrogated by the combined use of neutralizing antibodies against these three beta-chemokines. Interestingly, a complete inhibition of HIV-1 infection was observed in peripheral blood mononuclear cells on infection induced by adding the culture supernatant or a small number of HIV-1-resistant cell clones. Our results suggest the presence of a clonal self-defense mechanism within the CD4+ T cell population in vivo that involves the secretion of beta-chemokines.     

Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells

The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of beta-galactoside-binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4+CD25+ T cells. Reduced regulatory activity was observed in CD4+CD25+ T cells obtained from galectin-1-homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells.     

Inhibition of HIV strains by GB virus C in cell culture can be mediated by CD4 and CD8 T-lymphocyte derived soluble factors

OBJECTIVE: A number of studies concerning the pathogenesis of GB virus C (GBV-C) in HIV-infected people suggest a beneficial effect and improved survival for dually infected individuals. However there has remained controversy regarding the clinical relevance of these findings, as some studies have not confirmed these observations. To address the possibility of direct inhibitory mechanisms, we studied the impact of GBV-C on HIV-1 replication in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with sera from GBV-C positive individuals or transfected with GBV-C specific RNA and superinfected with HIV. Replication kinetics of HIV were studied by quantification of HIV-p24 release. Induction of soluble antiretroviral factors were monitored with an HIV infection assay and by quantification of chemokine secretion. Changes in chemokine receptor expression were analysed by flow cytometry. RESULTS: We demonstrate that GBV-C infection of PBMC leads to significant replication inhibition of R5- and X4-HIV isolates representing eight HIV clades. The inhibitory effect is mediated by GBV-C infection and also by expression of GBV-C structural glycoproteins and/or of non-structural proteins NS2/NS3. Upon GBV-C infection CD4 and CD8 T lymphocytes produce soluble HIV-suppression factors. Induction of stromal cell-derived factor (SDF)-1 and subsequent internalization of CXCR4 was not observed. CONCLUSIONS: CD4 and CD8 T lymphocytes are stimulated by GBV-C to secrete antiretroviral factors, inhibiting R5- and X4-HIV strains. As no induction of SDF-1 and no down-regulation of the respective receptor CXCR4 could be observed, it is likely that additional unidentified factors causing inhibition of X4-HIV strains are induced by GBV-C.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Virus-induced delayed-type hypersensitivity reaction is sequentially mediated by CD8+ and CD4+ T lymphocytes

After subcutaneous inoculation into the hind foot of a mouse, the lymphocytic choriomeningitis (LCM) virus multiplies locally, attaining 10(7)-10(8) mouse infectious units per g of tissue; elimination commences around day 7. About 1 day earlier, the foot begins to swell, which is regarded as a delayed-type hypersensitivity (DTH) reaction. To answer the question of whether the local inflammatory response is involved in virus clearance, we needed to known what cells mediate both these phenomena. With three different procedures--namely, depletion in vivo of defined cells by treatment of mice with monoclonal antibodies ("serologic surgery"), adoptive immunization with negatively selected cells, and adoptive immunization with cells from mice differing at the major histocompatibility gene complex--it is shown that the LCM virus-induced local DTH reaction consists of two phases that are sequentially mediated by (first) class I-restricted cytotoxic/suppressive CD8+ and (second) class II-restricted helper/inducer CD4+ T lymphocytes. In contrast, for virus elimination only the former subset of T lymphocytes was found to be needed. Thus, an association may exist between the CD8+ cell-mediated component of the local DTH response and control of the infection, but the CD4+ cell-mediated part appears to be of doubtful antiviral relevance.     

Proliferative pathways in CD1- CD3+ CD4+ CD8+ T-prolymphocytic leukemic cells: analysis with monoclonal antibodies and cytokines

The antigenic profile and the proliferative pathways in leukemic cells from the patient TRT with T-prolymphocytic leukemia (T-PLL) were analyzed using monoclonal antibodies (MoAbs) and cytokines. T-PLL cells expressed the phenotype CD1- CD3+ CD4+ CD8+. Incubation with the differentiating agent phorbol-12-myristate-13-acetate markedly increased the percentage of cells with the CD4- CD8+ phenotype, suggesting that leukemic cells were already committed towards a differentiated element with the CD4- CD8+ phenotype. T-PLL cells were induced to proliferate by anti-CD2 MoAb 9-1 + 9.6 and by anti-CD3 MoAb OKT3. The two pathways exhibited normal functional interactions and were susceptible to modulation by anti-HLA class I MoAbs. These results indicate that regulation of cell proliferation was preserved to a significant extent in the T-PLL cells analyzed. At variance with normal resting T cells that require previous activation to proliferate when incubated with interleukin-1 (IL-1) or interleukin-2 (IL-2), T-PLL cells proliferated vigorously when incubated with either interleukin. Furthermore, T-PLL cells proliferated when incubated with immune interferon (IFN-gamma). The latter finding parallels the enhancement by IFN-gamma of the proliferative response of lectin-activated murine T lymphocytes. These results suggest that T-PLL cells, which express a high constitutive level of c-myc mRNA, may be in an activated state. The antigenic phenotype and the characteristics of the proliferative pathways of T-PLL cells from the patient TRT are compatible with the possibility that they may be derived from an intermediate thymocyte.     

HIV-1 infection and expression in human colonic cells: infection and expression in CD4+ and CD4- cell lines

Three human colonic epithelial cell lines, SW620, HT29, and T84, were characterized with respect to HIV-1 infection and gene expression. SW620 and HT29, but not T84, could be infected with HIV-1. CD4 messenger RNA and its protein product were identified in SW620 cells but not in HT29 or T84 cells. Anti-CD4 antibody blocked infection of SW620 cells but had no effect on infection of HT29 cells. In SW620 and HT29 cells transfected with the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, an intact HIV-1 enhancer element was required for stimulation of CAT activity by tumor necrosis factor alpha (TNF alpha) and phorbol ester. T84 was not able to mediate a TNF alpha or phorbol ester response. These studies provide further evidence that HIV-1 can infect cells by mechanisms other than those mediated by the CD4 receptor and describe complementary models for analyzing HIV-1 infection and expression in colonic epithelial cells.     

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+ T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 <200 cell/mm3) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.     

A mouse mutant p53 product recognized by CD4+ and CD8+ T cells.

The T-cell response to mutated and normal p53 products of BALB/c-derived Meth A sarcoma was analyzed. Meth A p53 is known to have three missense point mutations in codons 132, 168, and 234, and 24 peptides containing wild-type or mutated sequences at the three mutation sites were constructed. Spleen cells from BALB/c or (BALB/c x C57BL/6)F1 mice immunized with p53 peptides were sensitized in vitro with the corresponding peptides. Because Meth A is resistant to cytotoxic T cells, the sensitive P1-HTR cell line, which expresses a low level of p53 lacking the Meth A p53 mutations, was chosen as a target, either pulse-labeled with p53 peptides or transfected with plasmids containing coding sequences from Meth A p53. One peptide, a nonamer containing the codon 234 mutation (234CM), induced CD8+ cytotoxic T cells that lysed 234CM-pulsed P1-HTR cells in an H-2Kd-restricted fashion. P1-HTR cells pulsed with the corresponding wild-type peptide were only weakly lysed by 234CM-reactive cytotoxic T cells. P1-HTR cells pulsed with other wild-type or mutated p53 peptides were not lysed by 234CM-reactive cytotoxic T cells, nor could these peptides, including 234CW (the wild-type counterpart to 234CM), elicit cytotoxic cells. P1-HTR cells transfected with plasmids coding for the 234CM sequence and expressing high p53 levels were weakly lysed by 234CM-reactive cytotoxic T cells. However, lysis of one of the transfectants was significantly increased by pretreatment with interferon gamma. A proliferative response of CD4+ T cells was elicited by immunization with 234CM and 234CW, but not with other p53-related peptides. The specificity of 234CM-induced CD4+ T cells for 234-region peptides was broader than the reactivity of 234CM-reactive cytotoxic T cells. Mice immunized with 234CM in incomplete Freund's adjuvant showed heightened resistance to Meth A challenge.     

CD8+CD38+ T cells but not HIV type 1 RNA viral load predict CD4+ T cell loss in a predominantly minority female HIV+ adolescent population

The purpose of this study was to evaluate predictors of HIV-1 disease progression in a cohort of predominantly female and minority adolescents who had acquired their HIV-1 infections through sexual risk behaviors. Subjects were identified from the REACH cohort who were not on antiretroviral therapy for at least 1 year and whose baseline CD4(+) T cells were >300 cells/mm(3). Biomedical and demographic characteristics of the subjects at the start of the study period were evaluated as predictors of CD4(+) T cell loss in univariate and multivariate models. Two-thirds of the 99 subjects meeting the selection criteria were female and 87% were black or Hispanic similar to the REACH cohort as a whole. Higher absolute CD8(+) CD38(+) T cell counts at the start of the assessment period were associated with a greater rate of loss of CD4(+) T cells. HIV-1 RNA viral load was among other potential predictors of HIV-1 disease progression that had no association with the rate of CD4(+) T cell loss in this cohort. This study extends the observed association of higher CD8(+) CD38(+) T cells numbers being predictive of HIV-1 disease progression into predominantly female, minority youth.     

Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection

Peripheral blood and thymic double-positive (DP) CD4(+)CD8(+) T cells from neonates have been described earlier, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here, we demonstrate the functional and immunophenotypic characteristics of DP cells in 6 different tissues, including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0 and 21 days of age. In general, intestinal DP T cells of neonates have higher percentages of memory markers (CD28(+)CD95(+)CD45RA(low)CD62L(low)) and proliferation compared with single-positive (SP) CD4(+) and CD8(+) T cells. In addition, percentages of DP T cells increase and CD62L expression decreases as animals mature, suggesting that DP cells mature and proliferate with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of CCR5 and are the primary targets in simian immunodeficiency virus (SIV) infection. Finally, DP T cells produce higher levels of cytokine in response to mitogen stimulation compared with SP CD4(+) or CD8(+) T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, activated memory cells and are likely involved in regulating immune responses, in contrast to immature DP T cells in the thymus.