Differing roles of inflammation and antigen in T cell proliferation and memory generation

Recent studies have demonstrated that viral and bacterial infections can induce dramatic in vivo expansion of Ag-specific T lymphocytes. Although presentation of Ag is critical for activation of naive T cells, it is less clear how dependent subsequent in vivo T cell proliferation and memory generation are upon Ag. We investigated T cell expansion and memory generation in mice infected alternately with strains of Listeria monocytogenes that contained or lacked an immunodominant, MHC class I-restricted T cell epitope. We found substantial differences in the responses of effector and memory T cells to inflammatory stimuli. Although effector T cells undergo in vivo expansion in response to bacterial infection in the absence of Ag, memory T cells show no evidence for such bystander activation. However, Ag-independent expansion of effector T cells does not result in increased memory T cell frequencies, indicating that Ag presentation is critical for effective memory T cell generation. Early reinfection of mice with L. monocytogenes before the maximal primary T cell response induces typical memory expansion, suggesting that the capacity for a memory T cell response exists within the primary effector population. Our findings demonstrate that T cell effector proliferation and memory generation are temporally overlapping processes with differing requirements for Ag.     

Mathematically modeling dynamics of T cell responses: predictions concerning the generation of memory cells

Mathematical models of T cell population dynamics after infection typically assume that T cells differentiate according to a linear process in which they first become effector cells, and then after some time, differentiate further into memory cells. In this paper, we offer a different mathematical model which can equally well capture T cell dynamics, using data from lymphocytic choriomeningitis (LCMV) infection. Our model assumes that memory cells are intermediates that further differentiate into effector cells only from additional or stronger antigenic stimulation. Our assumption naturally leads to a testable prediction about the generation of T cell memory-that the memory phenotype of T cells should be present in detectable numbers during the expansion phase of the response. We use our model to estimate a rate of differentiation from memory type cells to effectors. We argue that this differentiation assumption, where memory cells are intermediates, captures recent experimental work on T cell differentiation, and hence this new mathematical model could be helpful in doing further studies of T cell population dynamics. We also propose a method of distinguishing the models by examining the ratio of memory T cells detectable long after an infection to the peak numbers of T cells at the end of the expansion phase.     

Population dynamics of naive and memory CD8 T cell responses after antigen stimulations in vivo

The extent to which the progeny of one primary memory CD8 T cell differs from the progeny of one naive CD8 T cell of the same specificity remains an unresolved question. To explore cell-autonomous functional differences between naive and memory CD8 T cells that are not influenced by differences in the priming environment, an experimental model has been developed in which physiological numbers of both populations of cells were cotransferred into naive hosts before Ag stimulation. Interestingly, naive CD8 T cells undergo greater expansion in numbers than do primary memory CD8 T cells after various infections or immunizations. The intrinsic ability of one naive CD8 T cell to give rise to more effector CD8 T cells than one memory CD8 T cell is independent of the number and quality of primary memory CD8 T cells present in vivo. The sustained proliferation of newly activated naive CD8 T cells contributed to their greater magnitude of expansion. Additionally, longitudinal analyses of primary and secondary CD8 T cell responses revealed that on a per-cell basis naive CD8 T cells generate higher numbers of long-lived memory cells than do primary memory CD8 T cells. This enhanced "memory generation potential" of responding naive CD8 T cells occurred despite the delayed contraction of secondary CD8 T cell responses. Taken together, the data in this study revealed previously unappreciated differences between naive and memory CD8 T cells and will help further define the functional potential for both cell types.     

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1(58-66) can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.     

Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence

Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN-gamma plus single IFN-gamma) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN-gamma CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.     

Human T cell responses to gp63, a surface antigen of Leishmania

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.     

Discrete event modeling of CD4+ memory T cell generation

Studies of memory T cell differentiation are hampered by a lack of quantitative models to test hypotheses in silico before in vivo experimentation. We created a stochastic computer model of CD4+ memory T cell generation that can simulate and track 10(1)-10(8) individual lymphocytes over time. Parameters for the model were derived from experimental data using naive human CD4+ T cells stimulated in vitro. Using discrete event computer simulation, we identified two key variables that heavily influence effector burst size and the persistent memory pool size: the cell cycle dependent probability of apoptosis, and the postactivation mitosis at which memory T cells emerge. Multiple simulations were performed and varying critical parameters permitted estimates of how sensitive the model was to changes in all of the model parameters. We then compared two hypotheses of CD4+ memory T cell generation: maturation from activated naive to effector to memory cells (model I) vs direct progression from activated naive to memory cells (model II). We find that direct progression of naive to memory T cells does not explain published measurements of the memory cell mass unless postactivation expansion of the memory cell cohort occurs. We conclude that current models suggesting direct progression of activated naive cells to the persistent memory phenotype (model II) do not account for the experimentally measured size of the postactivation CD4+, Ag-specific, memory T cell cohort.     

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8(+) T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH(-/-) mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH(-/-)leucine aminopeptidase(-/-) progeny show a selective increase in CD8(+) T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.     

Lymphotoxin-alpha-deficient mice make delayed,but effective,T and B cell responses to influenza

Lymphotoxin-alpha(-/-) (LTalpha(-/-)) mice are thought to be unable to generate effective T and B cell responses. This is attributed to the lack of lymph nodes and the disrupted splenic architecture of these mice. However, despite these defects we found that LTalpha(-/-) mice could survive infection with a virulent influenza A virus. LTalpha(-/-) mice and normal wild-type mice infected with influenza A generated similar numbers of influenza-specific CD8 T cells that were able to produce IFN-gamma and kill target cells presenting influenza peptides. Furthermore influenza-infected LTalpha(-/-) mice produced high titers of influenza-specific IgM, IgG, and IgA. However, both CD8 and B cell immune responses were delayed in LTalpha(-/-) mice by 2-3 days. The delayed cellular and humoral immune response was sufficient to mediate viral clearance in LTalpha(-/-) mice that were infected with relatively low doses of influenza virus. However, when LTalpha(-/-) mice were infected with larger doses of influenza, they succumbed to infection before the immune response was initiated. These results demonstrate that neither LTalpha nor constitutively organized lymphoid tissues, such as lymph nodes and spleen, are absolutely required for the generation of effective immunity against the respiratory virus influenza A. However, the presence of LTalpha and/or lymph nodes does accelerate the initiation of immune responses, which leads to protection from larger doses of virus.     

T cell tolerance to a neo-self antigen expressed by thymic epithelial cells: the soluble form is more effective than the membrane-bound form

We have previously shown that transgenic (Tg) mice expressing either soluble or membrane-bound hen egg lysozyme (sHEL or mHEL, respectively) under control of the alphaA-crystallin promoter develop tolerance due to thymic expression of minuscule amounts of HEL. To further address the mechanisms by which this tolerance develops, we mated these two lines of Tg mice with the 3A9 line of HEL-specific TCR Tg mice, to produce double-Tg mice. Both lines of double-Tg mice showed deletion of HEL-specific T cells, demonstrated by reduction in numbers of these cells in the thymus and periphery, as well as by reduced proliferative response to HEL in vitro. In addition, the actual deletional process in thymi of the double-Tg mice was visualized in situ by the TUNEL assay and measured by binding of Annexin V. Notably, the apoptosis localized mainly in the thymic medulla, in line with the finding that the populations showing deletion and increased Annexin V binding consisted mainly of single- and double-positive thymocytes. Interestingly, the thymic deletional effect of sHEL was superior to that of mHEL in contrast to the opposite differential tolerogenic effects of these HEL forms on B cells specific to this Ag. Analysis of bone marrow chimeras indicates that both forms of HEL are produced by irradiation-resistant thymic stromal cells and the data suggest that sHEL is more effective in deleting 3A9 T cells due mainly to its higher accessibility to cross-presentation by dendritic APC.     

Dendritic cell-T cell interactions in the generation and maintenance of CD8 T cell memory

Dendritic cells (DCs) are the critical antigen-presenting cells involved in initiating CD8 T cell responses to microbial and viral pathogens. Hence the generation of memory T cells from naïve T cells is intricately intertwined with DCs at every level. This review broadly addresses DC-CD8 T cell interactions that result in the generation and maintenance of CD8 memory T cells.     

Suppression of memory CD8 T cell generation and function by tryptophan catabolism

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

Antigen-specific B cells efficiently present low doses of antigen for induction of T cell proliferation

Previous experiments suggested a role for specific B cells in the induction of antigen (SRBC)-specific T cell proliferation. Two models were proposed: in the first, B cells directly presented antigen to T cells; alternatively, B cells secreted antibody, which opsonized antigen for presentation by macrophages. Experiments to distinguish between these possibilities are presented here. Three lines of evidence support the conclusion that antigen is presented directly by specific B cells. First, nonimmune splenic adherent cells (SAC), which efficiently induced proliferation of appropriately primed T cells to antigens such as OVA and GAT, were unable to induce SRBC-specific proliferation. Secondly, a slope analysis of the logarithmic plot of T cell proliferation vs the number of irradiated B cells suggested that two cells were limiting within the presenting population. The addition of IL 1 or SAC reduced the slope to 1 (although in serum-free conditions, the addition of IL 1, but not SAC, reduced the slope of the line). Specificity of the B cells for the antigen continued to be required in the presence of exogenous IL 1 or SAC. These results suggested that presentation by specific B cells and the amount of IL 1 were the limiting requirements for the induction of SRBC-specific T cell proliferation. The third line of evidence was the demonstration of a restricted interaction between T cells and B cells. The addition of irradiated, allogeneic SRBC-specific B cells to T cell lines and syngeneic SAC failed to support proliferative responses. We further show that a GAT-specific T cell clone was triggered to proliferate by either SAC or B cells, but that antigen-specific B cells were necessary at low doses of antigen. This finding is important in two respects. First, the T cell clone previously has been shown to act as a helper; secondly, when low doses of antigen are used, the requirement for priming of the B cells to the specific antigen is true for a soluble, as well as a particulate, antigen. We propose that at low (physiologic) doses of antigen, presentation to secondary T cells takes place mainly at the surface of antigen-specific B cells. At high doses of antigen,h presentation can also be accomplished by nonspecific cells such as other B cells, macrophages, or dendritic cells.     

Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory

Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.     

Selection of similar naive T cell repertoires but induction of distinct T cell responses by native and modified antigen

To study the T cell responses induced by native and modified Ag, we have followed in vivo TCR selection and cytokine profile of T cells, as well as the isotype of induced Abs, in response to the model Ag hen egg-white lysozyme (HEL) and its reduced and carboxymethylated form (RCM-HEL). RCM-HEL induces in vivo a T cell response focused on the same immunodominant determinant characterizing the response to native HEL, but further skewed to the Th1 pathway. No difference between HEL and RCM-HEL could be observed in the efficiency of processing, nor in the type of APCs involved. In vivo experiments show that coimmunization with HEL and RCM-HEL generates distinct Th2 or Th1 responses in naive mice, but the two forms of Ag expand the same HEL-specific public clonotype, characterized by the Vbeta8.2-Jbeta1.5 rearrangement, indicating that the populations of naive T cells activated by the two Ag forms overlap. T cells primed by RCM-HEL are restimulated by soluble HEL in vivo, but divert the phenotype of the HEL-specific response to Th1, implying that priming of naive T cells by a structurally modified Ag can induce Th1-type memory/effector T cells more efficiently than native Ag.     

T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.