The helper cell function of primed T cells. I. Marked amplification of antibody formation by antigen-educated T cells carrying surface Ig 6 h after priming

These experiments deal with a subpopulation of T cells bearing adsorbed Ig, thus considered to be Fc-receptor positive. Such cells were collected from spleens of lethally irradiated mice reconstituted with thymocytes 6 h after priming with heterologous erythrocytes (sheep or horse red blood cells). The helper cell function of such cells was examined upon transfer to secondary hosts with normal bone marrow cells. Such educated thymocytes upon challenge generated a 2–3-fold increase of 19 S plaque-forming cells (PFC) and a 6–7-fold increase of 7 S PFC above the level given by unprimed thymocytes. Such amplification of antibody formation is highly specific. No helper cell function was detected in cells obtained similarly from nonreconstituted animals or from mice reconstituted with bone marrow cells. Carrier-primed thymocytes collected 6 h after priming are also able to enhance the primary 7 S anti-hapten response but only when the hapten is provided on the same carrier used for priming. The helper cell function of 6 h primed thymocytes was compared to that of more conventionally educated thymocytes collected 5 days after priming. Although both cell preparations induced similar enhancements of 19 S PFC, the 6 h primed cells gave more than twice the number of 7 S PFC as compared to 5 day primed cells. This difference is statistically highly significant (p < 0.001). Since the 6 h primed thymocytes contain a subpopulation which has acquired surface Ig, as shown by our previous work, their role in the mechanism of the enhancement of the helper cell function is discussed.     

Requirement for interactions between two subpopulations of T cells for helper cell induction in vitro.

The evidence for the interaction of 2 subpopulations of T cells, short-lived cells sensitive to adult thymectomy (T1 cells), and long-lived recirculating cells, sensitive to the action of antilymphocyte serum (T2 cells) in the induction of helper cells is presented. This T-T interaction occurred across a cell-impermeable nucleopore membrane, indicating that it did not depend on cell contact, but was mediated by subcellular factors. There was no genetic restriction on this T-T interaction, if it was performed across a nucleopore membrane. The implications of these results on our concepts of the mechanism of help are discussed.     

T-T cell interactions during cytotoxic T cell responses IV. Murine lymphoid dendritic cells are powerful stimulators for helper T lymphocytes

Enriched populations of Ia⁺ Fc receptor-negative dendritic cells were compared to other cell types for their stimulatory activity in primary mixed lymphocyte reactions to alloantigens and 2,4,6-trinitrophenylated syngeneic cells. Dendritic cells were 20-100 times more effective than unfrationated splenocytes. A second cell type exhibiting strong stimulatory activity was an Ia⁺ Fc receptor-positive transiently adherent cell. Both types of stimulatory cells were only effective when able to produce the monokine interleukin 1. Thus glutaraldehyde-fixed cells were not stimulatory unless extraneous interleukin 1 was added. Stimulation of helper cells by either dendritic cells or Ia⁺ Fc receptor-positive cells resulted in the production of interleukin 2. The data are discussed in view of the recently formulated interleukin model as a minimal scheme to explain T-T cell interactions during the in vitro induction of cytotoxic T lymphocytes.     

Stimulation of a memory B cell response does not require primed helper T cells

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross-reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non-overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.     

Recruitment of functional subpopulations of T lymphocytes. Initiator T lymphocytes recruit helper T cells for cytotoxic response

Initiator T (Ti) lymphocytes are defined by their ability to recruit other T cell populations in vivo. In this study the function of T cells recruited into draining lymph nodes following injection of Ti cells primed to alloantigens in mixed lymphocyte culture was examined. The results demonstrate that alloantigen-specific helper T cells that interact with cytotoxic T (Tc) lymphocyte precursors are recruited, as shown by the significantly higher frequencies of helper cells in draining lymph nodes compared with controls. However, neither Tc cells nor their precursors are recruited. Recruitment by Ti lymphocytes is therefore selective for certain T cell subsets. Proposals to explain the mechanism, specificity, and selectivity of recruitment are discussed. We suggest that Ti cells have a central role in both the initiation of T cell-dependent immune responses and in the maintenance of immunologic memory. Their function is the rapid mobilization of T cell subclasses to a regional lymphoid organ where the immune response subsequently develops.     

Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations.

Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-gamma) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL-10, and significantly less IFN-gamma, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-gamma production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Th1 subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss.     

Antigen presentation by a subset of T8+ Ia+ cells to T4+ helper cells.

The helper function of human T4+ cells acting on autologous peripheral blood B cells was elicited by the hapten-carrier conjugate DNP-streptococcal antigen (DNP-SA), in the presence of monocyte-enriched cells. The antigen presenting function of monocyte-enriched cells can be replaced by a subset of autologous T8+ Vicia villosa lectin adherent (T8VV+) cells. Cell depletion studies confirmed that the T8VV+ cell presentation was mediated by a T cell since killing with anti-T3 antibody and complement removed the antigen presenting capacity of T8VV+ cells but not of monocyte-enriched cells. Furthermore, killing with anti-Ia specific monoclonal antibody and complement abrogates antigen presentation by the T8VV+ cells, suggesting that the latter express an Ia gene product. The antigen presentation is specific to DNP-SA which elicits anti-DNP IgM antibodies, as the latter are not produced in response to tetanus toxoid. We suggest that interactions between T cells may occur not only by T4+ cells inducing T8+ suppressor cell functions but also the reverse activity of T8+ cell presenting antigen to T4+ cells to induce helper function in the absence of other accessory cells.     

Limiting dilution analysis of helper T-cell function. II. An approach to the study of the function of single helper T cells.

Procedures have been described which are suitable for experiments utilizing a microculture system whereby one can easily study the function of single helper T cells. These are: (a) the elimination of the background anti-hapten response by preliminary anti-theta + guinea-pig complement complement treatment of the 'hapten-primed' population; (b) the removal of any contaminating hapten-specific B cells from the carrier-primed population by passage through nylon wool; (c) irradiation of long-term primed 'nylon wool' enriched helper cells tp prevent cell division; (d) the partition of individual helper cells by limiting dilution.     

B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27⁺ and CD27^? cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27⁺ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor-related transmembrane ligand, CD70. Adult peripheral blood CD27⁺ B cells can be further subdivided into two discrete subtypes: IgD^?CD27⁺ and IgD⁺ CD27⁺ B cells. IgD^? CD27⁺ B cells produce IgG, IgM and IgA, whereas IgD⁺ CD27⁺ B cells predominantly produce IgM. The addition of activated CD4⁺ CD45RO T cells expressing CD70 caused down-regulation of CD27 expression on activated B cells, and this down-modulation was completely blocked by anti-CD70 monoclonal antibody, indicating direct T-B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin-2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27⁺ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.     

Autoreactive T cells in rheumatic disease. II. Function and specificity of an autoreactive T helper cell clone established from a HLA-B27+ reactive arthritis

T cell lines were established by limiting dilution of peripheral blood (PBL) and synovial fluid lymphocytes (SFL) of a patient with HLA-B27+ reactive arthritis. Among these cell lines, the CD4 phenotype was dominant. Functionally, the majority of these cell lines exhibited helper activity for the immunoglobulin production by autologous B cells and proliferated in response to autologous mononuclear cells. In most cases, this autoreactive response was associated with alloreactivity. Only one cell line, the autoreactive CD4+ T cell clone, UA-S2, which was derived from the synovial fluid, proliferated in a highly specific manner in response to a determinant associated with MHC class II products present on autologous mononuclear cells. The restriction element was shown to be associated with DR molecules by inhibition experiments with monoclonal antibodies. Within the patient's family, the capacity of mononuclear cells to stimulate a proliferative response of UA-S2 segregated together with the HLA haplotype A2 or 32, B27, Cw1, DRw11 which was contributed by the patient's mother. UA-S2 proved to be a functional helper T cell clone. In the absence of additional antigen or mitogen, it induced IgG and IgM synthesis of autologous and family members' B cells. This helper activity of UA-S2 showed the same MHC restriction as the proliferative response. Although the patient's father also typed DRw11, this haplotype was not recognized by UA-S2. It is suggested that this autoreactive T cell clone detects a microheterogeneity of the serologically defined DRw11 haplotype. Indeed, typing of the patient's family members with cellular reagents established a difference between the two DRw11 haplotypes.     

Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.     

The function of T cells carrying receptors for complexes of Ig and antigen.

Thymocytes exposed briefly in vitro to a variety of particulate substances (such as mycobacteria, erythrocytes of allogeneic cells) or to substances known to act in vivo as adjuvants (LPS or poly A:U), generate supernates which are able to induce cytophilic Ig in normal mouse serum in the presence of a foreign protein (antigen). This cytophilic Ig is taken up by 20-25% of splenic T cells. Hydrocortisone resistant thymocytes show the same property, while bone marrow cells are inactive. This activity is similar to that reported previously as being present in the 4S fraction of mouse serum, collected 6 hours after injection of complete Freund's adjuvant. It is proposed that this factor is responsible for the formation of complexes of Ig and antigen which have been detected in the serum 6 hours after immunization. Thymocytes collected 6 hours after priming in vivo with SRBC (when a subpopulation among them carries easily demonstrable surface Ig) are able to amplify markedly the antibody response particularly the 7S. It is postulated that the factor by generating the cytophilic Ig (complexes?) which is taken up by T cells, sets up a mechanism which markedly amplifies their helper cell function.     

T cell subpopulations in inflammatory bowel disease: evidence for a defective induction of T8+ suppressor/cytotoxic T lymphocytes.

Abnormal immune responses associated with inflammatory bowel disease may reflect a defect in immunoregulatory functions. To analyse T-T cell cooperation, we examined the influence of polyclonal activation with the mitogen phytohaemagglutinin on the distribution of the T4+ helper/inducer and the T8+ cytotoxic/suppressor T cell subset. Compared to controls, lymphocytes from patients with Crohn's disease displayed a slight reduction in T3+ cells; neither in patients with ulcerative colitis nor in patients with Crohn's disease a significant difference in T4+ and T8+ cells and the T4/T8 ratio was observed. Phytohaemagglutinin stimulation of normal lymphocytes resulted in a decrease of the T4 subset and a clear increase of the T8 subpopulation. In contrast, the subset distribution pattern was not changed by the mitogenic stimulation in any of the patients. This abnormal reaction pattern could not be influenced by the addition of interleukin-2. Small numbers of normal lymphocytes, however, were able to restore the predominant proliferation of T8+ cells. Thus, the reduced response of T8+ lymphocytes cannot be attributed to an altered composition of this subset in patient with inflammatory bowel disease; our results provide evidence for a defective induction of T8+ suppressor/cytotoxic T cells, which is independent from disease activity.     

Increased expression of CD40 on thymocytes and peripheral T cells in autoimmunity: a mechanism for acquiring changes in the peripheral T cell receptor repertoire.

CD40, a cell surface molecule found on B lymphocytes and other antigen presenting cells, can, when engaged by CD40 ligand (CD40L), induce gene rearrangements and isotype switching. We report here that CD40 is also expressed on thymocytes and on up to 50% of peripheral T cells from autoimmune prone strains of mice. In normal animals, CD40 is present on a small population of T cells and thymocytes. CD40 is expressed on most T cell hybridomas. We demonstrate that CD40 engagement on peripheral T cells, T cell hybridomas and thymocytes results in altered TCRValpha expression. That induced expression of different Valpha's results from the activity of the recombinase gene is implied by the observation that CD40 does not induce TCR changes in RAG knock-out mice. Total cell numbers remained unchanged between anti-CD40 treated and untreated populations of thymocytes or T cells indicating that treatment does not induce cell proliferation or cell death. The data presented here suggest a mechanism by which self reactive T cells accumulate peripherally and independently of selective processes of the thymus.     

HIV virus, prostaglandins and essential fatty acids; a suggested mechanism for T helper cell penetration

Prostaglandins and or its precursors are present in certain body fluids. It has been suggested that critical concentrations of PGE are essential for the HIV to penetrate a lymphocyte. Certain clinical implications of this hypothesis have been discussed.     

Angioimmunoblastic T‐cell lymphoma: more than a disease of T follicular helper cells

Angioimmunoblastic T‐cell lymphoma (AITL) is one of the most frequent entities of peripheral T‐cell lymphoma. An AITL has two components: the AITL tumour cells, which have a T follicular helper (TFH) cell phenotype, and a surrounding and extensive tumour microenvironment that is populated with various reactive cell types, including B cells. Recurrent TET2 mutations have been described in 50–80% of AITLs, possibly occurring in a haematopoietic progenitor cell. An article published recently in the Journal of Pathology describes the use of microdissection to isolate PD1⁺ AITL tumour cells and CD20⁺ B cells from the AITL microenvironment, and to show that TET2 mutations are actually more frequent in these diseases than previously thought. Whereas TET2 mutations were detected in only six of 13 AITLs, 12 of 13 samples of microdissected PD1⁺ AITL tumour cells possessed this mutation. Moreover, TET2 mutations were detected in CD20⁺ B cells from the AITL microenvironment in six of nine informative cases. These results confirm that TET2 mutation is an early event in the majority of AITL cases, and that the driving molecular anomalies are not restricted to the T lineage tumour cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Enrichment of nucleofected primary human CD4+ T cells: a novel and efficient method for studying gene function and role in human primary T helper cell differentiation

Identification of key factors mediating the differentiation of naïve CD4⁺ T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4⁺ T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2K^k cell surface marker followed by selection of H-2K^k positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2K^k positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4⁺ T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.     

Cell cooperation during in vivo anti-hapten antibody responses: V. Two synergistic Ly-1+23− helper T cells with distinctive specificities

Experiments were carried out to determine the antigen specificity of two distinct helper T cells (Th) that act synergistically in adoptive secondary in vivo anti-hapten antibody responses. Both Th were present in anti-Ly-2 and complement-treated spleen T cell populations, implying that both Th are Ly-1⁺,23^?. Adding normal T cells or T cells primed to other carriers to specific carrier-primed T cells, using a variety of different protocols did not affect the helper activity of the specifically primed Th. Thus, both Th apparently are antigen-specific. Furthermore, Th primed with one carrier and boosted with that carrier plus hapten linked to a noncross- reactive carrier cannot help B cells. However, if a mixture of Ly-1 T cells from mice primed with two different carriers is transferred along with B cells, and the mice are boosted with hapten coupled to one of the two priming carriers, then giving the other carrier induces a significant increase in antibody production. Thus, only one of the two Th (Th1) requires a hapten-carrier bridge, while the other does not (Th2). However, both Th 1 and Th2 are clearly antigen-specific and require stimulation with antigen to exert helper activity. Furthermore, these experiments strongly suggest that Th 2 cannot express helper function in vivo in the absence of Th 1. These findings, and the absence of Th2-like cells in agammaglobulinemic mice, were correlated with other studies in which two helper activities have been described. It was concluded that in vivo responses require an effective Th 1-B cell interaction, whereas Th2, if stimulated with antigen, will augment certain portions of the antibody response, such as idiotype or allotype, and thus influence the quality of the antibody response directly.     

Effect of cyclosporin A on T cell immunity. II. Defective thymic education of CD4 T helper cell function in cyclosporin A-treated mice

Cyclosporin A (CsA) is an immunosuppressive agent that is widely used in transplantation. Recent animal studies indicate that CsA can affect the development of immunity so that autoreactive T lymphocytes are generated. In this study, mice were treated with CsA prior to irradiation and transplantation of syngeneic bone marrow to determine whether CsA pretreatment would affect the ability of the bone marrow recipients to develop normal T cell function. Our results indicate that (a) thymuses of CsA-treated mice do not contain single-positive thymocytes (i.e. L3T4+Ly-2- or L3T4-Ly-2+) during i.p. treatment with 15 mg/kg/day of CsA; (b) both populations of single-positive thymocytes reappear within 2 weeks of termination of CsA and (c) irradiation and bone marrow reconstitution of these CsA-treated mice results in reconstitution of normal numbers of L3T4+ and Ly-2+ cells, but the L3T4+ T cells to not provide T helper function, as determined by interleukin 2 production and cytotoxic T lymphocytes generation. These findings indicate that CsA can affect thymic microenvironment and may be important as a model for investigating intrathymic T cell maturation. Our results may also have clinical implications for T lymphocyte development in transplant patients receiving CsA.