Dysregulation of TFH-B-TRM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR-mutant lung cancer.

Patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations exhibit an unfavorable response to PD-1 inhibitor through unclear mechanisms. Hypothesizing that EGFR mutations alter tumor-immune interactions, we compare tumor-infiltrating lymphocytes between EGFR mutant (EGFR-MT) and wild type (EGFR-WT) tumors through single-cell transcriptomic analysis. We find that B cells, CXCL13-producing follicular helper CD4+ T (TFH)-like cells, and tissue-resident memory CD8+ T (TRM)-like cells decreased in EGFR-MT tumors. The NOTCH-RBPJ regulatory network, which is vital for persistence of TRM state, is perturbed, and the interactions between TFH and B cells through the CXCL13-CXCR5 axis disappear in EGFR-MT tumors. Notably, the proportion of TRM-like cells is predictive for anti-PD-1 response in NSCLC. Our findings suggest that the impairment of TFH-B-TRM cooperation in tertiary lymphoid structure formation, accompanied by the dysregulation of TRM homeostasis and the loss of TFH-B crosstalk, underlies unfavorable anti-PD-1 response in EGFR-MT lung tumors.

The receptor Ly108 functions as a SAP adaptor-dependent on-off switch for T cell help to B cells and NKT cell development.

Humans and mice deficient in the adaptor protein SAP (Sh2d1a) have a major defect in humoral immunity, resulting from a lack of T cell help for B cells. The role of SAP in this process is incompletely understood. We found that deletion of receptor Ly108 (Slamf6) in CD4(+) T cells reversed the Sh2d1a(-/-) phenotype, eliminating the SAP requirement for germinal centers. This potent negative signaling by Ly108 required immunotyrosine switch motifs (ITSMs) and SHP-1 recruitment, resulting in high amounts of SHP-1 at the T cell:B cell synapse, limiting T cell:B cell adhesion. Ly108-negative signaling was important not only in CD4(+) T cells; we found that NKT cell differentiation was substantially restored in Slamf6(-/-)Sh2d1a(-/-) mice. The ability of SAP to regulate both positive and negative signals in T cells can explain the severity of SAP deficiency and highlights the importance of SAP and SHP-1 competition for Ly108 ITSM binding as a rheostat for the magnitude of T cell help to B cells.

CD26: a novel treatment target for T-cell lymphoid malignancies? (Review).

CD26 is a surface glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) enzyme activity with multiple biological roles, including being intricately involved in immunoregulation as a T-cell activation molecule and as a regulator of chemokine function. T-cell lymphoid malignancies represent a heterogeneous group of diseases that are generally aggressive and are for the most part resistant to current treatment modalities. Previous studies showed that CD26 is expressed on selected T-cell neoplasms, suggesting a potential role for CD26 in tumor development. We review herein recent classification schemes for T-cell lymphoid malignancies that take into account various facets of their clinical presentation. In addition, we discuss findings supporting the conclusion that CD26 has an essential role in human T-cell activation, as well as its ability to regulate the biological effects of selected chemokines through its DPPIV activity. Finally, we will present recent work from our laboratory that indicates a potential role for CD26 as a molecular target for novel treatment modalities for T-cell lymphoid malignancies.

Impaired CD40-signalling in CD19-deficient mice selectively affects Th2-dependent isotype switching.

Activation of B lymphocytes involves binding of antigen to the specific receptor and signalling through several membrane coreceptors, of which CD19 has been found to play a pivotal role as a response regulator. Although previous studies in CD19 gene knockout mice have demonstrated that antibody responses to T-cell-dependent antigens are strongly impaired in the absence of this coreceptor, little is known about the consequences of CD19 deficiency for the interaction between T and B cells. Here we report that Th2 co-ordinated B-cell differentiation is selectively impaired in CD19-deficient mice in response to mucosal or systemic immunizations or following an intestinal infection with Nippostrongylus brasiliensis. Whereas immunoglobulin (Ig)G1 or IgE antibody responses were low or absent, IgG2a responses were normal. The selective defect was not caused by a poor Th2-development or interleukin (IL)-4 responsiveness in CD19-deficient mice. Rather, it was the result of an impaired Th2-B cell interaction, owing to a substantially reduced ability to signal via CD40 in CD19-deficient B cells. Thus, our study in CD19-deficient mice suggests that CD40L-CD40-interactions are more important for Th2 than for Th1 co-ordinated B-cell differentiation.

Participación de los linfocitos Th2 en la enfermedad alérgica / Participation of lymphocites Th2 in allergic disease

Los padecimientos alérgicos son determinados genéticamente y afectan al 20 a 30 por ciento de la población de los países desarrollados. En la última década se ha observado un incremento en la prevalencia de los mismos, caracterizado por un aumento en la capacidad de los linfocitos B para producir anticuerpos inmunoglobulinas tipo E (IgE). Esta síntesis de la IgE humana resulta de la colaboración entre los subtipos de la células T auxiliadoras (Th) CD4+ y las células B. En años recientes se han estudiado intensamente las propiedades funcionales de la células Th, particularmente las tipo 2 en el mecanismo de unión de las células B productoras de IgE, células cebadas o basófilos y eosinófilos en reacciones alérgicas. Estos conceptos son conocidos en la actualidad e alergia como la teoría Th2

The logic of intercellular communication in the immune system.

The collaboration between T and B lymphocytes is used as an example to illustrate how the key features of immune regulation (cell interaction, reciprocal exchange of signals by cell contact, and dependence on soluble cytokines) serve as amplifying reactions. By linking cell-based amplifiers in sequence, the resulting immune response is made highly sensitive to small changes in the environment. Thus, intercellular communication in the immune system can be viewed as a higher level analogue to the kinase cascades that amplify intracellular signalling mechanisms.

Evidence for a critical role for IL-2 in CD40-mediated activation of naive B cells by primary CD4 T cells.

The interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.

Herpesvirus saimiri-transformed human CD4+ T cells can provide polyclonal B cell help via the CD40 ligand as well as the TNF-alpha pathway and through release of lymphokines.

We have developed human CD4+ T cell lines from the PBL of normal donors by infection with Herpesvirus saimiri (HVS), to evaluate functional properties of these immortalized lymphocytes. In this report, we characterize two such CD4+ T cell lines, CHCD4 and MHCD4, which were derived from two different donors. These cells grew independent of exogenous IL-2 stimulation for over 1 yr, and expressed surface markers (CD25+, CD69+, HLA-DR+, and B7+) associated with an activated T cell phenotype. Both lines constitutively produced and released IFN-gamma, but no IL-2 or IL-4. However, the surface expression of the two cell lines differed in that CHCD4 constitutively expressed CD40 ligand (CD40L) and membrane TNF-alpha, but MHCD4 did not. Also, CHCD4, but not MHCD4, potently induced polyclonal B cell activation and differentiation in the absence of PWM, in an MHC-unrestricted fashion. The B cell help afforded by CHCD4 included contact-dependent and soluble components. Contact-dependent help was strongly inhibited by mAb against CD40L (5C8) and to a lesser extent, by anti-TNF-alpha Ab. The CD40L-dependent helper function of CHCD4 contrasts with the recent description of other HVS-transformed CD4+ T cells that provide B cell help primarily via the membrane TNF-alpha and TNF-alphaR pathways. Furthermore, CHCD4 cells also secreted soluble factors that could mediate CD40-linked B cell differentiation into Ab-producing cells. Interestingly, this factor is not likely to be IL-2, IL-4, IL-6, IL-10, IL-15, TNF-alpha, or IFN-gamma as Abs against these cytokines were not able to inhibit the contact-independent B cell help by CHCD4. These results indicate that HVS-immortalization of CD4+ lymphocytes may produce T cell clones with a spectrum of important contact-dependent, as well as contact-independent, B cell helper function capacities.

A role for phosphatidylinositol 3-kinase in generating T cell help for B cell growth and differentiation.

Evidence supporting the importance of the 3-phosphoinositide signaling pathway in lymphocyte activation is rapidly accumulating. In our study, we assessed the effects of two PI 3-kinase inhibitors, wortmannin and LY294002, on T cells as a means to analyze the role of the PI 3-kinase-signaling pathway in the generation of T cell help for B cell growth and differentiation. For these studies, B cells were cocultured with CD3-activated mitomycin C-treated T cells to induce B cell responsiveness. Of interest, wortmannin or LY294002 pretreatment of the T cell population significantly inhibited T cell-dependent induction of B cell proliferation and differentiation. The failure of wortmannin-treated CD3-activated mitomycin C-treated T cells to provide help in driving the differentiation of B cells to Ig-secreting cells could not be corrected by the addition of exogenous IL-2. Further studies designed to elucidate the mechanism by which wortmannin-treated T cells failed to provide B cell help indicated that wortmannin and LY294002 significantly inhibited the induction of CD40 ligand and, to a lesser extent, intercellular adhesion molecule-1 expression. These results suggest that the PI 3-kinase-signaling pathway, or other wortmannin- and LY294002-sensitive pathways, may be important for the induction of expression of crucial interaction molecules, such as CD40 ligand, on T cells and thus indicates that D-3 phosphoinositides play a pivotal role in regulating T cell-dependent B cell activation.

Identification of distinct domains in CD40 involved in B7-1 induction or growth inhibition.

Binding of CD40 ligand to CD40 on a murine B lymphoma M12 induces B7-1 and inhibits cell growth. We have used M12 lymphomas transfected with wild-type and mutant human CD40 molecules to identify regions of the CD40 cytoplasmic tail involved in these signal transduction events. We find that threonine residues at positions 227 and 234 in the cytoplasmic domain play important role in B7-1 induction, but have lesser importance in CD40-mediated growth inhibition. In contrast, a deletion mutant with only six amino acids in the cytoplasmic tail retains some growth-inhibitory function, but has no detectable B7-1 induction capacity. We also find that cAMP synergizes with CD40 signaling to induce high level B7-1 induction, and that the synergistic signal through CD40 requires either T227 or T234, but not both. Thus, we provide evidence for at least two distinct signaling domains in the CD40 molecule.

Engagement of the adhesion receptor CD22 triggers a potent stimulatory signal for B cells and blocking CD22/CD22L interactions impairs T-cell proliferation.

The B-lymphocyte-restricted adhesion protein CD22 mediates sialic acid-dependent cell-cell interactions. Engagement of CD22 on B lymphocytes with a CD22 monoclonal antibody (MoAb) HB22.7 that blocks the binding of CD22 to its ligand(s) directly stimulated B-cell proliferation. In addition, the HB22.7 MoAb costimulated B-cell proliferation with either anti-IgM, interleukin-2 (IL-2), IL-4, or CD40 and triggered predominantly B-cell IgG secretion with IL-2. Even more striking levels of B-cell proliferation occurred with HB22.7 MoAb under culture conditions that enhanced B-B-cell interactions. In contrast, a nonblocking CD22 MoAb (CD22.5) poorly costimulated in similar experiments. The functional differences between the two antibodies likely result from differing abilities to trigger downstream signaling events as significant differences in CD22 tyrosine phosphorylation and the recruitment of the tyrosine kinase p53/56lyn and the tyrosine phosphatase SH-PTP1C were found. Besides their role in B-cell stimulation, CD22/CD22L interactions may also assist in regulating T-cell proliferation because inhibition of CD22-CD22L engagement with the HB22.7 MoAb impaired T-cell proliferation in a costimulatory assay. Thus, CD22/CD22L interactions result in stimulatory signals for both B and T lymphocytes.

The role of gp39 (CD40L) in immunity.

The function of CD40 and its ligand (CD40L;gp39) has provided a molecular understanding for cognate interactions and the basis for hyper IgM syndrome. Studies using an antibody that neutralizes gp39 function in vivo have demonstrated that interactions between CD40 and gp39 are essential for primary and secondary thymus-dependent humoral immune responses, as well as for the generation of memory B cells. In addition, gp39 is critical for B cells to upregulate important costimulatory molecules and acquire professional antigen-presenting cell status. If B cells do not receive the gp39 signal, they are rendered tolerogenic for T cells. Finally, gp39-CD40 interactions are important in thymic education since blockade of this interaction interrupts negative selection.

Th1/Th2 cross regulation.

We present and analyze a model for the cross-regulation of the Th1 and Th2 helper cell subsets during an immune response by the regulatory cytokines interferon-gamma (IFN)-gamma) and interleukin-10 (IL-10). IFN-gamma, secreted by Th1 cells, can inhibit the proliferation of Th2 cells. Interleukin-10, secreted by Th2 cells, inhibits cytokine production by Th1 cells. Based on these properties, the model shows that responses are expected to be dominated by either Th1 cells or Th2 cells but not both. Which type dominates is shown to depend principally on the relative efficiencies of activation of the responding Th1 and Th2 cells. However, our model, as well as numerous experiments, show that perturbations of the system allow one to switch from a Th2 to a Th1 response, or vice versa. Our model can account for observed outcomes of parasitic infection and may also contribute to our understanding of immune responses to HIV infection as well as to tolerance to self components. It also predicts that in certain parameter ranges vaccination with low doses of live parasites can provide protection against subsequent encounters with high doses that normally induce disease. Experiments by Bretscher et al. (1992, Science 257, 539) on Leishmania major infection are consistent with this prediction. A similar strategy may also be relevant for the design of an AIDS vaccine. Lastly, our results indicate that Th1/Th2 cross-regulation is capable of generating a "sneaking through" phenomenon, and hence it may play a role in tumor immunity.

B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation.

Small dense B cells are stimulated to proliferate by membranes prepared from activated helper T (Th) cell clones. In combination with Th2 lymphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th membrane requires the expression of CD40 ligand by the T cells, and initiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of the B cell response and found that Th membranes stimulated B cell proliferation and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of division rounds between day 2 and 5 of culture. IgM-secreting cells appeared in culture by day 3 and increased to reach a maximum, comprised of 30% of the viable cells, on day 5. IgG1-secreting cells appeared 12-24 h after IgM-secreting cells, and IgE-secreting cells did not appear until day 5. These data are consistent with a sequential model of isotype switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior to and during B cell proliferation for differentiation to Ig-secreting cells to proceed. This period corresponded closely to the time of maximum DNA synthesis. Consistent with sequential switching of Ig isotypes, differentiation to IgM secretion required the shortest exposure to lymphokines and IgE the longest. These experiments strongly suggest that the lymphokine-induced commitment to differentiate is made before DNA synthesis begins, although the lymphokine-delivered signals are necessary during DNA synthesis to support Ig class switching.

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.

Current concepts of B cell modulation.

Three interleukins with distinct functions, IL-4, IL-5 and IL-6, are involved in the regulation of B cell response into antibody producing cells. The studies with recombinant interleukins, however, demonstrated that the activities of these interleukins were not restricted to B lineage cells but showed a wide variety of biological functions on various tissues and cells. One of the most typical example of multifunctional interleukins is IL-6. It acts not only on B cells as B cell differentiation factor but also on T cells, hematopoietic stem cells, hepatocytes, nerve cells and myeloma cells. Deregulation of the expression of these interleukins was shown to be responsible for various diseases, such as i) IL-4 vs. immediate type hypersensitivity and ii) IL-6 vs. autoimmunity and multiple myelomas.