Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model.

Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.

Deletion of idiotype (Id)-specific T cells in multiple myeloma.

Mycloma cells secrete monoclonal immunoglobulin (Ig), called myeloma protein. The variable (V) regions of myeloma proteins are unique to each plasma cell tumor, and therefore contain highly tumor-specific antigenic determinants called idiotopes (Id). T cells with specificity for Id are thought to be of importance in eradication of multiple myeloma. In ongoing clinical trials, myeloma patients are vaccinated against the Id of their own myeloma protein, with the aim of inducing Id-specific T cells. However, this strategy will only succeed if Id-specific T cells are present in patients, and are able to respond. In an experimental animal model, we have shown that [d-specific T cells become progressively deleted as the myeloma protein serum concentration exceeds 50 microg/ml. This indicates that the ability of multiple myeloma patients to respond to Id-vaccination might be seriously handicapped. We suggest that Id-vaccination should be reserved for eradication of minimal residual disease, e.g. after high-dose chemotherapy and stem-cell transplantation.

Resting small B cells present endogenous immunoglobulin variable-region determinants to idiotope-specific CD4(+) T cells in vivo.

Antigenic determinants localized within the highly diversified V-regions of Ig are called idiotopes (Id). Processed Id-peptides can be presented on MHC class II molecules to CD4(+) T cells. If B cells present their endogenous Id-peptides, T cell activation could occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. To test this idea, we used mice made transgenic for a lambda2 L-chain (Id(+) mice). Another transgenic mouse strain expresses TCR transgenes with specificity for the Id (lambda2), presented on MHC class II molecules. When highly purified sorted Id(+) B cells and Id-specific T cells were sequentially injected into MHC syngeneic SCID host, T cell became blastoid, CD69(+) and proliferated. To exclude any role of host APC, MHC incompatible Rag2(- / -) mice (H-2(b)) were used as recipients for the Id(+) B and Id-specific T cells, with similar results. Exposure to extracellular Id(+) immunoglobulin (Ig) was not sufficient for Id priming of B cells in vivo, highlighting the preferential presentation of Id peptides derived from endogenous Ig, by B cells. The results suggest that B cells presenting Id self-peptides generated by V(D)J recombinations or somatic mutations may directly stimulate T cell in vivo in the absence of conventional antigen.

Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells.

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.

Transient overexpression of CD4 enhances allelic exclusion of T-cell receptor (TCR) alpha chains and promotes positive selection of class II-restricted TCR-transgenic thymocytes.

CD4 contributes to antigen recognition of T cells by binding to class II MHC molecules. There is heterogeneity in expression of CD4 coreceptor among CD4+CD8+ thymocytes. We have investigated whether the expression level of coreceptor influences positive selection. Thymocytes of mice expressing transgenic lambda2(315)-Ig-light-chain/I-Ed specific TCR are poorly positively selected because they fail to allelically exclude endogenous TCR alpha chain genes and because there is no skewing towards CD4. Transient overexpression of CD4 during thymocyte development, in mice transgenic for both TCR and CD4, resulted in skewing towards CD4 in the periphery, reduced rearrangement and expression of endogenous alpha-chains, and decreased levels of thymocyte RAG-1 transcripts. Kinetic BrdU labeling experiments showed that single CD4+ thymocytes developed faster, representing the predominant population even in the cortex of the double transgenic thymi. These results demonstrate that increased coreceptor expression can compensate for poorly selectable TCR, supporting avidity and instructional models for positive selection of thymocytes.

Dual T cell receptor T cells have a decreased sensitivity to physiological ligands due to reduced density of each T cell receptor.

A considerable fraction of T cells express two distinct T cell receptors (TCR), mainly due to expression of two TCR alpha chains. It has been suggested that such dual-TCR cells could have a role in autoimmunity. However, as such cells express less of each TCR, they could be less sensitive to their physiological ligand, i.e. peptide plus major histocompatibility complex molecules (MHC). We tested this hypothesis in a transgenic TCR model in which most T cells express different amounts of the transgene-encoded TCR, due to expression of endogenous TCR alpha chains. Five Th1 clones derived from lambda2(315) immunoglobulin light chain-specific TCR-transgenic mice expressed different levels of the transgene-encoded TCR, ranging from approximately 10,000 to approximately 50,000 TCR per cell. Cytosolic Ca2+ mobilization in single T cells from these clones elicited by lambda2(315) peptide-pulsed, I-Ed-expressing antigen-presenting cells, correlated linearly with the relative transgene-encoded TCR expression. The peptide requirement for half-maximal T cell proliferation showed a similar correlation, with low TCR levels requiring higher peptide concentration. Corroborative evidence was obtained by deployment of short-term polyclonal CD4+ lines from TCR-transgenic mice. Such lines had reduced early (Ca2+ mobilization) and late (lymphokine and proliferation) responses, compared with T cell lines from recombination-deficient TCR-transgenic severe combined immunodeficiency mice (which express only a single transgene-encoded TCR). Taken together, the Ca2+ responses increase gradually with increasing TCR expression per cell, similar to the previously described analog Ca2+ signaling elicited by increasing amounts of peptide/MHC [Røtnes et al., Eur. J. Immunol. 1994. 24: 851]. Surprisingly small reductions in TCR expression per cell reduce T cell responsiveness. This suggests that dual-TCR T cells are immunologically less effective than single-TCR T cells.

T cells with two Tcrbeta chains and reactivity to both MHC/idiotypic peptide and superantigen.

It is thought that Tcrbeta genes are effectively allelically excluded, while Tcralpha genes are not. We report here that endogenous Tcrbeta genes are expressed on as much as 5-7% of CD4+ T cells in 8-week-old Tcralphabeta-transgenic mice. In this model, the transgenic Tcr recognizes residues 91 - 101 of a lambda2(315) Ig light chain, presented on the I-Ed class II molecule. From such mice, a CD4+ T cell clone was isolated which not only responded to lambda2(315), but also mobilized Ca2+ and proliferated in response to Mls-1a. (Inadvertently we found that the C3H/Tif substrain, in contrast to C3H/HeJ, is Mls-1a positive.) The clone expressed the transgenic Tcr (Valpha1 and Vbeta8.2), and in addition an endogenous Vbeta6 chain, conferring the Mls-1a reactivity. On the population level, 1-2% of Tcr-transgenic lymph node cells displayed Vbeta6, in addition to the transgenic beta-chain. Such dual Tcrbeta expressor cells could be preferentially expanded in vitro by first stimulating with DBA/2 spleen cells and then with lambda2(315)-pulsed BALB/c antigen-presenting cells. In addition to demonstrating that allelic exclusion of Tcrbeta-chain genes is substantial but not complete in this model, the data show that the double beta-chain expressors can have two different specificities, and be signaled through both receptors, by physiological ligands. However, such dual-Tcr T cells appear to have reduced sensitivity to ligands, due to their decreased expression of each receptor. This holds true for both early (Ca2+ mobilization) and late (proliferation) T cells activation parameters. Dual Tcr cells may have a role in the pathogenesis of autoimmune diseases: If naive T cells are first stimulated by (infectious) superantigen, they could later, as activated T cells, respond to self-peptide/MHC on costimulation-deficient cells and cause autoimmunity. As a corollary, dual Tcr Id-specific T cells could, once activated, directly regulate Id+ B cells.

Preferential positive selection of T lymphocytes which express two different TCR alpha chains, an endogenous and a transgenic.

A hallmark of positive selection in T-cell receptor (TCR)-transgenic mice is a strong skewing towards the CD4+ or the CD8+ subset, depending on the class II or I restriction of the TCR, respectively. However, previous experiments in TCR transgenic mice specific for an Ig light chain (lambda 2(315)/I-Ed class II molecule did not fit into this scheme because the authors observed an anomalous skewing towards CD8. In this paper the authors show that endogenous TCR alpha chains are expressed on > 90% of CD4+ and CD8+ cells in this particular transgenic strain, even on a selecting H-2d haplotype. Endogenous TCR alpha chains are first detected when double-positive thymocytes down-regulate either CD4 or CD8. Endogenous V alpha seems to influence generation of T-cell subsets because CD4+ and CD8+ cells express different frequencies of endogenous V alpha 2 and V alpha 8. In the absence of endogenous TCR alpha chains in recombination-deficient TCR-transgenic severe combined immunodeficiency (SCID) mice, a strong skewing towards CD4+ T cells is seen, but such mice are severely T-cell deficient. As an explanation for these results, the authors suggest that the transgenic TCR has a too low affinity for efficient positive selection, therefore, TCR alpha gene rearrangements proceed. Endogenous TCR alpha paired with transgenic TCR beta could bind to class I or class II molecules, enhance positive selection and thereby production of CD4+ or CD8+ cells. Most of the 'mismatched' CD8+ cells are lambda 2(315)-specific and I-Ed class II restricted, and may function as idiotype-specific suppressors of B cells. These results may help explain the origin of dual TCR alpha T cells. Furthermore, the authors suggest that T cells 'mismatched' for co-receptor/TCR MHC-specificity may be enriched among dual TCR alpha T cells.