Darcy B. Wilson

Origin of immunoreactive lymphocytes in rats

Abstract The results of these studies show that in rats both thymus-derived and bone marrow-derived lymphocytes are required for the production of anti-SRBC immunoglobulin, and that the antibody-forming cells are derived from the bone marrow. On the other hand, bone marrow-derived cells are apparently not required for reactivity in mixed lymphocyte interaction (MLI) and approximately 90% of the cells reactive to histocompatability antigens in the MLI or to phytohemagglutinin are thymus-derived.

LYMPHOCYTES AND TRANSPLANTATION IMMUNITY.

Publisher Summary This chapter discusses different aspects of transplantation immunity and presents complete review of the lymphocyte and its role in different types of immunological reactions in experimental animals and in in vitro systems. The lymphoid tissues of a normal mammal constitute about 1% of its body weight and the predominant cellular component is the “small” lymphocyte. Lymphocytes serve as trephocytes and stem cells and even regulators of tissue growth and size. On the basis of their morphology and size, lymphocytes are subdivided into “large,” “medium,” and “small” varieties. The majority of the lymphocytes present within the lymphoid tissues as well as in the blood and lymph are of the “small” variety. The large and medium lymphocyte categories represent a much more heterogeneous population of cells, some of which possess a strongly basophilic cytoplasm. Electron microscope studies of the ultrastructure of these cells reveals two types, both distinguishable from small lymphocytes on the basis of their cytoplasmic organelles and their more abundant cytoplasm. Among the characteristics of small lymphocytes, their ubiquity, mobility, and longevity are desirable attributes of any cell type the role of which is to participate in the recognition, effector, and memory aspects of immunological phenomena. The recirculating small lymphocytes, rather than the sessile lymphoid cell population, play an important role in the primary response of rats to skin homografts.

Results of a phase I clinical trial of a T-cell receptor vaccine in patients with multiple sclerosis. II. Comparative analysis of TCR utilization in CSF T-cell populations before and after vaccination with a TCRVβ6 CDR2 peptide

We report here the results of a phase I trial of a T-cell receptor (TCR) V beta 6 CDR2 region peptide vaccine in 10 patients with multiple sclerosis who showed biased over-representations of V beta 6 mRNA among T-cells in their cerebrospinal fluids (CSF). One group of 5 patients was immunized twice during a four week period with 100 micrograms of the TCRV beta 6 peptide 39-LGQGPEF LTYFQNEAQLEKS-58 emulsified in incomplete Freunds adjuvant (IFA); the second group of 5 MS patients received 300 micrograms of the same peptide in IFA over a similar time period. Patients were monitored for adverse events, immunogenicity of the peptide and changes in their CSF T-cell populations. The results indicate that this peptide was immunogenic (T-cell proliferation assays and recall DTH responses) in some of the patients, although none of the immunized patients produced detectable anti-peptide antibodies. More importantly, we show that the 5 patients treated with higher doses of the vaccine displayed a slight decrease in CSF cellularity, a lack of growth of CSF cells in cytokine supplemented expansion cultures that implies a significant absence of a subset of activated CD4 T-cells and a marked diminution in V beta 6 mRNA levels among T-cells in these cultures. By comparison, in 5 patients receiving the lower dosage of the vaccine, CSF cellularity was the same or slightly increased over pre-vaccination levels, CSF cells from 1 patient failed to grow in expansion cultures and cultured CSF cells from 2 patients underwent a change from an oligoclonal V beta 6 pattern to one that was more polyclonal. These results justify a more through exploration of the use of TCR peptide vaccines as a possible therapeutic treatment for MS.

Results of a phase I clinical trial of a T-cell receptor peptide vaccine in patients with multiple sclerosis. I. Analysis of T-cell receptor utilization in CSF cell populations

To identify a panel of multiple sclerosis patients (MS) for a phase I clinical trial of a T-cell receptor (TCR) peptide vaccine we characterized the T-cell populations present in the cerebrospinal fluids (CSF) of a large group of patients with respect to surface phenotype and state of activation, TCR beta chain utilization, features of the CDR3 junctional region, the extent of clonality and persistence of selected clonotypes over time. These CSF cell populations consist of approximately 60% CD4+ T-cells, half of which bear IL-2 receptors, indicating these activated T-cells may be part of the pathogenic process in MS. When these activated CD4+ T-cells were selectively expanded in IL-2/IL-4 supplemented cultures, an over-representation of several TCRV beta families was noted in 39/47 patients, the most frequent being V beta 6.5, V beta 6.7, V beta 2, V beta 5 and V beta 4. Biased expression of various members of the V beta 6 family was seen in 21 of this group of 39 patients. Clonal analysis of TCR beta 6 CDR3 sequences, revealed two notable features: clonal dominance and clonal persistence. CSF cells from two-thirds of MS patients contained a dominant clone comprising 50% or more of sequences and the same patient-specific clone could be shown to persist for up to 18 months. This clonal prevalence and over representation of V beta 6+TCR raises the possibility that immunization with a V beta 6 peptide vaccine may produce a regulatory immune response leading to a clinical benefit.

Mixed Leukocyte Reactions and Histocompatibility in Rats

Mixed leukocyte culture tests have been carried out on two hetero geneous but genetically defined backcross populations of rats to determine whether the reactions observed can provide the basis for histocompatibility matching. The experiments were designed so that only-one-way reactions could occur. Only when the donors of individual leukocyte mixtures differed at the important Ag-B histocompatibility locus was there any in vitro reactivity, and differences at this locus were invariably associated with the prompt rejection of skin homografts. Determination of compatibility at this locus proved to be important in that it facilitated the prolongation of survival of skin homografts by immunosuppressive therapy.

Idiotypic regulation of T cells in graft-versus-host disease and autoimmunity.

As part of the theory of clonal selection, Burnet (Burnet 1959) proposed several years ago that self-reactive lymphocytes were both necessary and sufficient to cause autoimmune disease; these self-reactive clones were deemed forbidden and their elimination essential in order to avoid autoimmunity. However, as Cohen has pointed out earlier in these Reviews (Cohen 1986), the evidence is quite clear that substantial numbers of self-reactive cells do occur among the Tand B-cell repertoires of normal individuals, yet in most circumstances such individuals remain healthy without evidence of autoimmune disease. This finding forces the view that the underlying process leading to an autoimmune state is not the interaction between anti-self lymphocytic clones and self antigens, but rather how these ever-present self-reactive clones escape regulation or containment to become activated by self antigens. Current efforts to understand the basis for regulation of self-reactive clones and the failure of this regulatory process in autoimmunity now center around the very confusing phenomenology of suppressor cells on the one hand and, on the other, the possible role of regulatory cells predicted by Jerne in his network hypothesis (Jeme 1974) to have anti-idiotypic specificity for receptors of self-reactive clones. In this article we review our attempts over the past several years to understand the cellular mechanisms underlying three seemingly related models in which T cells with potent immunological reactivity to gene products of the major histocompatibility complex (MHC) can be specifically suppressed by a second Tcell population which seems to display idiotypic specificity for the receptors of MHC-reactive T cells. These models, studied extensively in rats, involve specifically induced resistance to graft-versus-host (GVH) disease, the inhibition of abolition of transplantation tolerance by adoptive transfer of syngeneic T cells.

Degeneracy and Repertoire of the Human HIV-1 Gag p1777–85 CTL Response

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db β2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer:Target cell interactions

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.

Mixed Lymphocyte Reactions and Tissue Transplantation Tolerance

The induction of tolerance of Lewis histocompatibility antigens in BN rats inoculated at birth with BNI Lewis F1 hybrid bone marrow cells, as revealed by the prolonged survival of Lewis skin grafts, is accompanied by markedly decreased reactivity in the mixed lymphocyte interaction. Blood lymphocytes from animals inoculated with lymph node cell suspensions also display diminished proliferative reactivity to hybrid BN/Lewis cells in the interaction. However, these recipients are not tolerant of Lewis skin grafts. Blood lymphocytes from BN rats inoculated neonatally with Lewis thymocytes fail to display any level of unresponsiveness in vitro, and such animals are not tolerant of Lewis skin grafts. The results suggest that in rats skin and marrow cells have histocompatibility antigens that are absent or poorly expressed on lymph node cells and thymocytes.

Inhibition of cell-mediated lympholysis by cloned and uncloned lines of natural killer cells and cytotoxic T lymphocytes with sugars and lectins☆

This study was designed to further understand the nature of the interaction between natural killer (NK) cells and their susceptible targets. To do this, a panel of sugars and two lectins was tested for the ability to inhibit the lysis of NK-sensitive targets by cloned and uncloned lines of human NK cells. Six of these sugars (beta-gentiobiose, sucrose, alpha-lactose, beta-lactose, N-acetylglucosamine, and N-acetylgalactosamine) and one lectin, wheat germ agglutinin (WGA), proved to be potent inhibitors of the lytic activity of NK cells as well as of cytotoxic T lymphocytes activated in mixed lymphocyte cultures. Both beta-gentiobiose and WGA were shown to inhibit lysis at the level of the killer cell. Finally, the inhibitory effect of WGA could be reversed by addition of its sugar ligand, N-acetylglucosamine, which is itself an inhibitor of lytic function. From these findings it is concluded that these inhibitors probably do not act at the recognition stage of lysis since all of the NK and CTL lines tested, regardless of specificity, were inhibited by the same panel of sugars and lectins. Instead, it appears more likely that these inhibitors block some postrecognition stage of the lytic mechanism. The common inhibition profile by these sugars on NK and CTL activity further suggests that these two cell types may share, at least partially, a common lytic mechanism.