Human eosinophils modulate peripheral blood mononuclear cell response to Schistosoma mansoni adult worm antigen in vitro.

High numbers of eosinophils are observed in parasitic infections and allergic diseases, where they are proposed to be terminally differentiated effector cells that play beneficial role in host defence, or cause harmful inflammatory response. Eosinophils have been associated with killing of schistosomulae in vitro, but there is growing evidence that eosinophils can play additional immuno-regulatory role. Here, we report results of a study that examines peripheral blood mononuclear cell (PBMC) cytokine responses to Schistosoma mansoni adult worm antigen (SWA) when stimulated alone or enriched with autologous eosinophils. Production of the Th-2 type cytokines interleukin (IL)-4, IL-5 and IL-13 was lower (P = 0·017, 0·018 and <0·001, respectively) in PBMC + eosinophil cultures than in PBMC-only cultures stimulated with SWA. Substantial levels of IL-13, IL-10, interferon gamma and tumour necrosis factor alpha were recorded in cultures of eosinophils, but none of these cytokines showed significant association with the observed eosinophil-induced drop in cytokine responses of PBMC. Transwell experiments suggested that the observed effect is due to soluble mediators that downmodulate production of Th-2 type cytokines. This study shows that eosinophils may down-modulate schistosome-specific Th-2 type cytokine responses in S. mansoni-infected individuals. The mechanism of this immune modulation remains to be elucidated.

Junctional biases in the naive TCR repertoire control the CTL response to an immunodominant determinant of HSV-1.

The enormous diversity of the T cell pool makes it difficult to determine whether inherent biases in the naive TCR repertoire can influence T cell responsiveness. In C57BL/6 mice the cytotoxic T lymphocyte response to an immunodominant HSV-1 determinant (gB) is characterized by a prominent bias in Vbeta element usage, associated with a conserved and preferentially D element-encoded CDR3 sequence. Comparison of naive and gB-specific T cell populations revealed a similar enrichment of germline D element-encoded CDR3 sequences in the preimmune repertoire. Strikingly, eliminating the germline coding of the gB-specific CDR3 sequence caused an almost complete loss of the dominant subset of gB-specific T cells, illustrating that CDR3 biases can significantly alter both the composition and strength of an immune response.

Herpes simplex virus type 1-specific cytotoxic T-lymphocyte arming occurs within lymph nodes draining the site of cutaneous infection.

Various studies have shown that major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL) can be isolated from lymph nodes draining sites of cutaneous infection with herpes simplex virus type 1 (HSV-1). Invariably, detection of this cytolytic activity appeared to require some level of in vitro culture of the isolated lymph node cells, usually for 3 days, in the absence of exogenous viral antigen. This in vitro "resting" period was thought to represent the phase during which committed CD8(+) T cells become "armed" killers after leaving the lymph nodes and prior to their entry into infected tissue as effector CTL. In this study we reexamined the issue of CTL appearance in the HSV-1 immune response and found that cytolytic activity can be isolated directly from draining lymph nodes, although at levels considerably below those found after in vitro culture. By using T-cell receptor elements that represent effective markers for class I-restricted T cells specific for an immunodominant glycoprotein B (gB) determinant from HSV-1, we show that the increase in cytotoxicity apparent after in vitro culture closely mirrors the expansion of gB-specific CTL during the same period. Taken together, our results suggest that HSV-1-specific CTL priming does not appear to require any level of cytolytic machinery arming outside the lymph node compartment despite the absence of any detectable infection within that site.

Diminished secondary CTL response in draining lymph nodes on cutaneous challenge with herpes simplex virus.

We have shown that C57BL/6-derived CD8(+) CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved Vbeta10/junctional sequence combination. This extreme T cell receptor beta-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of Vbeta10(+)CD8(+) CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of Vbeta10(+)CD8(+) T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of Vbeta10(+)CD8(+) activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B.

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.

Antigen-specific CD8+ T cell subset distribution in lymph nodes draining the site of herpes simplex virus infection.

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8+ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB-specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB-specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen-specific subset that normally makes up a small proportion of the naive T cell repertoire.

TCR alpha-chain usage can determine antigen-selected TCR beta-chain repertoire diversity.

There is considerable variation in the TCR repertoire diversity selected by different peptide Ags. Certain responses show limited V region bias with minimal restrictions in the remainder of the sequence while others can be dominated by a single TCR clonotype repeatedly isolated from different individuals. CTL specific for a Kb-restricted determinant from the herpes simplex virus glycoprotein B (gB) preferentially express a dominant TCRBV10 beta-chain subset with extensive conservation located at the V-D junction. However, unlike some biased responses, no single beta-chain V-D-J combination appears to dominate these CTL. Different animals respond with a large array of unique or "private" beta-chain sequences with little J region preference. Here we examine the contribution of the TCR alpha-chain to the gB-specific CTL diversity. The TCR alpha-chains from different TCRBV10-positive gB-specific CTL clones were found to exhibit extensive sequence variation. However, when T cells were forced to use a single alpha-chain in TCR alpha-chain transgenic mice, gB-specific CTL showed limited variation in their beta-chain selection. These T cells retained the TCRBV10 bias but were now dominated by a single beta-chain sequence that could be repeatedly isolated from different transgenic animals. This "public" TCR consisted of the transgenic alpha-chain and a common TCRBV10D2J2S6 beta-chain. These results suggest that preferential use of one TCR subunit can restrict the level of diversity in the other chain due to interchain interactions involving J-derived sequences.

Characterization of diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V beta bias.

The glycoprotein B (gB) from herpes simplex virus type I is a major target of cytotoxic T lymphocytes (CTL) in C57BL/6 mice. The majority of these T cells are directed to a single Kb-restricted determinant, gB498-505. We have analyzed the T-cell receptor (TCR) usage in gB-specific CTL lines derived shortly after virus infection. The CTL populations preferentially used two V beta regions, a dominant V beta 10 element and a subdominant V beta 8 element. Detailed sequence analysis revealed considerable TCR beta-chain heterogeneity despite a striking level of predicted amino acid conservation at the V beta-D beta junction. This junction forms part of the third hypervariable loop of the TCR thought to directly contact the major histocompatibility complex-bound antigenic peptide. The results reveal considerable diversity within the primary T cells responding to a single viral determinant while still maintaining a high degree of TCR V beta bias and sequence conservation at the V-D-J junction.

Expression of two T cell receptor alpha chains on the surface of normal murine T cells.

We have previously reported that a subset of T cells in T cell receptor (TCR)-transgenic mice may express two different alpha chains on their surface. The expression of two functional alpha chains has also been demonstrated for human peripheral blood T cells. In this report, we show that a proportion of normal murine lymph node T cells express two functional alpha chains on their surface. The extrapolated frequency of these cells present in the normal repertoire ranges from 7-21%, with an average of 15%. Our analysis of a small number of antigen-specific T cell clones suggests that the frequency of antigen-responsive cells expressing two surface alpha chains is relatively low. This raises the possibility that dual alpha chain T cells may have a selective disadvantage in responding to specific antigen.

Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I-bound determinant.

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of V beta 5.2+TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA257-264 determinant, suggesting that the TCR alpha-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.