Fidelity of the repertoire in T cell reconstituted athymic nude rats. Preservation of a deficit in alloresponsiveness over one year.

A single intravenous injection of a relatively small number of T cells contained in the population of rat thoracic duct lymphocytes (TDL) is sufficient to restore to normal the peripheral T cell pool of athymic PVG.rnu/rnu nude rats. The donor T cells expand greater than 10-15-fold, self-renew, and restore immunocompetency to nude recipients permanently (greater than 2 yr). We asked whether the T cell repertoire was affected by the expansion and self-renewal process. Nude recipients were injected with syngeneic PVG TDL that had been allospecifically depleted (negatively selected) by consecutive passage from blood to thoracic duct lymph through two irradiated (DAxPVG)F1 intermediate rats. Negatively selected TDL were tested before transfer by the P----F1 popliteal LN GVH assay and showed a greater than 90% depletion of specific reactivity to DA alloantigens. Surviving cells or their progeny were recovered from LN or TDL of nude recipients 8 and 12 mo after transfer. The deficit in GVH reactivity to the DA haplotype persisted, but normal GVH activity was demonstrated against a third party (AOxPVG)F1 alloantigen. The "hole" in the repertoire could not be attributed to tolerance induced by the co-transfer of contaminating irradiated F1 TDL. PVG TDL passaged consecutively through (AOxPVG)F1 and (DAxPVG)F1 intermediates and devoid of (AOxPVG)F1 cells remained specifically depleted to both AO and DA haplotypes when recovered from nude recipients 4 and 13 mo later, but displayed GVH activity to a third-party (BNxPVG)F1 alloantigen. Thus the exact specificity of the T cell repertoire of the original inoculum was faithfully maintained in nude recipients throughout the initial phase of rapid expansion and the continued self-renewal of the mature peripheral T cell pool.

Factors influencing the fate of 111indium-labelled lymphocytes after transfer to syngeneic rats.

Thoracic duct lymphocytes were labelled in vitro with 111indium-oxine or 111indium-acetylacetone in order to follow their migration after i.v. injection into syngeneic rats. Under certain conditions both preparations produced results with quantitatively confirmed data obtained by other approaches to the physiological pattern of lymphocyte recirculation. However, three significant difficulties were identified: (1) chemical toxicity by minor contaminants of the preparation; (2) radiation damage indicated by a progressive impairment of the recovery of radioactivity from lymph nodes. A labelling concentration of 20 microCi/10(8) cells was the highest compatible with survival of most lymphocytes for 24 h in vivo as confirmed by autoradiography; (3) rapid loss of 111In in vivo found at labelling concentrations below 1 microCi/10(8) cells. By one week after the injection of lymphocytes labelled at 20 Micro/Ci/10(8) cells most of the 111In had been transferred from lymphocytes to non-recirculating radioresistant cells within the spleen and lymph nodes.

Major histocompatibility complex control of NK-related allogeneic lymphocyte cytotoxicity in rats. The contributions of strong and medial transplantation antigens.

Allogeneic lymphocyte cytotoxicity (ALC) describes the elimination of allogeneic lymphocytes in vivo by an NK-related activity. There is evidence that ALC is demonstrable between donor and recipient when these are incompatible at MHC gene loci alone. Since ALC is a property of T cell-deficient nude rats, the role of the MHC in this rejection process needs further study. We have determined the contribution of the MHC to ALC using congenic and recombinant rats. In our analysis we have assumed that ALC involves the recognition of classic alloantigens by clonally distributed effector cells as for other examples of transplant rejection, although this is not yet proved. Strong ALC was measured between congenic rats that differed for MHC genes only. Non-MHC incompatibility alone did not elicit ALC. In the presence of MHC incompatibility the strength of ALC generated in a recipient was dependent on non-MHC genes. The PVG background generated high ALC responses whereas ALC was not measured in the DA rat. However ALC was measured in the congenic PVG-RT1avl (DA) rat. The contributions of classic class I (RT1.A), class II (RT1.B/D), and medial transplantation (RT1.C) regions of the rat MHC were determined by comparing different recombinant donors into the same recipient strain. Single region differences alone in any of these three MHC regions did not elicit full ALC. In two sets of transfers a combination of RT1.B/D and RT1.C region incompatibility was sufficient to generate a full allogeneic response. It can be concluded that the controlling element for allogeneic lymphocyte cytotoxicity is in the RT1.B/D-RT1.C region of the rat MHC.

Allograft rejection in athymic nude rats by transferred T cell subsets. II. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to an isolated MHC class I disparity.

Athymic PVG-rnu/rnu (RT1c) rats were grafted with skin bearing isolated MHC disparities 7-14 days in advance of cell transfer. The ability of naive CD4+ or CD8+ thoracic duct lymphocytes to induce rejection was assessed by adoptive transfer of one or both T cell subsets into nude recipients bearing congenic PVG.r1 (MHC class I-only disparity, Aa) or PVG.r19 (class I and II-only disparity, Aa B/Da) skin grafts. Recipients of purified CD4+ TDL always rejected r19 allografts, whereas CD8+ TDL were ineffective against this class I + II difference. Neither the injection of CD4+ TDL nor CD8+ TDL alone resulted in destruction of r1 skin grafts. However, rejection of r1 tissue was observed in 63% of cases (19/30) when both CD4+ and CD8+ TDL were present in the nude recipients. Rejection of r1 skin was also induced in some recipients when CD8+ TDL were transferred 8 weeks in advance of CD4+ TDL. In contrast, sequential transfer in the reverse order apparently induced tolerance in the CD4+ population--i.e., surviving r1 skin grafts on 8 week CD4+ T cell-reconstituted nude recipients were not rejected following the subsequent transfer of CD8+ TDL. We conclude that CD4+ T cells were required for rejection of both class I and class II differences. In the presence of a class II difference, CD4+ T cells function autonomously to initiate both the inducer and effector stages of rejection. When the disparity is confined to class I, CD8+ T cells are required (probably at the effector stage) but are dependent on CD4+ T cells for help. There was no evidence of CD4+ effector T cells that could recognize class I directly within the graft.

A comparison of immune responses against AG-B and non-AG-B antigens, presented alone or together.

By exploiting congenic rat strains (HO.B2 and PVG/c) cell-mediated immune responses against Ag-B antigens alone were measured and compared with responses against (1) non-Ag-B antigens and (2) Ag-B and non-Ag-B antigens in combination. It was confirmed that multiple non-Ag-B antigens provoke prompt first-set skin graft rejections, but are much weaker than Ag-B antigens in stimulating both graft-versus-host (GVH) and cytotoxic activity. No evidence of synergistic interaction was found between anti-Ag-B and anti-non-Ag-B responses either by GVH assay or in the generation of cytotoxic cells. Specific partitioning of cytotoxic cells on antigenic monolayers suggested that cytotoxic cells on antigenic monolayers suggested that cytotoxicity is predominantly directed against Ag-B antigens. The measurements of GVH activity consolidate previous work, which suggested that 4.5 to 12% of nonimmune T cells can respond to each Ag-B determined antigenic complex and eliminate the possibility that most of these cells were responding to non-Ag-B antigens. Two principles for measuring GVH activity were compared: (1) 3H-thymidine incorporation into donor lymphocytes at 24 hr after transfer to irradiated F1 hybrid recipients and (2) the popliteal lymph node assay, which depends on a secondary phase of host cell proliferation.

The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen.

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (>or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

Membrane CD45R isoform exchange on CD4 T cells is rapid, frequent and dynamic in vivo.

CD4 T cells bearing high (240-190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC-) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC- CD4 T cells (depleted of immature Thy-1+ CD45RC- recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30-60% of CD45RC- CD4 T cells became CD45RC+; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC- state. CD45RC- CD4 T cells declined in number more rapidly than the CD45RC+ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC+) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.

Interconversion of CD45R subsets of CD4 T cells in vivo.

T lymphocytes express multiple forms of the leukocyte common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exons 4-6. Species-specific monoclonal antibodies against restricted epitopes (CD45R) of the antigen subdivide CD4 T cells into reciprocal subsets expressing either the high molecular weight isoforms CD45RA or RB or a molecule in which exons 4-6 have been spliced out (CD45R0). CD45R+ or RB+ CD4 T cells are potent in graft-versus-host reactions, and interleukin-2 related activities, whereas the CD45R0+ subset responds in vitro to recall antigens and provides help for antibody synthesis. It is unclear whether CD45R subsets derive from separate lineages, or are products of unidirectional or reversible differentiation. We show by transferring CD45R+ or CD45R- allotype-marked CD4 T cells into athymic nude rats that both subsets routinely generate cells of the opposite phenotype with a function that follows phenotype, not parentage. The recent equation of CD45R subsets as maturation stages representing 'naive' and 'memory' T cells is difficult to reconcile with this finding.

The peripheral T-cell pool: regulation by non-antigen induced proliferation?

Despite continuous perturbations, the recirculating lymphocyte pool remains relatively constant. Expansion of the pool is compensated for by cell loss. When the T-cell pool is in deficit, either from a congenital defect or an acquired immunodeficiency, T-cell numbers are restored by extrathymic division-a response that occurs without deliberate provocation. We have considered how the recirculating pool may be stably maintained and how a T-cell deficit might be restored to equilibrium. Recent evidence suggests that depleted T-cell compartments are replenished by CD4 T-cell proliferation in the absence of specific antigen, a response that occurs without engaging the T-cell receptor.

Lymphocyte trafficking: CD4 T cells with a 'memory' phenotype (CD45RC-) freely cross lymph node high endothelial venules in vivo.

Antigen encounter not only induces a change in surface expression of CD45RC isoforms in the rat from a high (CD45RC+) to a low molecular weight molecule (CD45RC-), but also stimulates changes in expression of adhesion molecules that regulate CD4 T-cell migration. T cells with an activated or 'memory' phenotype (CD45RC-) are thought to enter lymph nodes almost exclusively via afferent lymphatics whereas T cells in a resting state (CD45RC+) migrate across high endothelial venules (HEV). The present study monitored the rapid recirculation from blood to lymph of allotype-marked CD45RC T-cell subsets. Surprisingly, we found that CD45RC- CD4 T cells entered the thoracic duct slightly faster and reached peak numbers 3 hr earlier (18 hr) than did the CD45RC+ subset. To determine whether the entrance of CD45RC+ and RC- subsets was restricted to HEV and afferent lymphatics, respectively, recirculation of CD4 T cells was monitored in mesenteric lymphadenectomized (MLNx) rats (on healing the intestinal afferent lymphatics are joined directly to the thoracic duct), or in recipients that had had the mesenteric lymph node (MLN) acutely (2-3 hr) deafferentized (entry would be restricted to HEV). In these studies CD45RC- CD4 T cells entered the MLN across HEV on an equal basis with T cells expressing a CD45RC+ phenotype. Contrary to currently held dogma the results showed that, in vivo, CD4 T cells with a memory phenotype freely enter lymph nodes (LN) across HEV as well as via afferent lymphatics.

CD45R CD4 T cell subset-reconstituted nude rats: subset-dependent survival of recipients and bi-directional isoform switching.

High and low molecular weight variants (CD45R) of the leukocyte common antigen (CD45) divide CD4 T helper cells into subpopulations which display distinct characteristics. In vitro and in vivo evidence suggested that the presence of the high molecular weight splice variants CD45RA or CD45RB distinguished naive CD4 T cells from memory T cells which underwent an irreversible switch to the low molecular weight isoform as a consequence of antigen encounter. In the rat monoclonal antibody MRC OX22 identifies an epitope on the CD45RB molecule. We investigated this proposed differentiation pathway by reconstituting athymic nude rats with highly purified OX22+ or OX22- CD4 T lymphocyte subsets obtained from the thoracic duct (TDL) of euthymic, congenic, allotype-marked donors. Injection of CD4+CD45RB- (45R-) cells ensured long-term survival of nude recipients; recipients of CD4+CD45R+ (45R+) cells died within 2-3 months of injection. Early after transfer (3-4 weeks) the progeny of both 45R+ and 45R- TDL were uniformly 45R-. With time (by 2 months) progeny of both parental types expressed the high molecular weight CD45RB isoform. Nude recipients of 45R- TDL always generated progeny a proportion of which bore the 45R+ phenotype; 3 months to 2 years post-injection, 30%-50% of the donor-derived CD4 T cells were 45R+, 45R- progeny isolated from primary recipients of either 45R- or 45R+ cells transferred into secondary nude recipients induced skin allograft rejection with equal effectiveness and also generated 45R+ offspring. The results indicated that CD4 T cell subsets switched between CD45R isoforms and that the change between high and low molecular weight expression was bi-directional. The splice variants apparently are not lineage or maturation markers, but rather identify CD4 T cells that exist transiently in different functional states.

Allograft rejection in athymic nude rats by transferred T-cell subsets. I. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to complete allogeneic incompatibilities.

PVG.rnu/rnu nude rats were pre-grafted with two allogeneic skin grafts, AO(RTlu) and BN(RTln), 6-14 days in advance of cell transfer. Cellular requirements for rejection were established by transferring graded numbers of B cell-depleted (Ig-) thoracic duct lymphocytes (TDL) or purified W3/25+ (CD4+) or OX8+ (CD8+) TDL subsets. Allografts were rejected by 10(5) to 5 x 10(6) Ig- TDL in a dose-dependent fashion. A similar dose-response relationship was found by transferring 5 x 10(5) to 5 x 10(6) Ig- OX8- TDL (purified by depletion of B cells and OX8+ cells). Larger numbers of Ig- OX8- TDL (10-30 x 10(6)) did not significantly accelerate rejection. W3/25+ TDL alone (10(5)), highly purified by fluorescence-activated cell sorting (FACS), were sufficient to induce allograft rejection in this athymic nude rat model. In contrast, 10 times more FACS purified OX8+ TDL (10(6)) were unable to initiate skin graft rejection despite the complete class I and class II MHC incompatibilities. Furthermore, the addition of 10(6) OX8+ cells did not accelerate or retard the rejection induced by 10(5) W3/25+ cells alone. Pre-grafted nude recipients, irradiated (500 R) 2 hr before W3/25+ TDL injection, in order to eliminate putative nude T cells, rejected allografts on the same day as unirradiated controls. We conclude that when confronted with complete MHC disparities, CD4+ T cells are necessary and sufficient to induce skin allograft rejection whereas CD8+ T cells do not appear to contribute.

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via alpha 4 integrin-VCAM-1 interaction.

The present investigation examines the localization and migration of purified T cell subsets in comparison with B cells, CD8 T cells and CD4+ CD8- single-positive thymocytes. CD4 T cell subsets in the rat are defined by mAb MRC OX22 (anti-CD45RC), which distinguishes resting CD4 T cells (CD45RC+) from those (CD45RC-) which have encountered antigen in the recent past--subpopulations often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 T cells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to the spleen was followed by a redistribution to mesenteric lymph nodes (MLN) and cervical lymph nodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization of these subpopulations to the small or large intestine [Peyer's patches (PP) excluded]. In contrast, CD45RC- CD4 T cells (indistinguishable in size from the CD45RC+ subset) localized in large numbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for at least 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC- CD4 T cells in the MLN could be accounted for entirely by afferent drainage from the intestine. Unexpectedly, CD45RC- CD4 T cells (but not other subsets) localized and accumulated in the thymus. In vivo treatment with mAb HP2/1 against the integrin alpha 4 subunit inhibited almost entirely CD45RC- CD4 T cell migration into the PP (98.1%), intestine (87.1%), MLN (89.1%) and thymus (93.5%); migration into the CLN was only reduced by half. To distinguish between recognition of MAdCAM-1 and VCAM-1 by alpha 4-containing integrins, recipients were treated with mAb 5F10 against rat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC- CD4 T cells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC- CD4 T cells alone showed enhanced localization to the gut and PP, probably via alpha 4 beta 7-MAdCAM-1 interaction; (ii) that many CD45RC- cells entered non-mucosal LN independently of alpha 4 integrin or VCAM-1; and (iii) that entry of mature recirculating CD45RC- CD4 T cells into the thymus across thymic endothelium was apparently regulated by alpha 4 integrin-VCAM-1 interaction.

The migration of lymphocytes across specialized vascular endothelium. III. Concanavalin A delays lymphocytes in normal traffic areas.

Radioactively labelled rat lymphocytes were treated in vitro with concanavalin A (con A) and injected intravenously into syngeneic recipients. By examining the blood and tissues at intervals from 30 min to 48 h after injection, it was confirmed that con A altered the distribution of lymphocytes. Comparison was made with the localization of alternatively labelled untreated lymphocytes injected into the same recipients and with untreated lymphocytes injected into other recipients. Within 1 h of injection there was a surplus of treated cells in the lungs and liver and deficits (of equal magnitude) in the blood, spleen and lymphonodes. By 24 h after injection there was a twofold surplus of treated cells in the spleen but a persisting deficit in lymphnodes. These perturbations can be ascribed to the prolonged retention of lymphocytes in normal sites of localization; there is no evidence that con A either hinders the migration of lymphocytes from the blood or diverts them to abnormal sites. It is not clear whether the delay in the recipients' tissues requires an active response to con A but, if so, then it does not proceed to blastic transformation.

The effects of interferon on the recirculation of lymphocytes in the rat.

Lymphocytes from the thoracic duct (TDL) were incubated with interferon (IFN) prior to i.v. injection into syngeneic or allogeneic recipient rats. The effect of IFN treatment on the ability of lymphocytes to migrate was studied using 'standard' TDL collected overnight at 4 degrees or an 'optimal' collection of passaged TDL which recirculate with an accelerated tempo (Smith & Ford, 1983). Interferon treatment resulted in an increase in early (30 min) localization of both standard and optimal TDL into lymph nodes. Entry of standard IFN-treated TDL was increased by 91% and 54% in cervical and mesenteric lymph nodes, respectively; increases of 50% and 22% in the same lymph nodes were recorded for optimal IFN-treated TDL. Enhanced entry of standard TDL was contrasted with a reduced ability of IFN-treated TDL to migrate out of lymph nodes; there was a reduced output into the thoracic duct and a surplus of IFN-treated lymphocytes in cervical lymph nodes despite 24 hr continuous thoracic duct drainage. Incubation with interferon did not, however, alter the ability of optimal TDL to reach the thoracic duct rapidly after injection. Allogeneic lymphocytes, which are eliminated soon after injection by an NK-like cytotoxicity, a phenomenon termed ALC, were unaffected by incubation with interferon, thus IFN-treated allogeneic lymphocytes were killed after i.v. injection as rapidly as untreated cells.

The stable and permanent expansion of functional T lymphocytes in athymic nude rats after a single injection of mature T cells.

Athymic nude rats (PVG.rnu/rnu) were injected at 6 to 10 wk of age with 1 to 200 million thoracic duct lymphocytes (TDL) containing 40 to 60% mature T cells. Thereafter TDL-injected nude recipients were monitored for evidence of T cell function for up to 2 yr. W3/25+ T helper (Th) cells in lymph nodes (LN) increased from 7% at 2 wk to 30% at 8 wk after TDL transfer. The percent of W3/25+ cells remained elevated for the life of the recipient (up to 2 yr), approximating normal levels. The total size of the recirculating pool expanded in TDL-injected nude rats to reach 2/3 the level of euthymic controls by 16 wk, an increase of 10-fold to 15-fold in W3/25+ cells. The expansion of the W3/25+ population was independent of initial TDL dose. With time spleen and LN acquired a normal histological appearance including the development of germinal centres and a marked increase in cellularity in T cell traffic areas. TDL-injected nude rats rejected skin allografts with near normal kinetics. In addition graft vs host (GVH) responsiveness, assessed by the popliteal LN assay, progressively increased reaching a level 9 mo to 1 yr after replacement that resembled the GVH activity in euthymic controls.

The origin of T cells in permanently reconstituted old athymic nude rats. Analysis using chromosome or allotype markers.

Athymic PVG-rnu/rnu rats receiving a single intravenous injection of syngeneic euthymic thoracic duct lymphocytes (TDL) develop normal levels of CD4+ T lymphocytes and survive for more than 2 years in a conventional animal house. We investigated the origin of the T cells (and B cells) in reconstituted nude recipients by transferring TDL carrying either the 3T chromosome marker or the RT6b + Igk-1b allotype or the RT7b (leucocyte-common) allotype markers. Karyotype analysis of spleen and lymph node (LN) cells from 1- to 2-year-old PVG-3T/3T-reconstituted nude recipients, stimulated in vitro with phytohaemagglutinin (PHA), unexpectedly revealed that a majority (79-97%) of dividing cells were of nude origin. However, extensive nude cell division was also recorded in PHA-stimulated cultures using mixtures of euthymic (PVG-3T/3T) and unreconstituted nude spleen cells; the assumption that only T cells divide in PHA-stimulated cultures thus appears to be erroneous. In contrast to the karyotype analysis, sIg- RT6b+ LN cells obtained from nude recipients reconstituted 2 years earlier with PVG-RT6b allotype-marked TDL, were all of donor origin with no indication of a nude-derived sIg- RT6a+ population. Igk-1b+ donor B cells were not found in these same recipients. Dual fluorescence analysis of TDL from 18- to 20-month RT7b-reconstituted nudes showed that 91-100% of CD4+ cells were donor-derived. When tested functionally, sIg- RT7b+ (donor) cells, but not sIg- RT7b- (nude-derived) cells, were able to reject skin allografts and induce local graft-versus-host (GVH) responses. Donor T cells, in contrast to CD4+ cells of nude origin, divided extensively in nude recipients; FACS-purified RT7b+ (donor) TDL retransferred from 17-month primary reconstituted nude rats, expanded further (60-100-fold) in secondary nude recipients. In conclusion, only the donor-derived CD4+ cells in reconstituted nude rats displayed T-cell function; evidence to the contrary from karyotype analysis was flawed. At no stage in their life did uninjected or T-cell reconstituted nude rats develop endogenous cells that in any way resembled CD4+ products of the thymus.

Both CD45R(low) and CD45R(high) "revertant" CD4 memory T cells provide help for memory B cells.

During a primary response to thymus dependent antigens, B cells undergo a number of qualitative changes to become memory B cells - processes that require co-stimulatory signals and cytokine help from CD4 T cells. The question of whether distinct, antigen-experienced memory CD4 T cells are subsequently needed to program memory B cells into antibody synthesis has not been clearly resolved. Using an adoptive transfer model in which memory but not naive B cells were stimulated, we evaluated CD4 T cell help using lymphocytes obtained from primed or unprimed thymectomized donors and expressing a naive (CD45R(high)) or a memory (CD45R(low)) phenotype. Memory B cells, most of which were committed to the IgG1 (Th2) subclass, could be stimulated to produce antibody using help transferred by the CD45R(high) naive subset of unprimed donors (slow onset of response), the CD45R(low) subset of 7 day primed donors (large, rapid antibody response) or by both the CD45R(low) and the CD45R(high) "revertant" subsets of 6 month primed donors. We found that antigen primed CD45R(low) CD4 T cells reverted (defaulted) with time to a CD45R(high) resting state, a change that was prevented by persisting antigen. The evidence suggests that CD4 memory T cells are partitioned into two different functional states (CD45R(high) and CD45R(low)) and that these determine the characteristics of the memory B cell response in terms of speed, size and longevity.

CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endothelial venules: within the tissue their migratory behavior differs.

It is thought that naive T cells predominantly enter lymphoid organs such as lymph nodes (LN) and Peyer's patches (PP) via high endothelial venules (HEV), whereas memory T cells migrate mainly into non-lymphoid organs. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. Such knowledge would be of considerable interest for understanding T cell memory in vivo. In the present study we separated naive and memory CD4+ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEV, and T and B cell areas of different LN and PP. Three major findings emerged. First, both naive and memory CD4+ T cells enter lymphoid organs via the HEV in comparable numbers. Second, naive and memory CD4+ T cells migrate into the B cell area, although in small numbers and continuously enter established germinal centers (GC) with a bias for memory CD4+ T cells. Third, memory CD4+ T cells migrate faster through the T cell area of lymphoid organs than naive CD4+ T cells. Thus, our study shows that memory CD4+ T cells are not excluded from the HEV route. In addition, "memory" might depend in part on the ability of T cells to specifically enter the B cell area and GC and to screen large quantities of lymphoid tissues in a short time.

The migration of lymphocytes across specialized vascular endothelium. II. The contrasting consequences of treating lymphocytes with tryspin or neuraminidase.

Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues were measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, tryspin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.